Keratinocyte growth factor-2

专利摘要:
The present invention relates to newly identified polynucleotides, polypeptides encoded by the polynucleotides, uses of such polynucleotides and polypeptides, and the production of such polynucleotides and polypeptides. More specifically, the polypeptide of the present invention is a keratinocyte growth factor, sometimes referred to herein as " KGF-2 ", previously also known as fibroblast growth factor 12 (FGF-12). The present invention further relates to the therapeutic use of KGF-2 for promoting wound healing. The present invention also relates to a novel mutant KGF-2 form with increased activity, increased stability, higher yield or better solubility.
公开号:KR20020026517A
申请号:KR1020027000040
申请日:2000-07-03
公开日:2002-04-10
发明作者:스티븐 엠 루벤；파블로 지메네즈；디. 록산느 두안；마크 에이. 람피；돈나 멘드릭；준 창；지안 니；폴 에이. 무어；티모시 에이. 콜만；조아킴 알. 그루버；패트릭 제이. 딜론；라이너 엘. 겐츠
申请人:추후제출；휴먼 게놈 사이언시즈, 인크.；
IPC主号:

专利说明:

Keratinocyte growth factor-2 {KERATINOCYTE GROWTH FACTOR-2}
[2] Fibroblast growth factor classes have emerged as a major class of growth factors associated with soft tissue growth and regeneration. It now includes several classes that share varying degrees of homology at the protein level and have a similar broad cell division promoting spectrum except for one. I. E., Promote the proliferation of various cells of mesodermal and / or neurovegetal origin and / or promote angiogenesis.
[3] The expression patterns of the different classes are very different from extremely limited expression of any developmental stage to ubiquitous expression in various tissues and organs. All classes appear to bind heparin and heparin sulfate proteoglycans and glycosaminoglycans and are strongly concentrated in the extracellular matrix. KGF was initially found to belong to the FGF class by sequence homology or by factor purification and cloning. The keratinocyte growth factor (KGF) has been isolated as a mitogen of the cultured murine keratinocyte cell line (Rubin, JS et al., Proc. Natl. Acad. Sci. USA 86: 802-806 1989). Unlike other members of the FGF family, it is less active on mesenchymal-derived cells but promotes the growth of epidermal cells. The keratinocyte growth factor encodes a 194-amino acid polypeptide (Finch, P. W. et al., Science 245: 752-755 (1989)). The N-terminal 64 amino acids are unique, but the remainder of the protein has about 30% homology with bFGF. KGF is the most divergent of the FGF classes. This molecule has a hydrophobic signal sequence and is secreted efficiently. Post-translational modifications include cleavage of the signal sequence and N-linked glycosylation at one site, thereby producing a 28 kDa protein. The keratinocyte growth factor is produced by fibroblasts derived from skin and fetal lungs (Rubin et al., (1989)). Keratinocyte growth factor mRNA has been shown to be expressed in adult kidney, colon and iliac, and not expressed in brain or lung (Finch, P. W. et al., Science 245: 752-755 (1989)). KGF represents the region conserved in the FGF protein class. KGF binds to the FGF-2 receptor with high affinity.
[4] Damaged wound healing is an important source of morbidity and can lead to complications such as fever, anastomotic breakdown, and non-healing wounds. In normal subjects, wound healing is achieved without complications. In contrast, impaired healing is associated with a number of diseases such as diabetes, infection, immunosuppression, obesity and malnutrition (Cruse, PJ and Foord, R., Arch. Surg. Surgery 97: 631 (1985); Irvin, GL et al., Am Surg 51: 418 (1985)).
[5] Wound healing is the result of complex interactions and biological processes. In normal wound healing, three stages have been described: acute inflammation stages, extracellular matrix and collagen synthesis, and remodeling (Peacock, E.E., Jr., Wound Repair, 2nd edition, WB Saunders, Philadelphia (1984)). This process involves interactions at the wound sites of keratinocytes, fibroblasts, and inflammatory cells.
[6] Tissue regeneration appears to be regulated by specific peptide factors that regulate cell migration and proliferation involved in the repair process (Barrett, TB et al., Proc. Natl. Acad. Sci. USA 81: 6772- 6774 (1985); Collins, T. et al., Nature 316: 748-750 (1985)). Thus, growth factors may be potential treatments for the treatment of wounds, burns and other skin disorders (Rifkin, DB and Moscatelli, J. Cell. Biol. 109: 1-6 (1989); Sporn, MB et al. , J. Cell. Biol. 105: 1039-1045 (1987); Pierce, GF et al., J. Cell. Biochem. 45: 319-326 (1991)). The sequence of the healing process is initiated by temporary tissue deposition during the acute inflammation phase. Thereafter, re-epithelization, collagen synthesis and deposition, fibroblast proliferation, and neurovascularization occur, all of which ultimately define the remodeling phase (Clark, RAF, J. Am. Acad. Dermatol. ). These events are affected by growth factors and cytokines secreted by inflammatory cells or by localized cells at the margins of the wound (Assoian, RK et al., Nature (Lond.) 309: 804 (1984); Nemeth, GG et al., "Growth Factors and Their Role in Wound and Fracture Healing," Growth Factors and Other Aspects of Wound Healing in Biological and Clinical Implications , New York (1988), pp.
[7] Several polypeptide growth factors have been identified that are involved in wound healing, including keratinocyte growth factor (KGF) (Antioniades, H. et al., Proc. Natl. Acad. Sci. USA 88: 565 (1991) , Platelet derived growth factor (PDGF) (Antioniades, H. et al., Proc. Natl. Acad. Sci. USA 88: 565 (1991); Staiano-Coico, L. et al., Jour. (BFGF) (Golden, MA et al., J. Clin. Invest. 87: 406 (1991)), acidic fibroblast growth factor (aFGF) (Mellin, TN et al., J. Invest. Dermatol. 104: 850-855 (1995)), epidermal growth factor (EGF) (Whitby, DJ and Ferguson, WJ, Dev. Biol. 147: 207 (TGF-alpha) (Gartner, MH et al., Surg., 42: 643 (1991); Todd, R. et al., Am. J. Pathol. Transformational growth factor-beta (TGF-beta) (Wong, DTW et al., Am. J. Pathol. 143: 622 (1987)), neu differentiation factor (rNDF) (Danilenko, DM et al., J. Clin. Invest. 95: 842-851 (1995)), insulin-like (IGF-I), insulin-like growth factor II (IGF-II) (Cromack, DT et al., J. Surg. Res. 42: 622 (1987)).
[8] It has been reported that rKGF-1 of the skin stimulates keratinocytes and epidermal keratinocytes in hair follicles and sebaceous glands (Pierce, GF et al., J. Exp. Med. 179: 831-840 (1994)).
[1] The present invention relates to newly identified polynucleotides, polypeptides encoded by the polynucleotides, use of the polynucleotides and polypeptides, and the production of the polynucleotides and polypeptides. More specifically, the polypeptide of the present invention is a keratinocyte growth factor, which will sometimes be referred to as " KGF-2 ", formerly known as fibroblast growth factor 12 (FGF-12). The present invention also relates to the therapeutic use of KGF-2 for promoting or accelerating wound healing. The present invention also relates to novel variant forms of KGF-2 that exhibit increased activity, increased stability, higher yield or better solubility. The present invention also relates to a method for purifying a KGF-2 polypeptide.
[9] The present invention provides a kit comprising an amino acid sequence encoded by a cDNA clone deposited as ATCC Accession No. 75977 on December 16, 1994 or a keratinocyte growth factor having an amino acid sequence as shown in Figure 1 [SEQ ID NO: 2] RTI ID = 0.0 > (KGF-2). ≪ / RTI > The nucleotide sequence determined by sequencing the deposited KGF-2 clone shown in Figure 1 (SEQ ID NO: 1) contains an open reading frame encoding a polypeptide of 208 amino acid residues, -3 and has an estimated leader sequence of about 35 or 36 amino acid residues and an estimated molecular weight of about 23.4 kDa. The amino acid sequence of mature KGF-2 is shown in FIG. 1, which is about amino acid 36 or 37 to 208 [SEQ ID NO: 2].
[10] The polypeptides of the present invention were presumed to belong to the FGF class, and more specifically the polypeptides were deduced as KGF-2 as a result of amino acid sequence homology with others of the FGF family.
[11] According to one aspect of the present invention, there are provided novel mature polypeptides which are KGF-2 as well as fragments, analogs and derivatives thereof which are biologically active and useful for diagnosis or treatment. The polypeptides of the invention are derived from human.
[12] According to another aspect of the present invention there is provided a separate nucleic acid molecule encoding human KGF-2, which comprises mRNA, DNA, cDNA, genomic DNA, as well as its antisense analogs and biologically active, Lt; RTI ID = 0.0 > useful < / RTI >
[13] According to another aspect of the present invention there is provided a recombinant vector comprising a recombinant vector such as a cloning and expression plasmid useful as a reagent in the recombinant production of a KGF-2 protein, as well as recombinant prokaryotic and / or eukaryotic host cells containing a human KGF- Lt; RTI ID = 0.0 > recombinant < / RTI > techniques.
[14] According to another aspect of the present invention there is provided a method of treating such a polypeptide or a polynucleotide encoding the polypeptide for therapeutic purposes, for example, stimulating epithelial cell proliferation for wound healing and basal keratinocytes, Lt; RTI ID = 0.0 > healing. ≪ / RTI > KGF-2 can be used to treat a wide range of conditions including, but not limited to, surgical wounds, ablative wounds, deep wounds including dermis and epidermal damage, eye tissue wounds, tooth tissue wounds, oral wounds, diabetic ulcers, skin ulcers, elbow ulcers, Or other abnormal wound healing, such as wound healing, steroids, radiation therapy and antineoplastic medicaments, complications associated with systemic treatment with metabolic antagonists and vitamin deficiency, uremia, malnutrition, etc., May be clinically useful. KGF-2 may also be used to promote dermal re-establishment following dermal loss.
[15] KGF-2 can also be used to increase adhesion of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed. The following are the types of implants in which KGF-2 can be used to increase adhesion to the wound bed: autograft, artificial skin, allograft, autologous dermal graft, autologous transplantation, vascular grafting, Blair-Brown Blair-Brown) transplantation, bone grafting, embryonic tissue grafting, skin grafting, delayed grafting, dermal grafting, epidermal grafting, fascia grafting, full thickness grafting, heterologous grafting, xenotransplantation (xenograft), a homologous graft, a hyperplastic transplant, a lamellar transplant, a mesh transplant, a mucosal transplant, an Ollier-Thiersch transplant, an omenpal transplant, a patch transplant, pedicle transplants, penetration transplants, split skin grafts, or thick split grafts. KGF-2 may also be used to increase skin strength and improve the appearance of aging skin.
[16] KGF-2 is also believed to produce changes in epithelial cell proliferation, and hepatocyte proliferation, in the lung, breast, stomach, stomach, small intestine, and large intestine. KGF-2 inhibits proliferation of epithelial cells such as sebum cells, hair follicles, liver cells, type II pulmonary cells, mucin-producing cells, and other epithelial cells as well as progenitor cells of the cells contained in the skin, lungs, liver, . KGF-2 can promote the proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
[17] KGF-2 can also be used to reduce the side effects of gut toxicity caused by radiation, chemotherapy treatment or viral infection. KGF-2 may have a cytoprotective effect on the small intestine mucosa. KGF-2 can also stimulate the healing of mucositis (mouth ulcers) caused by chemotherapy and viral infection.
[18] KGF-2 can also be used for the treatment of other skin lesions such as psoriasis, complete regeneration of the skin (i.e., re-aggregation of hair follicles, marine and sebaceous glands) of total thickness and partial thickness skin damage, including burns. KGF-2 can be used to treat epidermal blistering, impairment of adhesion of epidermis and underlying dermis with frequent, widespread and acute herpes, by promoting re-epithelization of damaged areas. KGF-2 can also be used to treat stomach ulcers and duodenal ulcers and to accelerate the healing of mucous membrane formation and regeneration of mucous and duodenal mucosal lining. Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis are diseases that destroy the mucosal surface of the small intestine or colon, respectively. Thus, KGF-2 may be used to promote resurfacing of mucosal surfaces to aid faster healing and to prevent the progression of inflammatory bowel disease. KGF-2 therapy is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and can be used to protect the intestinal mucus from postoperative or ingested harmful substances. KGF-2 can be used to treat diseases associated with the expression of KGF-2.
[19] Moreover, KGF-2 can also be used to prevent and cure lung damage caused by various pathological conditions. Growth factors such as KGF-2, which can stimulate proliferation and differentiation, can promote the repair of alveoli and bronchial epithelium, thereby preventing or treating acute or chronic lung injury. For example, emphysema that causes necrosis of bronchial epithelium and alveoli, inhalation damage resulting from smoking and fire, and loss of progression of the alveoli can be effectively treated with KGF-2. In addition, KGF-2 can be used to stimulate the proliferation and differentiation of type II lung cells, which may be helpful in treating or preventing diseases such as infant respiratory distress syndrome and bronchopulmonary dysplasia, have.
[20] KGF-2 can stimulate hepatocyte proliferation and differentiation, and thus can be used for the treatment of rapidly progressive liver disease, viral hepatitis and toxins caused by cirrhosis (i.e., acetaminophen, carbon tetrachloride, ), ≪ / RTI > such as liver damage caused by < RTI ID = 0.0 >
[21] Additionally, KGF-2 may be used to treat or prevent the onset of diabetes. In patients with newly diagnosed Type I and II diabetes with some islet cell function remaining, KGF-2 can be used to maintain islet function, alleviate, delay or prevent the persistent expression of the disease. In addition, KGF-2 can be used as an adjuvant in islet cell transplantation to improve or promote islet cell function.
[22] According to another aspect of the present invention, an antibody against the polypeptide is provided.
[23] According to another aspect of the present invention there is provided a nucleic acid probe comprising a nucleic acid molecule of sufficient length that specifically hybridizes to a human KGF-2 sequence.
[24] According to a further aspect of the present invention there is provided a mimetic peptide of KGF-2 which can be used as a therapeutic peptide. The imitation KGF-2 peptide is a short peptide that mimics the biological activity of the KGF-2 protein by binding to and activating the cognate receptor of KGF-2. In addition, imitation KGF-2 peptides may inhibit binding to the kinase receptors of KGF-2.
[25] According to another aspect of the present invention, there is provided an antagonist of said polypeptide, which can be used to inhibit the action of said polypeptide. For example, it can be used to reduce traces during the wound healing process and to prevent and / or treat tumor growth, diabetic retinopathy, rheumatoid arthritis, osteoarthritis and tumor growth. KGF-2 antagonists may also be used to treat diseases associated with overexpression of KGF-2.
[26] According to another aspect of the present invention there is provided a diagnostic assay for detecting a disease associated with, or susceptibility to, a mutation of a KGF-2 nucleic acid sequence or an overexpression of a polypeptide encoded by the sequence.
[27] According to another aspect of the present invention, there is provided a method of using the polypeptide, or a polynucleotide encoding the polypeptide, for in vitro purposes related to scientific research, synthesis of DNA, and DNA vector production.
[28] Accordingly, one aspect of the present invention provides a separate nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) the complete amino acid sequence of SEQ ID NO: 2 (B) a nucleotide sequence encoding a mature KGF-2 polypeptide having an amino acid sequence at position 36 or 37 to 208 in Figure 1 (SEQ ID NO: 2); (c) a nucleotide sequence encoding a KGF-2 polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC Accession No. 75977; (d) a nucleotide sequence encoding a mature KGF-2 polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Accession No. 75977; And (e) a nucleotide sequence complementary to any of the nucleotide sequences of (a), (b), (c), or (d) above.
[29] A further embodiment of the present invention is a nucleotide sequence which is at least 80% identical to any of the nucleotide sequences of (a), (b), (c), (d) or (e) above, more preferably at least 85% (A), (b) or (c), wherein the polynucleotide has a nucleotide sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97% , a polynucleotide that hybridizes under stringent hybridization conditions to a polynucleotide of (c), (d), or (e). These hybridizing polynucleotides do not hybridize under stringent hybridization conditions with polynucleotides having nucleotide sequences that consist only of A residues or consist only of T residues. A further nucleic acid embodiment of the invention is a separate nucleic acid construct comprising a polynucleotide sequence encoding an amino acid sequence of an epitope-containing portion of KGF-2 having the amino acid sequence of (a), (b), (c) Nucleic acid molecule.
[30] The present invention further provides isolated KGF-2 polypeptides having an amino acid sequence selected from the group consisting of: (a) a complete 208 (including the leader sequence shown in SEQ ID NO: 2) (B) an amino acid sequence of a mature KGF-2 polypeptide (without leader) having an amino acid sequence at position 36 or 37 to 208 in Figure 1 [SEQ ID NO: 2] ; (c) an amino acid sequence of a KGF-2 polypeptide having a complete amino acid sequence, including a leader, encoded by a cDNA clone contained in ATCC Accession No. 75977; (d) the amino acid sequence of a mature KGF-2 polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Accession No. 75977. The polypeptide of the present invention may also have a similarity of 80% or more, more preferably 90% or more, 95% or more, 96% or more, 97% or more, 97% or more, , More preferably at least 85%, even more preferably at least 90%, at least 91%, at least 95%, at least 95%, at least 98%, at least 99% , 92% or more, 93% or more, 94% or more, 95% or more, 97% or more, 98% or more or 99% or more.
[31] Another aspect of the present invention relates to a peptide or polypeptide having an amino acid sequence of an epitope-containing portion of KGF-2 polypeptide having the amino acid sequence of (a), (b), (c) or (d) above. The peptide or polypeptide having the amino acid sequence of the epitope-containing part of the KGF-2 polypeptide of the present invention may be an epitope-containing polypeptide having the entire amino acid sequence of any length up to the entire amino acid sequence of the polypeptide of the present invention described above, But includes at least 6 or 7, preferably at least 9, more preferably at least about 30 amino acids to about 50 amino acids. In another embodiment, the invention provides isolated antibodies that specifically bind KGF-2 polypeptides having the amino acid sequences of (a), (b), (c), or (d) above.
[32] According to another aspect of the present invention, new variants of KGF-2 are also provided. This may be produced by deletion or substitution of one or more amino acids of KGF-2. Natural mutations are called allelic variations. The opposition mutation may be silent (no change in the polypeptide to be encoded) or may have a changed amino acid sequence. Protein engineering can be used to attempt to improve or alter the properties of native KGF-2. Recombinant DNA techniques known in the art can be used to create new polypeptides. Muteins and deletion mutants can, for example, exhibit increased activity or increased stability. In addition, it exhibits better solubility at least under certain purification and storage conditions and can be purified to higher yields.
[33] These aspects of the invention and other aspects of the invention will be apparent to those skilled in the art from the following description.
[34] The accompanying drawings illustrate embodiments of the invention and are not to be construed as limiting the scope of the invention as claimed.
[35] Brief Description of Drawings
[36] Figures 1A-1C show the cDNA and corresponding amino acid sequence of the polypeptide of the present invention. The first 35 or 36 amino acid residues represent an assumed leader sequence (not shown). For amino acids, standard one letter notation was used. Sequencing Inaccuracy is a common problem when attempting to determine polynucleotide sequences. Sequencing was performed using a 373 automated DNA sequencer (Applied Biosystems, Inc.). Sequencing accuracy was expected to be greater than 97%. [SEQ ID NO: 1]
[37] 2A-2D shows a comparison of the amino acid sequences of the polypeptide of the present invention with other fibroblast growth factors [SEQ ID NO: 13-22]
[38] 3A-3D shows the mRNA and amino acid sequence of the full length of KGF-2 gene [SEQ ID NO: 23 and 24]
[39] 4A-4E shows the analysis of the KGF-2 amino acid sequence. Alpha, beta, turn and coil regions; hydrophilic and hydrophobic; Both amphiphilic domains; flexible domains; antigenicity indices and surface probabilities are shown. In the "Antigenic Index-Jameson-Wolf" graph, the amino acid residues 41-109 of the first degree [SEQ ID NO: 2] are located in the high antigenic region of the KGF-2 protein . The hydrophobic region (Hope-Woods Plot) is found below the median line (negative value) and the hydrophilic region (kite-doolittle Plot) is found above the median line (positive value, 41-109). The curve spans the entire 208 amino acid ORF.
[40] 5 shows the evaluation of KGF-2 for wound closure of diabetic mice. The wounds were measured immediately after the injury and daily for 5 consecutive days and around 8 days. Percent wound closure was calculated using the following equation: [area around 1 day] - [area around 8] / [area around one day]. Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[41] 6 shows the evaluation of KGF-2 for wound closure of non-diabetic mice. The wounds were measured immediately after the injury and daily for 5 consecutive days and around 8 days. Percent wound closure was calculated using the following equation: [area around 1 day] - [area around 8] / [area around one day]. Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[42] FIG. 7 shows the time course of wound closure in diabetic mice. The wounds were measured immediately after the injury and daily for 5 consecutive days and around 8 days. The value is given as the total area (mm ² ). Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[43] Figure 8 shows the time course of wound closure in non-diabetic mice. The wounds were measured immediately after the injury and daily for 5 consecutive days and around 8 days. The value is given as the total area (mm ² ). Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[44] FIG. 9 shows a histopathological evaluation of KGF-2 in diabetic mice. Numerical values were given by a blind observer. Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[45] 10 shows a histopathological evaluation of KGF-2 in non-diabetic mice. Numerical values were given by a blind observer. Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[46] FIG. 11 shows the effect of keratinocyte growth on diabetic mice. Numerical values were given by a blind observer. Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[47] FIG. 12 shows the effect of keratinocyte growth on non-diabetic mice. Numerical values were given by a blind observer. Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[48] Figure 13 shows the effect of skin proliferation in diabetic mice. Numerical values were given by a blind observer. Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[49] 14 shows the effect of skin proliferation in non-diabetic mice. Numerical values were given by a blind observer. Statistical analysis was performed using an unpaired t test (mean +/- SEM, n = 5).
[50] Figure 15 plots the protein and DNA sequences expressed from the pQE60-Cys37 construct [SEQ ID NO: 29 and 30]. The expressed KGF-2 protein contains the sequence from position 37 cysteine to position 208 serine and has a 6X (his) tag attached to the N-terminus of the protein.
[51] FIG. 16 shows the effect of methyl-prednisolone on wound healing in rats. Male SD adult rat (n = 5) was injected with 5 mg of methylprednisolone on the wounded day. The animals were exposed to dermal punch wounds (8 mm) and treated daily with KGF-2 solution or buffer solution in 50 μl buffer for 5 consecutive days. Scratches were measured daily on grades 1-5 and 8 with a graduated Jameson caliper. Values represent measurements taken around 8 days (mean +/- SEM).
[52] Figure 17 shows the effect of KGF-2 on wound closure. Dendritic punch wounds (8 mm) were applied to male SD adult rats (n = 5) and injected with 5 mg of methylprednisolone on the wounded day. The animals were treated daily with KGF-2 or buffer solution in 50 [mu] l buffer solution for 5 consecutive days starting from the day of injury. Measurements were taken daily for 5 consecutive days and around 8 days. Wound closure was calculated by the following formula: [area around 8] - [area around 1] / [area around 1]. The area of day 1 produced by dermal punches was determined to be 64 mm ² . Statistical analysis was performed using the unpaired t test (mean +/- SEM).
[53] Figure 18 shows the time course of wound healing in the glucocorticoid-damaged model of wound healing. Male SD adult rats (n = 5) were exposed to dermal punch wounds (8 mm) around day 1, wounded and treated daily with 50 μl of KGF-2 solution or buffer for 5 consecutive days. The animals were administered 5 mg of methylprednisolone on the day of injury. Wounds were measured with a Jameson caliper graduated from the day of injury to the animals, daily for 5 consecutive days and around 8 days. Statistical analysis was performed using the unpaired t test (mean +/- SEM).
[54] Figure 19 (A) shows the effect of KGF-2 on the wound site in a rat model of wound healing in the absence of methyl-prednisolone 5 days after challenge. Male SD rats (n = 5) were exposed to dermal punch wounds (8 mm) around day 1 and treated daily with 50 ul of KGF-2 solution or buffer solution for wounded, scarred and then five consecutive days. Scratches were measured daily with graduated Jameson calipers. Statistical analysis was performed using the unpaired t test (mean +/- SEM). Figure 19 (B) shows the evaluation of PDGF-BB and KGF-2 in male SD rats (n = 6). Methylprednisolone (MP) (17 mg / kg) was administered to 8 mm wounds on all rats and to injure wound healing. Wounds were treated daily with various concentrations of PDGF-BB and KGF-2 or buffer. Scars were measured on scales 2, 4, 6, 8, and 10 using scaled Jameson calipers. Statistical analysis was performed using the unpaired t test (mean +/- SEM). ^※ Comparison with buffer. 1 ^{ml of} PDGF-BB vs. 1 μg of KGF-2 / E3.
[55] 20 shows the effect of KGF-2 on wound healing in the glucocorticoid-damaged model of wound healing. Dendritic punch injuries (8 mm) were made in male SD adult rats (n = 5) and 17 mg / kg methyl-prednisolone was administered to the wounded days. The animals were treated daily with KGF-2 or buffer solution in 50 [mu] l buffer for 5 consecutive days and 8 days. And measured with a scaled micrometer under a scratched optical microscope. Statistical analysis was performed using the unpaired t test (mean +/- SEM).
[56] 21 (A) shows the stimulation of normal primary epidermal keratinocyte proliferation by KGF-2. (B) shows the stimulation of normal primary epidermal keratinocyte proliferation by KGF-2 33. (C) shows stimulation of normal primary epidermal keratinocyte proliferation by KGF-2 28. Human primary normal epidermal keratinocytes were incubated with various concentrations of KGF-2, KGF-2 33, KGF-2 28 for 3 days. In all three experiments, alamarBlue was added for 16 hours and the intensity of the red color converted from the Alemar blue by the cells was measured by the difference between O.D.570 nm and O.D.600 nm. For each KGF-2 protein, the positive control of the full keratinocyte growth medium (KGM) and the negative control of the keratinocyte basal medium (KBM) were included in the same assay plate.
[57] Figure 22 (A) shows the stimulation of thymidine incorporation by KGF-2 and FGF7 in Baf3 cells transfected with FGFR1b and FGFR2. The effects of KGF-2 (right panel) and FGF7 (left panel) on the proliferation of BF3 cells transfected with FGFR1iiib (open circles) or FGFR2iiib / KGFR (solid circles) were investigated. The Y-axis represents the amount of [3H] thymidine insertion (cpm) into the DNA of Baf3 cells. The X-axis represents the final concentration of KGF-2 or FGF7 added to the tissue culture medium. (B) shows the stimulation of thymidine insertion by KGF-2 33 in Baf3 cells transfected with FGFR2iiib. (C) shows the stimulation of thymidine insertion by KGF-2 (white rod), KGF-2 33 (black rod) and KGF-2 28 (gray rod) in BF3 cells transfected with FGFR2iiib.
[58] 23 shows the DNA and protein sequence of E. coli optimized full length KGF-2 [SEQ ID NO: 38 and 39].
[59] 24A and 24B show DNA and protein sequences of E. coli optimized mature KGF-2 [SEQ ID NO: 42, 43, 54 and 55].
[60] 25 shows DNA and encoded protein sequences of KGF-2 deletion constructs comprising amino acids 36 to 208 of KGF-2 (SEQ ID NOS: 65 and 66).
[61] 26 shows DNA and encoded protein sequences of KGF-2 deletion constructs comprising amino acids 63 to 208 of KGF-2 (SEQ ID NO: 67 and 68).
[62] 27 shows DNA and encoded protein sequences of KGF-2 deletion constructs comprising amino acids 77 to 208 of KGF-2 (SEQ ID NOS: 69 and 70).
[63] FIG. 28 shows DNA and encoded protein sequences of KGF-2 deletion constructs comprising amino acids 93 to 208 of KGF-2 (SEQ ID NOS: 71 and 72).
[64] 29 shows DNA and encoded protein sequences of KGF-2 deletion constructs comprising amino acids 104 to 208 of KGF-2 (SEQ ID NO: 73 and 74).
[65] 30 shows DNA and encoded protein sequences of KGF-2 deletion constructs comprising amino acids 123 to 208 of KGF-2 (SEQ ID NO: 75 and 76).
[66] FIG. 31 shows DNA and encoded protein sequences of KGF-2 deletion constructs comprising amino acids 138 to 208 of KGF-2 (SEQ ID NO: 77 and 78).
[67] 32 shows DNA and encoded protein sequences of KGF-2 deletion constructs comprising amino acids 36 to 153 of KGF-2 (SEQ ID NOS: 79 and 80).
[68] 33 shows DNA and encoded protein sequences of KGF-2 deletion constructs comprising amino acids 63 to 153 of KGF-2 (SEQ ID NOS: 81 and 82).
[69] 34 shows the DNA sequence of a serine mutant construct of KGF-2 cysteine-37 [SEQ ID NO: 83].
[70] 35 shows the DNA sequence of a serine mutant construct of KGF-2 cysteine-37 / cysteine-106 [SEQ ID NO: 84].
[71] FIG. 36 shows the evaluation of KGF-2 33 effect on wound healing in male SD rats (n = 5). The animals were scratched 6 mm on the back and treated with various concentrations of buffer, or KGF-2 33, for 4 consecutive days. Scratches were measured daily with Jameson calipers. Statistical analysis was performed using the unpaired t-test. (Mean +/- SE) ^* Comparison with buffer.
[72] 37 shows the effect of KGF-2 [Delta] 33 on wound healing in normal rats. Male, SD, 250-300 g, total thickness of 6 mm in rats (n = 5), and the like. The wound was measured with a caliper for 4 days starting from the day of surgery and treated with various concentrations of KGF-2 33 and buffer. The wound was collected on the last day. Statistical analysis was performed with an unpaired t-test. ^* Values are compared with untreated controls. The † values were compared to the buffer controls.
[73] Figure 38 shows the effect of KGF-2 [Delta] 33 on the breaking strength of the incision wound. The male adult SD rats (n = 10) were subjected to a 2.5 cm total thickness incisional wound at about 1 day, wounded, and the buffer or KGF-2 (delta 33) (1, 4, Lt; / RTI > Animals were sacrificed at 5 days and 0.5 cm wound samples were cut for normal histology and burst strength analysis. Biomechanical tests were performed using an Instron skin tensiometer with applied force across the wound. The rupture strength was defined as the maximum force that was blocked by each wound before rupture. Statistical analysis was performed using the unpaired t-test (mean +/- SE)
[74] FIG. 39 shows the effect of KGF-2 (delta 33) on the epidermal thickness of the incision wound. The male adult SD rats (n = 10) were subjected to a 2.5 cm total thickness incisional wound at about 1 day, wounded, and the buffer or KGF-2 (delta 33) (1, 4, Lt; / RTI > Animals were sacrificed at 5 days and 0.5 cm wound samples were cut for normal histology and burst strength analysis. Epidermal thickness was determined by taking the mean of six measurements taken around the wound. Measurements were taken by a blind observer on a Masson Trichrome stained area under an optical microscope using a calibrated lens micrometer. Statistical analysis was performed using the unpaired t-test (mean +/- SE)
[75] FIG. 40 shows the effect of KGF-2 33 on epidermal thickness after a single intradermal injection. Male adult SD rats (n = 18) were injected intradermally at day 0 with 6 μg of buffer or KGF-2 at a concentration of 1 μg and 4 μg in 50 μl. Animals were sacrificed 24 hours and 48 hours after injection. The epidermal thickness was measured from the granular layer to the bottom of the basal layer. Approximately 20 measurements were made along the injection site and the mean thickness was determined. Measurements were made on the Masson Trichrome stained area under a light microscope using a calibrated micrometer. Statistical analysis was performed using the unpaired t-test (mean +/- SE)
[76] 41 shows the effect of KGF-2 (delta 33) on BrdU scoring. Male adult SD rats (n = 18) were injected intradermally at day 0 with 6 μg of placebo or KGF-2 at a concentration of 1 μg and 4 μg in 50 μl. Animals were sacrificed 24 hours and 48 hours after injection. Animals were injected with 5-2'-bromo-deoxyuridine (100 mg / kg ip) 2 hours before sacrifice. The ratings were made by blind observer under light microscope using the following rating system: 0-3 no or minimal BrdU labeled cells, 4-6 medium labeling, 7-10 strongly labeled cells. Statistical analysis was performed using the unpaired t-test (mean +/- SE)
[77] FIG. 42 shows the anti-inflammatory effect of KGF-2 on PAF-induced foot edema.
[78] 43 shows the anti-inflammatory effect of KGF-2 [Delta] 33 on PAF-induced foot edema in Lewis rats.
[79] 44 shows the effect of KGF-2 33 on the survival of whole body irradiated Balb / c mice. Balb / c male mice (n = 5), 22.1 g, were irradiated with 519 RADS. The animals were treated with buffer or KGF-2 (1 & 5 mg / kg, sq.) Daily 2 days prior to and for the next 7 days.
[80] FIG. 45 shows the effect of KGF-2 33 on body weight of the irradiated mice. 22.1 g of Balb / c male mice (n = 5) were injected with buffer or KGF-2 33 (1, 5 mg / kg) two days before irradiation at 519 RAD / min. The animals were weighed and dosed daily for 7 days after irradiation.
[81] 46 shows the effect of KGF-2 33 on the survival of whole body irradiated Balb / c mice. Balb / c male mice (n = 7), 22.1 g, were irradiated with 519 RADS. The animals were treated with buffer or KGF-2 (1 & 5 mg / kg, sq.) Daily 2 days prior to and for the next 7 days.
[82] FIG. 47 shows the effect of KGF-2 33 on wound healing in a glucocorticoid-injured rat model.
[83] Figure 48 shows the effect of KGF-2 [Delta] 33 on cell proliferation when determined using BrdU labeling.
[84] 49 shows the effect of KGF-2 [Delta] 33 on localized collagen content at the anastomotic site of the rat colon.
[85] Figure 50 shows a schematic representation of the pHE4-5 expression vector (SEQ ID NO: 147) and the subcloned KGF-2 cDNA encoding sequence. The position of the kanamycin resistance marker gene, the KGF-2 coding sequence, the oriC sequence, and the lac Iq coding sequence.
[86] Figure 51 shows the nucleotide sequence (SEQ ID NO: 148) of the regulatory elements of the pHE promoter. Two lac operator sequences, a Shine-Delgarno sequence (S / D), and a terminal HindIII and NdeI restriction site (italic part).
[87] 52 shows the proliferation of the bladder epidermis after ip or sc administration of KGF-2 33.
[88] 53 shows the proliferation of prostate epidermal cells after systemic administration of KGF-2 33.
[89] 54 shows the effect of KGF-2 33 on bladder wall ulcers of cyclophosphamide-induced hemorrhagic cystitis in rats.
[90] 55 shows the effect of KGF-2 33 on bladder wall thickness in a cyclophosphamide-induced cystitis rat model.
[91] Figure 56 provides an overview of the study design to determine whether KGF-2 [Delta] 33 induces normal epidermal proliferation in rats when systemically administered using the SC and IP pathways.
[92] In FIG. 57, normal Sprague Dawley rats were injected daily with KGF-2 33 (5 mg / kg, HG03411-E2) or buffer, and after the final injection, they were sacrificed one day later. Blind observers were magnified 10X to count cells proliferating in 10 randomly chosen fields per animal. SC administration of KGF-2 33 caused significant proliferation after 1 day and then returned to normal until about 2 days. The ip-administered KGF-2 33 stimulated proliferation from day 1 to 3, but only the results at day 1 and day 3 were statistically significant.
[93] In FIG. 58, normal Sprague Dawley rats were injected daily with KGF-2 33 (5 mg / kg, HG03411-E2) or buffer and sacrificed one day after the last injection. Blind observers were magnified 10X to count cells proliferating in 10 randomly chosen fields per animal. SC administration of KGF-2 Δ33 did not increase proliferation at any time points, but ip-administered KGF-2 Δ33 stimulated proliferation throughout the study period.
[94] In FIG. 59, normal Sprague Dawley rats were sacrificed one day after injection with KGF-2 33 (5 mg / kg, HG03411-E2) or buffer daily and final injection. Blind observers were magnified 10X to count cells proliferating in one cross section per animal. SC administration of KGF-2 33 caused a significant increase in proliferation after 1, 2, and 3 days after daily administration. The ip-administered KGF-2 33 showed proliferation only after 2 and 3 days.
[95] FIG. 60 shows KGF-2 33 induced proliferation in normal rat lung.
[96] According to an aspect of the present invention, there is provided a polypeptide having an amino acid sequence of the first aspect (SEQ ID NO: 2), or a polypeptide having an amino acid sequence deduced from a polypeptide of the present invention, A polypeptide encoded by the cDNA of the clone deposited as ATCC Accession No. 75977 on the 16th day of September, or as ATCC accession number 75901 on September 29, 1994, in the Manassas University College Boulevard 10811 American Type Culture Collection patent depot, Virginia, USA, 20110-2209 A separate nucleic acid (polynucleotide) encoding the polypeptide encoded by the cDNA of the deposited clone is provided.
[97] Nucleic acid molecule
[98] Unless otherwise indicated, all nucleotide sequences determined by sequencing the DNA molecule here were determined using an automated DNA sequencer (Model 373 from Applied Biosystems, Inc., etc.), wherein the polypeptide encoded by the determined DNA molecule All amino acid sequences were deduced by translation of the determined DNA sequences. Thus, as is known in the art for any DNA sequence determined by this automated method, certain nucleotide sequences determined herein may contain some errors. The nucleotide sequence determined by automation is typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical, to the actual nucleotide sequence of the DNA molecule being sequenced. The actual sequence can be determined more accurately by other methods including manual DNA sequencing methods known in the art. Also, as is well known in the art, a single insertion or deletion in the nucleotide sequence determined relative to the actual sequence can cause a frame shift in the translation of the nucleotide sequence so that the putative amino acid sequence encoded by the determined nucleotide sequence can be inserted Or will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule starting at the deletion point.
[99] Unless otherwise indicated, each "nucleotide sequence" described herein is given as a sequence of deoxyribonucleotides (denoted as A, G, C, and T). However, the "nucleotide sequence" of a nucleic acid molecule or polynucleotide is a sequence of a deoxyribonucleotide in the case of a DNA molecule or polynucleotide, and a sequence of a ribonucleotide (A, G, C and U) in the case of an RNA molecule or a polynucleotide Wherein the thymidine deoxyribonucleotide (T) of the deoxyribonucleotide sequence specified herein is replaced by a ribonucleotide uridine (U). For example, an RNA molecule having the sequence of SEQ ID NO: 1 described using the deoxyribonucleotide designation can be obtained by introducing a ribonucleotide A, G, or C of each corresponding deoxyribonucleotide A, G, or C of SEQ ID NO: C < / RTI > and each deoxyribonucleotide T is replaced by a ribonucleotide U.
[100] &Quot; Separated " nucleic acid molecule (s) refers to nucleic acid molecules, DNA or RNA removed from the natural environment. For example, recombinant DNA molecules contained in a vector are considered isolated for purposes of the present invention. Additional examples of isolated DNA molecules include purified (partially or substantially) solution-phase DNA molecules or recombinant DNA molecules maintained in heterologous host cells. The isolated RNA molecule comprises an in vivo or in vitro RNA transcript of the DNA molecule of the present invention. Isolated nucleic acid molecules according to the invention also include those molecules produced by synthesis.
[101] The isolated nucleic acid molecule of the present invention comprises a DNA molecule comprising an open reading frame (ORF) having an initiation codon at positions 1-3 of the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1); A DNA molecule comprising the coding sequence of the mature KGF-2 protein (last 172 or 173 amino acid) [SEQ ID NO: 2] shown in Figure 1; And DNA molecules that still contain sequences substantially different from those described above but still encode the KGF-2 protein because of the degeneracy of the genetic code. Of course, genetic codes are well known in the art. Therefore, it is easy for a person skilled in the art to generate the above-described dehydrated variant.
[102] Polynucleotides encoding the polypeptides of the invention can be obtained from human prostate and fetal lungs. The cDNA fragment encoding the polypeptide was initially isolated from a library derived from normal human prostate glands. The open reading frame encoding the full length protein was then separated from the randomly primed human fetal lung cDNA library. It is structurally related to the FGF class. It contains an open reading frame that encodes a protein of 208 amino acid residues, since the first 35 or 36 amino acid residues are the predicted leader sequence, so the mature protein will contain 173 or 172 amino acids. The protein has the highest homology with human keratinocyte growth factors and has a similarity of 45% identity and 82% over the 206 amino acid stretch. It is also important that the sequence conserved in the FGF family is found to be conserved in the protein of the present invention.
[103] In addition, the nested PCR results of the KGF-2 cDNA from the library indicated that there is a possible selective splicing form of KGF-2. Specifically, PCR products of 0.2 kb and 0.4 kb were obtained from various cDNA libraries using primers at the N-terminal periphery of the open reading frame of KGF-2. The 0.2 kb size was the expected product of KGF-2, but a 0.4 kb size can result from the selectively spliced form of KGF-2. The 04kb product was observed from a library of gastric cancer, adult testis, duodenum and interest.
[104] The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, including cDNA, genomic DNA, and synthetic DNA. DNA can be double-stranded or single-stranded, and if single-stranded it can be a coded strand or a non-coded (anti-sense) strand. The coding sequence encoding the mature polypeptide may be identical to that of the coding sequence or deposited clone shown in Figure 1 (SEQ ID NO: 1), or the coding sequence may be identical to the coding sequence [SEQ ID NO: 1] or another coding sequence encoding the same mature polypeptide as the deposited cDNA.
[105] The polynucleotide encoding the putative mature polypeptide encoded by the putative cDNA or the putative mature polypeptide of the first figure [SEQ ID NO: 2] may comprise: the coding sequence of the mature polypeptide only; the leader or secretory The coding sequence of the mature polypeptide, the coding sequence of the mature polypeptide (and optionally the additional coding sequence) and the 5 'and / or 3' ratio of the coding sequence of the mature polypeptide, - non-coding sequences such as coding sequences or introns. Also, full-length mRNA including the 5 'and 3' untranslated regions of the gene was obtained (FIG. 3 (SEQ ID NO: 23)).
[106] As will be appreciated by those skilled in the art, due to the sequencing errors described above as well as the variety of cleavage sites for the leader in other known proteins, the actual KGF-2 polypeptide encoded by the deposited cDNA contains about 208 amino acids, And the actual leader sequence of this protein is about 35 or 36 amino acids, but can be anywhere in the range of about 30 to about 40 amino acids.
[107] Thus, the term " polynucleotide encoding a polypeptide " encompasses not only polynucleotides that contain only the coding sequence of the polypeptide, but also polynucleotides that contain additional coding and / or non-coding sequences.
[108] The present invention also relates to polypeptides having the deduced amino acid sequence of Figure 1 (SEQ ID NO: 2), or variants of the polynucleotides encoding fragments, analogs and derivatives of the polypeptides encoded by the cDNA of the deposited clones do. Variants of polynucleotides may be variants of naturally occurring polynucleotides or variants of naturally occurring polynucleotides.
[109] Accordingly, the present invention provides a polynucleotide encoding an estimated mature polypeptide identical to that shown in Figure 1 (SEQ ID NO: 2) or an encoded mature polypeptide encoded by a cDNA of a deposited clone, Derivatives or analogues of the polypeptide of SEQ ID NO: 2, or variants of said polypeptide which encode a polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion mutants, substitution mutants, additions or insertion mutants.
[110] The present invention includes a polynucleotide encoding a mimetic peptide of KGF-2 that can be used as a therapeutic peptide. Imitation KGF-2 paphed is a short peptide that binds to and activates the cognate receptors of KGF-2 to mimic the biological activity of the KGF-2 protein. In addition, the imitation KGF-2 peptide may bind to and inhibit the cognate receptors of KGF-2. KGF-2 receptors include, but are not limited to, FGFR2iiib and FGFRliiib. Such mimetic peptides are obtained from, but are not limited to, methods such as phage display or combinatorial chemistry. For example, there is a method disclosed by Wrighton et al., Science 273: 458-463 (1996) for producing a bovine KGF-2 peptide.
[111] As indicated above, the polynucleotide may have a coding sequence which is a naturally occurring opposing variant of the coding sequence of the first degree [SEQ ID NO: 1] or of the coding sequence of the deposited clone. As is known in the art, an allelic variant is another form of a polynucleotide sequence that may have substitution, deletion, or addition of one or more nucleotides and that does not substantially alter the function of the encoding polypeptide.
[112] The invention also encompasses polynucleotide sequences that aid in the expression and secretion of a polypeptide from a host cell, such as a leader sequence that functions as a secretory sequence that controls the transport of the polypeptide from the host, and a coding sequence for the mature polypeptide that are identical And includes polynucleotides that can be fused. The polypeptide having the leader sequence is a preprotein and the leader sequence can be cleaved by the host cell to form a mature form of the polypeptide. The polynucleotide can also encode a mature protein plus an additional 5 'amino acid residue, proprotein. The mature protein with prosequence is proprotein and is an inactive form of the protein. When the prosequence is cleaved, the active mature protein remains.
[113] Thus, for example, a polynucleotide of the invention can encode a mature protein, encode a protein having a prosequence, or encode a protein having a prosequence and a presequence (leader sequence).
[114] The polynucleotides of the present invention may also have a coding sequence fused to match the frame with a marker sequence that facilitates purification of the polypeptide of the invention. The marker sequence may be hexahistidine supplied by pQE-9 to provide a purification of the mature polypeptide fused to the marker in the case of a bacterial host, or such as a mammalian host, such as when COS-7 cells are used, Tag may be a maglutinin (HA) tag. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I. et al., Cell 37: 767 (1984)).
[115] The term " gene " refers to a DNA segment involved in generating a polypeptide chain, which includes intervening sequences (introns) between each coding sequence (exon) as well as regions (leader and trailer) before and after the coding region.
[116] A fragment of the full-length gene of the present invention can be used as a hybridization probe of a cDNA library in isolating a full-length cDNA and isolating other cDNA having high sequence similarity with the gene or similar biological activity. Probes of this type preferably have at least 30 bases and may comprise, for example, 50 or more bases. The probe can also be used to identify genomic clones or clones containing the complete gene, including the regulatory and promoter regions, exons and introns, and cDNA clones corresponding to full length transcripts. An example of a screen includes separating the coding region of a gene by using a known DNA sequence to synthesize an oligonucleotide probe. A labeled oligonucleotide having a sequence complementary to that of the gene of the present invention is used to screen a library of human cDNA, genomic DNA or cDNA to determine which of the libraries the probe hybridizes to.
[117] (B) a nucleotide sequence encoding a full length KGF-2 polypeptide having the complete amino acid sequence of the first figure [SEQ ID NO: 2], including the putative leader sequence; A nucleotide sequence encoding a mature KGF-2 polypeptide (full length polypeptide with leader removed) having an amino acid sequence at position 36 or 37 to 208 in Figure 1 (SEQ ID NO: 2); (c) a nucleotide sequence encoding a full-length KGF-2 polypeptide having a complete amino acid sequence including a leader encoded by a cDNA clone contained in ATCC Accession No. 75977; (d) a nucleotide sequence encoding a mature KGF-2 polypeptide having an amino acid sequence encoded by a cDNA clone contained in ATCC Accession No. 75977; And (e) a nucleotide sequence encoding any of the KGF-2 analogs or deletion mutants described below; Or (f) a nucleotide sequence that is at least 80% identical, more preferably at least 85%, 90%, or 90% identical to a nucleotide sequence complementary to any of the nucleotide sequences of (a), (b), (c) 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical nucleotide sequences.
[118] A polynucleotide having a nucleotide sequence that is at least, for example, 95% " identical " with a reference nucleotide sequence encoding a KGF-2 polypeptide is a polynucleotide having a polynucleotide sequence of 5 or fewer nucleotides per 100 nucleotides of the reference nucleotide sequence encoding a KGF- Means that the nucleotide sequence of the polynucleotide is the same as the reference sequence, except that it may include a point mutation. That is, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides of the reference sequence may be deleted or substituted with other nucleotides, or a plurality of up to 5% of the entire nucleotide of the reference sequence The nucleotide can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5 'or 3' terminal position of the reference nucleotide sequence or can be located within one or more adjacent groups within the reference sequence or between these terminal positions individually spotted between the nucleotides in the reference sequence It can happen in places.
[119] As a practical matter, it is contemplated that any particular nucleic acid molecule may be at least 80%, 85%, 90%, 91%, or 90% identical to the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) or the nucleotide sequence of the deposited cDNA clone, , 92%, 93%, 94%, 95%, 97%, 98%, or 99% identical can typically be determined using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetic Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Best-fit uses the homology homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981) to find the best homology region between two sequences. When using a best-fit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to the reference sequence according to the present invention, the parameters as well as the percent identity are calculated over the entire length of the reference nucleotide sequence, Gt; 5% < / RTI > homology gap of the total number of nucleotides of the nucleotide sequence of SEQ ID NO.
[120] A preferred method for determining the best overall match between a subject sequence and a query sequence (also known as a sequence of the invention), also referred to as global sequence alignment, is described in Brutlag et al. (Comp. App. Biosci. : 237-245) using the FASTDB computer program. In the sequence alignment, both the query sequence and the target sequence are DNA sequences. RNA sequences can be compared by converting U to T. The result of the global sequence alignment is percent identity. The preferred parameters used for FASTDB alignment of DNA sequences to calculate percent identity are: Matrix = Unitary, k-tuple = 4, Mismatch Fernity = 1, Joining Fernity = 30, Random The length of the subject nucleotide sequence is shorter than that of the target group length = 0, the cut-off score = 1, the gap penalty = 5, the gap size penalty = 0.05, the window size =
[121] If the target sequence is not due to an internal deletion and is shorter than the query sequence due to a 5 'or 3' deletion, a manual correction should be made to the results. This is because the FASTDB program fails to account for the 5 'and 3' truncations of the target sequence when computing percent identity. For the subject sequence truncated at the 5 'or 3' end compared to the query sequence, the base number of the query sequence of 5 'and 3' of the target sequence that is not matched / ordered is calculated as a percentage of the total base of the query sequence, The identity is corrected. Whether the nucleotides are matched / aligned is determined by the result of the FASTDB sequence alignment. The percentage is then subtracted from the percentage identity calculated by the FASTDB program using a particular parameter to arrive at the final percent identity score. This corrected score is used for the purpose of the present invention. Only those bases outside the 5 'and 3' bases of the subject sequence are calculated for the purpose of manually adjusting the percent identity score, as indicated by the FASTDB alignment, which is not matched / aligned with the query sequence.
[122] For example, to determine percent identity, a 90 base sequence is aligned to a 100 base sequence. Deletions occur at the 5 ' end of the subject sequence, thus FASTDB alignment does not represent the first 10 bases match / alignment at the 5 ' end. The 10 unpaired bases represent 10% of the sequence (total number of bases in the unmatched bases at the 5 'and 3' ends / base of the query sequence) and therefore 10% from the percent identity score calculated by the FASTDB program Subtract. If the remaining 90 bases are completely matched, the final percent identity will be 90%. In another example, a 90 base sequence is compared to a 100 base sequence. This time, the deletion is an internal deletion, so there is no 5 'or 3' base of the target sequence that is not matched / aligned with the query sequence. In this case, the percent identity calculated by FASTDB is not manually corrected. Again, only the 5 'and 3' bases of the target sequence that are not matched / aligned with the query sequence are manually corrected. For the purposes of the present invention, no other passive correction is made.
[123] The present application discloses a nucleic acid sequence which is at least 80%, 85%, 90% identical to the nucleic acid sequence shown in the first figure [SEQ ID NO: 1] or the deposited cDNA, regardless of whether the polypeptide encodes a polypeptide having KGF- , 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical nucleic acid molecules. This is because even when a particular nucleic acid molecule does not encode a polypeptide having KGF-2 activity, one skilled in the art will still know how to use the nucleic acid molecule, e.g., as a hybridization probe or polymerase chain reaction (PCR) primer. The use of a nucleic acid molecule of the invention that does not encode a polypeptide having KGF-2 activity, in particular (1) separating the KGF-2 gene or its allelic variant from a cDNA library; (2) Verma et al., Human (E.g., "FISH") with a mid-term chromosome spread to provide the correct chromosomal location of the KGF-2 gene, as described in Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York ; And Northern blot analysis to detect KGF-2 mRNA expression in specific tissues.
[124] However, the nucleic acid sequence shown in Figure 1 (SEQ ID NO: 1) coding for a polypeptide having KGF-2 protein activity is at least 80%, 85%, 90%, 91% Nucleic acid molecules having 92%, 93%, 94%, 95%, 97%, 98% or 99% identical sequences are preferred. "Polypeptide having KGF-2 activity" refers to an activity of wild-type KGF-2 protein of the invention or an increase in the activity of wild-type KGF-2 protein (full-length protein, or preferably a mature protein) (Not necessarily the same) activity of the polypeptide.
[125] An assay of KGF-2 activity is described, for example, in Examples 10 and 11 below. These assays can be used to measure the KGF-2 activity of partially purified or purified native or recombinant proteins.
[126] KGF-2 stimulates the proliferation of epidermal keratinocytes but not mesenchymal cells such as fibroblasts. Thus, a " polypeptide having KGF-2 protein activity " includes a polypeptide exhibiting KGF-2 activity in the keratinocyte proliferation assay disclosed in Example 10, which includes FGF receptor isoforms 1-iiib and 2-iiib (Example 11). Preferably, the " polypeptide having KGF-2 protein activity " will exhibit substantially similar activity as compared to the KGF-2 protein (i.e., the candidate polypeptide Will exhibit greater activity, or less than about 10-fold, preferably less than about 2-fold, activity for the reference KGF-2 protein.
[127] Of course, owing to the degeneracy of the genetic code, those skilled in the art will understand that the nucleotide sequence of the deposited cDNA or 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, It is instantly known that a number of nucleic acid molecules having a sequence of greater than 93%, greater than 94%, greater than 95%, greater than 97%, greater than 98%, or greater than 99% will encode a peptide having "KGF-2 protein activity" Will recognize. In fact, since all of the degenerate variants of these nucleotide sequences encode the same polypeptide, it will be apparent to those skilled in the art without performing the above-described comparative analysis. In addition, it will be appreciated by those skilled in the art that for such nucleic acid molecules that are not degenerate variants, a modest number will encode polypeptides that possess KGF-2 protein activity. This is because a person skilled in the art will fully appreciate amino acid substitutions (for example, substituting one aliphatic amino acid with a secondary aliphatic amino acid) that are unlikely or likely to have a significant effect on protein function.
[128] For example, a guide on how to make phenotypically silent adult amino acid substitutions is provided by Bowie, JU et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247: 1306-1310 Here, the authors point out two main approaches to study the tolerance of amino acids to be altered. The first method relies on the process of evolution, where mutations are accepted or rejected by natural selection. The second approach uses genetic techniques to introduce amino acid changes at specific positions of the cloned gene and screen or screen to identify sequences that retain functionality. As the authors noted, these studies surprisingly revealed that the protein tolerates amino acid substitutions. In addition, the authors point out what amino acid changes may be acceptable at specific locations in the protein. For example, most amino acid residues in the deep need to be non-polar side chains, usually only a few surface side chain features are conserved. Other such phenotypically silent substitutions are described in Bowie, J. U. et al., Supra, and are herein incorporated by reference.
[129] Further, the present invention relates to a method for producing a nucleic acid having a homology of at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, 91%, 92%, 93%, 94% , 98%, or 99% identity to a polynucleotide that hybridizes with the sequences described herein above. The present invention relates to polynucleotides that hybridize with the polynucleotides described herein above under particularly stringent conditions. As used herein, " stringent conditions " means that hybridization occurs when there is at least 95% identity, preferably at least 97% identity, between sequences. In a preferred embodiment, a polynucleotide that hybridizes with a polynucleotide described herein above is a polypeptide having substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure 1 (SEQ ID NO: 1) or the deposited cDNA (s) .
[130] For example, "stringent hybridization conditions" include 50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10 % Dextran sulfate, and 20 ug / ml denatured sheared salmon sperm DNA at 42 < 0 > C, followed by washing the filter in 0.1 x SSC at about 65 < 0 > C. Alternatively, the polynucleotide may hybridize with the polynucleotide of the present invention as described hereinabove, and may have at least 20 bases, preferably at least 30 bases, more preferably at least 50 bases, which may or may not have activity, Base or more. For example, such polynucleotides can be used to recover polynucleotides, for example, as probes of polynucleotides of SEQ ID NO: 1 or as diagnostic probes or as PCR primers.
[131] Also, nucleic acid molecules that hybridize with KGF-2 polynucleotides under moderately high stringency hybridization conditions are also expected. Changes in the stringency of the hybridization and signal detection are predominantly the formamide concentration (low percent formamide yields low stringency); Salt conditions or temperature. For example, moderately stringent conditions include 6 x SSPE (20 x SSPE = 3 M NaCl; 0.2 M NaH ₄ PO ₄ ; 0.02 M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug / Lt; 0 > C overnight at 3 [deg.] C in a solution containing sperm DNA, followed by washing with 1 x SSPE, 0.1% SDS at about 50 < 0 > C. In addition, washing performed after stringent hybridization to achieve lower stringency can be performed at higher salt concentration (e.g., 5 x SSC).
[132] It should be noted that modifications in the above conditions can be carried out through the inclusion and / or substitution of alternative blocking reagents used to inhibit background in hybridization experiments. Normally blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA and commercially available agents. The inclusion of a specific blocking reagent may require variation of the hybridization conditions described above due to compatibility problems.
[133] Of course, larger polynucleotides (e.g., deposited cDNA clones), such as polynucleotides that hybridize with a reference polynucleotide of a full length, for example, 50-750 nt in length, or nucleotide sequences of the deposited cDNA, Is useful as a probe according to the present invention, which is a polynucleotide corresponding to most of the depicted nucleotide sequence [SEQ ID NO. 1], but not all. A portion of a polynucleotide of "greater than or equal to 20 nt in length" includes, for example, 20 or more contiguous nucleotides from a nucleotide sequence of a reference polynucleotide (eg, the deposited cDNA or the nucleotide sequence shown in Figure 1 [SEQ ID NO: 1] it means. As set forth, such sites may be used as probes according to conventional DNA hybridization techniques or as probes, for example as described in Molecular cloning, A Laboratory Manual, 2nd Ed., Sambrook, J., Fritsch, Diagnostics as primers for amplification of a target sequence by polymerase chain reaction (PCR) as described in, for example, Wiley, C. and Maniatis, T. (1989), Cold Spring Harbor Laboratory Press, useful.
[134] It is routine to those skilled in the art to construct a polynucleotide that hybridizes to a portion of a KGF-2 cDNA molecule, since the KGF-2 cDNA clone has been deposited and its determined nucleotide sequence is provided in FIG. 1 [SEQ ID NO: 1]. For example, restriction endonuclease digestion of KGF-2 cDNA clones or shearing by sonication can be used to form DNA fragments of various sizes that are polynucleotides that hybridize to a portion of the KGF-2 cDNA molecule have. Alternatively, the hybridized polynucleotides of the present invention may be synthesized according to known techniques. Of course, polynucleotides that hybridize only to the complementary stretch of the polyA sequence (e.g., the 3 'terminal poly (A) track of the KGF-2 cDNA [SEQ ID NO: 1] shown in Figure 1) or the T (or U) Are not included in the polynucleotides of the present invention used to hybridize to some of the nucleic acids of the invention. Because the polynucleotide will hybridize to any nucleic acid molecule containing a poly (A) stretch or a complement thereof (e.g., virtually any double-stranded cDNA clone).
[135] Furthermore, the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding an epitope-bearing region of a KGF-2 protein. In particular, there is provided a separate nucleic acid molecule encoding a polypeptide comprising the amino acid residues shown in Figure 1 (SEQ ID NO: 2), wherein the inventor has determined the antigenic region of the KGF-2 protein:
[136] 1. Gly41-Asn71: GQDMVSPEATNSSSSSFSSPSSAGRHVRSYN [SEQ ID NO: 25];
[137] 2. Lys91-Ser109: KIEKNGKVSGTKKENCPYS [SEQ ID NO: 26];
[138] 3. Asn135-Tyr164: NKKGKLYGSKEFNNDCKLKERIEENGYNTY [SEQ ID NO: 27]; And
[139] 4. Asn181-Ala199: NGKGAPRRGQKTRRKNTSA [SEQ ID NO: 28].
[140] Also, there are two additional predicted shorter antigenic regions in Figure 1 [SEQ ID NO: 2], Gln74-Arg78 and Gln170-Gln175. The method of forming the epitope-containing region of KGF-2 is described below.
[141] The deposit referred to herein shall be maintained under the buttress treaty on the international recognition of microbial deposits for patent purposes. These deposits are merely provided for the convenience of those skilled in the art and deposits are not provided as the required authorization under 35 USC §112. The amino acid sequences of the polypeptides encoded thereby as well as the polynucleotide sequences contained in the deposited material are also incorporated herein by reference and control any conflict events with any description of the sequences herein. Licenses are required for the manufacture, use or sale of the deposited material, and this license does not grant this license.
[142] KGF-2 polypeptides and fragments
[143] Furthermore, the present invention relates to polypeptides having the amino acid sequence encoded by the cDNA retained or deposited with the deduced amino acid sequence [SEQ ID NO. 2] of FIG. 1, as well as fragments, analogs and derivatives of such polypeptides.
[144] As will be appreciated by those skilled in the art, due to the diversity of cleavage sites for the leader of different known proteins as well as the possibility of sequence analysis errors discussed above, the actual KGF-2 polypeptide encoded by the deposited cDNA contains about 208 amino acids , Somewhere in the range of about 200-220 amino acids, and the actual leader sequence of the protein may be anywhere from about 35 or 36 amino acids, but in the range of about 30 to about 40 amino acids.
[145] The terms " fragment, " " derivative, " and " analogue " when referring to the polypeptide of Figure 1 [SEQ ID NO: 2] or the polypeptide encoded by the deposited cDNA, Lt; / RTI > polypeptide. Thus, analogs include pro-proteins that are activated by cleavage of the pro-protein moiety to produce an active mature polypeptide.
[146] The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
[147] Fragments, derivatives or analogs of polypeptides encoded by the polypeptides of Figure 1 [SEQ ID NO: 2] or the deposited cDNAs are useful for the detection of (i) one or more amino acid residues that are conservative or non- conservative amino acid residues (preferably conservative amino acid residues) And such substituted amino acid residues are not encoded or encoded by the genetic code; Or (ii) one or more amino acid residues comprise a substituent; Or (iii) a mature polypeptide is fused with another compound, e. G., A compound that increases the half-life of the polypeptide (e. G., Polyethylene glycol), or (iv) the additional amino acid is fused to a mature polypeptide, A leader or secretory sequence or a sequence used for the purification of a mature polypeptide or proprotein sequence. Such fragments, derivatives and analogs may be determined within the scope of those skilled in the art from those taught herein.
[148] The terms " peptide " and " oligopeptide " are to be considered synonymous (as commonly recognized), and each term is intended to mean a linkage between two or more amino acids, as required herein to indicate a chain of two or more amino acids paired by peptide bonds It is possible to use it. &Quot; Polypeptide " is used herein in the case of a chain comprising at least 10 amino acid residues. All oligopeptides and polypeptide form sequences herein are recorded from left to right and from amino terminus to carboxy terminus.
[149] It will be appreciated by those skilled in the art that some amino acid sequences of KGF-2 polypeptides may be modified without significant effect on the structure or function of the protein. It should be remembered that if this difference in sequence is expected, there will be a significant region of the protein that determines activity. Typically, if a moiety that performs a similar function is used, the moiety that forms the tertiary structure may be replaced. In another example, the type of residue may not be entirely important if the modification occurs in non-critical regions of the protein.
[150] Accordingly, the present invention further comprises a variant of a KGF-2 polypeptide comprising a region of the KGF-2 protein, such as a protein region which exhibits substantial KGF-2 polypeptide activity or is discussed below. Such variants include deletions, insertions, inversions, repetitions, and substitution types (e.g., substituting hydrophilic residues for another, but not substituting strong hydrophilic residues with strong hydrophobic residues). Small changes or such " neutral " amino acid substitutions will usually have little effect on activity.
[151] Substituting one of the aliphatic amino acids Ala, Val, Leu and Ile for another, as is usually the case for conservative substitutions; The exchange of hydroxyl residues Ser and Thr, the exchange of acidic residues Asp and Glu, the substitution between the amide residues Asn and Gln, the exchange of the basic residues Lys and Arg and the replacement of the aromatic residues Phe and Tyr.
[152] As indicated above, additional guidance on what amino acid changes may be phenotypically silent (i.e., not likely to have significant adverse effects on function) can be found in Bowie, JU et al., Deciphering the &Quot; Message in Protein Sequence: Tolerance to Amino Acid Substitutions, " Science 247: 1306-1310 (1990).
[153] The present invention includes a mimetic peptide of KGF-2 that can be used as a therapeutic peptide. Mimetic KGF-2 peptides are short peptides that mimic the biological activity of the KGF-2 protein by binding to and activating the homologous receptor of KGF-2. Mimetic KGF-2 peptides can also bind to and inhibit the homologous receptor of KGF-2. The KGF-2 receptor includes, but is not limited to, FGFR2iiib and FGFR1iiib. Such mimetic peptides are obtained from a method such as, but not limited to, phage display and combinatorial chemical reactions. For example, the method disclosed in Wrighton et al., Science 273: 458-463 (1996) can be used to form a mimetic KGF-2 peptide.
[154] The polypeptides and polynucleotides of the present invention are preferably provided in isolated form, more preferably homogeneously purified.
[155] The polypeptides of the invention are preferably in isolated form. By "isolated polypeptide" is meant a polypeptide that has been removed from its natural environment. Thus, polypeptides produced in recombinant host cells and / or contained within recombinant host cells are considered isolated for purposes of the present invention. Also refers to polypeptides that are partially or substantially purified from recombinant host cells or natural sources.
[156] A polypeptide of SEQ ID NO: 2, as well as a polypeptide of SEQ ID NO: 2, as well as polypeptides of SEQ ID NO: 2, as well as polypeptides of SEQ ID NO: 2 with greater than 80%, 85%, 90%, 91%, 92% (More preferably at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 97% , 95% or more, 97% or more, 98% or more, or 99% or more identity), and the polypeptide portion is usually part of a polypeptide containing 30 or more amino acids, more preferably 50 or more amino acids (E. G., Deletion mutants described below).
[157] As is known in the art, " similarity " between two polypeptides is determined by comparing the amino acid sequence of the first polypeptide and its conservative amino acid substitution with the sequence of the second polypeptide.
[158] The "% similarity " of the two polypeptides was calculated using the BestPlug program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Medison WI 5311) Quot; means the similarity score provided by comparing the amino acid sequence of the polypeptide. Best uses the local homology algorithm of Smith and Waterman (Advances in Applied mathematics 2: 482-489, 1981) to find the best similarity segment between two sequences.
[159] A polypeptide having an amino acid sequence that is " identical " for example at least 95% identical to a reference amino acid sequence of a KGF-2 polypeptide means that the polypeptide sequence comprises no more than 5 amino acid modifications per 100 amino acids in the reference amino acid sequence of the KGF- Quot; means that the amino acid sequence of the polypeptide is the same as the reference sequence. On the other hand, up to 5% of the amino acid residues in the reference sequence may be deleted or replaced with another amino acid, or 5% or more of the total amino acid residues in the reference sequence may be substituted for the polypeptide having the same amino acid sequence as the reference amino acid sequence, May be added to the reference sequence. These modifications of the reference sequence can occur at any of the amino or carboxy terminal positions of the reference amino acid sequence or between any of these terminal positions and are spotted among the residues in the reference sequence or within one or more adjacent groups within the reference sequence .
[160] In practice, it is contemplated that any particular polypeptide may be, for example, at least 80%, at least 85%, at least 90%, at least 91% identical to the amino acid sequence encoded by the amino acid sequence [SEQ ID NO: 2] , 92%, 93%, 94%, 95%, 97%, 98%, or 99% ≪ / RTI > University Research Park, 575 Science Drive, Medison WI 53111). When determining whether a particular sequence is, for example, 95% identical to the reference sequence according to the invention using the bests or any other sequence alignment program, the percent identity (%) for the full length of the reference amino acid sequence is of course calculated The parameters are set so that a homology difference of 5% or less of the total number of amino acid residues in the reference sequence is allowed.
[161] A preferred method for determining the best overall match between a query sequence (a sequence of the invention) and a target sequence referred to as a global sequence alignment is described by Brutlag et al. [Comp. App. Biosci. 6: 237-245 (1990)] using the FASTDB computer program. In a sequence listing, both the query and subject sequences are either nucleotide sequences or both amino acid sequences. The result of the global sequence alignment is identity (%). The preferred parameters used in the FASTDB amino acid alignment are matrix = PAM 0, k-tuple = 2, mismatch penalty point = 1, binding penalty point = 20, randomization group length = 0, cutoff score = Length, gap penalty point = 5, gap size penalty point = 0.05, window size = 500, or target amino acid sequence length (whichever is shorter).
[162] If the target sequence is shorter than the suspect sequence due to an N-terminal or C-terminal deletion rather than an internal deletion, manual correction of the result should be performed. This is because the FASTDB program can not account for the N- and C-terminal truncations of the target sequence when computing the global identity (%). In the case of the subject sequence truncated at the N- and C-termini, identity (%) compared to the query sequence is the identity of the residues of the query sequence, N- and C-termini of the subject sequence not matched / The number is corrected by calculating the percentage of total residues in question. Whether or not the residues are matched / aligned is determined as the result of FASTDB sequence alignment. This ratio is then subtracted from the identity (%) calculated with the FASTDB using a specific parameter to arrive at the final identity (%) score. A final% identity score is used in the present invention. Only the residues for the N- and C-termini of the target sequence that are not matched / aligned with the query sequence are considered to manually adjust the percent identity (%) score. In other words, only the positions of the query residues outside the N- and C-terminal residues at the most remote positions in the target sequence are considered.
[163] For example, to determine identity (%), the 90 amino acid residue sequence aligns with the query sequence of 100 residues. Because deletions occur at the N-terminus of the target sequence, FASTDB alignment does not indicate the match / alignment of the first 10 residues at the N-terminus. Since 10 non-paired residues represent 10% of the sequence (10% of the total number of residues / mismatches at the unconjugated N-terminus and C-terminus), the percentage identity (%) calculated with the FASTDB program . If the remaining 90 residues are fully matched, the final identity (%) is 90%. As another example, the target sequence of 90 residues is compared to the query sequence of 100 residues. There is no deletion at the N- or C-terminus of the target sequence that is internalized and therefore not aligned / aligned with the query sequence. In this case, the identity (%) calculated by FASTDB is not manually corrected. Again, only the positions of residues outside the N- and C-termini of the target sequence indicated in the FASTDB alignment, which are not matched / aligned with the query sequence, are manually corrected. For the purposes of the present invention, there is no other manual correction.
[164] Polypeptides of the invention can be used to produce polyclonal antibodies and monoclonal antibodies, as described in detail below, which antibodies are useful for diagnostic assays to detect KGF-2 protein expression, 2 < / RTI > protein function. Furthermore, such polypeptides can be used in enzyme double-hybrid systems to " capture " KGF-2 protein binding proteins that are candidate agents and antagonists in accordance with the present invention. The yeast dual hybrid system is described in Fieds and Song, Nautre 340: 245-246 (1989).
[165] In yet another aspect, the invention provides a peptide or polypeptide comprising an epitope-bearing site of a polypeptide of the invention. The epitope of this polypeptide moiety is an immunogenic or antigenic epitope of the polypeptide of the present invention. An " immunogenic epitope " is defined as a portion of a protein that elicits an antibody response if the whole protein is an immunogenic agent. These immunogenic epitopes are believed to be confined to some loci on the molecule. On the other hand, the region of a protein molecule to which an antibody can bind is defined as an " antigenic epitope ". The number of immunogenic epitopes of a protein is generally less than the number of antigenic epitopes. See, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983).
[166] With respect to the selection of peptides or polypeptides bearing an antigenic epitope (i. E. Containing a region of a protein molecule to which the antibody is capable of binding), relatively short synthetic peptides that mimic a portion of the protein sequence are generally partially reactive with the mimic protein Lt; RTI ID = 0.0 > antagonist < / RTI > See, e.g., Sutcliffe, J.G. Shinnick, T. M., Green, N. and Learner, R.A. See " Antibodies that react with selected sites on proteins ", Science 219: 660-666 (1983). Peptides capable of inducing protein reactive sera are provided primarily in the primary sequence of the protein, can be characterized by simple chemical rules, and can be characterized by amino or carboxy terminus (e. G. . Peptides that are extremely hydrophobic and peptides with six or fewer residues are ineffective in inducing antibodies that bind to mimetic proteins, and peptides that contain longer, water soluble peptides, particularly proline residues, are usually effective [Sutcliffe et al., Supra , At 661]. For example, 18 of the 20 peptides, designed according to these guidelines and containing 8-39 residues, accounting for 75% of the sequence of the influenza virus hemagglutinin HA1 polypeptide chain, contained antibodies that reacted with HA1 protein or complete virus ; The 12/12 peptide from the MuLV polymerase and the 18/18 peptide from the rabbit glycoprotein induced antibodies that precipitate each protein.
[167] Antigenic epitope-bearing peptides and polypeptides of the present invention are useful for producing antibodies, including monoclonal antibodies, that specifically bind to the polypeptides of the invention. Thus, a high proportion of hybridomas obtained by fusion of splenocytes from a donor immunized with an antigen-epitope-bearing peptide normally secrete antibodies reactive with the native protein. At 663 of the document Sutcliffe et al. Antibodies generated by antigenic epitope-bearing peptides or polypeptides are useful for detecting mimetic proteins, and antibodies to different peptides can be used to track the fate of various regions of the protein precursor that undergo post-translational processing. Peptide and anti-peptide antibodies can be used for a variety of qualitative or quantitative analyzes (e. G., Competitive assays) on mimetic proteins, which may involve binding of larger peptides in the immunoprecipitation assays even for short peptides Because it shows that you can do it instead. (See, for example, Wilson et al., Cell 37: 767-778 (1984), 777). In addition, the anti-peptide antibodies of the present invention are useful for purifying mimetic proteins, for example, by adsorption chromatography using methods well known in the art.
[168] The antigenic antibody-bearing peptides and polypeptides of the present invention devised in accordance with the above manual are preferably contained in the amino acid sequence of the polypeptide of the present invention, preferably 7 or more, more preferably 9 or more, and most preferably about 15 to about It contains 30 amino acids. However, it is contemplated that a polypeptide comprising an amino acid sequence of any length up to about 30, 40, 50, 60, 70, 80, 90, 100 or 150 amino acids, or the entire amino acid sequence of a polypeptide of the invention, Peptides or polypeptides comprising the amino acid sequence of the larger region of the polypeptide of the invention are also considered epitope-bearing peptides or polypeptides of the invention and are also useful for inducing antibodies that react with the mimetic protein. It is preferred that the amino acid sequence of the epitope-bearing peptide be selected to provide substantial solubility in aqueous solvents (e. G., This sequence includes relatively hydrophilic residues and avoids high hydrophobic sequences); Sequences containing proline residues are particularly preferred.
[169] Non-limiting examples of antigenic polypeptides or peptides that can be used to produce KGF-2-specific antibodies include the following.
[170] 1. Gly41-Asn71: GQDMVSPEATNSSSSSFSSPSSAGRHVRSYN [SEQ ID NO: 25];
[171] 2. Lys91-Ser109: KIEKNGKVSGTKKENCPYS [SEQ ID NO: 26];
[172] 3. Asn135-Tyr164: NKKGKLYGSKEFNNDCKLKERIEENGYNTY [SEQ ID NO: 27]; and
[173] 4. Asn181-Ala199: NGKGAPRRGQKTRRKNTSA [SEQ ID NO: 28].
[174] Also, there are two additional predicted shorter antigenic regions in Figure 1 [SEQ ID NO: 2], Gln74-Arg78 and Gln170-Gln175.
[175] The epitope-bearing peptides and polypeptides of the present invention may be produced by conventional means of peptide or polypeptide production, including recombinant methods using nucleic acid molecules of the invention. For example, short epitope-bearing amino acid sequences can be fused to larger polypeptides that act as a carrier during immunization to produce anti-peptide antibodies, as well as during recombinant production and purification. In addition, epitope-bearing peptides may be synthesized using known methods of chemical synthesis. For example, Houghten disclosed a simple method for the synthesis of large numbers of peptides, such as 248 distinct 13 residue peptides 10-20 mgs representing 1 amino acid variation of a segment of the HA1 polypeptide, (By ELISA-type binding studies) and characterized. (Houghten, R. A. (1985)). General methods for rapid solid phase synthesis of multiple peptides: specificity of antigen-antibody interaction at the level of individual amino acids. (Proc. Natl. Acad. Sci. USA 82: 5131-5135). This " simultaneous plural peptide synthesis (SMPS) " method is further described in U.S. Patent No. 4,631,211 (1986) to Hughton et al. In this method, each resin for the solid-phase synthesis of various peptides is contained in a separate solvent-permeable packet to suitably utilize many of the same repetitive steps involved in the solid-phase method. A complete passive method allows simultaneous synthesis of 500-1000 or more (Houghten et al., 5134, supra)
[176] The present invention encompasses an epitope of a polypeptide having the amino acid sequence of SEQ ID NO: 2, or a polypeptide encoded by a polynucleotide sequence contained in ATCC Accession No. 75977 or under the hybridization conditions of the above-mentioned stringent hybridization conditions or lower stringency A polypeptide comprising or consisting of an epitope of a polypeptide sequence encoded by a polynucleotide that hybridizes to a sequence contained in ATCC Deposit No. 75977 or a complement of the sequence of SEQ ID NO: 1. The invention encompasses polynucleotide sequences encoding the epitopes of the polypeptide sequences of the invention (e. G., Sequences set forth in SEQ ID NO: 1), polynucleotide sequences of the complementary strand of the polynucleotide sequences encoding the epitopes of the invention, And further comprises a polynucleotide sequence hybridizing to the complementary strand under stringent hybridization conditions or hybridization conditions of lower stringency.
[177] As used herein, an " epitope " refers to a polypeptide moiety having an antigenic or immunogenic activity in an animal, preferably a mammal, most preferably a human. In a preferred embodiment, the invention comprises a polypeptide comprising an epitope and a polynucleotide encoding said polypeptide. An " immunogenic epitope " as used herein is defined as a region of a protein that induces an antibody response in an animal when determined by any method known in the art, such as, for example, the method of antibody generation described below (Geysen et al , Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983)). As used herein, " antigenic epitope " refers to a region of a protein that an antibody can specifically bind to its antigen when determined by any method known in the art, such as immunoassays disclosed herein. Immunospecific binding excludes non-specific binding, but does not necessarily exclude cross-reactivity with other antigens. An antigenic epitope does not necessarily have to be immunogenic.
[178] Fragments that act as epitopes may be produced by any conventional method (see Houghten, Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985), further in U.S. Patent 4,631,211 )
[179] In the present invention, the antigenic epitope preferably has a sequence of at least 4, at least 5, at least 6, at least 7 amino acids, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14 , At least 15, at least 20, at least 25, at least 30, at least 40, at least 50 amino acids, and most preferably between about 15 and about 30 amino acids. Preferred polypeptides comprising an immunogenic or antigenic epitope comprise at least 10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 Amino acid residue length. An additional preferred antigenic epitope comprises or consists of the following residues of SEQ ID NO: 2: M-1 to H-15; W-2 to L-16; K-3 to P-17; W-4 to G-18; I-5 to C-19; L-6 to C-20; T-7 to C-21; H-8 to C-22; C-9 to C-23; A-10 to F-24; S-11 to L-25; A-12 to L-26; F-13 to L-27; P-14 to F-28; H-15 to L-29; L-16 to V-30; P-17 to S-31; G-18 to S-32; C-19 to V-33; C-20 to P-34; C-21 to V-35; C-22 to T-36; C-23 to C-37; F-24 to Q-38; L-25 to A-39; L-26 to L-40; L-27 to G-41; F-28 to Q-42; L-29 to D-43; V-30 to M-44; S-31 to V-45; S-32 to S-46; V-33 to P-47; P-34 to E-48; V-35 to A-49; T-36 to T-50; C-37 to N-51; Q-38 to S-52; A-39 to S-53; L-40 to S-54; G-41 to S-55; Q-42 to S-56; D-43 to F-57; M-44 to S-58; V-45 to S-59; S-46 to P-60; P-47 to S-61; E-48 to S-62; A-49 to A-63; T-50 to G-64; N-51 to R-65; S-52 to H-66; S-53 to V-67; S-54 to R-68; S-55 to S-69; S-56 to Y-70; F-57 to N-71; S-58 to H-72; S-59 to L-73; P-60 to Q-74; S-61 to G-75; S-62 to D-76; A-63 to V-77; G-64 to R-78; R-65 to W-79; H-66 to R-80; V-67 to K-81; R-68 to L-82; S-69 to F-83; Y-70 to S-84; N-71 to F-85; H-72 to T-86; L-73 to K-87; Q-74 to Y-88; G-75 to F-89; D-76 to L-90; V-77 to K-91; R-78 to I-92; W-79 to E-93; R-80 to K-94; K-81 to N-95; L-82 to G-96; F-83 to K-97; S-84 to V-98; F-85 to S-99; T-86 to G-100; K-87 to T-101; Y-88 to K-102; F-89 to K-103; L-90 to E-104; K-91 to N-105; I-92 to C-106; E-93 to P-107; K-94 to Y-108; N-95 to S-109; G-96 to I-110; K-97 to L-111; V-98 to E-112; S-99 to I-113; G-100 to T-114; T-10l to S-115; K-102 to V-116; K-103 to E-117; E-104 to I-118; N-105 to G-119; C-106 to V-120; P-107 to V-121; Y-108 to A-122; S-109 to V-123; I-110 to K-124; L-111 to A-125; E-112 to I-126; I-113 to N-127; T-114 to S-128; S-115 to N-129; V-116 to Y-130; E-117 to Y-131; I-118 to L-132; G-119 to A-133; V-120 to M-134; V-121 to N-135; A-122 to K-136; V-123 to K-137; K-124 to G-138; A-125 to K-139; I-126 to L-140; N-127 to Y-141; S-128 to G-142; N-129 to S-143; Y-130 to K-144; Y-131 to E-145; L-132 to F-146; A-133 to N-147; M-134 to N-148; N-135 to D-149; K-136 to C-150; K-137 to K-151; G-138 to L-152; K-139 to K-153; L-140 to E-154; Y-141 to R-155; G-142 to I-156; S-143 to E-157; K-144 to E-158; E-145 to N-159; F-146 to G-160; N-147 to Y-161; N-148 to N-162; D-149 to T-163; C-150 to Y-164; K-151 to A-165; L-152 to S-166; K-153 to F-167; E-154 to N-168; R-155 to W-169; I-156 to Q-170; E-157 to H-171; E-158 to N-172; N-159 to G-173; G-160 to R-174; Y-161 to Q-175; N-162 to M-176; T-163 to Y-177; Y-164 to V-178; A-165 to A-179; S-166 to L-180; F-167 to N-181; N-168 to G-182; W-169 to K-183; Q-170 to G-184; H-171 to A-185; N-172 to P-186; G-173 to R-187; R-174 to R-188; Q-175 to G-189; M-176 to Q-190; Y-177 to K-191; V-178 to T-192; A-179 to R-193; L-180 to R-194; N-181 to K-195; G-182 to N-196; K-183 to T-197; G-184 to S-198; A-185 to A-199; P-186 to H-200; R-187 to F-201; R-188 to L-202; G-189 to P-203; Q-190 to M-204; K-191 to V-205; T-192 to V-206; R-193 to H-207; And / or R-194 to S-208. Polynucleotides encoding these polypeptide fragments are also included in the present invention.
[180] Additional non-exclusive preferred antigenic epitopes include those as well as the antigenic epitopes disclosed herein. An antigenic epitope is useful, for example, to generate an antibody comprising a monoclonal antibody that specifically binds to the epitope. Preferred antigenic epitopes include any of the antigenic epitopes disclosed herein as well as any combination of two, three, four, five, or more of these antigenic epitopes. Antigenic epitopes can be used as target molecules in immunoassays (see, for example, Wilson et al., Cell 37: 767-778 (1984); Sutcliffe et al., Science 219: 660-666 ) Reference).
[181] Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods known in the art (Sutcliffe et al., Supra; Wilson et al., Supra; Chow et al., Proc Natl. Acad. Sci. USA 82: 910-914; and Bittle et al., J. Gen. Virol.66: 2347-2354 (1985)). Preferred immunogenic epitopes comprise the immunogenic epitopes disclosed herein and include any combination of two, three, four, five, or more of these immunogenic epitopes. Polypeptides comprising one or more immunogenic epitopes are provided to an animal system (rabbit or mouse) to induce an antibody response with a carrier protein such as albumin, or if the polypeptide is of sufficient length (at least about 25 amino acids) May be provided without a carrier. However, immunogenic epitopes comprising from 8 to 10 small amino acids have been shown to be sufficient to generate antibodies capable of binding to a minimal linear epitope of a modified polypeptide (e. G., Western blot).
[182] The epitope-bearing peptides and polypeptides of the present invention may be used to induce antibodies according to methods known in the art. For example, Sutcliffe et al., Supra; Wilson et al., Supra; Chow et al., Proc. Natl. Acad. Sci. USA 82: 910-914; And Bittle et al., J. Gen. Virol. 66: 2347-2354 (1985). Usually an animal can be immunized with a free peptide. However, the anti-peptide antibody potency may be increased by binding the peptide to a macromolecular carrier (e.g., keyhole limpet hemacyanin (KLH) or Tetanus toxin). For example, peptides containing cysteine may be coupled to a carrier using a linker such as m-maleimido benzoyl-N-hydroxysuccinimide ester (MBS), and other peptides may be conjugated to a carrier such as the more general Or may be bound to the carrier using a linking agent. Animals such as rabbits, mice and mice are immunized with free or carrier-bound peptides by peritoneal injection and / or endothelial injection of about 100 g of peptide or carrier protein and emulsion containing Freund's adjuvant. Some intensive injections may be needed at about two week intervals to provide useful potency of anti-peptide antibodies that can be detected, for example, by ELISA assays using free peptides adsorbed to solid surfaces. The potency of the anti-peptide antibodies in the serum of the immunized animal may be increased by selection of anti-peptide antibodies, for example, by adsorption to a peptide on a solid support and eluting the selection antibody according to methods known in the art.
[183] Such portions of the immunogenic epitope-bearing peptides of the invention, i. E., Proteins that induce antibody responses when the whole protein is an immunogen, are identified according to methods known in the art. For example, Geyset et al. Disclose procedures for rapid and concurrent synthesis of hundreds of peptides on a solid support with sufficient purification to react in enzyme-linked immunosorption assays. The interaction of the synthesized peptides with the antibodies is then easily detected without removing them from the support. Peptides carrying an immunogenic epitope of the desired protein in this manner can be identified by routine methods to those skilled in the art. For example, an immunologically important epitope in the coat protein of foot-and-mouth viruses has been identified by Geysen et al. In 7 amino acid resolution by synthesizing a nested set of all 208 possible hexapeptides, including the entire 213 amino acid sequence of the protein . Subsequently, a complete set of substitutions, in which all 20 amino acids were substituted in turn at all positions in the epitope, were synthesized to determine specific amino acids that conferred specificity for reaction with the antibody. Thus, peptide analogs of the epitope-bearing peptides of the invention can be routinely produced by this method. US Patent No. 4,708,781 (1987) to Geysen further describes a method for identifying peptides carrying an immunogenic epitope of a desired protein.
[184] Further, US Patent 5,194,392 (1990) of Geysen discloses that monomers that are topologically equivalent to an epitope (i.e., " mimotope ") complementary to a particular paratope (antigen binding site) ) Or to determine its sequence. More generally, US Patent No. 4,433,092 (1989) by Geysen describes a method for detecting or sequencing a monomer that is topographically equivalent to a ligand that is complementary to the ligand binding site of a particular receptor of interest. Similarly, U. S. Patent 5,480, 971 (1996) (pertaining to " Peralkylated Oligopeptide Mixture ") by Houghen, RA discloses linear C ₁ -C ₇ -alkylperalkylated oligopeptides and sets and libraries of such peptides Describes methods for using such oligopeptide sets and libraries to determine the sequence of a peralkylated oligopeptide that binds preferentially to a recipient molecule of interest. Thus, non-peptide analogs of the epitope-bearing peptides of the invention can also be routinely produced by these methods.
[185] As will be appreciated by those skilled in the art, the KGF-2 polypeptides of the invention and their epitope-bearing fragments can be combined with a portion of the constant domain of an immunoglobulin (IgG) to produce a chimeric polypeptide. These fusion proteins facilitate purification and exhibit increased in vivo half-life. This has been confirmed, for example, in the case of chimeric proteins consisting of the first two domains of the human CD4-polypeptide and the various domains of the heavy or light chain constant domains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331: 84-86 1988). Fusion proteins with disulfide-linked dimeric structures by the IgG moiety are more effective at neutralizing binding to other molecules than the monomeric KGF-2 protein or protein fragment itself (Fountoulakis et al., J Biochem 270: 3958-3964 (1995) .
[186] In accordance with the present invention, novel KGF-2 variants are also described. These can be produced by deletion or substitution of one or more amino acids of KGF-2. Natural mutations are referred to as allelic variants. The allelic variation may be silent or have an altered amino acid sequence (no change in the encoded polypeptide).
[187] Protein manipulation can be used to improve or alter the properties of native KGF-2. Recombinant DNA techniques known to those skilled in the art can be used to generate new polypeptides. Muteins and deletions can indicate, for example, enhanced activity or increased stability. In addition, they can be purified in high yields and may be more soluble at least under certain purification and storage conditions. Examples of mutations that can be made are described below.
[188] The KGF-2 polypeptide of the present invention can be a monomer or a multimer (e.g., dimer, trimer, tetramer or larger multimer). Accordingly, the present invention relates to monomers and oligomers of KGF-2 polypeptides of the invention, formulations thereof and compositions (preferably therapeutic compositions) containing them. In certain embodiments, the polypeptide of the invention is a monomer, dimer, trimer, or tetramer. In a further embodiment, the oligomer of the invention is at least a dimer, at least a trimer or at least a tetramer.
[189] The oligomers encompassed by the present invention may be homomultimers or heteromers. As used herein, the term homomultimer (homomer) refers to a polypeptide encoded by a cDNA contained in a clone corresponding to or corresponding to the amino acid sequence of SEQ ID NO: 2 (fragments, variants, Splice variants, and fusion proteins). These homomultimeters may comprise KGF-2 polypeptides having the same or different amino acid sequences. In certain embodiments, homomultimeters of the invention are multimers comprising only the KGF-2 polypeptide having the same amino acid sequence. In yet another embodiment, the alleles of the present invention are oligomers comprising a KGF-2 polypeptide having a different amino acid sequence. As a specific example, the oligomers of the present invention may be homodimers (e.g., including KGF-2 polypeptides having the same or different amino acid sequences) or homotrimeric (e.g., KGF-2 polypeptides having the same and / or different amino acid sequences ). In another embodiment, the homomultimer of the present invention is at least a homodimer, at least a homomeric trimer, or at least an enantiomer thereof.
[190] As used herein, the term heterodimer means a multimer containing, in addition to the KGF-2 polypeptide of the present invention, one or more heterologous polypeptides (i. E., Polypeptides of different proteins). As a specific example, the oligomer of the present invention is a heterodimer, a heterotrimer, or a heteromer. In a further embodiment, the heterodimers of the invention are at least heterodimers, at least heterodimers, or at least heterodimers.
[191] The oligomers of the present invention may result in hydrophobic, hydrophilic, ionic and / or covalent bonds and / or may be indirectly linked by, for example, liposomal formation. Thus, in one embodiment, a multimer of the invention, such as a homodimer or homotrimer, is formed when the polypeptides of the invention are in contact with each other in solution. In another embodiment, the heterodimers of the invention, e. G., Heterotetramers or heterodimers, can be constructed such that the polypeptide of the invention binds the antibody to a polypeptide of the invention (including an antibody to a heterologous polypeptide sequence in the fusion protein of the invention) In the solution. In another embodiment, the oligomers of the invention are formed by covalent bonds between and / or between the KGF-2 polypeptides of the invention. The covalent bond will comprise one or more amino acid residues contained within a polypeptide sequence (eg, as shown in SEQ ID NO: 2, or clone HPRCC57, or included in a polypeptide encoded by a clone contained in ATCC Accession No. 75977 or 75901). As an example, a covalent bond is a cross-link between cysteines present in a polypeptide sequence that interacts in a native (i.e. naturally occurring) polypeptide. As another example, covalent bonds are the result of chemical or recombinant manipulations. Alternatively, the covalent bond may comprise one or more amino acid residues contained within the heterologous polypeptide sequence of the KGF-2 fusion protein. As an example, covalent bonds exist between heterologous sequences contained in the fusion proteins of the present invention (see, for example, U.S. Patent No. 5,478,925). In certain embodiments, the covalent bond is between the heterologous sequences contained in the KGF-2-Fc fusion protein of the invention (as described above). In yet another embodiment, the covalent attachment of the fusion protein of the present invention forms a covalently bonded multimer such as, for example, osteoptrogerenine (see, e.g., WO 98/49305, incorporated herein by reference) Lt; RTI ID = 0.0 > polypeptide < / RTI > In yet another embodiment, two or more polypeptides of the invention are linked through a peptide linker. Examples include peptide linkers as disclosed in U.S. Patent No. 5,073,627, incorporated herein by reference. Proteins comprising the multiple polypeptides of the invention separated by a peptide linker can be generated using conventional recombinant DNA techniques.
[192] Another method of producing the multimeric polypeptides of the present invention involves the use of the polypeptides of the invention fused to a leucine zipper or isoleucine zipper polypeptide sequence. Leucine zipper and isoleucine zipper domains are polypeptides that promote the formation of multimers of the proteins in which they are found. Leucine zipper was first identified in several DNA-binding proteins (Landshulz et al., Science 240: 1759, (1988)). It has also been found in a variety of different proteins. There are naturally occurring peptides and derivatives thereof which dimerize or trimerize in the known leucine zipper. Examples of leucine zipper domains suitable for the production of soluble multimeric proteins of the present invention are described in PCT application WO 94/10308 (herein incorporated by reference). A recombinant fusion protein comprising a polypeptide of the invention fused to a polypeptide sequence that is dimerized or trimerized in solution is expressed in a suitable host cell and the resulting soluble multimer fusion proteins are isolated from the culture supernatant using well known techniques Is recovered.
[193] The trimeric polypeptides of the present invention will provide the advantage of improved biological activity. Preferably, the leucine zipper portion and the isoleucine zipper portion form a trimer. One example is the use of pulmonary surfactant protein D (SPD) as described in Hopper et al. (FEBS Letters 344: 191, (1994)) and US patent application Ser. No. 08 / 446,922 It is derived from leucine zipper. Other peptides derived from naturally occurring trimer proteins will be used to prepare the trimeric polypeptides of the present invention.
[194] In another embodiment, a protein of the invention is linked by interaction between Flag (TM) polypeptide sequences contained in a fusion protein of the invention, including a Flag (TM) polypeptide sequence. In a further embodiment, the protein binding of the present invention is linked by the interaction between the Flag < (R) > fusion protein of the present invention and a heterologous polypeptide sequence comprised in the anti-Flag < (R) >
[195] The oligomers of the present invention can be produced using chemical techniques known in the art. For example, polypeptides that are preferably included in the oligomers of the invention can be chemically cross-linked using linker molecule and linker molecular length optimization techniques known in the art (see, for example, U.S. Patent No. 5,478,925, The entirety of which is hereby incorporated by reference). In addition, the oligomers of the present invention can be produced using techniques known in the art to form one or more intermolecular cross-linkages between cysteine residues located within the sequence of a polypeptide that is preferably contained in the mass (see, for example, U. S. Patent No. 5,478, 925, which is hereby incorporated by reference in its entirety). In addition, the polypeptides of the present invention can be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide, and the known techniques are applicable to the generation of oligomers comprising one or more of the above modified polypeptides (See, for example, U.S. Patent No. 5,478,925, which is hereby incorporated by reference in its entirety). In addition, the known techniques can be applied to produce liposomes comprising a polypeptide component which is preferably included in the multimers of the present invention (see, for example, U.S. Patent No. 5,478,925, which is incorporated herein by reference in its entirety).
[196] Alternatively, the oligomers of the present invention may be produced using genetic engineering methods known in the art. In one embodiment, the polypeptides included in the oligomers of the present invention may be prepared using the fusion protein techniques described herein or other known techniques (see, e.g., U.S. Patent No. 5,478,925, which is incorporated herein by reference in its entirety) Recombinantly produced. As a specific embodiment, a polynucleotide encoding a homodimer of the present invention can be obtained by ligating a polypeptide sequence encoding the polypeptide of the present invention to a sequence encoding a linker polypeptide, then ligating the polypeptide sequence encoding the polypeptide of the present invention in the reverse direction of the N- (See, for example, U.S. Patent No. 5,478,925, which is incorporated herein by reference in its entirety) to a synthetic polynucleotide encoding the translated product of the polypeptide (leader sequence deletion). As another embodiment , Recombinant techniques described herein, or other techniques known in the art, include a transmembrane domain (or hydrophobic or signal peptide) and can be modified by liposomal membrane reconstitution techniques (see, e.g., U.S. Patent No. 5,478,925, which is hereby incorporated by reference in its entirety) ) To produce recombinant polypeptides of the present invention.
[197] Polynucleotides and polypeptide fragments
[198] The invention also relates to a fragment of the isolated nucleic acid molecule described herein. For example, a nucleotide sequence of the deposited cDNA (clone HPRCC57), a nucleotide sequence encoding a polypeptide encoded by the deposited cDNA, a nucleotide sequence encoding the polypeptide sequence shown in Figure 1 (SEQ ID NO: 2) The fragment of the isolated nucleic acid molecule carrying the nucleotide sequence (SEQ ID NO: 1), or a complementary sequence thereof, has a length of at least 15 nt, preferably at least about 20 nt, more preferably at least 30 nt, , 100, 150, 200, 250, 300, 325, 350, 375, 400, 450, 500, 550 or 600 nt or more. These fragments have a number of uses including, but not limited to, diagnostic probes and primers discussed herein. Of course, larger fragments, such as fragments with a length of 501-1500 nt, which are not the most of the nucleotide sequence of the deposited cDNA (clone HPRCC57) or the nucleotide sequence shown in Figure 1 (SEQ ID NO: 1) Are also useful according to the invention. For example, a fragment having a length of 20 nt or more includes fragments containing, for example, a nucleotide sequence of the deposited cDNA or a nucleotide sequence (SEQ ID NO: 1) shown in Fig. 1 containing 20 or more adjacent bases.
[199] In addition, representative examples of KGF-2 polynucleotide fragments include, for example, the nucleotide numbers of the nucleotides 1-50, 51-100, 101-150, 151-200, 201 of the cDNA contained in SEQ ID NO: 1 or its complementary strand or deposited clone -250, 251-300, 301-350, 351-400, 401-450, 451-500, 501-550, 551-600, 601-650, 651-700, 701-750, 751-800, 801-850 , 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451 -1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000 and / And fragments that retain the sequence. As used herein, " about " includes a range specifically cited at one end or both ends, and includes many or fewer nucleotides than the range (5, 4, 3, 2, or 1) nucleotides.
[200] Preferably, the polynucleotide fragment of the invention encodes a polypeptide exhibiting KGF-2 functional activity. KGF-2 A polypeptide that exhibits " functional activity " means a polypeptide capable of exhibiting one or more functional activities associated with a full-length (complete) KGF-2 protein. These functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with) a KGF-2 polypeptide for binding to an anti-KGF-2- polypeptide], immunogenicity [an antibody that binds to a KGF- Ability to form multimers with the KGF-2 polypeptides of the invention, and the ability to bind receptors or ligands to KGF-2 polypeptides.
[201] The functional activity of KGF-2 polypeptides and fragments, variants, derivatives and analogs thereof can be assayed by a variety of methods.
[202] For example, in one embodiment for analyzing the ability to bind or compete with full-length KGF-2 polypeptide when bound to an anti-KGF-2 antibody, various immunoassays known in the art that can be used include, but are not limited to, Analysis, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunodiffusion assay, gel diffusion settling reaction, immunodiffusion assay, in situ immunoassay (eg using colloidal gold, enzyme or radioisotope labels) Competitive assays that employ techniques such as Western blot, sedimentation, flocculation assays (eg, gel flocculation assays, hemagglutination assays), complement-fixation assays, immunofluorescence assays, protein A assays and immunoelectrophoresis assays . In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting that the primary antibody is secondary encompassed or reacted to the primary antibody. In another embodiment, the secondary antibody is labeled. A number of methods for detecting binding in immunoassays are known in the art and are within the scope of the present invention.
[203] In other embodiments in which the KGF-2 ligand is identified or the ability of the polypeptide fragment, variant or derivative of the invention to form a multimer is assessed, the binding may be accomplished, for example, by methods well known in the art (e. G., Reducing and non- , Protein affinity chromatography and affinity blotting). Phizichy, E et al., Microbiol. Rev. 59: 94-123 (1995). In other embodiments, physiological correlates of KGF-2 binding to its substrate (signal transduction) can be analyzed.
[204] In addition, KGF-2 polypeptides and fragments thereof, variants (e. G., In vitro and in vivo) that induce KGF-2 related biological activity (in vitro or in vivo) using the assays described herein , Derivatives and analogs thereof. Other methods will be known to those skilled in the art and fall within the scope of the present invention.
[205] The present invention relates to fragments of KGF-2 polypeptides described herein. For example, the isolated KGF-2 polypeptide encoded by the deposited cDNA (clone HPRCC57), the polypeptide sequence encoded by the deposited cDNA, the fragment of the polypeptide sequence shown in Figure 1 (SEQ ID NO: 2) Or a polypeptide fragment encoded by the cDNA contained in the deposited clone. The protein fragment may be " free-standing ", or the fragment may be contained within a larger polypeptide forming part or region, and most preferably may be present as a single continuous region. Representative examples of the polypeptide fragment of the present invention include amino acid residues of about 1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180, 181 -200, 201-220, 221-240, 241-260, 261-280, or 281 through fragments derived from the end of the cipher region. In addition, the polypeptide fragment may be an amino acid length of 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, Herein, " approximately " includes a range specifically cited at one end or both ends, and includes many or fewer amino acids (5, 4, 3, 2, or 1) than the range.
[206] Although deletion of one or more amino acids from the N-terminus of a protein yields a modification that deletes one or more biological functions of the protein, other functional activities (e.g., biological activity, ability to form multimers, binding of KGF-2 ligand Ability) can still be retained. For example, the ability of a shortened KGF-2 mutein to induce and / or bind an antibody that recognizes the complete or mature form of the polypeptide will usually be retained when the majority of the full or mature polypeptide residues are not removed from the N-terminus . Whether a particular polypeptide lacking the N-terminal residue of the complete polypeptide retains such immunogenic activity can be readily ascertained by routine methods described herein. KGF-2 muteins with multiple deleted N-terminal amino acid residues may retain some biological or immunogenic activity. In fact, peptides composed of as little as six KGF-2 amino acid residues can often cause an immune response.
[207] Thus, the polypeptide fragment includes not only the secretory KGF-2 protein but also the mature form. Further, preferred polypeptide fragments include secretory KGF-2 proteins or mature forms, comprising a sequence of amino or carboxy residues or residues deleted from both ends. For example, any number of amino acids in the 1-60 range can be deleted from the secreted KGF-2 polypeptide or amino terminal of the mature form. Similarly, any number of amino acids in the 1-30 range can be deleted from the secreted KGF-2 polypeptide or the carboxy terminus of the mature form. In addition, a combination of amino and carboxy terminal deletion is preferable. Similarly, polynucleotide fragments encoding these KGF-2 polypeptide fragments are also preferred.
[208] In particular, the N-terminal deletion of the KGF-2 polypeptide can be represented by the general formula m-208, wherein m is from 2 to 207 and m corresponds to the position of the amino acid residue identified in SEQ ID NO: 2. Specifically, the present invention includes polypeptides comprising or consisting of the following amino acid residues: W-2 to S-208 of SEQ ID NO: 2; K-3 to S-208; W-4 to S-208; I-5 to S-208; L-6 to S-208; T-7 to S-208; H-8 to S-208; C-9 to S-208; A-10 to S-208; S-11 to S-208; A-12 to S-208; F-13 to S-208; P-14 to S-208; H-15 to S-208; L-16 to S-208; P-17 to S-208; G-18 to S-208; C-19 to S-208; C-20 to S-208; C-21 to S-208; C-22 to S-208; C-23 to S-208; F-24 to S-208; L-25 to S-208; L-26 to S-208; L-27 to S-208; F-28 to S-208; L-29 to S-208; V-30 to S-208; S-31 to S-208; S-32 to S-208; V-33 to S-208; P-34 to S-208; V-35 to S-208; T-36 to S-208; C-37 to S-208; Q-38 to S-208; A-39 to S-208; L-40 to S-208; G-41 to S-208; Q-42 to S-208; D-43 to S-208; M-44 to S-208; V-45 to S-208; S-46 to S-208; P-47 to S-208; E-48 to S-208; A-49 to S-208; T-50 to S-208; N-51 to S-208; S-52 to S-208; S-53 to S-208; S-54 to S-208; S-55 to S-208; S-56 to S-208; F-57 to S-208; S-58 to S-208; S-59 to S-208; P-60 to S-208; S-61 to S-208; S-62 to S-208; A-63 to S-208; G-64 to S-208; R-65 to S-208; H-66 to S-208; V-67 to S-208; R-68 to S-208; S-69 to S-208; Y-70 to S-208; N-71 to S-208; H-72 to S-208; L-73 to S-208; Q-74 to S-208; G-75 to S-208; D-76 to S-208; V-77 to S-208; R-78 to S-208; W-79 to S-208; R-80 to S-208; K-81 to S-208; L-82 to S-208; F-83 to S-208; S-84 to S-208; F-85 to S-208; T-86 to S-208; K-87 to S-208; Y-88 to S-208; F-89 to S-208; L-90 to S-208; K-91 to S-208; I-92 to S-208; E-93 to S-208; K-94 to S-208; N-95 to S-208; G-96 to S-208; K-97 to S-208; V-98 to S-208; S-99 to S-208; G-100 to S-208; T-101 to S-208; K-102 to S-208; K-103 to S-208; E-104 to S-208; N-105 to S-208; C-106 to S-208; P-107 to S-208; Y-108 to S-208; S-109 to S-208; I-110 to S-208; L-111 to S-208; E-112 to S-208; I-113 to S-208; T-114 to S-208; S-115 to S-208; V-116 to S-208; E-117 to S-208; I-118 to S-208; G-119 to S-208; V-120 to S-208; V-121 to S-208; A-122 to S-208; V-123 to S-208; K-124 to S-208; A-125 to S-208; I-126 to S-208; N-127 to S-208; S-128 to S-208; N-129 to S-208; Y-130 to S-208; Y-131 to S-208; L-132 to S-208; A-133 to S-208; M-134 to S-208; N-135 to S-208; K-136 to S-208; K-137 to S-208; G-138 to S-208; K-139 to S-208; L-140 to S-208; Y-141 to S-208; G-142 to S-208; S-143 to S-208; K-144 to S-208; E-145 to S-208; F-146 to S-208; N-147 to S-208; N-148 to S-208; D-149 to S-208; C-150 to S-208; K-151 to S-208; L-152 to S-208; K-153 to S-208; E-154 to S-208; R-155 to S-208; I-156 to S-208; E-157 to S-208; E-158 to S-208; N-159 to S-208; G-160 to S-208; Y-161 to S-208; N-162 to S-208; T-163 to S-208; Y-164 to S-208; A-165 to S-208; S-166 to S-208; F-167 to S-208; N-168 to S-208; W-169 to S-208; Q-170 to S-208; H-171 to S-208; N-172 to S-208; G-173 to S-208; R-174 to S-208; Q-175 to S-208; M-176 to S-208; Y-177 to S-208; V-178 to S-208; A-179 to S-208; L-180 to S-208; N-181 to S-208; G-182 to S-208; K-183 to S-208; G-184 to S-208; A-185 to S-208; P-186 to S-208; R-187 to S-208; R-188 to S-208; G-189 to S-208; Q-190 to S-208; K-191 to S-208; T-192 to S-208; R-193 to S-208; R-194 to S-208; K-195 to S-208; N-196 to S-208; T-197 to S-208; S-198 to S-208; A-199 to S-208; H-200 to S-208; F-201 to S-208; L-202 to S-208; P-203 to S-208. Polynucleotides encoding these polypeptides are also encompassed by the present invention.
[209] S69-S208; A63-S208; Y70-S208; V77-S208; E93-S208; E104-S208; V123-S208; G138-S208; R80-S208; A39-S208; S69-V178; S69-G173; S69-R188; S69-S198; S84-S208; V98-S208; A63-N162; S69-N162; And M35-N162 are particularly preferred.
[210] Also, as mentioned above, although deletion of one or more amino acids from the C-terminus of a protein yields a modification that deletes one or more biological functions of the protein, other functional activities (e.g., biological activity, ability to form multimers , Ability to bind KGF-2 ligand) can still be retained. For example, the ability of a shortened KGF-2 mutein to induce and / or bind an antibody that recognizes the complete or mature form of the polypeptide will usually be retained when the majority of the full or mature polypeptide residues are not removed from the C-terminus . Certain polypeptides lacking the C-terminal residue of the complete polypeptide can be readily identified by routine methods described herein or by other methods known in the art. KGF-2 muteins with multiple deleted C-terminal amino acid residues may retain some biological or immunogenic activity. In fact, peptides composed of as little as six KGF-2 amino acid residues can often cause an immune response.
[211] Thus, the present invention also provides a polypeptide having one or more residues deleted from the carboxy terminus of the KGF-2 polypeptide amino acid sequence shown in Figure 1 (SEQ ID NO: 2), represented by the general formula 1-n, 2 to 207 and corresponds to the position of the amino acid residue identified in SEQ ID NO: 2. More specifically, the present invention relates to a pharmaceutical composition comprising M-1 to H-207 of SEQ ID NO: 2; M-1 to V-206; M-1 to V-205; M-1 to M-204; M-1 to P-203; M-1 to L-202; M-1 to F-201; M-1 to H-200; M-1 to A-199; M-1 to S-198; M-1 to T-197; M-1 to N-196; M-1 to K-195; M-1 to R-194; M-1 to R-193; M-1 to T-192; M-1 to K-191; M-1 to Q-190; M-1 to G-189; M-1 to R-188; M-1 to R-187; M-1 to P-186; M-1 to A-185; M-1 to G-184; M-1 to K-183; M-1 to G-182; M-1 to N-181; M-1 to L-180; M-1 to A-179; M-1 to V-178; M-1 to Y-177; M-1 to M-176; M-1 to Q-175; M-1 to R-174; M-1 to G-173; M-1 to N-172; M-1 to H-171; M-1 to Q-170; M-1 to W-169; M-1 to N-168; M-1 to F-167; M-1 to S-166; M-1 to A-165; M-1 to Y-164; M-1 to T-163; M-1 to N-162; M-1 to Y-161; M-1 to G-160; M-1 to N-159; M-1 to E-158; M-1 to E-157; M-1 to I-156; M-1 to R-155; M-1 to E-154; M-1 to K-153; M-1 to L-152; M-1 to K-151; M-1 to C-150; M-1 to D-149; M-1 to N-148; M-1 to N-147; M-1 to F-146; M-1 to E-145; M-1 to K-144; M-1 to S-143; M-1 to G-142; M-1 to Y-141; M-1 to L-140; M-1 to K-139; M-1 to G-138; M-1 to K-137; M-1 to K-136; M-1 to N-135; M-1 to M-134; M-1 to A-133; M-1 to L-132; M-1 to Y-131; M-1 to Y-130; M-1 to N-129; M-1 to S-128; M-1 to N-127; M-1 to I-126; M-1 to A-125; M-1 to K-124; M-1 to V-123; M-1 to A-122; M-1 to V-121; M-1 to V-120; M-1 to G-119; M-1 to I-118; M-1 to E-117; M-1 to V-116; M-1 to S-115; M-1 to T-114; M-1 to I-113; M-1 to E-112; M-1 to L-111; M-1 to I-110; M-1 to S-109; M-1 to Y-108; M-1 to P-107; M-1 to C-106; M-1 to N-105; M-1 to E-104; M-1 to K-103; M-1 to K-102; M-1 to T-101; M-1 to G-100; M-1 to S-99; M-1 to V-98; M-1 to K-97; M-1 to G-96; M-1 to N-95; M-1 to K-94; M-1 to E-93; M-1 to I-92; M-1 to K-91; M-1 to L-90; M-1 to F-89; M-1 to Y-88; M-1 to K-87; M-1 to T-86; M-1 to F-85; M-1 to S-84; M-1 to F-83; M-1 to L-82; M-1 to K-81; M-1 to R-80; M-1 to W-79; M-1 to R-78; M-1 to V-77; M-1 to D-76; M-1 to G-75; M-1 to Q-74; M-1 to L-73; M-1 to H-72; M-1 to N-71; M-1 to Y-70; M-1 to S-69; M-1 to R-68; M-1 to V-67; M-1 to H-66; M-1 to R-65; M-1 to G-64; M-1 to A-63; M-1 to S-62; M-1 to S-61; M-1 to P-60; M-1 to S-59; M-1 to S-58; M-1 to F-57; M-1 to S-56; M-1 to S-55; M-1 to S-54; M-1 to S-53; M-1 to S-52; M-1 to N-51; M-1 to T-50; M-1 to A-49; M-1 to E-48; M-1 to P-47; M-1 to S-46; M-1 to V-45; M-1 to M-44; M-1 to D-43; M-1 to Q-42; M-1 to G-41; M-1 to L-40; M-1 to A-39; M-1 to Q-38; M-1 to C-37; M-1 to T-36; M-1 to V-35; M-1 to P-34; M-1 to V-33; M-1 to S-32; M-1 to S-31; M-1 to V-30; M-1 to L-29; M-1 to F-28; M-1 to L-27; M-1 to L-26; M-1 to L-25; M-1 to F-24; M-1 to C-23; M-1 to C-22; M-1 to C-21; M-1 to C-20; M-1 to C-19; M-1 to G-18; M-1 to P-17; M-1 to L-16; M-1 to H-15; M-1 to P-14; M-1 to F-13; M-1 to A-12; M-1 to S-11; M-1 to A-10; M-1 to C-9; M-1 to H-8; Or a polynucleotide encoding the polypeptide comprising the amino acid sequence of the M-1 to the T-7 residue or consisting of the sequence. Polynucleotides encoding these polypeptides are also encompassed by the present invention.
[212] Likewise, the C-terminal deletion of the KGF-2 polypeptide of the present invention represented by SEQ ID NO: 2 corresponds to S-69 to H-207 of SEQ ID NO: 2; S-69 to V-206; S-69 to V-205; S-69 to M-204; S-69 to P-203; S-69 to L-202; S-69 to F-201; S-69 to H-200; S-69 to A-199; S-69 to S-198; S-69 to T-197; S-69 to N-196; S-69 to K-195; S-69 to R-194; S-69 to R-193; S-69 to T-192; S-69 to K-191; S-69 to Q-190; S-69 to G-189; S-69 to R-188; S-69 to R-187; S-69 to P-186; S-69 to A-185; S-69 to G-184; S-69 to K-183; S-69 to G-182; S-69 to N-181; S-69 to L-180; S-69 to A-179; S-69 to V-178; S-69 to Y-177; S-69 to M-176; S-69 to Q-175; S-69 to R-174; S-69 to G-173; S-69 to N-172; S-69 to H-171; S-69 to Q-170; S-69 to W-169; S-69 to N-168; S-69 to F-167; S-69 to S-166; S-69 to A-165; S-69 to Y-164; S-69 to T-163; S-69 to N-162; S-69 to Y-161; S-69 to G-160; S-69 to N-159; S-69 to E-158; S-69 to E-157; S-69 to I-156; S-69 to R-155; S-69 to E-154; S-69 to K-153; S-69 to L-152; S-69 to K-151; S-69 to C-150; S-69 to D-149; S-69 to N-148; S-69 to N-147; S-69 to F-146; S-69 to E-145; S-69 to K-144; S-69 to S-143; S-69 to G-142; S-69 to Y-141; S-69 to L-140; S-69 to K-139; S-69 to G-138; S-69 to K-137; S-69 to K-136; S-69 to N-135; S-69 to M-134; S-69 to A-133; S-69 to L-132; S-69 to Y-131; S-69 to Y-130; S-69 to N-129; S-69 to S-128; S-69 to N-127; S-69 to I-126; S-69 to A-125; S-69 to K-124; S-69 to V-123; S-69 to A-122; S-69 to V-121; S-69 to V-120; S-69 to G-119; S-69 to I-118; S-69 to E-117; S-69 to V-116; S-69 to S-115; S-69 to T-114; S-69 to I-113; S-69 to E-112; S-69 to L-111; S-69 to I-110; S-69 to S-109; S-69 to Y-108; S-69 to P-107; S-69 to C-106; S-69 to N-105; S-69 to E-104; S-69 to K-103; S-69 to K-102; S-69 to T-101; S-69 to G-100; S-69 to S-99; S-69 to V-98; S-69 to K-97; S-69 to G-96; S-69 to N-95; S-69 to K-94; S-69 to E-93; S-69 to I-92; S-69 to K-91; S-69 to L-90; S-69 to F-89; S-69 to Y-88; S-69 to K-87; S-69 to T-86; S-69 to F-85; S-69 to S-84; S-69 to F-83; S-69 to L-82; S-69 to K-81; S-69 to R-80; S-69 to W-79; S-69 to R-78; S-69 to V-77; S-69 to D-76; And includes a polypeptide comprising the amino acid sequence of S-69 to G-75 residues.
[213] In addition, any of the N-terminal or C-terminal deletions described above can be combined with each other to generate N-terminal and C-terminal deleted KGF-2 polypeptides. The present invention also provides polypeptides having one or more amino acids deleted from both the amino and carboxyl termini, wherein the ends are represented as having mn residues in SEQ ID NO: 2, wherein n and m are as described above It is an integer. In addition, the N-terminal or C-terminal deletion mutants may also include position-specific amino acid substitutions. Polynucleotides encoding these polypeptides are also encompassed by the present invention.
[214] Also included is a nucleotide sequence encoding a polypeptide consisting of a portion of the complete KGF-2 amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75977, said portion of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 75977 Or any integer number of amino acid residues in the 1 to about 198 amino acids from the carboxy terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Accession No. 75977, Any combination of the amino terminal and carboxy terminal deletions is excluded. Polynucleotides encoding all of the deletion mutant polypeptide forms are also provided.
[215] The present specification discloses that mn is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98%, or 99% sequence identity with the KGF- To a protein comprising the same polypeptide. In a preferred embodiment, the present disclosure encompasses a polypeptide having 80% or more, 85%, 90%, 91%, 92%, 93%, 94% or more of a polypeptide having the amino acid sequence of the specific KGF-2 and the C- , 95%, 97%, 98%, or 99% identical to a polypeptide comprising the same polypeptide. Polynucleotides encoding these polypeptides are also included in the present invention.
[216] Fragments characteristic of the functional or structural properties of KGF-2 are one of the preferred fragments of the present invention. Such fragments include the alpha-helix and alpha-helix forming sites (" alpha-site "), beta-sheet, beta-sheet forming site (" beta- (&Quot; coil-site "), a hydrophilic site, a hydrophobic site, an alpha amphipathic site, a beta amphipathic site, a surface forming site, And an amino acid residue comprising a high cost region (i. E., Identified using a Jameson-Wolf program defect parameter, including four or more adjacent amino acids with an antigenicity of 1.5 or higher). Specific preferred regions are those shown in Figure 4 and include, but are not limited to, regions of the above-described type identified by analysis of the amino acid sequence shown in Figure 1 (SEQ ID NO: 2) Alpha-site, beta-site, turn-site, and coil site of the a-Robson prophecy; Alpha-site, beta-site, turn-site and coil-site of Shu-Parsman Prophecy; The hydrophobic and hydrophilic parts of the Kite-Doolittle prophecy; Eisenberg alpha and beta amphipathic sites; Emissive surface-forming regions; And a Jamesson-Wolf high antigen ratio site, as predicted using the defect parameter of the computer program. Polynucleotides encoding the polypeptide are also encompassed by the present invention.
[217] In a further embodiment, the polypeptide of the invention encodes a functional property of KGF-2. In this regard, preferred embodiments of the present invention are directed to the use of KGF-2 in the manufacture of a medicament for the treatment and / or prophylaxis and / or treatment of KGF-2 comprising an alpha -helical and alpha-helix forming site ("alpha-site"), a beta-sheet, (&Quot; coil-site "), a hydrophilic site, a hydrophobic site, an alpha amphipathic site, a beta amphipathic site, a flexible site, a surface- And a fragment containing a high-cost region.
[218] Data representing the structural or functional properties of KGF-2 shown in Figure 1 and / or Table 1 as described above were generated using various moduli and algorithms of DNA * STAR set with defect parameters. In a preferred embodiment, the data set forth in columns VIII, IX, XIII and XIV of Table 1 can be used to determine the site of KGF-2 exhibiting a high antigenic potential. A highly antigenic site may be selected from the data set forth in columns VIII, IX, XIII and / or IV, a value indicative of the site of the polypeptide where antigen recognition is susceptible to exposure on the surface of the polypeptide in the environment that occurs during the initiation of the immune response .
[219] Specific preferred regions in this regard are shown in Fig. 4 as shown in Table 1, but can be expressed and identified using those calculated in the table of data shown in Fig. The DNA * STAR computer algorithm (used as a conventional defect parameter) used to create Figure 4 was used to represent the data of Figure 4 in tabular form (see Table 1). The table format of the data in Fig. 4 can be used to easily determine a specific region of the desired region.
[220] The preferred sites described above in FIG. 4 and Table 1 include, but are not limited to, sites of the above-identified type identified by analysis of the amino acid sequence shown in FIG. As set forth in Figure 4 and Table 1, the preferred sites include the Ganer-Robson alpha-site, the beta-site, the turn-site, and the coil site; Shu-Pasman alpha-site, beta-site and coil-site; A kite-dirty hydrophobic region and a hydrophilic region; Eisenberg alpha and beta amphipathic sites; CAPlus-Schulz-Flex region; Emissive surface-forming regions; And the Jamesson-Wolfe antigen ratio region. The column was labeled with headings "Res", "Position" and Roman numeral I-XIV. Column heading refers to the following features of the amino acid sequence shown in Figure 3 and Table 1: " Res ": the amino acid residue of SEQ ID NO: 2 and Figures 1A and 1B; &Quot; Position ": the position of amino acid residues corresponding to SEQ ID NO: 2 and FIGS. 1A and 1B; I: alpha, site-ganny-robson; II: Alpha, site - Shu-Pasman only; III: Beta, site-ganier-Robson; IV: Beta, site-Shu-Pasman only; V: Turn, part - Gannier - Robson; VI: Turn, site - Shu-Pasman only; VII: coil, part - ganier - Robson; VIII: hydrophilic plot-kite-two liters; IX: hydrophobic plot-hops-wood; X: Alpha, amphipathic site - Eisenberg; XI: Beta, amphipathic region - Eisenberg; XII: Flexible moiety -Car Plus-Schultz; XIII: Antigen ratio - Jameson-Wolf; And XIV: surface probability plots - Emini.
[221]
[222]
[223]
[224]
[225]
[226]
[227]
[228]
[229]
[230]
[231] In this connection, it is a highly desirable fragment to include the KGF-2 site, which combines several structural features, some of which are described above.
[232] In addition, the technique of gene-shuffling, motif-shuffling, exon-shuffling, and / or codon-shuffling (collectively, "DNA shuffling") modulates the activity of KGF- Can be used to effectively prepare antagonists. Generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; And 5,837,458; And Patten, P.A. , Et al., Curr. Opinion Biotechnol. 8: 724-33 (1997); Harayama, S., Trends Biotechnol. 16 (2): 76-82 (1998); Hansson, L.O. Et al., J. Mol. Biol. 287: 265-276 (1999); And Lorenzo, M.M. And Blasco, R., Biotechniques 24 (2): 308-313 (1998), each of which is incorporated herein by reference.
[233] In one embodiment, modification of the KGF-2 polynucleotide and its corresponding polypeptide is performed by DNA shuffling. DNA shuffling involves a combination of two or more DNA segments introduced into the molecule of a given KGF-2 by homologous or site-specific recombination. In another embodiment, the KGF-2 polynucleotide and the corresponding polypeptide are altered by performing random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In other embodiments, one or more components, motifs, sections, fragments, domains, fragments, etc. of KGF-2 can be recombined into one or more components, motifs, sections, fragments, domains, fragments, etc. of one or more heterologous molecules. In a preferred embodiment, the heterologous molecule is a member of KGF-2. In a more preferred embodiment, the heterologous molecule is selected from the group consisting of platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor (TGF) BMP-5, BMP-6, BMP-7, Actibin A and B, decapentaplegic (dpp) , 60A, OP-2, dorsalin, growth differentials (GDFs), nodules, MIS, inhivin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, -Induced neurotrophic factor (GDNF). Another preferred fragment is the biologically active KGF-2 fragment. Biologically active fragments exhibit similar activity to the activity of a KGF-2 polypeptide, but are not necessarily the same. The biological activity of the fragment may improve the desired activity or reduce the undesirable activity.
[234] In addition, the present invention provides a method of screening for compounds to identify compounds that modulate polypeptide activity of the invention. Examples of such assays include combining fibroblasts of mammals, polypeptides of the present invention, compounds to be screened and 3 [H] thymidine under cell culture conditions in which fibroblasts are routinely able to proliferate. Regulatory analysis can be conducted in the absence of the compound to compare the amount of fibroblast proliferation by screening in the presence of the compound to determine the uptake of 3 [H] thymidine in each case and determine whether the compound will stimulate proliferation have. The amount of fibroblast proliferation is determined by liquid scintillation chromatography, which measures the infusion of 3 [H] thymidine. Both the agonist and the antagonist compound can be identified in this way.
[235] In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the invention is incubated with the polypeptide of the invention labeled in the presence of the compound. Thus, the ability of a compound to enhance or prevent this interaction can be measured. Alternatively, after measuring the response of a known second messenger system according to the interaction of the compound to be screened with the KGF-2 receptor, the ability of the compound to bind to the receptor and reveal the second messenger response is measured, Whether it is agonist or antagonist. Such a second messenger system includes, but is not limited to, cAMP guanylate cyclase, ion channel or phosphoinositide hydrolisis. All of these assay methods can be used as diagnostic or prognostic markers. Molecules discovered using these analytical methods can be used to either cause specific outcomes to the patient (e.g., angiogenesis) or to treat disease by activating or inhibiting KGF-2 molecules. In addition, agents that can inhibit or enhance the production of KGF-2 from cells or tissues that have been suitably engineered by this assay method can be found.
[236] Accordingly, the present invention provides a method for producing a recombinant protein comprising the steps of: (a) incubating a candidate binding compound with KGF-2; And (b) determining whether binding has occurred. The method comprises identifying a compound that binds to KGF-2. In addition, the present invention relates to a method for determining the biological activity of KGF-2 comprising (a) incubating a candidate compound with KGF-2, (b) analyzing the biological activity, and (c) Lt; / RTI > agent / antagonist.
[237] In addition, molecules binding to KGF-2 can be identified experimentally using the beta-pleated sheet site as shown in FIG. 4 and Table 1. Particular embodiments of the invention thus relate to polynucleotides encoding polypeptides comprising or consisting of the amino acid sequence of each beta-pleated sheet site as set forth in Figure 3 / Table 1.
[238] A further embodiment of the invention is directed to a polynucleotide encoding a KGF-2 polypeptide comprising or consisting of all or any combination of the beta-pleated sheet sites disclosed in Figure 4 / A further preferred embodiment of the invention relates to a polypeptide comprising or consisting of the KGF-2 amino acid sequence of each of the beta-pleated sheet portions disclosed in Figure 4 / A further embodiment of the present invention relates to KGF-2 polypeptides comprising or consisting of all or any combination of the beta-pleated sheet sites disclosed in Figure 4 /
[239] Another preferred embodiment of the invention relates to a KGF-2 fragment that binds to a KGF-2 receptor. A fragment that binds to the KGF-2 receptor may be useful as an agonist or antagonist of KGF-2. For example, a fragment of KGF-2 that binds to a receptor can prevent binding to KGF-2 and activate a portion thereof. Other fragments may specifically bind to the receptor to specifically inactivate receptor and receptor activity, or may specifically deactivate antibodies capable of recognizing the receptor-ligand complex and preferably bind to an unbound receptor or a binding Lt; RTI ID = 0.0 > ligand. ≪ / RTI > Similarly, fragments that activate the receptor are included in the present invention. These fragments can act as receptor antagonists, i.e., a subset or all of the biological activity of ligand-mediated receptor activation can be enhanced or activated by, for example, inducing dimerization of the receptor. Such fragments may be embodied as agonists, antagonists or inverse agonists for biological activity, including the specific biological activity of the inventive peptides disclosed herein.
[240] Examples of KGF-2 fragments that bind to the KGF-2 receptor include the amino acids 147-155, 95-105, 78-94, 119-146, 70-94, 78-105, 114-146, 70-105 , 86-124, 100-139, 106-146, 160-209, and / or 156-209. Also preferred are polynucleotides encoding said polypeptides.
[241] Another preferred fragment is the biologically active KGF-2 fragment. The biologically active fragment is not necessarily identical to the activity of the KGF-2 polypeptide, but exhibits similar activity. The biological activity of the fragment may improve the desired activity or reduce the undesirable activity.
[242] However, many polynucleotide sequences (such as the EST sequence) are publicly available and available from sequence databases. Some of these sequences are associated with SEQ ID NO: 1 and would have been publicly available prior to the invention. It is preferred that such related polynucleotides are specifically excluded from the scope of the present invention. It is annoying to list all related sequences. Accordingly, it is preferred that one or more polynucleotides comprising the nucleotide sequence represented by the general formula ab are excluded from the present invention, wherein a is an arbitrary integer between 1 and 613 of SEQ ID NO: 1 and b is an integer between 15 and 627 And a and b correspond to the positions of the nucleotide residues shown in SEQ ID NO: 1, wherein b is a + 14 or larger.
[243] Amino terminal and carboxy terminal deletion
[244] Various members of FGF have been modified using recombinant DNA technology. Positively charged molecules were deleted or replaced in both aFGF and bFGF which are important in heparin binding. The modified molecule had decreased heparin binding activity. Thus, it is known that reducing the amount of modified molecules isolated by heparin in a patient will increase the efficacy of FGF to reach appropriate receptors (EP 0 298 723).
[245] Natural KGF-2 is relatively unstable in the aqueous state and is chemically physically denatured, impairing its biological activity during processing and storage. In addition, natural KGF-2 is inactivated under acidic conditions because it is easy to agglomerate when the temperature rises in aqueous solution.
[246] Protein engineering can be used to improve or modify one or more properties of native KGF-2. Ron et al., J. Biol. Chem., 268 (4): 2984-2988 (1993) disclose altered KGF proteins with heparin binding activity even when the amino acid residues at the 3, 8, have. Deletions of 3 and 8 amino acids have total activity. A more severe deletion of KGF is disclosed in PCT / IB / 00971. Deletion of the carboxy terminal amino acid can enhance the activity of the protein. One example is interferon gamma, which shows up to 10-fold higher activity by deleting 10 amino acid residues from the carboxy terminus of the protein (Dobeli et al., J. of Biotechnology 7: 199-216 (1998)). Accordingly, one aspect of the present invention provides a method of screening for a polypeptide homologue of KGF-2 exhibiting enhanced stability (e.g., when exposed to conventional pH, thermal conditions or other storage conditions) to a native KGF-2 polypeptide, Lt; RTI ID = 0.0 > nucleotides < / RTI >
[247] Particularly preferred KGF-2 polypeptides are as follows (starting from the first amino acid (Met) of the protein) (Figure 1 (SEQ ID NO: 2)):
[248]
[249] (SEQ ID NO: 68) and Ser (69) -Ser (208) (KGF-2 33) (SEQ ID NO: No. 96). Other preferred N-terminal and C-terminal deletion mutants are described in Examples 13 and 16 (c) of the specification and include: Ala (39) -Ser (208) (SEQ ID NO: 116); Pro (47) -Ser (208) (SEQ ID NO: 2) of FIG. 1; Val (77) -Ser (208) (SEQ ID NO: 70); Glu (93) -Ser (208) (SEQ ID NO: 72); Glu (104) -Ser (208) (SEQ ID NO: 74); Val (123) -Ser (208) (SEQ ID NO: 76); And Gly (138) -Ser (208) (SEQ ID NO: 78). Other preferred C-terminal deletion mutants include Met (1), Thr (36), or Cys (37) -Lys (153) of Figure 1 (SEQ ID NO: 2).
[250] The present invention includes deletion mutants having amino acids deleted from both the N-terminus and the C-terminus. Such mutants include a combination of an N-terminal deletion mutant and a C-terminal deletion mutant, such as Ala (39) -His (200), Figure 1 (SEQ ID NO: 2) (63) -Lys (153) of Figure 1 (SEQ ID NO: 2), Ser (69) -Lys (153) of Figure 1 . These combinations can be made using recombinant techniques known to those skilled in the art.
[251] Thus, in one embodiment, an N-terminal deletion mutant is provided by the present invention. This mutant has a deletion below the first N-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) (i.e., deletion of Met (1) -Gin (38) (SEQ ID NO: 2). Alternatively, the deletion region may consist of less than the first N-terminal amino acid residue (Met (1) - Gln (38) or more) above the first 38 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2). Alternatively, the deletion can consist of less than the first 46 N-terminal amino acid residue of the Figure 1 (SEQ ID NO: 2) and less than the first 137 N-terminal amino acid residue. Alternatively, the deletion region can consist of less than the first 62 N-terminal amino acid residue of the Figure 1 (SEQ ID NO: 2) and less than the first 137 N-terminal amino acid residue. Alternatively, the deletion region may consist of the first 68 N-terminal amino acid residues above the first 137 N-terminal amino acid residues in Figure 1 (SEQ ID NO: 2). Alternatively, the deletion can consist of less than the first 76 N-terminal amino acid residue of the Figure 1 (SEQ ID NO: 2) and less than the first 137 N-terminal amino acid residue. Alternatively, the deletion region can consist of less than the first 92 N-terminal amino acid residue and less than the first 137 N-terminal amino acid residue of Figure 1 (SEQ ID NO: 2). Alternatively, the deletion region can consist of less than the first 103 N-terminal amino acid residue of the Figure 1 (SEQ ID NO: 2) and less than the first 137 N-terminal amino acid residue. Alternatively, the deletion region can consist of less than the first 122 N-terminal amino acid residue of the Figure 1 (SEQ ID NO: 2) and less than the first 137 N-terminal amino acid residue.
[252] In addition to the range of N-terminal deletion mutants described above, the present invention relates to all combinations of the above-mentioned ranges, for example as follows: the first 62 N-terminal amino acid residues in Figure 1 (SEQ ID NO: 2) A deletion site below the terminal amino acid residue; A deletion of the first 62 N-terminal amino acid residues above the first 76 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion of the first 62 N-terminal amino acid residues above the first 92 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion in the first 62 N-terminal amino acid residues above the first 103 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion of the first 68 N-terminal amino acid residues above the first 76 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion of the first 68 N-terminal amino acid residues above the first 92 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion in the first 68 N-terminal amino acid residues above the first 103 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion region from the first 46 N-terminal amino acid residue to the first 62 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion site beyond the first 46 N-terminal amino acid residue and the first 68 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); Terminal amino acid residue in Figure 1 (SEQ ID NO: 2), and a deletion in the first 76 N-terminal amino acid residue or less.
[253] In another embodiment, a C-terminal deletion mutant is provided by the present invention. The N-terminal amino acid residue of the C-terminal deletion mutant is preferably the amino acid residue 1 (Met), 36 (Thr), or 37 (Cys) of FIG. 1 (SEQ ID NO: 2). This mutant lacks the deletion of the final C-terminal amino acid residue (Ser (208)) above the final 55 C-terminal amino acid residue (i.e., deletion of the amino acid residue Glu (154) -Ser (208) 2). ≪ / RTI > Alternatively, the deletion region may consist of more than the final C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) or less than the final 65 C-terminal amino acid residue. Alternatively, the deletion region may consist of less than the final 10 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) or less than the final 55 C-terminal amino acid residue.
[254] Alternatively, the deletion can consist of more than the final 20 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) or less than the final 55 C-terminal amino acid residue. Alternatively, the deletion region can consist of a final more than the last 30 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) or less than the last 55 C-terminal amino acid residue. Alternatively, the deletion region may consist of less than the final 40 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) or less than the final 55 C-terminal amino acid residue. Alternatively, the deletion can consist of more than the last 50 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) or less than the last 55 C-terminal amino acid residue.
[255] In addition to the aforementioned range of C-terminal deletion mutants, the present invention relates to all combinations of the above-mentioned ranges, for example as follows: more than the final C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) A deletion part of the terminal amino acid residue or less; A deletion in the final C-terminal amino acid residue or less of the final 20 C-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion in the final C-terminal amino acid residue or less of the final 30 C-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion site beyond the last C-terminal amino acid residue in Figure 1 (SEQ ID NO: 2) or below the last 40 C-terminal amino acid residue; A deletion in the final 10 C-terminal amino acid residue or less of the final 20 C-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion in the final 10 C-terminal amino acid residue or less of the final 30 C-terminal amino acid residue in Figure 1 (SEQ ID NO: 2); A deletion site beyond the last 10 C-terminal amino acid residue of Figure 1 (SEQ ID NO: 2) below the last 40 C-terminal amino acid residue; Terminal amino acid residue of Figure 1 (SEQ ID NO: 2), or a deletion of the final 30 C-terminal amino acid residue or less.
[256] In another embodiment, deletion mutants having amino acids deleted from both N-terminal and C-terminal residues are also included in the present invention. The mutants include all combinations of the N-terminal deletion mutants and the C-terminal deletion mutants described above. The mutant has a deletion in the last first 137 N-terminal amino acid residue from the first 46 N-terminal amino acid residue in Figure 1 (SEQ ID NO: 2) and a final 55 amino acid residue in the final C- 1 (SEQ ID NO: 2) except for a deletion site below the C-terminal amino acid residue. Alternatively, the deletion is the first 62, 68, 76, 92, 103 or 122 N-terminal amino acids of Figure 1 (SEQ ID NO: 2) , 20, 30, 40, or 50 C-terminal amino acid residues and less than or equal to the final 55 C-terminal amino acid residue. And all combinations of the above-mentioned ranges are included.
[257] Amino acid substitution
[258] A further aspect of the invention also includes amino acid substitutions. Natural mature KGF-2 contains 44 charged residues, 32 of which have positive charge. Substituting these one or more clustered residues with an amino acid having a negative or neutral charge depending on the position of the residue in the tertiary structure of the protein can change the electrostatic interactions of neighboring residues and reduce the aggregation of the protein It is useful for increasing stability. Protein aggregation may be immunogenic and thus not only reduces activity but also becomes a problem in the manufacture of pharmaceutical preparations (Pinckard et al., Clin. Exp. Immunol. 2: 331-340 (1967), Robbins et al., Diabetes 36: 838 -845 (1987), Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10: 307-377 (1993)). Efforts should be made to minimize charge repulsion in the tertiary structure of the protein molecule with constant modification. Thus, the replacement of the charged amino acid with another charge and a neutral or negatively charged amino acid should be specifically considered. Substitution with neutral or negatively charged amino acids improves the properties of KGF-2 by reducing both charges in the protein. This improvement increases homologous stability and reduces aggregation as compared to native KGF-2 protein.
[259] Substitution of amino acids can selectively alter binding to cell surface receptors. Ostade et al., Nature 361: 266-268 (1993) describe that a particular TNF alpha mutation selectively binds TNF alpha to either of two known TNF receptors.
[260] Embodiments of the present invention also include amino acids of a KGF-2 polypeptide having an amino acid sequence with an amino acid substitution of at least 1 and at most 50, more preferably at most 40, more preferably at most 30, most preferably at most 20 amino acids ≪ / RTI > sequence. Of course, it is also possible to select one or more, preferably 20 or less, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, Most preferably a polypeptide or peptide having an amino acid sequence comprising an amino acid sequence of a KGF-2 polypeptide substituted with an amino acid of SEQ ID NO: 1. In embodiments, the number of additions, substitutions, and / or deletions of the amino acid sequence of Figure 1 or a fragment thereof (e.g., mature form and / or other fragment described herein) is 1-5, 5-10, 5-50, 10-50 or 50-150, conservative amino acid substitutions being preferred.
[261] KGF-2 molecules include one or more amino acid substitutions, deletions or additions by both natural mutations or human manipulation. The mutation can be made with whole KGF-2, mature KGF-2, any other suitable KGF-2 fragment, such as A63-S208, S69-S208, V77-S208, R80-S208 or E-93-S208. Examples of certain preferred mutations are as follows: Ala (49) Gln, Asn (51) Ala, Ser (54) Val, Ala (63) Pro, Gly Lys (91) Arg, Ser (99) Lys, Lys (102) Gln, Lys (103) Val, Arg (80) Lys, Lys Glu (104) Met, Asn (105) Lys, Pro (107) Asn, Ser (109) Asn, Leu (111) Met, Thr Val (123) Ile, Ala (125) Gly, Ile (126) Val, Asn (127) Glu, Asn (127) Gln, Tyr Leu (152) Phe, Glu (154) Gly, Glu (154) Glu, Gly (142) Ala, Ser (143) Lys, Phe Asp, Arg (155) Leu, Glu (157) Leu, Gly (160) His, Phe (167) Ala, Asn (168) Lys, Gln Gln, Gln (190) Lys, Lys (195) Glu, Thr (197) Lys, Ser (185) Leu, Ala Arg (188) Glu, Lys (183) Glu, Arg (194) Glu, Lys (191) Glu, Lys Ala, Arg (178) Ala, Lys (183) Ala, Lys (144) Ala, Lys (151) s (137) Ala, and Lys (139) Ala.
[262] For example, Ala (49) Gln is intended to indicate that Ala at position 49 of Figure 1 (SEQ ID NO: 2) is substituted with Gln.
[263] In addition, the following mutants are particularly preferred: S69-S208 with a point mutation at R188E; S69-S208 with point mutation in K191E; S69-S208 with point mutation at K149E; S69-S208 with point mutation in K183Q; S69-S208 with point mutation in K183E; S63-S208 with point mutation in R68G; A63-S208 with point mutation in R68S; A63-S208 with point mutation at R68A; A63-S208 with point mutations in R78A, R80A and K81A; A63-S208 with point mutations in K81A, K87A and K91A; A63-S208 with point mutations in R78A, R80A, K81A, K87A and K91A; A63-S208 with point mutations in K136A, K137A, K139A and K144A; A63-S208 with point mutations in K151A, K153A and K155A; A63-S208 with point mutations in R68G, R78A, R80A and K81A; A63-S208 with point mutations in R68G, K81A, K87A and K91A; A63-S208 with point mutations in R68G, R87A, R80A, K81A, K87A and K91A; A63-S208 with point mutations in R68G, K136A, K137A, K139A and K144A; A63-S208 with point mutations in R68G, K151A, K153A and R155A; A63-S208 with point mutations in R68S, R78A, R80A and K81A; A63-S208 with point mutations in R68S, K81A, R87A and K91A; A63-S208 with point mutations in R68S, R78A, R80A, K81A, K87A and K91A; R68S, K136A, K137A A63-S208 with point mutations in K139A and K144A; A63-S208 with point mutations in R68S, K151A, K153A and R155A; A63-S208 with point mutations in R68A, R78A, R80A and K81A; A63-S208 with point mutations in R68A, K81A, K87A and K91A; A63-S208 with point mutations in R68A, R78A, R80A, K81A, K87A and K91A; A63-S208 with point mutations in R68A, K136A, K137A, K139A and K144A; A63-S208 with point mutations in R68A, K151A, K153A and R155A. Also, A63-S208 with a charged residue in the amount of R68 to K91 is replaced with alanine [A63-S208 (R68-K91A)]; Length KGF-2 with charged residues in the amounts of R68 to K91 is replaced by alanine [KGF-2 (R68-K91A)]; A63-S208 having a charged residue in the amount of R68 to K91 is substituted with a neutral residue, such as G, S, and / or A; Length KGF-2 with a charged moiety of the amount of R68 to K91 is substituted with a neutral moiety, such as G, S, and / or A; A63-S208 having a charged moiety of the amount of R68 to K91 is substituted with a negatively charged moiety, such as D and / or E; Length KGF-2 with charged residues in the amount of R68 to K91 is substituted with negatively charged moieties such as D and / or E; Total length KGF-2 with point mutations in R78A, R80A and K81A; Total length KGF-2 with point mutations in K81A, K87A and K91A; Total length KGF-2 with point mutation at R68G; Total length KGF-2 with point mutation in R68S; Total length KGF-2 with point mutation at R68A; A63-S208 with point mutations in R174A and K183A; And A63-S208 with point mutations in R187A and R188A.
[264] Also preferred are A63-S208 with a point mutation in R188E, K191E, K149E, K183Q or K183E; S69-S208 with point mutations in R78A, R80A and K81A; S69-S208 with point mutations in K81A, K87A and K91A; S69-S208 with point mutations in R174A and K183A; S69-S208 with point mutations in R187A and R188A; V77-S208 with point mutations in R188E, K191E, K149E, K183Q or K183E; V77-S208 with point mutations in R78A, R80A and K81A; V77-S208 with point mutations in K81A, K87A and K91A; V77-S208 with point mutations in R174A and K183A; V77-S208 with point mutations in R187A and R188A; R80-S208 with point mutations in R188E, K191E, K149E, K183Q or K183E; R80-S208 with point mutations in R174A and K183A; R80-S208 with point mutations in R187A and R188A; E93-S208 with point mutations in R188E, K191E, K149E, K183Q or K183E; E93-S208 with point mutations in R174A and K183A; Or E93-S208 with point mutations in R187A and R188A.
[265] All of the above point mutations can also be prepared with full length KGF-2, mature KGF-2, or any other fragment KGF-2 described herein. In the above symbol, R188E indicates that arginine at position 188 is replaced with glutamic acid.
[266] In addition, site directed mutants can be produced at each amino acid of KGF-2, preferably between amino acids A63 and E93. Each amino acid can be substituted by one of the remaining 19 remaining amino acids. For example, preferred mutants include: C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W , Or A63 substituted with Y; G64 substituted with A, C, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; R65 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; H66 substituted with A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W or Y; V67 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y; R68 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; S69 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y; Y 70 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; N71 substituted with A, C, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y; H72 substituted with A, C, D, E, F, G, I, K, L, M, N, P, Q, R, S, T, V, W or Y; L73 substituted with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; Q74 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y; D76 substituted with A, C, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; V77 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, W or Y; R78 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; W79 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or Y; R80 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y; K81 substituted with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y; L82 substituted with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; F83 substituted with A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; S84 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W or Y; F85 substituted with A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; T86 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; K87 substituted with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y; Y88 substituted with A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or W; F89 substituted with A, C, D, E, G, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y; L90 substituted with A, C, D, E, F, G, H, I, K, M, N, P, Q, R, S, T, V, W or Y; K91 substituted with A, C, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y; I92 substituted with A, C, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y; And / or E93 substituted with A, C, D, F, G, H, I, K, L, M, N, P, Q, R, S, T, V,
[267] These mutants can be prepared from the above-described N-terminal deletion constructs, in particular those starting with the amino acids M1, T36, C37 or A63. In addition, one or more amino acids (e.g., 2,3, 4,5, 6,7, 8,9, and 10 species) may be substituted with other amino acids in these regions (A63 to E93). The resulting constructs can be screened for loss of heparin binding, loss of KGF-2 activity, and / or enzymatic cleavage between amino acids R68 and S69.
[268] Preferred mutants are the N-terminal deletion constructs M1, T36, C37 and A63 as well as mutants in the heparin binding domain of all of the N-terminal mutants listed above, particularly T36, C37, A63, S69, V77, R80 or E93 Lt; RTI ID = 0.0 > R68 < / RTI > The heparin binding domain is between Arg 174 and Lys 183. Preferred Arg 68 mutants are those in which arginine is replaced by Gly, Ser or Ala; A preferred Arg 187 mutant replaces arginine with alanine.
[269] Two ways in which mutants can be made are site directed mutagenesis or accelerating mutagenesis [Kuchner and Anold, Tibtech 5: 523-530 (1997); Crameri et al., Nature (1998); And Christian et al., Nature Biotechnology 17: 259264 (1999)]. These methods are well known in the art.
[270] Preferably, the alteration is for minor properties such as conservative amino acid substitutions that do not significantly affect protein superposition or activity. Examples of conservative amino acid substitutions known to those skilled in the art are as follows:
[271] Aliphatic amino acids: phenylalanine
[272] Tryptophan
[273] Tyrosine
[274] Hydrophobic amino acid: leucine
[275] Isoleucine
[276] Balin
[277] Polar amino acid: glutamine
[278] Asparagine
[279] Basic amino acids: arginine
[280] Lysine
[281] Histidine
[282] Acidic amino acid: Aspartic acid
[283] Glutamic acid
[284] Small amounts of amino acids: alanine
[285] Serine
[286] Threonine
[287] Methionine
[288] Glycine
[289] Of course, the number of amino acid substitutions that a person skilled in the art can make depends on a number of factors, including those mentioned above. Generally, the number of substitutions for any given KGF-2 polypeptide varies depending on purpose, but can not be 50, 40, 30, 20, 10, 5 or 3 or more. For example, a number of substitutions made at the C-terminus of KGF-2 that can improve stability are described above and in Example 22.
[290] Particularly preferred are KGF-2 molecules with conservative amino acid substitutions, including: M1 substituted with A, G, I, L, S, T or V; W2 substituted with F or Y; K3 substituted with H or R; W4 substituted with F or Y; I5 substituted with A, G, L, S, T, M or V; T7 substituted with A, G, I, L, S, M or V; S11 substituted with A, G, I, L, T, M or V; G, I, L, S, T, M or V; F13 substituted with Y or W; H15 substituted with K or R; L16 substituted with A, G, I, S, T, M or V; G18 substituted with A, I, L, S, T, M or V; L25 substituted with A, G, I, S, T, M or V; L26 substituted with A, G, I, S, T, M or V; L27 substituted with A, G, I, S, T, M or V; F28 substituted with W or Y; L29 substituted with A, G, I, S, T, M or V; V30 substituted with A, G, I, L, S, T or M; S31 substituted with A, G, I, L, T, M or V; S32 substituted with A, G, I, L, T, M or V; V33 substituted with A, G, I, L, S, T or M; V35 substituted with A, G, I, L, S, T or M; G, I, L, S, T, M or V; L40 substituted with A, G, I, S, T, M or V; G41 substituted with A, I, L, S, T, M or V; Q42 substituted with N; D43 substituted with E; M44 substituted with A, G, I, L, S, T or V; V45 substituted with A, G, I, L, S, T or M; S46 substituted with A, G, I, L, T, M or V; E48 substituted with D; G, I, L, S, T, M or V; T50 substituted with A, G, I, L, S, M or V; N51 substituted with Q; S52 substituted with A, G, I, L, T, M, or V; S53 substituted with A, G, I, L, T, M or V; S54 substituted with A, G, I, L, T, M or V; S55 substituted with A, G, I, L, T, M or V; S56 substituted with A, G, I, L, T, M or V; F57 substituted with W or Y; S58 substituted with A, G, I, L, T, M or V; S59 substituted with A, G, I, L, T, M or V; S61 substituted with A, G, I, L, T, M or V; S62 substituted with A, G, I, L, T, M or V; G, I, L, S, T, M or V; G64 substituted with A, I, L, S, T, M or V; R65 substituted with H or K; H66 substituted with K or R; V67 substituted with A, G, I, L, S, T or M; R68 substituted with H or K; S69 substituted with A, G, I, L, T, M or V; Y 70 substituted with F or W; N71 substituted with Q; H72 substituted with K or R; L73 substituted with A, G, I, S, T, M or V; Q74 substituted with N; G75 substituted with A, I, L, S, T, M or V; D76 substituted with E; V77 substituted with A, G, I, L, S, T or M; R78 substituted with H or K; W79 substituted with F or Y; R < 80 > K81 substituted with H or R; L82 substituted with A, G, I, S, T, M or V; F83 substituted with W or Y; S84 substituted with A, G, I, L, T, M or V; F85 substituted with W or Y; T86 substituted with A, G, I, L, S, M or V; K87 substituted with H or R; Y88 substituted with F; F89 substituted with W or Y; L90 substituted with A, G, I, S, T, M or V; K91 substituted with H or R; I92 substituted with A, G, L, S, T, M or V; E93 substituted with D; K94 substituted with H or R; N95 substituted with Q; G96 substituted with A, I, L, S, T, M or V; K97 substituted with H or R; V98 substituted with A, G, I, L, S, T or M; S99 substituted with A, G, I, L, T, M or V; G100 substituted with A, I, L, S, T, M or V; T101 substituted with A, G, I, L, S, M or V; K102 substituted with H or R; K103 substituted with H or R; E104 substituted with D; N105 substituted with Q; Y108 substituted with F or W; S109 substituted with A, G, I, L, T, M or V; I110 substituted with A, G, L, S, T, M or V; L111 substituted with A, G, I, S, T, M or V; E112 substituted with D; I113 substituted with A, G, L, S, T, M or V; T114 substituted with A, G, I, L, S, M or V; S115 substituted with A, G, I, L, T, M or V; V116 substituted with A, G, I, L, S, T or M; E117 substituted with D; I118 substituted with A, G, L, S, T, M or V; G119 substituted with A, I, L, S, T, M or V; V120 substituted with A, G, I, L, S, T or M; V121 substituted with A, G, I, L, S, T or M; G, I, L, S, T, M or V; V123 substituted with A, G, I, L, S, T or M; K124 substituted with H or R; G, I, L, S, T, M or V; I126 substituted with A, G, L, S, T, M or V; N127 substituted with Q; S128 substituted with A, G, I, L, T, M or V; N129 substituted with Q; Y130 substituted with F or W; Y131 substituted with F or W; L132 substituted with A, G, I, S, T, M or V; G, I, L, S, T, M or V; M134 substituted with A, G, I, L, S, T or V; N135 substituted with Q; K136 substituted with H or R; K137 substituted with H or R; G138 substituted with A, I, L, S, T, M or V; K139 substituted with H or R; L140 substituted with A, G, I, S, T, M or V; Y141 substituted with F or W; G142 substituted with A, I, L, S, T, M or V; S143 substituted with A, G, I, L, T, M or V; K144 substituted with H or R; E145 substituted with D; F146 substituted with W or Y; N147 substituted with Q; N148 substituted with Q; D149 substituted with E; K151 substituted with H or R; L152 substituted with A, G, I, S, T, M or V; K153 substituted with H or R; E154 substituted with D; R155 substituted with H or K; I156 substituted with A, G, L, S, T, M or V; E157 substituted with D; E158 substituted with D; N159 substituted with Q; G160 substituted with A, I, L, S, T, M or V; Y161 substituted with F or W; N159 substituted with Q; G162 substituted with A, I, L, S, T, M or V; T163 substituted with A, G, I, L, S, M or V; Y164 substituted with F or W; G, I, L, S, T, M or V; S166 substituted with A, G, I, L, T, M or V; F167 substituted with W or Y; N168 substituted with Q; W169 substituted with F or Y; Q170 substituted with N; H171 substituted with K or R; N172 substituted with Q; G173 substituted with A, I, L, S, T, M or V; R174 substituted with H or K; Q175 substituted with N; M176 substituted with A, G, I, L, S, T or V; Y177 substituted with F or W; V178 substituted with A, G, I, L, S, T or M; A179 substituted with G, I, L, S, T, M or V; L180 substituted with A, G, I, S, T, M or V; N181 substituted with Q; G182 substituted with A, I, L, S, T, M or V; K183 substituted with H or R; A, I, L, S, T, M or V; G, I, L, S, T, M or V; R187 substituted with H or K; R188 substituted with H or K; G189 substituted with A, I, L, S, T, M or V; Q190 substituted with N; K191 substituted with H or R; T192 substituted with A, G, I, L, S, M or V; R193 substituted with H or K; R194 substituted with H or K; K195 substituted with H or R; N196 substituted with Q; T197 substituted with A, G, I, L, S, M or V; S198 substituted with A, G, I, L, T, M or V; G, I, L, S, T, M or V; H200 substituted with K or R; F201 substituted with W or Y; L202 substituted with A, G, I, S, T, M or V; M203 substituted with A, G, I, L, S, T or V; V205 substituted with A, G, I, L, S, T or M; V206 substituted with A, G, I, L, S, T, or M; H207 substituted with K or R; Or S208 substituted with A, G, I, L, T, M or V.
[291] However, also KGF-2 molecules with non-conservative amino acid substitutions, including the following: M1 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; W, substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; K3 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; W is substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; I5 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L6 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T7 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; C9 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; A10 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S, substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; D, E, H, K, R, N, Q, F, W, Y, P or C; F13 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; P14 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; H, substituted by D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L16 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P17 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; G18 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C19 substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C21 substituted by D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C22 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; C 23 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; F24 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L25 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L26 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L27 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F, substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L29 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V30 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S, substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; S, substituted by D, E, H, K, R, N, Q, F, W, Y, P or C; V33 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P34 substituted with D, E, H, K, R, N, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; V35 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T36 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; C37 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; Q38 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; A39 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L40 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G41 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q 42 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D43 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; M44 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V45 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S, substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P47 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; E48 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A49 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T50 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N51 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S52 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S53 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S54 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S55 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S5, substituted with D, E, H, K, R, A, N, Q, F, W, Y, P or C; F57 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S58 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S59 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P60 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; S61 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S62 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A63 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G64 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R65 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; H66 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V67 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; S69 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y 70 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N71 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; H72 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L73 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q74 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G75 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D76 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V77 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R78 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; W79 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K81 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L82 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F83 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S84 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S84 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F85 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; T86 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K87 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Y88 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; F89 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L90 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K91 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I92 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E93 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K94 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N95 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G96 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K97 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; V98 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S99 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G100 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T101 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K102 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K103 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E104 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N105 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; P107 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; Y108 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; S109 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I110 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L111 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E112 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I113 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; T114 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S115 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V116 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E117 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I118 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; G119 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V120 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V121 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A122 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V123 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K124 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; A125 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; I126 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N127 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; S128 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N129 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; Y130 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Y131 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L132 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A133 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; M134 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N135 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K136 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K137 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G138 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K139 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L140 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y141 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; G142 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; D, E, H, K, R, N, Q, F, W, Y. S143 substituted with P or C; K144 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E145 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; F146 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; N147 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N148 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; D149 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or P; K151 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; L152 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K153 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E154 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; I156 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; E157 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; E158 substituted with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N159 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G160 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y161 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; N162 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; T163 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y164 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; A165 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S166 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; F167 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; N 168 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; W169 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; Q170 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; H171 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N172 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G173 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R174 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Q175 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; M176 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Y177 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; V178 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A179 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; L180 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; N181 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; G182 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; K183 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G184 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A185 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P186 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; R187 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R188 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; G189 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; Q190 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; K191 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; T192 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; R193 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; R194 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; K195 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; N196 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P or C; T197 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; S198 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; A199 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H200 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; F201 substituted with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P or C; L202 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; P203 substituted with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y or C; M204 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V205 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; V206 substituted with D, E, H, K, R, N, Q, F, W, Y, P or C; H207 substituted with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P or C; Or S208 substituted with D, E, H, K, R, N, Q, F, W, Y,
[292] One of the assays described herein can be used to test the activity of a substitution mutant. KGF-2 molecules with conservative substitutions maintain the activity and properties of wild-type proteins; As long as all other activities or characteristics are retained, have enhanced activity or properties relative to the wild-type protein; It is particularly preferred to have one or more enhanced activities or properties relative to the wild-type protein. In contrast, KGF-2 molecules with non-conservative substitutions lack activity or properties of the wild-type protein, as long as all other activities and characteristics are maintained; Or wild type protein.
[293] For example, the activity or properties of KGF-2 that can be altered in KGF-2 molecules with conservative or non-conservative substitutions include, but are not limited to, keratinocytes, epithelia Growth stimulation of cells, hair follicles, hepatocytes, kidney cells, breast tissue, bladder cells, prostate cells, pancreatic cells; Differentiation stimulation of muscle cells, nerve tissue, prostate cells, lung cells, hepatocytes, kidney cells, and breast tissue; Promotion of wound healing; Angiogenic stimulation; Reduction of inflammation; Cell protection; Heparin binding; Ligand binding; stability; Solubility; And / or properties affecting the tablet.
[294] Amino acids in KGF-2 that are essential for function can be identified by methods known in the art, such as site directed mutagenesis or alanin-scanning mutagenesis [Cunningham and Wella, Science 244: 1081-1085 (1989) have. The latter method introduces a single alanine mutation into all residues in the molecule. The resulting mutant molecule is then tested for biological activity, such as receptor binding, or in vitro and in vivo proliferative activity (see, for example, Examples 10 and 11). In addition, sites that are important for ligand-receptor binding can be measured by structural analysis, such as crystallization, nuclear magnetic resonance or optical affinity labeling (see, e.g., Smith et al., J. Mol. Biol. 224: 899-904 1992)).
[295] Another aspect of the invention relates to the substitution of cysteine with serine at amino acid positions 37, 106 and 150. An odd number of cysteine means that one or more cysteine residues can be used for intermolecular crosslinking or linkage, which allows the protein to adopt an undesirable three-dimensional structure. In general, the novel KGF-2 protein, which causes one or more cysteines to be replaced by serine or, for example, alanine, is purified to a high yield of soluble precisely overlaid proteins. Although not proved, the cysteine residue at position 106 is believed to be important for function. This cysteine residue is more highly conserved than all other FGF family members.
[296] Another aspect of the present invention is the use of a fusion protein of KGF-2 with another protein or fragment thereof such as fusion with other FGF proteins such as KGF (FGF-7), bFGF, aFGF, FGF-5, FGF- Or a hybrid. Published PCT Application No. 90/08771 describes the production of chimeric proteins consisting of the first 40 amino acid residues of KGF and the C-terminal portion of aFGF. Although chimeras have been reported as target keratinocytes such as KGF, chimeras lack sensitivity to heparin, features of aFGF, whereas KGF does not. Fusion with some of the constant domains of immunoglobulins (IgG) often exhibits an increased half-life in vivo. For example, it represents a chimeric protein consisting of the first two domains of a human CD4-polypeptide having various domains of the constant region of the heavy or light chain of mammalian immunoglobulin [European Patent Application, publication number 394827, Traunecker et al. Nature 331: 84-86 (1988)]. Fusion proteins having a disulfide-bonded dimer structure may also be more effective in binding monomer molecules alone (Fountoulakis et al., J. of Biochemistry, 270: 3958-3964, (1995)).
[297] Additional fusion proteins of the present invention can be used for gene-shuffling, motif-shuffling, exon-shuffling and / or codon shuffling (collectively referred to as "DNA- ). ≪ / RTI > DNA splicing can be used to modulate the activity of the polypeptides of the invention, which can be used to generate polypeptides with altered activity as well as working substances and antagonists of the polypeptides. Generally, U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; And Patten et al., Curr. Opinion Biotechnol. 8: 724-33 (1997), Harayama, Trends Biotechnol. 16 (2): 76-82 (1998), Hansson et al., J. Mol. Biol. 287: 265-76 (1999) and Lorenzo and Blasco, Biotechniques 24 (2): 308-13 (1998), all of which patents and publications are incorporated herein by reference. In one embodiment, alteration of a polynucleotide corresponding to SEQ ID NO: 1 and a polypeptide encoded by the polynucleotide can be achieved by DNA replication. DNA supple- ments involve mutations in the polynucleotide sequence, including a set of two or more DNA segments by homologous or site-specific recombination. In another embodiment, polynucleotides and encoded polypeptides of the invention can be altered by other methods prior to random disruption, random nucleotide insertion or recombination by error prone PCR. In another embodiment, one or more of the constituents, motifs, sections, fragments, fragments, etc. of a polynucleotide encoding a polypeptide of the invention may comprise one or more components of one or more heterologous molecules, Motifs, sections, fragments, fragments, and the like.
[298] The antigenic / hydrophilic part of KGF-2
[299] As illustrated in Figures 4A-4E, there are four major highly hydrophilic regions within the KGF-2 protein. The amino acid residues are Gly 41-Asn 71, Lys 91-Ser 109, Asn 135-Tyr 164 and Asn 181-Ala 199 [SEQ ID NOS 25-28]. Two additional antigenic regions are shorter and predetermined antigenic regions, Gly 74-Arg 78 (SEQ ID NO: 2) in Figure 1 and Gln 170-Gln 175 (SEQ ID NO: 2) in Figure 1. Hydrophilic moieties are predominantly located outside (on the surface) of the protein and are therefore known to be useful for antibodies that recognize these regions. In addition, these regions are prone to association with KGF-2 and its receptor (s). Synthetic peptides derived from these regions can interfere with the binding of KGF-2 to its receptor (s) and thus block the function of the protein. In addition, synthetic peptides derived from the hydrophilic portion of a protein may be an agonistic, or mimic, function of KGF-2.
[300] Thus, the invention also relates to a discrete polypeptide comprising a hydrophilic region of KGF-2, said polypeptide comprising at least one of the aforementioned KGF-2 hydrophilic regions and having a length of up to 150 amino acids, 100, 75, or 50 amino acids.
[301] The epitope-containing portion of KGF-2
[302] In another aspect, the invention provides polypeptides and peptides comprising an epitope-containing portion of a polypeptide of the invention. These epitopes are immunogenic or antigenic epitopes of the polypeptides of the invention. An " immunogenic epitope " is defined as a portion of a protein that elicits an antibody response in vivo when the entire polypeptide of the invention, or a fragment thereof, is an immunogen. On the other hand, the region of the polypeptide to which the antibody can bind is defined as an " antigenic determinant " or " antigenic epitope ". In general, the number of in vivo immunogenic epitopes of a protein is less than the number of antigenic epitopes. See, e.g., Geysen et al., Proc. Natl. Acad. Sci. USA 81: 3998-4002 (1983). However, by using a phage display, antibodies against an antigenic epitope can be made, whether or not the epitope is immunogenic epitopes. See, for example, Petersen G. et al., Mol. Gen. Genet. 249: 425-431 (1995). Thus, both immunogenic epitopes and antigenic epitopes are encompassed by the present invention.
[303] Exemplary amino acid sequence listings including immunogenic epitopes are shown in Table 1 below. Table 1 provides a summary of the results obtained in the literature [Jameson and Wolf, (1988) Comp. Appl. Biosci. 4: 181-186] (the entire contents of which are incorporated herein by reference), to enumerate the amino acid residues containing the epitope intended to have the highest antigenicity. Jameson-Wolf antigen analysis was performed using a computer program PROTEAN (Power Macintosh version 3.11 from DNASTAR, Inc., 1228 South Park Street, Madison, Wis.) Using a sparse parameter. Polypeptide moieties not listed in Table 1 and Table 1 are considered non-immunogenic. The immunogenic epitopes in Table 1 are exemplary and not exhaustive because other immunogenic epitopes are not themselves recognized by the particular algorithm used. In general, amino acid residues containing other immunogenic epitopes can be measured using in vivo antigenic reaction assays using algorithms similar to Jameson-Wolf assays, or using methods known in the art. See, for example, the above cited documents by Geysen et al., And U.S. Patent Nos. 4,708,781, 5,194,392, 4,433,092 and 5,480,971, all of which are incorporated herein by reference in their entirety.
[304] The antigenic epitope-containing peptides and polypeptides of the present invention preferably contain at least 7, more preferably at least 9, and most preferably at least 15 to 30 amino acids contained within the amino acid sequence of the polypeptide of the invention . Examples of antigenic polypeptides or peptides that can be used for KGF-2 specific antibodies include, but are not limited to, the following: SEQ ID NO: 2 [about Gly 41-Asn 71; Lys 91-Ser 109; Asn 135-Tyr 164; Asn 181-Ala 199; Gln74-Arg 78; And Gln170-Gln175]. These polypeptide fragments can be assayed for the presence of an antigenic epitope of the KGF-2 protein by analysis of the Jameson-Wolf antigenicity index as shown in FIG.
[305] In particular, it indicates that the amino acid sequence of Table 1 contains an immunogenic epitope. Table 1 lists only the important residues of the immunogenic epitope measured by Jameson-Wolf analysis. Thus, additional flanking residues on the N-terminus, C-terminus, or N-terminus and C-terminus and C-terminus terminus are added to the sequences in Table 1 to generate the epitope- . Thus, the immunogenic epitopes of Table 1 may include additional N-terminal or C-terminal amino acid residues. Additional flanking amino acid residues may be contiguous flanking N-terminal and / or C-terminal or heterologous polypeptide sequences derived from the polypeptides of the invention, or both contiguous flanking sequences and heterologous polypeptide sequences derived from the polypeptides of the invention . ≪ / RTI > A polypeptide of the invention comprising an immunogenic or antigenic epitope corresponds to at least seven amino acid residues in length. The term " above " may be any integer between 7 amino acid residues in length of the polypeptide of the invention comprising an immunogenic or antigenic epitope, or between 7 and the entire polypeptide length of the invention . Preferred polypeptides comprising an immunogenic or antigenic epitope are 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more than 100 amino acid residues. However, it indicates that each integer and all integers between the number of amino acid residues of the polypeptide of 7 and the full length are included in the present invention.
[306] The immunogenic and antigenic epitope containing fragments may be specified by the adjacent amino acid residues as described above or by the N-terminal or C-terminal positions of these fragments on the amino acid sequence of SEQ ID NO: 2. All combinations of N-terminal or C-terminal positions occupied by the fragments corresponding to, for example, 7 or more contiguous amino acid residues in length on the amino acid sequence of SEQ ID NO: 2 are encompassed by the present invention. In addition, " having a length of at least 7 contiguous amino acid residues " corresponds to a length of 7 amino acid residues, or corresponds to any integer number of amino acid residues between 7 and the total number of amino acid residues of the polypeptide length of the invention . Specifically, each integer and all integers between 7 and the number of amino acid residues of the full length polypeptide are included in the present invention.
[307] For example, the immunogenic and antigenic epitope-containing polypeptides of the invention are useful for specifically binding polypeptides of the invention and are useful in immunoassays for detecting the polypeptides of the invention. For example, antibodies are useful for affinity purification of the polypeptides of the present invention. In addition, antibodies can be routinely used for qualitative or quantitative immunoassays, particularly qualitative or quantitative immunoassays for polypeptides of the invention using known methods. See, for example, Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Second Edition, Cold Spring Harbor, New York (1988).
[308] The epitope-containing polypeptides of the present invention can be produced by any conventional means of producing polypeptides, including synthetic and recombinant methods known in the art. For example, epitope-containing peptides can be synthesized using known chemical synthesis methods. For example, Houghten refers to single amino acid variants of a plurality of peptides, e. G., HA1 polypeptide fragments, all of which are produced within 4 weeks (by ELISA type binding studies) and characterized by 248 individuals of 10-20 mg and A simple method for the synthesis of a separate 13 residue peptide has been described [Houghten, RA, Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985)). Such " Simultaneous Multiple Peptide Synthesis (SMPS) " is further described in U.S. Patent No. 4,631,211 to Houghten and co-workers (1986). In this method, individual resins for the solid phase synthesis of various peptides are contained in separate solvent permeable packets which enable optimal use of a number of identical iterative processes associated with solid phase processes. A fully manual process enables 500-1000 or more syntheses to be performed simultaneously (Houghten et al., (1985) Proc. Natl. Acad. Sci. 82: 5131-5135, especially 5134].
[309] The epitope-containing polypeptides of the present invention can be used to induce antibodies according to methods well known in the art, including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, for example, Sutcliff et al., Wilson et al., And Bittle et al. [J. Gen. Virol., 66: 2347-2354 (1985). If in vivo immunization is used, the animal can be immunized with a free peptide; However, the anti-peptide antibody titer may be further administered by coupling the peptide to a macromolecular carrier [e.g., keyhole limpet hemocyanin (KLH) or tetanus toxoid). For example, cysteine residue-containing peptides may be coupled to a carrier using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide (MBS), while other peptides may be coupled to a more common binding agent (E. G., Glutaraldehyde). ≪ / RTI > For example, by intraperitoneal and / or intradermal injection of an emulsion containing about 100 [mu] g of peptide or carrier protein and Freund ' s adjuvant or any other adjuvant known to stimulate the immune response (E. G., Rabbit, rat and mouse) with a free peptide or carrier coupled peptide. For example, some booster injections may be needed at about two week intervals to provide useful potency of the anti-peptide antibodies that can be detected by ELISA assays using free peptides adsorbed to the solid surface. The potency of the anti-peptide antibody in the immunized animal serum can be increased by selection of the anti-peptide antibody (e.g., by adsorption to a peptide on a solid support and elution of the selected antibody according to methods well known in the art).
[310] Those skilled in the art will appreciate that the polypeptides of the invention containing an immunogenic or antigenic epitope as described above may be fused to other polypeptide sequences. For example, fused polypeptides of the present invention with constant domains of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3 or any combination thereof and portions thereof) to generate chimeric polypeptides . The fusion protein can promote purification and increase in vivo half-life. This occurs in the case of chimeric proteins consisting of the first two domains of the human CD4-polypeptide and the various domains of the constant region of the heavy or light chain of the mammalian immunoglobulin. See, for example, EP 394,827 and Traunecker et al., Nature, 331: 84-86 (1988). (See, for example, PCT Publication Nos. WO 96/22024 and WO 99 (see, for example, PCT Publication Nos. WO 96/20224 and WO 99/1995), in the case of an antigen fused to an FcRn binding partner (e.g., IgG) or an Fc fragment, such as insulin, / 04813). In addition, IgG fusion proteins having a disulfide-bonded dimer structure due to the disulfide bond of the IgG moiety have been found to be more effective in binding and neutralizing molecules than other monomer polypeptides or fragments thereof. See, e.g., Fountoulakis et al., J. Biochem., 270: 3958-3964 (1995). In addition, the nucleic acid encoding the epitope can be recombined into an important gene as an epitope label (e.g., a red blood cell agglutination ("HA") label or a flag tag) to facilitate detection and purification of the expressed polypeptide. For example, the system described by Janknecht et al. Allows rapid and easy purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-897). In this system, the critical gene is subcloned into a vaccinia recombinant plasmid, so that the open reading-decoding structure of the gene is transcribably fused to an amino terminal label consisting of six histidine residues. The label serves as a matrix binding domain for the fusion protein. Extracts from cells infected with recombinant Vaccinia virus can be loaded onto Ni ²⁺ nitriloacetic acid-agarose columns and the histidine-labeled protein selectively eluted with an imidazole-containing buffer.
[311] Chemical transformation
[312] In addition, KGF wild type and analogs can be modified to contain additional chemical moieties that are not conventional protein moieties. Such derivatized moieties can enhance solubility, biological half-life or protein adsorption. In addition, the derivatized portion may reduce or eliminate any desirable side effects, etc., of the protein. An overview of the derivatized moiety can be found in REMINGTON'S PHARMACEUTICAL SCIENCE, 18th Edition, Mack Publishing Co., Eastton, Pennsylvania, USA. (1990). Polyethylene glycol (PEG) is a chemical moiety useful in the preparation of therapeutic proteins. PEG attachment to proteins appears to protect from proteolysis [Sada et al., J. Fermentation Bioengineering 71: 137-139 (1991)]. Various methods are useful for attachment of specific PEG moieties. For a review of this, see Abuchowski et al., In Enzymes as Drugs. (Holcerberg and Roberts, eds.) Pp. 367-383 (1981). A number of published patents (e.g., U.S. Patent No. 5,342,940 to Ono et al .; U. S. Patent No. 5,089, 261 to Nitecki et al; US Patent No. 5,349,052 to Delgado et al. Describe PEG derivatives and methods for their preparation. Generally, a PEG molecule is linked to a protein by a reactive group found on the protein. The amino terminus of the amino group (e. G., The amino group on the lysine) or protein is more convenient for attachment than any other.
[313] The entire disclosure of each of the documents cited in this section with respect to " polypeptides and peptides " is incorporated herein by reference.
[314] The polypeptides of the present invention may also be chemically synthesized using techniques known in the art (see, for example, Creighton, 1983, Proteins: Structures and Molecular Principles, WH Freeman & , Nature, 310: 105-111 (1984).) For example, a polypeptide corresponding to a fragment of KGF-2 polypeptide can be synthesized by use of a peptide synthesis apparatus. -2 polypeptide sequence, a nonclassical amino acid or a chemical amino acid analog can be introduced as a substitution or addition to the polypeptide sequence. In general, nonclassical amino acids include, but are not limited to, the following: Aminobutyric acid, Abu, 2-aminobutyric acid, g-Abu, e-Ahx, 6-aminohexanoic acid, Aib, 2-aminobutyric acid, Ah Butyric acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, isobutyric acid, amino acids such as b-alanine, fluoro-amino acid, designer amino acids such as b-methyl amino acid, Ca-methyl amino acid, Na-methyl amino acid and amino acid derivatives. ).
[315] The present invention also relates to the use of a KGF which is differentially modified during or after translation, for example by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, binding to antibody molecules or other cellular ligands, -2 < / RTI > polypeptide. Specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH _4; Acetylation, formylation, oxidation, reduction; Any number of chemical transformations can be performed by known techniques including, but not limited to, metabolic synthesis in the presence of tunicamycin.
[316] Examples of further post-translational modifications included in the present invention include treatment of N-linked or O-linked hydrocarbon chains, N-terminal or C-terminal ends, attachment of chemical moieties to amino acid backbones, N- Chemical modification of O-linked hydrocarbon chains, addition or deletion of N-terminal methionine residues as a result of prokaryotic host cell expression. In addition, the polypeptide can be modified with a detectable label (e.g., an enzyme label, fluorescent label, isotope or affinity label) to enable detection and isolation of the protein.
[317] The present invention also provides derivatives of chemically modified polypeptides of the invention that can provide additional advantages (e.g., increased solubility, stability and cycle time of the polypeptide, or decreased immunogenicity) 4,179,337). The chemical moiety for derivatization may be selected from water soluble polymers (e.g., polyethylene glycol, ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, and the like). The polypeptide may be modified at any position in the molecule or at a predetermined position in the molecule, and may include one, two, three or more attached chemical moieties.
[318] The polymer may have any molecular weight and may be branched or unbranched. In the case of polyethylene glycol, the preferred molecular weight for easy manipulation and preparation is from about 1 kDa to about 100 kDa (the term " about " refers to the molecular weight of some molecules in the production of polyethylene glycol, it means). Depending on the desired therapeutic profile (eg, the duration of the given western, non-biological activity, ease of manipulation, degree or lack of antigenicity, other known effects of polyethylene glycol on therapeutic proteins or analogs) Molecules can be used.
[319] The polyethylene glycol molecule (or other chemical moiety) must be attached to the protein, taking into account the effect on the protein's functional or antigenic domains. There are a number of attachment methods available to those skilled in the art, such as EP 0 401 384 (a method of coupling PEG to G-CSF), which is incorporated herein by reference, and others, Malik et al., Exp. Hematol. 20: 1028-1035 (1992); (Reported for pegylation of GM-CSF using trisyl chloride). For example, the polyethylene glycol may be covalently bonded through an amino acid residue by a reactive group (e.g., a free amino group or a carboxyl group). The reactive group is a group to which an activated polyethylene glycol molecule can be bonded. The amino acid residue having a free amino group may include a lysine residue and an N-terminal amino acid residue; The amino acid residue having a free carboxyl group may comprise an aspartic acid residue, a glutamic acid residue and a C-terminal amino acid residue. Further, a sulfhydryl group can be used as a reactive group for attaching the polyethylene glycol molecule. Attachment to an amino group (e.g., attachment to the N-terminal or lysine group) is preferred for therapeutic purposes.
[320] In particular, proteins chemically modified at the N-terminus may be preferred. When polyethylene glycol is used as an example of the composition of the present invention, the ratio of the polyethylene glycol to the protein (polypeptide) molecule in the reaction mixture, the type of PEGylation performed, Can be selected from the method of obtaining a PEGylated protein at its terminal. The method of obtaining an N-terminal PEGylated agent (i. E., The method of separating this moiety from other monopegylated moieties, if necessary) can be used for the purification of N-terminal PEGylated material from a number of PEGylated protein molecules can do. Selectively modified proteins chemically modified in the N-terminal modification can be achieved by reductive alkylation utilizing different reactivity of different types of primary amino groups (lysine to N-terminal) useful for derivatization in certain proteins. Under suitable reaction conditions, a substantially selective derivatization of the protein at the N-terminus is achieved with the carbonyl group containing the polymer.
[321] Antibody
[322] Additional polypeptides of the present invention are directed to polypeptides, polypeptide fragments or variants of SEQ ID NO: 2, and / or antibodies and T-cell antigen receptors (TCR) that immunospecifically bind epitopes of the invention (specificity As measured by immunoassays well known in the art as a method of analyzing antibody-antigen binding). The antibody of the present invention is produced by a polyclonal antibody, a monoclonal antibody, a multispecific antibody, a human antibody, a humanized antibody or a chimeric antibody, a single chain antibody, a Fab fragment, an F (ab ' (Including anti-Id antibodies to the antibodies of the present invention), and any epitope binding fragments thereof. The term " anti-Id antibody " As used herein, the term " antibody " means an immunoglobulin molecule and an immunologically active portion of an immunoglobulin, i.e., a molecule that contains an antigen binding site that binds an antigen specifically. The immunoglobulin molecules of the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), classes (e.g., IgGl, IgG2, IgG3, IgG4, IgA1 and IgA2) have.
[323] Most preferably, the antibody is a human antigen-binding antibody fragment of the present invention. Examples of the antibody include Fab, Fab ', F (ab') 2, Fd, single chain Fvs (scFv), single chain antibody, disulfide- (sdFv) and fragments comprising a V _L or V _H domain. Antigen-binding antibody fragments, including single chain antibodies, may comprise the variable region (s), alone or in combination with: hinge regions, CH1, CH2 and CH3 domains. Also included in the invention are antigen-binding antibody fragments comprising any combination of hinge region, CHl, CH2 and CH3 domains and variable region (s). The antibodies of the present invention can be derived from animals, including birds and mammals. The antibody is preferably human, murine (e.g. mouse and rat), monkey, ship rabbit, goat, guinea pig, horse or chicken. As used herein, a " human " antibody comprises an antibody having an amino acid sequence of human immunoglobulin and comprises an antibody isolated from a human immunoglobulin library or an animal transformed against one or more human immunoglobulin , And do not express the endogenous immunoglobulin as described, for example, in U.S. Patent No. 5,939,598 to Kucherlapati et al.
[324] The antibodies of the present invention may be monospecific, bispecific, trispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of the polypeptides of the present invention or may have specificity for the polypeptides and heterologous epitopes of the invention (e. G., Heterologous polypeptides or solid support materials). For example, PCT publications WO 93/17715, WO 92/08802, WO 91/00360, WO 92/05793; Tutt et al., J. Immunol. 147: 60-69 (1991); U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819; And Kostelny et al., J. Immunol. 148: 1547-1553 (1992).
[325] The antibodies of the invention may be described or embodied in terms of epitope (s) or polypeptide portion (s) of the invention that recognize and specifically bind. The epitope (s) or polypeptide portion (s) can be described in the detailed description, for example, by N-terminal and C-terminal positions, size within adjacent amino acid residues, have. Preferred epitopes of the invention include polynucleotides encoding these epitopes as well as amino acids 41-71, 91-109, 135-164, 181-199, 74-78 and 170-175 of SEQ ID NO: 2. In addition, antibodies that specifically bind any epitope or polypeptide of the invention may be excluded. Accordingly, the present invention includes an antibody that specifically binds to the polypeptide of the present invention, and it is also possible to exclude the antibody.
[326] In addition, the antibodies of the present invention can be described or embodied in terms of cross reactivity. Antibodies that do not bind any other analogs, orthologs or analogs of the polypeptides of the present invention. In addition, the polypeptide of the present invention has an identity of not less than 95%, not less than 90%, not less than 85%, not less than 80%, not less than 75%, not less than 70%, not less than 65%, not less than 60%, not less than 55% (Which are known in the art and have been calculated using the methods described herein) are also encompassed by the present invention. In certain embodiments, the antibodies of the invention cross-react with murine, rat and / or rabbit homologues of human proteins, and their corresponding epitopes. In addition, the polypeptides of the present invention may also have identity of less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55% ) (Calculated using methods known in the art and described herein). Antibodies that do not bind polypeptides are also encompassed by the invention. In certain embodiments, the cross-reactivity is selected from any single specific antigenic or immunogenic polypeptide, or a combination of 2, 3, 4, 5, or more of the specific antigenic or immunogenic polypeptides described herein . Also included in the invention are antibodies that bind polypeptides encoded by polynucleotides that form hybrids to the polynucleotides of the invention under stringent hybridization conditions (as described herein). In addition, the antibodies of the present invention can be described or embodied in terms of the binding affinity for the polypeptides of the present invention. The preferred binding affinity is less than 5 × 10 ^-2 M, 10 ^-2 M or less, 5 × 10 ^-3 M or less, 10 ^-3 M or less, 5 × 10 ^-4 M or less, 10 ^-4 M or less, 5 × 10 ^{- 5} M or less, 10 ^-5 M or less, 5 × 10 ^-6 M or less, 10 ^-6 M or less, 5 × 10 ^-7 M or less, 10 ^-7 M or less, 5 × 10 ^-8 M or less, 10 ^-8 M or less, 5 × 10 ^-9 M or less, 10 ^-9 M or less, 5 × 10 ^-10 M or less, 10 ^-10 M or less, 5 × 10 ^-11 M or less, 10 ^-11 M or less, 5 × 10 ^-12 M or less, 10 ^-12 M or less, 5 × 10 ^-13 M or less, 10 ^-13 M or less, 5 × 10 ^-14 M or less, 10 ^-14 M or less, 5 × 10 ^-15 M or less, or 10 ^-15 M or less And having a dissociation constant.
[327] The present invention also contemplates competitive inhibition of binding of an antibody to an epitope of the invention when measured by any method known in the art (e. G., Immunoassays described herein) as a method of measuring competitive binding Lt; / RTI > In a preferred embodiment, the antibody competitively inhibits binding to the epitope by greater than 95%, greater than 90%, greater than 85%, greater than 80%, greater than 75%, greater than 70%, greater than 60%, or greater than 50%.
[328] The antibodies of the present invention include, but are not limited to, methods known in the art as methods for purifying, detecting, and targeting polypeptides of the invention, including in vitro and in vivo diagnostic and therapeutic methods. For example, the antibody has use in immunoassays to qualitatively and quantitatively measure the level of a polypeptide of the invention in a biological sample. See, for example, Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, Second Edition, 1988), incorporated herein by reference in its entirety.
[329] The antibodies of the present invention may be used alone or in combination with other compositions. In addition, the antibody may be fused recombinantly to the heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated (including shared and non-covalent) to the polypeptide or other composition. For example, an antibody of the invention can be fused or conjugated to a label in a detection assay, and to an effector molecule (e. G., A heterologous polypeptide, drug or toxin) in a recombinant manner. For example, WO 92/08495, WO 91/14438, WO 89/12624; U.S. Patent No. 5,314,995; And EP 0 396 387.
[330] The antibodies of the present invention can be produced by any suitable method known in the art. For example, the polypeptide of the invention or an antigenic fragment thereof may be administered to an animal to induce the production of serum containing polyclonal antibodies. The term " monoclonal antibody " is not limited to antibodies produced through hybridoma techniques. The term " monoclonal antibody " refers to an antibody derived from a eukaryotic clone, a prokaryotic clone, or a phage clone, and does not mean that the antibody is produced.
[331] Hybridoma techniques are well known in the art and are described in Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988) and Hammerling et al .: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563 -681 (Elsevier, NY, 1981), all of which are incorporated herein by reference. Fab and F (ab ') 2 fragments can be produced by proteolytic cleavage using enzymes such as papain (for Fab fragment production) or pepsin (for F (ab') 2 fragment production).
[332] Alternatively, the antibodies of the present invention can be produced through application of recombinant DNA and phage display techniques, or through synthetic chemistry using methods known in the art. For example, the antibodies of the present invention can be produced using various phage display techniques known in the art. In a phage display method, the functional antibody domain displays on the surface of a phage particle carrying a polynucleotide sequence encoding said domain. A phage with certain binding properties selects a repertoire or a combinatorial antibody library (e.g., human or murine) by direct selection with the antigen, generally a solid surface or an antigen bound or captured in a bead. Generally, the phage used in these methods is a fibrous phage comprising M13 or fd having Fab, Fv or disulfide stabilized Fv antibody domains fused recombinantly to phage gene III or gene VIII protein. Examples of phage display methods that can be used to produce antibodies of the invention are described in Brinkman U., et al. (1995), J. Immunol. Methods 182: 41-50], [Ames, R.S. Et al. (1995), J. Immunol. Methods 184: 177-186], [Kettleborough, C.A. Et al. (1994), Eur. J. Immunol. 24: 952-958], [Persic, L. et al. (1997), Gene 187: 9-18], [Burton, D.R. Et al. (1994), Advances in Immunology 57: 191-280; PCT / GB91 / 011334; WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401; And U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 (the entire contents of which are incorporated herein by reference).
[333] As described in the above references, after the selection of the phage, the antibody-encoding region from the phage is separated and used to generate all antibodies, including human antibodies or any other desired antigen-binding fragment, Cells, plant cells, yeast, and bacteria. For example, methods known in the art, for example, WO 92/22324; Mullinax, R.L. (1992), BioTechniques 12 (6): 864-869, [Sawai, H. et al. (1995), AJRI 34: 26-34], and [Better, M. et al. Techniques for the recombinant production of Fab, Fab 'and F (ab') 2 fragments can also be used, using the methods described in US Pat. have.
[334] Examples of techniques that can be used to produce single chain Fvs and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; (Schu, L. et al. (1993), PNAS 90: 7995-7999 and Skerra, A. et al. (1988), Science 240: 1038 -1040. ≪ / RTI > For some uses, including in vivo use of human antibodies and in vitro detection assays, it is preferred to use chimeric, humanized or human antibodies. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229: 1202 (1985), Oi et al., BioTechniques 4: 214 (1986), Gillies, Et al. (1989), J. Immunol. Methods 125: 191-202; And U.S. Patent No. 5,807,715. The antibodies can be used in the treatment of CDR transplantation (EP 0 239 400, WO 91/09967, US 5,530,101 and 5,585,089), veneering or resurfacing (EP 0 592 106, EP 0 519 596; [Studnicka GM et al. (1994), Protein Engineering 7 (6): 805-814], [Roguska MA et al. (1994), PNAS 91 : 969-973, and Chuck Schupling (US Patent No. 5,565,332). Human antibodies can be produced by a variety of methods known in the art, including the phage display methods described above See also U.S. Patent Nos. 4,444,887, 4,716,111, 5,545,806 and 5,814,318; WO 98/50433, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741 The entire contents of which are incorporated herein by reference).
[335] The antibody of the present invention may act as an agonist or antagonist of the polypeptide of the present invention. For example, the invention includes antibodies that disrupt the receptor / ligand interaction of the polypeptide of the invention in whole or in part. It is preferred that an antibody of the invention binds an antigenic epitope as described herein or a portion thereof. The present invention is characterized by both receptor-specific antibodies and ligand-specific antibodies. In addition, the invention features receptor-specific antibodies that do not prevent ligand binding but prevent receptor activation. Receptor activation (i. E., Signaling) can be measured by techniques described herein or other techniques known in the art. For example, receptor activation may be detected by immunoprecipitation followed by phosphorylation of the receptor or its surface (e.g., tyrosine or serine / threonine) by western blot analysis (e.g., as described in the references) . In certain embodiments, the present invention provides a method of modulating the activity of a ligand or receptor in the absence of an antibody, wherein the activity is at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70% Lt; / RTI >
[336] The invention also features a receptor-specific antibody that recognizes a receptor-ligand complex and preferably prevents both ligand binding and receptor activation as well as antibodies that do not specifically recognize non-binding receptors or non-binding ligands. Likewise, the present invention includes antibodies that bind ligands and prevent ligand binding, but also bind to ligands and prevent ligand binding to the receptor, as well as antibodies that prevent ligand binding to the receptor. The present invention also includes an antibody that activates the receptor. These antibodies may act as receptor agonists. Thus, for example, the dimerization of the receptor can be induced to elevate or activate the entire population or subset of the biological activity of ligand-mediated receptor activation. The antibody may be embodied as an agonist, antagonist or reverse-acting agent for a biological activity, including the specific biological activity of the peptides of the invention described herein. The antibody working materials can be produced using methods known in the art. See, for example, PCT Publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92 (6): 1981-1988 (1998), Chen et al., Cancer Res. 58 (16): 3668-3678 (1998), Harrop et al., J. Immunol. 161 (4): 1786-1794 (1998)], [Zhu et al., Cancer Res. 58 (15): 3209-3214 (1998), Yoon et al., J. Immunol. 160 (7): 3170-3179 (1998), Prat et al., J. Cell. Sci. 111 (Pt2): 237-247 (1998), Pitard et al., J. Immunol. Methods 205 (2): 177-190 (1997), Liautard et al., Cytokine 9 (4): 233-241 (1997), Carlson et al., J. Biol. Chem. Muller et al., Structure 6 (9): 1153-1167 (1998)], [Taryman et al., Neuron 14 (4): 755-762 Bartunek et al., Cytokine 8 (1): 14-20 (1996)), all of which are incorporated herein by reference.
[337] The antibodies of the invention can be used to purify, detect, and target the polypeptides of the invention, including, for example, in vitro and in vivo diagnostic and therapeutic methods, but are not limited thereto. For example, the antibodies are used in immunoassays for qualitatively and quantitatively measuring the polypeptide levels of the invention in biological samples (see, e.g., Harlow et al., Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press (2nd Edition) (1998)]. In a preferred embodiment, the level of KGF-2 is detected using a chicken and chicken antibody in a purified sample (see Example 50, described below).
[338] As discussed in more detail below, the antibodies of the invention may be used alone or in combination with other compositions. The antibody may further be recombinantly fused to a heterologous polypeptide at the N-terminus or C-terminus, or chemically conjugated (including covalent and noncovalent) to the polypeptide or other composition. For example, an antibody of the invention may be recombinantly fused or conjugated to a molecule and effector molecule, such as a heterologous polypeptide, drug, radionuclide or toxin, useful as a label in a detection assay (see, for example, PCT International Publication No. WO 92/08495; WO 91/14438; WO 89/12624; US Patent No. 5,314,995; and EP No. 396,387).
[339] Antibodies of the present invention include modified derivatives, i.e., derivatives that are modified by covalent attachment of any type of molecule to the antibody, such covalent linkage does not prevent the antisense from producing an anti-genetic response. For example, the antibody derivatives may be used in conjunction with, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, And the like, including, but not limited to, Any variation of any of a number of chemical modifications may be accomplished by known techniques, such as, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis such as tonicamycin, and the like. In addition, the derivative may contain one or more unconventional amino acids.
[340] The antibodies of the invention can be produced by any suitable method known in the art. Polyclonal antibodies to the antigen of interest can be generated by a variety of methods known in the art. For example, the polypeptides of the present invention may be added to a variety of host animals, such as, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen . Depending on the host species, various adjuvants may be used to increase the immunological response. Examples of such adjuvants include, but are not limited to, fractions (complete adjuvant and incomplete adjuvant), mineral gel (eg, aluminum nitrate) Such as BCG (bacille Calmette-Guerin) and Corynebacterium paratum, such as lycopene, lycopene, lycoricin, lycoriclitin, pluronic polyols, polyvalent anions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol and potentially useful human adjuvants Corynebacterium parvum ). ≪ / RTI > Such adjuvants are also well known in the art.
[341] Monoclonal antibodies can be prepared using a variety of techniques known in the art, examples of which include hybridomas, recombinant and phage display techniques, or combinations thereof. For example, monoclonal antibodies can be generated using hybridoma techniques, which techniques are well known in the art and are described, for example, in Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., In: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMA 563-681 (Elsevier, N. Y.1981), all of which are incorporated herein by reference in its entirety. The term " monoclonal antibody ", as used herein, is not limited to antibodies generated through hybridoma techniques. The term " monoclonal antibody " refers to an antibody derived from a monoclonal comprising an eukaryotic clone, a prokaryotic clone or a phage clone, and does not mean a method of producing a monoclonal antibody.
[342] Methods for generating and screening specific antibodies using hybridoma techniques are common, are well known in the art, and are discussed in detail in the Examples. In a non-limiting example, a mouse can be immunized with a peptide of the invention or a cell encoding the peptide. When a general immune response is detected, for example, an antigen-specific antibody is detected in mouse serum, collecting mouse spleen and isolating splenocytes. The splenocytes are then fused to cells derived from any suitable melanoma cell, for example the cell line SP20, which is readily obtainable in the ATCC, by well known techniques. Hybridomas were selected and cloned by limiting dilution. The hybridoma clones were then analyzed for the cells that secrete antibodies capable of binding the antibodies of the present invention by methods known in the art. Generally, ascites containing high levels of antibody can be generated by immunizing mice with a positive hybridoma clone.
[343] Accordingly, the present invention provides a method of producing a monoclonal antibody and an antibody produced by the method, wherein the method comprises culturing a hybridoma cell secreting an antibody of the invention, Chopping board harves a spleen cell isolated from a mouse immunized with an antigen of the present invention with a melanoma cell) and then hybridizes to a hybridoma clone that secretes an antibody capable of binding the polypeptide of the present invention Screening the breeder.
[344] Antibody fragments that recognize a particular epitope can be generated by known techniques. For example, the Fab and F (ab ') 2 fragments of the invention can be prepared by using an enzyme such as papain (to produce Fab fragment) or pepsin (to produce F (ab') fragment) It can be created by cutting into pirates. The F (ab ') 2 fragment contains a variable region, a light chain constant region and a heavy chain CH1 domain.
[345] For example, the antibodies of the present invention can be generated using various phage display methods known in the art. In a phage display method, functional antibody domains are displayed on the surface of phage particles bearing a polynucleotide sequence that encodes them. In certain embodiments, such phages can be used to display antigen binding domains expressed from a repertoire or a combinatorial antibody library (e. G., Human or murine animal). A phage that expresses an antigen binding domain that binds an antigen of interest can be selected or identified using an antigen, e. G., A labeled antigen or solid surface, or an antigen bound or captured in a bead. The phage used in these methods is a typical pseudo phage comprising the fd and M13 binding domains expressed from phages harboring Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to phage gene III or gene VIII protein. Examples of phage display methods that can be used to prepare antibodies of the present invention are described in Brinkman et al., J. Immunol. Methods 182: 41-50 (1995); Ames et al., J. Immunol. Methods 184: 177-186 (1995); Kettleborough et al., Eur. J. Immunolo. 24: 952-958 (1994); Persic et al., Gene 187: 9-18 (1997); Burton et al., Advances in Immunology 57: 191-280 (1994); PCT / GB91 / 01134; WO 90/02890; WO91 / 10737; WO92 / 01047; WO 92/18619; WO93 / 11236; WO95 / 15982; WO95 / 20401; And US Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108 .
[346] After the selection of the phage, the antibody coding region derived from the phage is separated and used as described in the above document, and the preferred and preferred preferred embodiments thereof, including mammalian cells, insect cells, plant cells, yeast and bacteria, A whole antibody comprising a human antibody or any other desired antigen-binding fragment expressed in a host can be generated. For example, techniques for recombinantly producing Fab, Fab 'and F (ab') 2 fragments can be found in PCT International Publication No. WO 92/22324, which is incorporated herein by reference in its entirety. Mullinax et al., BioTechniques 12 (6): 864-869 (1992); And Sawai et al., AJRI 34: 26-34 (1995); And Better et al., Science 240: 1041-1043 (1988)).
[347] Examples of techniques that can be used to generate single chain Fvs and antibodies are described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203: 46-88 (1991); Shu et al., PNAS 90: 7995-7999 (1993); And Skerra et al., Science 240: 1038-1040 (1988). In some cases, including in vivo use of the antibody in humans and in vitro detection assay, it may be desirable to use a chimeric, humanized or human antibody. A chimeric antibody refers to an antibody having a variable region derived from a different species of animal, for example, a murine monoclonal antibody, and a human immunoglobulin constant region, wherein the different portions of the antibody are different. Methods of generating chimeric antibodies Are known in the art (see, e.g., Morrison, Science 229: 1202 (1985); Oi et al., BioTechniques 4: 214 (1986); Gillies et al., J. Immunol. Methods 125: 191-202 (1989); U.S. Patent No. 5,807,715; 4,816, 567; And 4,816,397; Quot; incorporated herein by reference in its entirety]. Humanized antibodies are antibody molecules derived from non-human antibodies that bind to one or more complementarity determining regions (CDRs) derived from a non-human species and certain antigens carrying framework regions derived from human immunoglobulin molecules. Often, framework residues within the human framework region will be replaced with corresponding residues from the CDR donor antibody to alter antigen binding, preferably to improve it. These framework substitutions may be made by methods known in the art, for example, modeling the interactions of CDRs and framework residues to identify important framework residues for antigen binding and sequence comparisons to identify unusual framework residues at specific sites (See, for example, Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332: 323 (1988); Quot; incorporated herein by reference in its entirety]. Antibodies can be produced by a variety of techniques known in the art, such as CDR-grafting (see EP 239,400; PCT International Publication No. 91/09967; US 5,225,539; 5,530,101; and 5,585,089) Veneering and re-surface treatment (see EP 592,106; EP 519,596; Padlan, Molecular Immunology 28 (4/5): 489-498 (1991); Studnicka et al., Protein Engineering 7 (6): 805-814 (1994); Roguska et al., PNAS 91: 969-973 (1994)) and chain shuffling (US Pat. No. 5,565,332).
[348] Fully human antibodies are particularly preferred for therapeutic treatment of humans. Human antibodies can be prepared by a variety of methods known in the art including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences (see, for example, U.S. Patent Nos. 4,444,887 and 4,716,111 number; And PCT International Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16645, WO 96/34096, WO 96/33735 and WO 91/10741; All of which are incorporated herein by reference in their entirety].
[349] In addition, a human antibody can not express a functional endogenous immunoglobulin, but can be produced using a transgenic mouse capable of expressing an interspecific immunoglobulin gene. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells by random or heterologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by heterologous recombination. Specifically, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells expand and are microinjected into blastosites to produce chimeric mice. The chimeric mice are then raised to produce homozygous progeny expressing human antibodies. Transgenic mice are immunized in the usual manner with the selected antigen, e. G., All or a portion of the polypeptide of the invention. The monoclonal antibodies detected for the antigens can be obtained from immunized transgenic mice using conventional hybridoma techniques. The human immunoglobulin transgene is fixed by transgenic mouse rearrangement during B cell differentiation and subsequently undergoes class switching and somatic mutation. Thus, using this technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. An overview of this technique for generating human antibodies can be found in Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995). A detailed description of this technique for generating human antibodies and human monoclonal antibodies and the protocols for generating such antibodies is described in PCT International Publication No. WO 98/24893, which is incorporated herein by reference in its entirety. WO 92/01047; WO 96/34096; WO 96/33735; European Patent 0 598 877; U.S. Patent No. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661, 016; 5,545,806; 5,814,318; 5,885, 793; 5,916,771; And 5,939,598. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Zenpam (San Jose, CA) may be involved in providing human antibodies directed against selected antigens using methods similar to those described above.
[350] A complete human antibody recognizing a selected epitope can be generated using a technique called " guided selection ". In this method, selected non-human monoclonal antibodies, e. G. Mouse antibodies, can be used to induce selection of complete human antibodies that recognize the same epitope (Jespers et al., Bio / technology 12: 899-903 )].
[351] In addition, antibodies against the polypeptides of the invention can be used to generate anti-genetic antibodies that "prone" the polypeptides of the invention using techniques well known in the art (see, eg, Greenspan & Bona, FASEB J. 7 (5): 437-444 (1989); And Nissinoff, J. Immunol. 147 (8): 2429-2438 (1991)]. For example, antibodies that competitively inhibit polypeptide dimerization and / or binding of a polypeptide of the invention to a ligand in combination may be used to generate a " tandem " antagonist of a polypeptide multimerization and / or binding domain , Which consequently binds to neutralize the polypeptide and / or its ligand. Such neutralizing antigens or Fab fragments of such antigens can be used in therapeutic methods to neutralize the polypeptide ligands. For example, such an anti-genetic antibody may block its biological activity by binding the polypeptide of the invention and / or using it to bind its ligand / receptor.
[352] The invention also relates to an antibody that acts as an agonist or antagonist of the polypeptide of the invention. Examples of antibodies that act as agonists or antagonists of the polypeptides of the present invention include antibodies that partially or completely destroy receptor / ligand interactions with the polypeptides of the present invention. For example, the invention encompasses antibodies that disrupt the ability of the protein of the invention to be massimulated. In another example, the invention includes antibodies that disrupt the ability of the proteins of the invention to bind to one or more KGF-2 receptor (s) / ligand (s) of the invention, while allowing the proteins of the invention to become massimetric. In another example, the present invention provides a method of making a protein of the present invention capable of being mass-polymerized and which binds the KGF-2 receptor (s) / ligand (s), but blocks the biological activity associated with the KGF-2 / receptor / ligand complex Lt; / RTI >
[353] In addition, antibodies acting as agonists or antagonists of the polypeptides of the invention include both receptor-specific antibodies and ligand-specific antibodies. Receptor-specific antibodies that do not interfere with ligand binding but interfere with receptor activation. Receptor activation (i. E., Signal transduction) can be measured using techniques described herein or other techniques known in the art. Also included are receptor-specific antibodies that interfere with both ligand binding and receptor binding. Similarly, it includes neutralizing antibodies that bind the ligand, interfere with the binding of the ligand to the receptor, and antibodies that interfere with receptor activation by binding the ligand, but do not prevent the ligand from binding the receptor. Also included are antibodies that activate the receptor. These antibodies may act as agonists for all or part of the biological activity that is affected by the ligand-mediated receptor activation. The antibody may be characterized as an agonist or antagonist of biological activity, including the specific activity disclosed herein. Such antibody actives can be prepared using methods known in the art (see, for example, WO 96/40281; U.S. Patent No. 5,811,097; Deng, B, et al., Blood 92 (6): 1981-1988 (1998); Chen, Z. et al., Cancer Res. 58 (16): 3668-3678 (1998); Harrop, J.A. Et al., J. Immunol. 161 (4): 1786-1794 (1998); Zhu, Z. et al., Cancer Res. 58 (15): 3209-3214 (1998); Yoon, D.Y. Et al., J. Immunol. 160 (7): 3170-3179 (1998); Prat, M. et al., J. Cell. Sci. 111 (Pt 2): 237-247 (1998); Pitard, V. et al., J. Immunol. Methods 205 (2): 177-190 (1997); Liautard, J et al., Cytokinde 9 (4): 233-241 (1997); Carlson, N.G. Et al., J. Biol. Chem. 272 (17): 11295-11301 (1997); Taryman, R.E. Neuron 14 (4): 755-762 (1995); Muller, Y.A. , Structure 6 (9): 1153-1167 (1998); Bartunek, P. et al., Cytokine 8 (1): 14-20 (1996); Quot; incorporated herein by reference in its entirety].
[354] As noted above, antibodies to the KGF-2 protein of the invention can be used to generate anti-genetic antibodies that "prone" KGF-2 using techniques well known to those skilled in the art For example, Greenspan & Bona, FASEB J. 7 (5): 437-444 (1989); And Nissinoff, J. Immunol. 147 (8): 2429-2438 (1991)]. For example, an antibody that binds to KGF-2 and that completely inhibits KGF-2 multimerization and / or binding to the ligand is used to " consume " the KGF-2 multimerization and / -2 < / RTI > and / or its ligands to neutralize it. Such neutralizing anti-genetic factors or Fab fragments of such anti-genetic factors can be used in therapy for neutralizing KGF-2 ligand. For example, KGF-2 biological activity can be blocked by binding KGF-2 using such an anti-genetic antibody or by binding KGF-2 ligand / receptor.
[355] Polynucleotides encoding antibodies
[356] The present invention further provides a nucleotide sequence encoding the antibody of the invention and fragments thereof. The present invention also encompasses antibodies that specifically bind to the polypeptides of the invention, preferably amino acid sequences of SEQ ID NO: 2, as described above, under stringent or less stringent hybridization conditions, Lt; RTI ID = 0.0 > polynucleotides < / RTI >
[357] Any method known in the art can be used to obtain a polynucleotide, and the nucleotide sequence of the polynucleotide can be determined. For example, where the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (see, for example, Kutmeier et al., BioTechniques 17: 242 (1994) , Roughly, the method involves synthesis of overlapping oligonucleotides containing a portion of the sequence encoding the antibody, annealing and linking of these oligonucleotides and amplification of the linked oligonucleotides by PCR.
[358] Alternatively, the polynucleotide encoding the antibody may be generated from a nucleic acid derived from a suitable source. If the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin can be obtained by using a synthetic primer capable of hybridizing to the 3 ' and 5 ' ends of the sequence PCR amplification or chemical synthesis, for example, by cloning using an oligonucleotide probe specific for a particular gene sequence to identify a cDNA clone derived from a cDNA library encoding the antibody, or by using a suitable source (for example, an antibody cDNA Library or any tissue or cell expressing the antibody, such as a nucleic acid isolated from a hybridoma cell selected to express the antibody of the invention, or a cDNA library derived therefrom). The amplified nucleic acid generated by PCR can then be cloned into a cloning vector that can be cloned using any method known to those skilled in the art.
[359] Once the nucleotide sequence and the corresponding amino acid sequence of the antibody have been determined, the nucleotide sequence of the antibody may be determined by methods well known in the art, for example, recombinant DNA techniques, locally designated mutagenesis methods, PCR (See, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual (Second Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel; For example, to produce amino acid substitutions, deletions, and / or insertions, by manipulating the amino acid sequence of SEQ ID NO: 1, which is herein incorporated by reference in its entirety.
[360] In certain embodiments, the amino acid sequences of the heavy and / or light chain variable domains are determined by methods known in the art, for example, by comparison with known amino acid sequences of other heavy and light chain variable regions to determine the hypervariable region And the sequence of the complementarity determining region (CDR) can be identified. Using conventional recombinant DNA techniques, one or more CDRs can be inserted into a framework region, e. G., A human framework region, to humanize the non-human antibody, as described above. The framework region may be a naturally occurring or common framework region, and preferably a human framework region (see, for example, Chothia et al., J. Mol. Biol. 278: 457-479 (1998); List of human framework areas]. Preferably, the polynucleotide generated by the combination of the framework region and the CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as described above, one or more amino acid substitutions can be made within the framework region, and amino acid substitutions preferably improve binding of the antibody to its antigen. In addition, amino acid substitutions or deletions of one or more variable region cysteines participating in intramolecular disulfide bonds can be performed using this method to produce antibody molecules lacking one or more intramolecular disulfide bonds. Other modifications to the polynucleotides are within the purview of those skilled in the art and are included in the present invention.
[361] Also, techniques developed to generate " chimeric antibodies " by ligating genes derived from human antibody molecules with suitable biological activity with genes from mouse antibody molecules with appropriate antigen specificity (Morrison et al., Proc. Natl. Acad. Sci. 81: 851-855 (1984); Neuberger et al., Nature 312: 604-608 (1984); Takeda et al., Nature 314: 452-454 (1985)]. As noted above, chimeric antibodies are molecules, such as humanized antibodies, that retain a variable region and a human immunoglobulin constant region derived from a molecule derived from a different animal species, such as a murine animal mAb, at different portions.
[362] Alternatively, techniques described for generating single-chain antibodies (see U.S. Patents 4,946,778; Bird, Science 242: 423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); And Ward et al., Nature 334: 544-54 (1989)] can be applied to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region by an amino acid bridge, resulting in a single chain polypeptide. Also, this. Techniques for assembly of functional Fv fragments in collages are also available (Skerra et al., Science 242: 1038-1041 (1988)).
[363] How to generate antibodies
[364] The antibodies of the present invention can be produced by any method known in the art for antibody production, specifically by chemical synthesis, preferably by recombinant expression techniques.
[365] Recombinant expression of an antibody of the invention or a fragment, derivative or analogue thereof (e.g., a heavy or light chain of an antibody of the invention, or a single chain of an antibody of the invention) may be achieved by using an expression vector containing a polynucleotide encoding the antibody . Once a heavy or light chain, or a portion thereof (preferably containing a heavy chain or light chain variable region) of the antibody molecule or antibody of the invention is obtained, the vector for generating the antibody molecule can be constructed using techniques well known in the art Can be generated by recombinant DNA techniques that utilize them. Thus, a method for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody is described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. Examples of these methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo gene recombination techniques. Thus, the present invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain, or heavy or light chain variable region thereof, operably linked to a promoter. Such a vector may comprise a nucleotide sequence encoding the constant region of the antibody molecule (see, for example, PCT International Publication No. WO 86/05807; WO 89/01036; And U.S. Patent No. 5,122,464), the variable region of the antibody can be cloned into such a vector for expression of the entire heavy or light chain.
[366] The expression vector is transferred into a host cell in a conventional manner, and the transfected cells are then cultured by conventional techniques to produce an antibody of the present invention. Accordingly, the present invention includes a host cell that encodes an antibody of the invention, a heavy or light chain thereof, or a single chain antibody of the invention, and which comprises a polynucleotide operably linked to a heterologous promoter. In a preferred embodiment for expression of a double-chain antibody, the vector encoding both the heavy chain and the light chain can be expressed simultaneously in a host for expression of the whole immunoglobulin molecule, as described below.
[367] A variety of host-expression vector systems can be used to express the antibody molecule of the invention. Such a host-expression system means a vehicle in which the coding sequence of interest is generated and can be subsequently purified, but also expresses the antibody molecule of the invention in situ when transfected or transfected with a suitable nucleotide coding sequence It also means cells that can be made. Examples of these include microorganisms, for example, bacteria that have been transfected with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences (e.g., E. coli, B. subtilis); Yeast transformed with a recombinant yeast expression vector containing an antibody coding sequence (e.g., Saccharomyces Pichia ); An insect cell system infected with a recombinant virus expression vector (e.g., baculovirus) containing an antibody coding sequence; Transformation with a recombinant plasmid expression vector (e.g., a Ti plasmid) infected with a recombinant virus expression vector (e.g., Cauliflower Mosaic Virus (CaMV), tobacco mosaic virus (TMV)) containing an antibody coding sequence or containing an antibody coding sequence Plant cell system; Or a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., late adenovirus promoter; vaccinia virus 7.5K promoter) But are not limited to, mammalian cell systems (e. G., COS, CHO, BHK, 293, 3T3 cells). Preferably, bacterial cells such as Escherichia coli and more preferably eukaryotic cells, particularly eukaryotic cells for expression of precursor recombinant antibody molecules, are used for the expression of recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary cells (CHO) are effective expression systems for antibodies with a vector, such as a major stem promoter element derived from human cytomegalovirus (see Foecking et al., Gene 45 : 101 (1986); Cockett et al., Bio / Technology 8: 2 (1990)].
[368] In bacterial systems, multiple expression vectors may be advantageously selected for the intended use for the expressed antibody molecule. For example, if a large amount of such a protein is to be produced, in the production of a pharmaceutical composition consisting of an antibody molecule, a vector which induces expression of a high-level fusion protein product that is easily purified may be preferred. These vectors include: The coli expression vector pUR278 (Ruther et al., EMBO J. 2: 1791 (1983)), in which the antibody coding sequences are individually ligated into a vector in the frame carrying the lacZ coding sequence to generate a fusion protein; pIN vector (see Inouye & Inouye, Nucleic Acids Res. 13: 3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem. 24: 5503-5509 (1989)], but are not limited thereto. In addition, the pGEX vector can be used to express an exogenous polypeptide as a fusion protein with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can be readily purified from the lysed cells by adsorption and elution in the presence of free glutathione after binding to the matrix glutathione-agarose beads. The pGEX vector may be constructed to include a thrombin or Xa factor protease cleavage position so that the cloned target gene product is free from the GST portion.
[369] In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences are individually cloned into non-essential regions of the virus (e. G., The polyhedrin gene) and located under the control of an AcNPV promoter (e. G., A polyhedrin promoter).
[370] In mammalian host cells, a number of viral expression systems can be used. When using adenovirus as an expression vector, the antibody coding sequence of interest may be linked to adenovirus transcription / translation regulatory complexes, such as late promoter and tripartite leader sequences. This chimeric gene can then be inserted into the adenoviral genome by in vitro or in vivo recombination. Insertion in non-essential regions of the viral genome (e. G., Regions E1 or E3) will survive in the infected host and will produce recombinant viruses capable of expressing antibody molecules (see, e. G., Logan &Amp; Shenk, Proc. Natl. Acad. Sci. USA 81: 355-359 (1984)]. In addition, a specific initiation signal may be required for efficient translation of the inserted antibody-encoding sequence. These sequences include the ATG start codon and adjacent sequences. Also, the initiation codon must be present in the phase with the leading frame of the desired cryptographic sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons may be of various origins, natural or synthetic. Expression efficiency can be enhanced by including appropriate transcription enhancing elements, transcription terminators, etc. (Bittner et al., Methods in Enzymol. 153: 51-544 (1987)).
[371] In addition, a host cell strain is selected that modulates the expression of the inserted sequence, or alters and processes the desired particular abbreviated gene product. Such modifications (e. G., Glycosylation) and processing (e. G., Cleavage) of the protein product may be important for protein fusion. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. A suitable cell line or host system can be selected to ensure correct modification and processing of the expressed foreign protein. For this purpose, eukaryotic host cells may be used which have suitable processing of the primary transcript, glycosylation of the gene product and cellular devices for phosphorylation. Examples of such mammalian host cells are CHO, VERA, BHK, Hela, COS, MDCK, 293, 3T3, WI38 and specifically breast cancer cell lines such as BT483, Hs578T, HTB2, BT20 and T47D, For example, CRL7030 and Hs578Bst.
[372] In order to produce a recombinant protein with high yield over a long period of time, stable expression is preferable. For example, a cell line that stably expresses an antibody molecule can be manipulated. Rather than using an expression vector containing a viral replication origin, the host cell can be transformed with DNA regulated by appropriate expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.) And may have a selectivity marker. After introduction of the foreign DNA, the engineered cells are grown for 1-2 days in an enriched medium and then switched to a selective medium. Selective markers in recombinant plasmids provide resistance to selection, allowing cells to stably integrate plasmids into their chromosomes, grow and clone and form points that can be expanded into cell lines. This method is advantageously used to manipulate cell lines expressing the antibody molecule. Such engineered cell lines are particularly useful for screening and evaluation of compounds that directly or indirectly interact with the antibody molecules.
[373] A number of selection systems are available, examples of which include herpes simplex virus thymidine kinase (Wigler et al., Cell 11: 223 (1997); Hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48: 202 (1992)] and adenine phosphoribosyltransferase [Lowy et al., Cell 22: 817 (1980)] genes. Or aprt-cells. In addition, antimetabolite resistance can be used as a basis for selection for the following genes: dhfr conferring resistance to methotrexate (Wiegler et al., Natl. Acad. Sci. USA 77: 357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78: 1527 (1981); Gpt which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (1981); Neo which confers resistance to aminoglycoside G-418 (Goldspiel et al., Clinical Pharmacy 12: 488-505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, Ann. Res. Pharmacol. Toxicol. 32: 573-596 (1993); Mulligan, Science 260: 926-932 (1993); And Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); TIB TECH 11 (5): 155-215 (1993.5)]; Hygro (Santerre et al., Gene 30: 147 (1984)) which confers resistance to hygromycin. Recombinant DNA techniques well known in the art can be generally applied to select the desired recombinant clones, including those described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons , NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, New York Stockton Publishers (1990); And Dracopoli et al. (Eds.), Current Protocols in Human Genetics Ch. 12 & 13, John Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol. 150: 1 (1981); All incorporated herein by reference.
[374] The level of expression of the antibody molecule can be increased by vector amplification (Bebbington and Hentschel, The use of vectors based on gene expression for cloned genes in mammalian cells in DNA cloning, Vol. 3, New York Academy Press (1987)]. If the marker in the vector system expressing the antibody is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is bound to the antibody gene, the production of the antibody will also be increased (Crouse et al., Mol. Cell. Biol. 3: 257 (1983)].
[375] Host cells can be co-transfected using the two expression vectors of the invention, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. The two vectors may contain the same selectable marker to enable the same expression of the heavy chain and light chain polypeptides. Alternatively, a single vector capable of encoding and expressing both heavy and light chain polypeptides may be used. In this situation, the light chain must be located before the heavy chain to avoid excess toxic glass heavy chain (Proudfoot, Nature 322: 52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77: 2197 (1980)]. The coding sequence for the heavy and light chains may comprise cDNA or genomic DNA.
[376] Once an antibody molecule of the invention is produced by an animal, chemically synthesized, or recombinantly expressed, any method known in the art for purification of immunoglobulin molecules, such as chromatography (e. , Ion exchange chromatography, affinity chromatography, in particular affinity chromatography, size column chromatography on specific antigens after protein A), centrifugation, differential dissolution, or any other standard technique for protein purification . In addition, the antibodies or fragments thereof of the present invention may be fused to heterologous polypeptide sequences disclosed herein or known in the art to facilitate purification.
[377] The present invention relates to a polypeptide of the present invention (or a portion thereof, preferably a polypeptide (or a portion thereof, preferably a polypeptide having at least 10, at least 20, at least 30, at least 40, At least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids). The fusion does not necessarily occur directly, , Or through linker sequences. The antibody may be a polypeptide of the invention (or a portion thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60 of the polypeptides , Greater than 70, greater than 80, greater than 90, or greater than 100 amino acids.) For example, an antibody may be specific for an individual cell surface in vitro or in vivo Antibodies fused or conjugated to the polypeptides of the present invention can be used to target antibodies of the present invention to a particular cell type by fusing or conjugating the polypeptides of the present invention to antibodies specific for a sieve. (See, e.g., Harbor et al., Supra and PCT International Publication No. WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39: 91- Gillies et al., PNAS 89: 1428-1432 (1992); Fell et al., J. Immunol. 146: 2446-2452 (1991);
[378] The invention further comprises a composition comprising a polypeptide of the invention fused or conjugated to an antibody domain other than a variable region. For example, a polypeptide of the invention can be fused or conjugated to an antibody Fc region or a portion thereof. The antibody portion fused to a polypeptide of the invention may comprise a constant region, a hinge region, a CH1 domain, a CH2 domain and a CH3 domain or an entire domain or any combination thereof. In addition, the polypeptide may be fused or conjugated to the antibody portion to form a multimer. For example, an Fc portion fused to a polypeptide of the invention can form a dimer through a disulfide bond between the Fc portions. Higher multimeric forms can be produced by fusing the above polypeptides to portions of IgA and IgM. Methods of fusing or conjugating the polypeptides of the present invention to the antibody portion are known in the art (see, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT International Publication No. WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al., J. Immunol. 154: 5590-5600 (1995); And Vil et al., Proc. Natl. Sci. USA 89: 11337-11341 (1992); Quot; incorporated herein by reference in its entirety].
[379] As described above, the polypeptide corresponding to the polypeptide, the polypeptide fragment, or the variant of SEQ ID NO: 2 may be fused or conjugated to the antibody portion to increase the in vivo half-life of the polypeptide, or to an immunoassay using methods known in the art Can be used. In addition, the polypeptide corresponding to SEQ ID NO: 2 may be fused or conjugated to the antibody portion to facilitate purification. One reported example describes a chimeric protein consisting of the first two domains of a human CD4-polypeptide and the various domains of the heavy or light chain constant region of a mammalian immunoglobulin (see EP 394,827; Traunecker et al., Nature 331: 84-86 (1988)]. In addition, polypeptides of the present invention that are fused or conjugated to an antibody having a disulfide-linked dimer structure may be more efficient in binding and neutralizing other molecules than monomelic secretory proteins or protein fragments alone (Fountoulakis et al. J. Biochem. 270: 3958-3964 (1995)). In many cases, the Fc portion of the fusion protein is amenable to therapy and diagnosis, and thus can exhibit, for example, improved pharmacokinetic properties (EP A 232,262). Alternatively, it is preferred that the fusion protein be expressed, detected, purified and then the Fc part deleted. For example, the Fc portion may interfere with therapy and diagnosis when the fusion protein is used as an antigen for immunization. For example, in drug development, human proteins such as the hIL-5 receptor are fused with the Fc portion for high-throughput screening assays to identify hIL-5 antagonists (Bennett et al., J. Molecular Recognition 8: 52- 58 (1995); Johanson et al., J. Biol. Chem. 270: 9459-7471 (1995)).
[380] In addition, the antibody or fragment thereof of the invention can be fused to a marker sequence, such as, for example, a peptide that facilitates purification. In a preferred embodiment, the marker amino acid sequence is a hexa-histidine peptide, for example, a tag provided in a pQE vector of a number of commercially available (Qiagenen, 9259, 9959, Catsworthington Avenue, CA, USA). See, e.g., Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824 (1989), hexa-histidine is provided for easy purification of the fusion protein. Examples of other peptide tags useful for purification include the " HA " tag (Wilson et al., Cell 37: 767 (1984)) and the " flag " tag, which correspond to epitopes derived from the influenza hemagglutinin protein , But is not limited thereto.
[381] The invention encompasses antibodies or fragments thereof conjugated to a diagnostic agent or therapeutic agent. The antibody can be used, for example, to diagnose, for example, to monitor the development or progression of a tumor as part of a clinical testing procedure to determine the efficacy of a given therapy. Detection may be performed by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, complementary molecular groups, fluorescent substances, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography, and non-radioactive paramagnetic metal ions. The detectable substance can be coupled or conjugated directly to the antibody (or fragment thereof), or indirectly coupled or conjugated via an intermediate (e. G., A linker known in the art) using techniques known in the art . See U.S. Patent No. 4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents in accordance with the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; Examples of suitable complementary molecular homologues include streptavidin / biotin and avidin / biotin; Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorocaine isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol ; Examples of bioluminescent materials include luciferase, luciferin and aequorin; Examples of suitable radioactive materials include ¹²⁵ I, ¹³¹ I, ¹¹¹ In or ⁹⁹ Tc.
[382] The antibody or fragment thereof may also be conjugated to a therapeutic agent such as a cytotoxin, for example, a cell division inhibitor or cytotoxic agent, a therapeutic agent or a radioactive metal ion such as an alpha-releasing agent (e.g., ²¹³ Bi). Cytotoxins or cytotoxic agents include any agent that has a deleterious effect on the cell. Examples thereof include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenofoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, di Dihydroestosterone, glucocorticoid, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologues thereof, may be used in combination with any of the above- . Examples of therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e. G., Mechlorethamine, (II) (")< / RTI > (II) < RTI ID = 0.0 > (II) < / RTI & DDP) cisplatin), anthracyclines (e.g., daunorubicin (former: daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (former actinomycin), bleomycin, (AMC), and anti-mitotic agents (e.g., vincristine and vinblastine).
[383] The conjugates of the invention can be used to modify a given biological response, and the therapeutic or drug moiety is not understood to be limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide having a predetermined biological activity. Such proteins include, for example, toxins (e.g., avrins, lysine A, Pseudomonas exotoxin, or diphtheria toxin); TNF-a, TNF-beta, AIM I (e. G., Tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, (International Publication No. WO 97/33899), AIM II (International Publication WO 97/34911), Fas ligand (Takashima et al., Int. Immunol. 6: 1567-1574 (1994)], VEGI Interleukin-1 (" IL-1 ") such as angiotensin or endostatin; or a biological response modifier such as, for example, ("IL-2"), interleukin-6 ("IL-6"), granulocyte colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor (" , Or other growth factors.
[384] Antibodies can also be attached to solid supports, which are particularly useful for immunoassay or purification of target antigens. Examples of such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
[385] Techniques for conjugating such therapeutic moieties to antibodies are known (Arnon et al., &Quot; Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy ", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds), pp. Alan R. Liss, Inc. 1985); Hellstrom et al., &Quot; Antibodies For Drug Delivery ", in Controlled Drug Delivery (2nd ed), Robinson et al. (Eds), pp. 633-53 (Marcel Dekker, Inc. 1987); Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review ", Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (Eds), pp. 475-506 (1985); Baldwin et al. (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al., &Quot; Analysis, Results, and Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy ", in Monoclonal Antibodies For Cancer Detection And Therapy , &Quot; The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates ", Immunol. Rev. 62: 119-58 (1982)].
[386] Alternatively, the antibody may be linked to a second antibody to form an antibody heterozygote, as described by Segal in U.S. Pat. No. 4,676,980 (herein incorporated by reference in its entirety).
[387] The antibody may be administered alone or in combination with a cytotoxic agent and / or cytokine, with or without a therapeutic moiety attached thereto.
[388] Determination of immune phenotype
[389] The antibody of the present invention may be used for determining the immunophenotype of a cell line and a biological sample. The translation product of the gene of the invention may be useful as a cell-specific marker, or more specifically as a cell marker that is differentially expressed at various stages of differentiation and / or maturation of a particular cell type. A monoclonal antibody raised against a particular epitope, or combination of epitopes, will enable the detection of a population of cells expressing the marker. A variety of techniques using monoclonal antibodies can be used to search for cell populations expressing the markers, including magnet separation using antibody-coated magnetic beads, " panning " by antibodies attached to solid matrices panning " and flow cytometry (see U.S. Patent Nos. 5,985,660; And Morrison et al., Cell 96: 737-49 (1999)).
[390] These techniques include specific cell populations such as those that may be found in hematologic tumors (e.g., minimal residual disease (MRD) in patients with acute leukemia) and " non-magnetic " "Allows screening of cells. On the other hand, these techniques allow the screening of hematopoietic stem cells and progenitor cells that can undergo proliferation and / or differentiation, such as can be found in human umbilical cord blood.
[391] Analysis of antibody binding
[392] The antibodies of the present invention may be analyzed for immunospecific binding by any known method. Immunoassays that may be used include, but are not limited to, Western blot, radioimmunoassay, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassay, immunoprecipitation assay, sedimentation reaction, gel diffusion settling reaction, But are not limited to, competitive and non-competitive assays using techniques such as complement-fixation, immunoradiometric assays, fluorescence immunoassays, and protein A immunoassays. Such assays are routine and well known in the art (see, Ausubel et al., Eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, incorporated herein by reference). Hereinafter, an exemplary immunoassay is briefly described (immunoassay is not limited to being described).
[393] Immunoprecipitation protocols generally involve contacting the cell population with RIPA buffer (1% NP-40 or Triton X-100, 1% sodium < RTI ID = 0.0 > (0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate (pH 7.2), 1% Trasylol), the antibody of interest is added to the cell lysate and incubated at 4 ° C for a period of time , 1-4 hours), protein A and / or protein G sepharose beads are added to the cell lysate, incubated at 4 ° C for about 1 hour or longer, the beads in the lysis buffer are washed, SDS / sample buffer. The ability of an antibody of interest to immunoprecipitate a particular antigen can be assessed, for example, by Western blot analysis. Those skilled in the art will know about variables that can be modified to increase antibody binding to antigen and reduce background (e. G., Pre-wash cell lysate with sepharose beads). See Ausubel et al., Eds., 1998, Current Protocols in Molecular Biology, 10.16.1 of John Wiley & Sons, NY for further explanation of immunoprecipitation protocols.
[394] Western blot analysis generally involves the preparation of protein samples, electrophoresis of protein samples in polyacrylamide gels (eg, 8% -20% SDS-PAGE depending on the molecular weight of the antigen), nitrocellulose, PVDF, Or membranes such as nylon, blocking membranes in blocking solutions (eg, PBS or skimmed milk with 3% BSA), washing membranes in washing buffer (eg, PBS-Tween 20), diluting the primary antibody Blocking of the membrane using an antibody of interest), washing of the membrane in the wash buffer, coupling to an enzymatic substrate (horseradish peroxidase or alkaline phosphatase) or radioactive molecule (such as ³² P or ¹²⁵ I) Blocking membranes with secondary antibodies (recognizing primary antibodies, anti-human antibodies), washing membranes in washing buffer, and detecting the presence of antigens. Those skilled in the art will know about variables that can be modified to increase the detected signal and reduce background noise. See Ausubel et al., Eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY 10.8.1 for a further description of western blotting.
[395] ELISA can be used for antigen production, addition of the antibody of interest linked to a detectable compound such as an enzymatic substrate (horseradish peroxidase or alkaline phosphatase) to a well of a 96-well microtiter plate well, , And detection of antigen presence. In ELISA, the antibody of interest need not be linked to a detectable compound. Instead, a second antibody (which recognizes an antibody of interest) linked to a detectable compound may be added to the well. Moreover, instead of coating the well with the antigen, the antibody may be coated onto the well. In this case, a second antibody linked to a detectable compound may be added after adding the antigen of interest to the coated well. Those skilled in the art will know other variables of the ELISA and variables that can be modified to increase the signal being detected. See Ausubel et al., Eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY 10.8.1 for further description of ELISA. The binding affinity of the antibody to the antigen and the separation rate of the antibody-antigen interaction can be determined by competitive binding assay. One example of a competitive binding assay is a radioimmunoassay that involves incubating the labeled antigen with the antibody of interest in the presence of an increased amount of non-labeled antigen and detecting the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding separation rate can be determined from the data by means of a Scatchard plot analysis. Competition with the second antibody may also be determined using radioimmunoassay. In this case, the antigen is incubated with the antibody of interest linked to the labeling compound (e.g., ³ H or ¹²⁵ I) in the presence of increasing amounts of the unlabeled second antibody.
[396] Vector and host cell
[397] The invention also relates to a vector comprising a separate DNA molecule of the invention, a host cell genetically engineered with said recombinant vector, and the production of a KGF-2 polypeptide or fragment thereof by recombinant techniques.
[398] Fragments or portions of the polypeptides of the invention may be used to generate corresponding full-length polypeptides by peptide synthesis; Thus, the fragment can be used as an intermediate for generating a full-length polypeptide. The full-length polynucleotide of the present invention can be synthesized using the polynucleotide fragment or portion of the present invention. The present invention also relates to a vector comprising a polynucleotide of the invention, a host cell genetically engineered with a vector of the invention, and a polypeptide of the invention by recombinant techniques.
[399] The host cell is genetically engineered (transduction or transformation or transfection) using a vector of the invention, which may be, for example, a cloning vector or an expression vector. The vector may be in the form of, for example, a plasmid, a viral particle, a phage, or the like. The engineered host cell may be cultured in a conventional nutrient medium suitably modified to activate the promoter, select the transformant, or amplify the KGF-2 gene. The culture conditions such as temperature, pH and the like can be the conditions used in culturing the host cells selected for expression, which are well known to those skilled in the art.
[400] Polypeptides of the present invention can be produced by recombinant techniques using the polynucleotides of the present invention. Thus, for example, the polynucleotide may be contained within any of a variety of expression vectors for expressing the polypeptide. Such vectors include chromosomal DNA sequences, non-chromosomal DNA sequences and synthetic DNA sequences, such as derivatives of SV40; Bacterial plasmids; Phage DNA; Baculovirus; Yeast plasmids; Vectors derived from plasmids and phage DNA, and viral DNAs (e.g., vaccinia, adenovirus, pharynx virus, and pseudorabies). However, any other vector that can replicate and survive within the host may be used.
[401] Suitable DNA sequences can be inserted into vectors in a variety of ways. Generally, the DNA sequence is inserted into a suitable restriction endonuclease site (s) by methods known in the art. These and other processes are considered to be within the scope of those skilled in the art.
[402] The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence (promoter) to induce cDNA synthesis. Representative examples of such promoters include the LTR or SV40 promoter, E. coli lac or trp, the phage lambda P _L promoter, and other promoters known to regulate the expression of genes in prokaryotic or eukaryotic cells or their viruses. In addition, the expression vector contains a ribosome binding site for translation initiation and transcriptional endurance. In addition, the vector may contain a suitable sequence to amplify the expression.
[403] It is also preferred that the expression vector contains one or more selectable marker genes to provide phenotypic characteristics for selection of transformed host cells, such as dihydrofolate reductase or neomycin for eukaryotic cell culture Tolerance, or this. Tetracycline or ampicillin resistance in the coli.
[404] A suitable host may be transformed with a suitable DNA sequence as described above and a vector containing the appropriate promoter or regulatory sequence to allow the host to express the protein.
[405] As indicated, the expression vector will preferably comprise at least one selectable marker. Such markers include neomycin resistance to dihydrofolate reductase, G418 or eukaryotic cell culture, ≪ / RTI > tetracycline, kanamycin or ampicillin resistance for cultivation in E. coli and other bacteria. Representative examples of suitable hosts include bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells; Fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC deposit no. 201178)); Insect cells such as Drosophila S2 and Spodoptera Sf9 cells; Animal cells such as CHO, COS, 293, and bovine melanoma cells; And plant cells. Suitable culture media and conditions for the host cells described above are known in the art.
[406] In addition to the use of expression vectors in the practice of the present invention, the present invention further comprises a novel expression vector comprising an operator and a promoter element operably linked to a nucleotide sequence encoding a protein of interest. An example of such a vector is pHE4-5 described below.
[407] As summarized in Figures 50 and 51, the component of the pHE4-5 vector (SEQ ID NO: 147) was 1) a neomycin phosphotransferase gene as a selectable marker, 2) 3) a T5 phage promoter sequence, 4) two lac promoter sequences, 5) a Shine-Dalgano sequence, and 6) a lactose operon inhibitor gene (lacIq). The origin of replication (oriC) is derived from pUC19 (LTI of Gettysburg, Maryland). Promoter sequences and operator sequences are prepared synthetically. Synthetic generation of nucleic acid sequences is well known in the art. Clone Tech 95/95 catalog 215-216, Clone Tech, Palo Alto East Meadow Circle 1020, California, USA 94303 California. The nucleotide sequence encoding KGF-2 (SEQ ID NO: 1) is operably linked to the promoter and operator by inserting the nucleotide sequence between the NdeI and Asp718 positions of the pHE4-5 vector.
[408] As described above, the pHE4-5 vector contains the lacIq gene. LacIq is an allele of the lacI gene that confers a clear control of the lac operator. Amann, E et al., Gene 69: 301-315 (1988); Stak, M., Gene 51: 255-267 (1987). The lacIq gene encodes a repressor gene that binds to the lac operator sequence and blocks electrons in the downstream (i. e., 3 ') sequence. However, the lacIq gene product is isolated from the lac operator in the presence of lactose or a specific lactose analog, i. E. Isopropyl B-D-thiogalactopyranoside (IPTG). METH1 or METH2 is therefore not produced in significant amounts in the untreated host cell containing the pHE4-5 vector. However, the induction of these host cells by the addition of agents such as IPTG results in the expression of the METH1 or METH2 coding sequence.
[409] The promoter / operator sequence of the pHE4-5 vector (SEQ ID NO: 148) contains the T5 phage promoter and 24 lac operator sequences. One operator is located 5 'of the transcription initiation site and the other operator is 3' of the same site. Such operators, when present in combination with the lacIq gene product, confer precise inhibition of downstream sequences in the absence of a lac operon inducing factor, such as IPTG. Expression of operatively linked sequences downstream from the lac operator may be induced by the addition of a lac operon inducer such as IPTG. When the lac inducible factor binds to the lacIq protein, they are released from the lac operator sequence and the operably linked sequence is transcribed. Lac operon regulation of gene expression is presented in Devlin, T., TEXTBOOK OF BIOCHEMISTRY WITH CLINICAL CORRELATIONS, Fourth Edition, pp. 802-807 (1997).
[410] A series of pHE4 vectors contain all of the components of the pHE4-5 vector except for the METH1 or METH2 coding sequence. The characteristics of the pHE4 vector include optimized synthetic T5 phage promoter, lac promoter and Shine-Delano gene sequence. In addition, since these sequences are optimally spaced apart, the expression of the inserted gene is precisely regulated, leading to the induction of high levels of expression.
[411] Among the known bacterial promoters suitable for use in the production of the protein of the present invention include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter. Suitable eukaryotic promoters include, but are not limited to, the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as the promoters of raus subcomarvirus (RSV) and metallothionein promoters, Metallothionein-I promoter.
[412] The pHE4-5 vector also contains the 5 'Shine-Delano gene sequence of the AUG start codon. The Shine-Delano sequence is a short sequence that is generally located about 10 nucleotides upstream (i.e., 5 ') from the AUG start codon. This sequence essentially positions the prokaryotic liposome at the AUG start codon.
[413] Accordingly, the present invention also relates to an expression vector useful for the production of the protein of the present invention. This aspect of the invention is exemplified by the pHE4-5 vector (SEQ ID NO: 147). The pHE4-5 vector containing the cDNA insert encoding KGF-2 [Delta] 33 was obtained from ATCC No. 209575, deposited with the ATCC on January 9, 1998.
[414] More particularly, the present invention also includes recombinant constructs comprising one or more sequences as broadly described above. Constructs of the present invention include vectors in the forward or reverse direction in which the sequences of the present invention are inserted, such as plasmidic or viral vectors. According to a preferred embodiment of this embodiment, the construct further comprises a regulatory sequence comprising, for example, a promoter operably linked to a sequence. A large number of suitable vectors and promoters are known to those skilled in the art and are commercially available. The following vectors are examples. Bacterial vectors: pQE70, pQE60, pQE-9 (quiagen), pBS, pDIO, phagiscript, psiX174, pbyscript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (pharmacia); Eukaryotic vectors: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (stratagene), pSVK3, pBPV3, pMSG, pSVL (pharmacia). However, any other plasmid or vector may be used as long as it is replicable and viable in the host.
[415] Bektes preferred for use in bacteria include pQE70, pQE60 and pQE-9 available from QIAGEN, Inc.; PF2H8A, pNH16a, pNH18A, pNH46A, which are available from Stratagene Cloning Systems, Inc .; And ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Preferred expression vectors for use in the yeast system include pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, pPIC9K and PAO815 (All available from Invitrogen, Calvad, USA), but are not limited thereto. Other suitable vectors will be readily apparent to those skilled in the art.
[416] The promoter region may be selected from any desired gene using a CAT (chloramphenicol transferase) vector or another vector with a selectable marker. Two suitable vectors are pKK232-8 and pCM7. Specifically named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda P _R , P _L and trp. Eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase, early and late SV40, LTR from retrovirus and mouse metallothionein-I. Selection of suitable vectors and promoters can be done in the common sense of those skilled in the art.
[417] Insertion of constructs into host cells may be performed by calcium phosphate infusion, DEAE-dextran mediated infection, cationic lipid-mediated infection, electrophoresis, transduction, transmission or other methods. Such methods are described in a number of standard laboratory manuals, for example, Davis et al., Basic Methods in Molecular Biology (1986). Specifically, the KGF-2 polypeptide may actually be expressed by a recombinant vector defective host cell.
[418] According to another embodiment, the present invention relates to a host cell containing the construct described above. The host cell may be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell may be a prokaryotic cell, such as a bacterial cell. Introduction of constructs to host cells can be performed by calcium phosphate infusion, DEAE-dextran mediated infection, or electrophoresis (Davis et al., Basic Methods in Molecular Biology (1986)].
[419] Constructs within the host cell can be used in a conventional manner to produce a gene product encoded by the recombination sequence. Alternatively, the polypeptides of the present invention can be synthetically produced by conventional peptide synthesizers.
[420] The mature protein can be expressed under the control of appropriate promoters in mammalian cells, yeast, bacteria or other cells. Cellular exclusion translation systems may also be used to produce such proteins using RNAs derived from the DNA constructs of the present invention. Cloning and expression vectors suitable for use with prokaryotic and eukaryotic hosts are described in Sambrook et al., Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY (1989) Reference is made to the specification.
[421] Transcription of DNA encoding the polypeptide of the present invention by higher eukaryotic cells is increased by inserting an enhancer sequence into the vector. Enhancers usually act on the promoter as a cis-acting component of DNA of about 10 to 300 bp and increase its transcription. Examples include the SV40 enhancer for the rear side of the replication origin of bp 100-270, the cytomegalovirus early promoter enhancer, the polyoma enhancer for the rear side of the replication origin, and the adenovirus.
[422] In order to secrete the translated protein into the lumen of the inner plasma reticulum, into the cell membrane space or into the extracellular space, a suitable secretory signal can be introduced into the expressed polypeptide. The signal may be endogenous or heterologous to the polypeptide.
[423] The polypeptide may be expressed in a modified form, e. G., A fusion protein, and may include additional heterologous action regions as well as secretory signals. For example, additional amino acid regions, specifically charged amino acids, may be added to the N-terminus of the polypeptide during purification or during subsequent processing and storage for stability and persistence in the host cell. In addition, the peptide component may be added to the polypeptide to facilitate purification. Such regions may be removed prior to the final manufacturing step of the polypeptide. It is well known in the art to add peptide components to the polypeptide to induce secretion or release, improve stability, and promote purification. A preferred fusion protein comprises a heterologous region from an immunoglobulin useful for stabilizing the receptor. For example, EP-AO 464 533 (corresponding to Canadian Patent No. 2045869) discloses a fusion protein comprising a different human protein or a portion thereof, in addition to various parts made up of certain regions of the immunoglobulin molecule. In many cases, the Fc portion of the fusion protein is highly advantageous for therapeutic and diagnostic purposes, thus providing improved drug bio-reactivity properties (EP-A 0232 262). On the other hand, for some applications, it is preferred that the fusion protein can be expressed, detected and purified, and then the Fc portion deleted, by the advantageous method as described above. This is the case where the Fc portion is hindered from being used for therapy and diagnosis, for example, when the fusion protein is used as an antigen for immunization. Upon drug discovery, human proteins, such as, for example, the shIL5-receptor, were fused with the Fc portion for high-throughput screening assays to identify hIL-5 antagonists. Bennett et al . , J. Mol. Recognition , Vol. 8, 52-58 (1995) and Kay. Johnson et al . , J. Biol. Chem., 270 (16): 9459-9471 (1995)].
[424] Generally, recombinant expression vectors contain a source of replication, a selectable marker that transforms the host cell, such as the ampicillin resistance gene of E. coli and the S. cerevisiae TRPI gene, and a high expression gene that directs transcription of downstream structural sequences ≪ / RTI > Such a promoter may be derived from an operon encoding for example a glycolytic enzyme such as 3-phosphoglycerate kinase (PGK), alpha-factor, acid phosphatase, or heat shock protein. The heterologous structural sequences are assembled at appropriate steps using the leader sequence and the termination sequence, preferably using leader sequences capable of directing the secretion of the transcription protein into the cell membrane space or extracellular medium. Optionally, the heterologous sequence may encode a fusion protein comprising an N-terminal identifying peptide that confers desirable properties, e. G., Stabilization or simple purification characteristics of the expressed recombinant product.
[425] An expression vector useful for bacteria is constructed by inserting a DNA structure sequence encoding a given protein with an appropriate translation initiation and termination signal on an available read with a functional promoter. The vector comprises one or more phenotypic selectable markers and a replication origin to ensure maintenance of the vector and, if necessary, to provide amplification in the host. Prokaryotic hosts suitable for transformation include the. Coli, Bacillus subtilis, Salmonella typhimurium, and Pseudomonas sp., Streptomyces sp. And Staphylococcus sp., But other species may be used if necessary.
[426] Representatively, expression vectors useful for bacteria may include, but are not limited to, bacterial replication origin derived from commercially available plasmids comprising a selectable marker and the gene component of the known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotech, Madison, Wis.). These pBR322 " backbone " moieties are combined with the appropriate promoter and structural sequence to be expressed.
[427] After the host cells are transformed to have appropriate host strain transformation and appropriate cell density, the selected promoter is induced by appropriate methods (e.g., temperature changes and chemical induction) and the cells are cultured for an additional period of time.
[428] Cells are generally collected by centrifugation and after cleavage by physical or chemical means, the obtained crude extract is stored for further purification.
[429] The microbial cells used for expression of the protein can be cleaved by any conventional method including freeze-thaw cycle, sonication, mechanical cleavage, or by a method using a cytolytic agent, which methods are well known to those skilled in the art have.
[430] Various mammalian cell culture systems may also be used to express the recombinant protein. Examples of mammalian expression systems include the COS-7 cell line of monkey kidney fibroblasts described in Glutsman, Cell 23 : 175 (1981) and other cell lines capable of expressing a compatible vector, such as C127, 3T3, CHO, HeLa and BHK cell lines. The mammalian expression vector comprises a replication origin, a suitable promoter and enhancer and also any necessary ribosome binding site, polyadenylation site, splice donor and acceptor site, transcription termination sequence, and 5 'side non-transcription sequence. DNA sequences derived from the SV40 splice and polyadenylation sites can be used to provide the necessary non-specific gene components.
[431] KGF-2 polypeptides are known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography And recovered from the recombinant cell culture. The most preferred for use in tablets is high performance liquid chromatography (" HPLC ").
[432] The polypeptides of the present invention may be prepared by recombinant techniques from naturally purified products or products by chemical synthetic methods or from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plants, insects and mammalian cells in culture) Lt; / RTI > Depending on the host used in the recombinant production method, the polypeptides of the present invention may be glycosylated or non-glycosylated. The polypeptides of the present invention may also comprise an initial methionine amino acid residue.
[433] KGF-2 polypeptides and preferably secreted forms include products purified from natural sources, including body fluids, tissues and cells, whether directly isolated or cultured, products by chemical synthetic methods, such as bacteria, yeast, higher plants , Recombinant techniques from prokaryotic or eukaryotic hosts, including insect and mammalian cells. Depending on the host used in the recombinant production method, the KGF-2 polypeptide may be glycosylated or non-glycosylated. In addition, the KGF-2 polypeptide may also optionally include an initially modified methionine residue derived from the host mediated method. That is, it is known in the art that the N-terminal methionine encoded by the translation initiation codon is generally highly efficiently removed from any post-translational protein present in all eukaryotic cells. N-terminal methionine or most proteins are efficiently removed from most prokaryotic cells, but in the case of some proteins, this prokaryotic cell removal process is inefficient depending on the type of amino acid to which N-terminal methionine is covalently linked.
[434] In one embodiment, the yeast Pichia pastoris is used to express the KGF-2 protein in a eukaryotic system. Pichia pastoris is a methylrotrophic yeast capable of metabolizing methanol as its single carbon source. The main step in the methanol metabolic pathway is the oxidation of methanol to formaldehyde using O ₂ . This reaction is catalyzed by an enzyme alcohol oxidase. In order to metabolize methanol to its single carbon source, Pichia pastoris must generate a high concentration of alcohol oxidase, partly because the affinity of alcohol oxidase to O ₂ is relatively low. As a result, in the growth medium according to the main carbon source methanol, the promoter region of one of the two alcohol oxidase genes ( AOXI ) is more active. In the presence of methanol, the alcohol oxidase prepared from the AOXI gene contains less than about 30% total soluble protein in P. pastoris (Ellis S. et al . ratio. Et al . , Mol. Cell. Biol. 5: 1111-21 (1985); Kauts. Ph. J. et al., Yeast 5: 167-77 (1989); Tsutsumi, Jay. F. Et al. , Nucl. Acids Res. 15 : 3859-76 (1987)). That is, heterologous coding sequences, such as the KGF-2 polynucleotides of the present invention, are expressed with exceptionally high efficiency in the presence of methanol under the transcriptional control of all or part of the AOXI regulatory sequences.
[435] In one embodiment, the plasmid vector pPIC9K is selected from the group consisting of d. egg. Higgins and J. In a Pichia yeast system as disclosed in Kragh's Pichia Protocols: Methods in Molecular Biology, The Hughmanapress, Inc., Toto, NJ, USA, the KGF of the present invention, as described herein, -2 < / RTI > polypeptide. This expression vector can express and secrete the KGF-2 protein of the present invention by a strong AOXI promoter coupled to a P. pastoris alkaline phosphatase (PHO) secretory signal peptide (i.e., leader) located upstream of the multiple cloning site .
[436] As long as the proposed expression construct provides an appropriately located signal for transcription, translation, secretion (if necessary), etc., as is readily apparent to those skilled in the art, as long as it provides an in-frame AUG as required, Other yeast vectors can be used, for example, pYES2, pYD1, pTEF1 / Zeo, pYES2 / GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K and PAO815 .
[437] In another embodiment, high concentration expression of the heterologous coding sequence may be achieved, for example, by the KGF-2 polynucleotide of the invention cloning the heterologous polynucleotide of the invention into an expression vector, such as pGAPZ or pGAPZ alpha, Can be achieved by growing yeast cultures.
[438] In addition to containing host cells containing the vector constructs described herein, the present invention also includes vertebrate origin, especially primary, secondary, and immobilized host cells of mammalian origin. The host cell may be engineered to delete or replace an endogenous genetic material (e.g., the KGF-2 coding sequence), and / or operably linked to a KGF-2 polynucleotide of the invention and to activate the endogenous KGF-2 polynucleotide (E. G., A heterologous polynucleotide sequence) that directs, transforms, and / or amplifies. For example, the known methods can be used to form new transcription units by operatively linking with heterologous regulatory sequences (e. G., Promoters and / or enhancers) and endogenous KGF-2 polynucleotide sequences by homologous recombination (See, for example, U.S. Patent No. 5,641,670 issued on June 24, 1997; U.S. Patent No. 5,733,761 issued on March 31, 1998; International Publication No. WO 96/29411, published on September 26, 1996; USA, 86 : 8932-8935 (1989); and Zijlstra et al., Nature 342 : 435-438 (1989), published in WO 94/12650, published Aug. 4, 1994; Koller et al . , Natl. Acad. The disclosure of which is incorporated herein by reference in its entirety).
[439] Diagnostic and Therapeutic Uses of KGF-2
[440] As described in the section below, " KGF-2 " refers to the full length and mature form of KGF-2 disclosed herein and the KGF-2 analogs, derivatives and mutants disclosed herein. The present invention also relates to the use of the KGF-2 gene as part of a diagnostic method for detecting the susceptibility or susceptibility to disease associated with the presence of a mutation in the KGF-2 nucleic acid sequence.
[441] Individuals that mediate mutations in the KGF-2 gene can be detected from DNA levels by a variety of techniques. The nucleic acid for diagnosis can be obtained from the cells of the subject, for example, blood, urine, saliva, biopsy or autopsy. Genomic DNA can be used directly for detection or enzymatically amplified using PCR prior to analysis (Sakai et al ., Nature 324 : 163-166 (1986)). In addition, RNA or cDNA can be used in a similar manner. For example, a PCR primer complementary to a nucleic acid encoding KGF-2 may be used to identify and analyze KGF-2 mutations. Deletion and insertion can be detected by comparing the size change of the amplified product with the normal genome type. The point mutation can be identified by hybridizing the amplified DNA with the radiolabeled KGF-2 RNA or the radiolabeled KGF-2 antisense DNA sequence. It is possible to distinguish a sequence that is completely matched by RNase A digestion or by melting temperature difference from a mismatched duplex.
[442] Genetic testing based on DNA sequence differences can be performed by detecting changes in electrophoretic mobility of DNA fragments in the gel, with or without the use of a denaturing agent. Small amounts of sequence deletions and insertions can be visualized by high resolution gel electrophoresis analysis. DNA fragments of differing sequence can be distinguished using modified polyamide gradient gels which delay the migration of other DNA fragments in the gel depending on the specific melting temperature or partial melting temperature [Myers et al., Science 230 : 1242 (1985).
[443] Sequence changes at specific positions can also be confirmed using nucleic acid degrading enzyme protection assays such as RNas and SI protection or chemical degradation methods (eg Cotton et al., PNAS, USA, 85 : 4397-4401 (1985)).
[444] That is, deletion of a particular DNA sequence may be accomplished by methods such as hybridization, RNas protection, chemical degradation, direct DNA sequencing or using restriction enzymes (for example, restriction fragment length polymorphism (RFLP)) and Southern blotting of genomic DNA .
[445] In addition to conventional gel electrophoresis and DNA sequencing, mutations can also be detected by in situ analysis.
[446] The present invention also provides a diagnostic method for detecting the level of KGF-2 protein change in various tissues because overexpression of the protein as compared with the normal control tissue sample can detect disease or disease susceptibility, for example, ≪ / RTI > Methods used to detect the concentration of KGF-2 protein in a sample derived from a host are well known to those skilled in the art and include radioimmunoassays, competitive binding assays, Western blot assays, ELISA assays and " sandwich " assays. ELISA assays [Coligan et al. , Current Protocols in Immunology 1 (2): Chapter 6 (1991), comprises first preparing an antibody, preferably a monoclonal antibody, specific for a KGF-2 antigen. In addition, reporter antibodies to monoclonal antibodies are prepared. Reporter antibodies are conjugated with radioactive, fluorescent, or detectable reagents such as horseradish peroxidase in this example. Samples are taken from the host and incubated on a solid support, for example a polystyrene dish, to bind the proteins in the sample. Followed by incubation with a nonspecific protein such as bovine serum albumin to cover any free protein binding site on the dish. The monoclonal antibody is then attached to any KGF-2 protein attached to the polystyrene dish. The unbound monoclonal antibody is thoroughly washed and removed using a buffer. The reporter antibody conjugated to wasp peroxidase is placed on a dish to bind the reporter antibody to any monoclonal antibody conjugated to KGF-2. Unbound reporter antibody cleans and removes. The peroxidase substrate is then added to the dish, where the amount of color expressed within a given time is indicative of the amount of KGF-2 protein present at a given sample volume from the patient as compared to the standard curve.
[447] An antibody specific for KGF-2 is attached to a solid support, the labeled KGF-2 and the host-derived sample are passed through a solid support, and then the amount of label detected by liquid scintillation chromatography, for example, A competitive method can be used that can be associated with an amount of -2.
[448] The "sandwich" assay is similar to the ELISA assay. In the " sandwich " assay, KGF-2 is passed through a solid support to bind to the antibody attached to the solid support. The second antibody is then conjugated to KGF-2. A third antibody that is labeled and specific to the second antibody may be bound to the second antibody by passing through a solid support, and then the amount thereof may be quantified.
[449] Polypeptides, fragments or other derivatives thereof, or analogs thereof, or cells expressing them may be used as immunogens to produce antibodies thereto. The antibody may be, for example, a polyclonal or monoclonal antibody. The present invention also encompasses products of Fab fragments, or Fab expression libraries as well as chimeric antibodies, single chain antibodies and humanized antibodies. Such antibodies and fragments can be prepared using a variety of methods known in the art.
[450] Antibodies generated against polypeptides corresponding to the sequences of the present invention can be obtained by directly injecting the polypeptide into the animal or by administering the polypeptide to an animal, preferably a non-human animal. The antibody so obtained is then conjugated to the polypeptide itself. In such a manner, even when a sequence encoding only the polypeptide fragment is used, an antibody that binds to the entire native polypeptide can be produced. Such antibodies can be used to isolate polypeptides from tissues expressing the polypeptide.
[451] Any technique that provides antibodies produced by continuous cell line cultures can be used to produce monoclonal antibodies. Examples include the hybridoma technique (Kohler & Milstein, Nature 256 : 495-497 (1975)), the trioma technique, the human B-cell hybridoma technique [Kozbor et al., Immunology Today 4 : 72 (1983) , And EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, Inc., pp. 77-96 (1985)) for producing human monoclonal antibodies.
[452] Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be applied to produce single chain antibodies to the immunogenic polypeptide products of the invention. Transgenic mice can also be used to express humanized antibodies to the immunogenic polypeptide products of the invention.
[453] The polypeptides of the present invention have been found to stimulate epithelial growth. That is, the polypeptide of the present invention can be used to stimulate epithelial growth. The term " epithelium " is a term encompassing the endothelium and epithelium of the body including the inner membrane of blood vessels and other small intestines. It consists of cells bound by a small amount of binding material. The epithelium is divided into types based on the number of layers, depth, and surface cell morphology. The epithelial cells include epithelial cells of the cornea, epithelium of bavet epithelium, epithelium of the ciliated epithelium, columnar epithelium, corneal epithelium, cubic epithelium, ciliated epithelium, enamel epithelium, caustic epithelium, epithelium, gingival epithelium, , Epithelium of the lens, mesenchymal epithelium, olfactory epithelium, pustular epithelium, papillary epithelium, protective epithelium, polythermal epithelium, vertebral epithelium, respiratory epithelium, hepatic epithelium, rectal tube epithelium, sensory epithelium, monolayer epithelium, squamous epithelium, The epithelium, mucous epithelium, transition epithelium, and epithelium of the eye, tongue, gland, oral mucosa, duodenum, ileum, plant, cecum, nasal passages, esophagus, tubules, There are epithelial cells.
[454] "Line" is a generic term for cells with special functions that secrete or release substances that are not related to the need for normal metabolism. Examples of lines that may include eukaryotic cells include, but are not limited to, absorption lines, barriers, phallocytes, acid glands, parotid gland, adrenal gland, A biliary tract, a biliary tract, a blood vessel, a blood vessel, and a blood vessel, and a blood vessel line, an arterial line, an arterial line, a tear line, an asiali line, an abian senna line, The main line is the bow line, the upper arm line, the bronchial line, the bruch line, the brunner line, the narrow line, the corpus callosum line, the paranchymal line, the carotid artery line, the abdominal line, the ear wax line, the cervical line, Ancillary lines, cholecal vessels, kobelin lines, coccyx lines, coiled lines, composite lines, spheroids, conjunctival lines, cooper lines, skin lines, cell glands, endocrine glands, duodenal lines, duburn lines, Gastric, endocrine, intraepithelial, esophageal, exocrine, exocrine glands, gastric, gastric, hypogastric, Glacier line, Glacier line, Glaine line, Glacier line, Dune merchant line, Improvement of sludge, Gergen line, Throat line, Haller line, Haul line, Haas line, Pleasure line, Vessel line, Vascular lymphatic line, Hematopoietic line, Hemolymph line, , Trunk, heterozygous gland, hibernating gland, exocrine gland and endocrine gland.
[455] Another example of the line is the carotid artery trunk line, median line, scapular trunk line, epilepsy line, intestinal line, intraepithelial line, dorsal extension line, jugular line, crowus line, laryngoscope, lacrimal line, Lens line, Lens line, Lieber queen line, Snow line, Legend line, Litet line, Lucicus line, lymphatic line, parotid lymph node, narrow line, wired line, submandibular line, Mandu line, Meliss line, Mybal line, partial gland line, Mucous glands, mucous glands, mucus glands, mucous glands, mucinous mucus, duodenal mucus, eustachian tubes, mucinous glands, multicellular glands, uterus, mucosa, Parotid gland, parathyroid gland, stanchion line, parotid gland, parotid gland, thoracic gland, stomach line, stomach line, There are hypocrisy, one line, fire line, pharynx, Philip line, pineal gland and sewer.
[456] Another example of a line is a polygonal line, a multilayer line, a line, a pregnancy line, a legend bone line, a foreskin line, a prostate line, a pubic line, a Yongun line, a dam line, a Husserin line, , An abdominal salivary gland, an external salivary gland, an internal salivary gland, a zander line, a Schiller line, an sebaceous gland, a conjunctival gland, a first gland, The paranasal sinuses, the paranasal sinuses, the paranasal sinuses, the sinuses, the sinuses, the synovial lines, the target lines, the tile lines, the thymus, the thyroid, the parathyroid, the larynx, the tracheal line , Tubular line, supernumerary line, high solid line, tyson line, unicellular line, yodon line, female yodon line, meridian line, uterine line, ovoid line, vine line, Daejeon Choonsoon line, Sound line, foot tire line, Weber line, ball flight line, Zais line, and Jukker line.
[457] That is, KGF-2 may be used to stimulate the growth of the cell or cells in the line.
[458] The polypeptides of the present invention may be used to stimulate new blood vessel growth or angiogenesis. Specifically, the polypeptide of the present invention can stimulate the growth and proliferation of keratinocytes. Thus, the present invention is directed to therapeutic polypeptides or polypeptides that encode such polypeptides to stimulate epithelial cell proliferation and basal keratinocytes to heal, for example wound healing, and to stimulate the formation of hair follicles and the healing of skin wounds A method of using a polynucleotide is provided.
[459] As described above, the polypeptide of the present invention can be used to stimulate epithelial cell proliferation to heal skin wounds. Such wounds can be in the epidermis or deep and include damage to the dermis and epidermis of the skin. That is, the present invention provides a method for promoting wound healing, including administering an effective amount of KGF-2 to a subject.
[460] Individuals receiving KGF-2 may have a wound healing at normal speed or healing. When administered to individuals who are not inhibited by healing, KGF-2 is administered to promote normal healing processes. When administered to individuals with impaired healing, KGF-2 is administered to promote healing of wounds that are slowly healed or not cured. As described below, many diseases and symptoms inhibit wound healing. Such diseases and symptoms include diabetes (e.g., type II diabetes), treatment with both steroids and other drugs, and ischemic blockade or inhibition. Steroids that have been shown to inhibit wound healing include cortisone, hydrocortisone, dexamethasone, and methylprednisolone.
[461] Nonsteroidal compounds, such as octreotide acetate, have also been found to inhibit wound healing [Waddell, B. et al., Am. Surg. 63 : 446-449 (1997)). The present invention is believed to promote wound healing in an individual being treated with such non-steroidal agents.
[462] Many growth factors have been found to promote wound healing in healing-impaired individuals (see, for example, Steed, D. et al., J. Am. Coll. Surg. 183 : 61-64 (1996); Richard, J. et al., Diabetes Care 18 : 64-69 (1995); Steed, D., J. Vasc. Surg. 21 : 71-78 (1995); Kelley, S. et al., Proc. Soc. Exp. Biol. 194 : 320-326 (1990)]. Such growth factors include growth hormone-releasing factors, platelet-derived growth factors, and basal fibroblast growth factors. That is, the present invention also relates to administering KGF-2 in combination with one or more additional growth factors or other drugs that promote wound healing.
[463] The present invention also provides a method of promoting healing of other wounds caused by suturing and surgery in an individual having wound healing or inhibition of healing at normal speed. The method comprises administering an effective amount of KGF-2 prior to, after, and / or during a suture or other operation. Suture is the connection of two tubular structures, for example, when the mid section of the intestine is removed and the remaining sections are connected to each other to reconstruct the intestinal tract. Unlike skin healing, the healing process of suture wounds is generally difficult to see with the naked eye. In addition, at least wound healing in the gastrointestinal tract occurs rapidly without complications, but complications may require further surgical correction [Thotnton, F. and Barbul, A., Surg. Clin. North Am . 77 : 549-573 (1997)). As described in Examples 21 and 28, treatment with KGF-2 significantly reduces peritoneal leakage and seal stenosis after coronary suture. KGF-2 is thought to promote such a process by reducing the likelihood of complications following such procedures.
[464] That is, the present invention also provides a method for promoting healing after a suture or other surgical procedure in an individual who heals the wound at normal speed or is healed, including administering an effective amount of KGF-2.
[465] The polypeptides of the present invention can also be used to stimulate the differentiation of cells, such as muscle cells, cells that make up neural tissue, prostate cells, and lung cells.
[466] KGF-2 is a systemic product of normal individuals and individuals who are in a condition where wound healing is in an abnormal state, such as uremia, malnutrition, vitamin deficiency, hyperplasia, infectious disease, immunosuppression, and steroids, radiation therapy, In the case of a complication-related complication, the patient may be treated with a surgical wound, a cut wound, a deep wound including damages of the dermis and epidermis, eye tissue wound, tooth tissue wound, oral wound, diabetic ulcer, skin ulcer, It may be clinically useful to stimulate healing of wounds including vein arrest ulcers and burns by heat exposure or chemical agents. KGF-2 is also useful for promoting ischemic and ischemic injury, for example, wound healing associated with chronic venous leg ulcers due to circulatory inhibition and insufficiency of the venous circulation system.
[467] KGF-2 is also useful for promoting skin recovery after skin loss. KGF-2 can also be used to increase the tensile strength and skin thickness of the epidermis.
[468] KGF-2 can be used to increase the adherence of skin grafts to wound sites and to stimulate epithelial formation from wound sites. Types of grafts that can use KGF-2 to increase adhesion to the wound site include: grafts, artificial skin, allografts, autologous epidermal grafts, autologous dermal grafts, vascular grafts, - Brown grafts, bone grafts, embryonic tissue grafts, dermal grafts, delayed grafts, dermal grafts, epidermal grafts, fascia grafts, full thickness skin grafts, xenografts, genitoplasts, allografts, proliferative grafts, lamellar grafts, retinal grafts, mucosa Grafts, oliver-tirhys grafts, aortic grafts, adhesive grafts, transplant grafts, penetrating grafts, partial-layer skin grafts, and posterior-layer grafts. KGF-2 can be used to promote skin strength and improve the appearance of aged skin.
[469] KGF-2 will also alter proliferation of hepatocytes and proliferation of epithelial cells in the lung, breast, pancreas, stomach, small intestine, and large intestine. KGF-2 is a proliferation of epithelial cells such as sebum cells, hair follicles, hepatocytes, type II alveolar cells, mucin-producing goblet cells, and other epithelial cells and their precursors involved in the skin, lung, liver, . ≪ / RTI > As described in Example 31, KGF-2 stimulates hepatocyte proliferation. That is, KGF-2 can also be used to treat acute viral hepatitis and chronic viral hepatitis as well as acute viral hepatitis, cirrhosis, drug-induced hepatitis and toxic hepatitis (i.e., acetaminophen, carbon tetrachloride, methotrexate, Other liver toxins known in the art), autoimmune chronic active hepatitis, hepatic transplantation, and hepatic partial hepatectomy (Cotran < RTI ID = 0.0 > et al., Pathologic basis of disease , 5th Edition, WB Saunders Company, Philadelphia, USA). KGF-2 can also be used to promote liver regeneration and to patients with alcoholic liver disease. KGF-2 can also be used to treat fibrosis of the liver.
[470] About 80% of cases of acute pancreatitis are associated with bile duct disease and alcoholism [Rattner DW, Scand J Gastroenterol 31 : 6-9 (1996); Cotran et al. Pathologic basis of disease , 5th Edition, WB Saunders Company, Philadelphia, 1994]. Acute pancreatitis is an important clinical problem with considerable mortality [Banerjee et al., British Journal of Surgery 81 : 1096-1103 (1994)]. The pathogenesis of this disease is still somewhat unknown, but the pancreatic enzyme is secreted in the pancreas and is widely recognized as causing proteolysis, epileptic infection, fatty necrosis and hemorrhage. Acute pancreatitis may cause interstitial coagulopathy, adult respiratory distress syndrome, shock, and acute renal tubular necrosis [Cotran et al. Pathologic basis of disease , 5th Edition, WB Saunders Company, Philadelphia, 1994]. As a reminder, about 5% of the patients die from shock during the first week of the clinical phase. There are pancreatic abscesses, pseudocysts, and duodenal obstruction in survivors [Cotran et al. Pathologic basis of disease , 5th Edition, WB Saunders Company, Philadelphia, 1994]. Chronic pancreatitis may also be a progressive pancreatic disorder caused by recurrent recurrence of acute gastritis. Chronic pancreatitis appears to cause a rather high-risk pancreatic carcinoma [Cotran et al. Pathologic basis of disease , 5th Edition, WB Saunders Company, Philadelphia, 1994].
[471] As described above and in Example 31, KGF-2 also promotes proliferation of pancreatic cells. That is, KGF-2 can be used for preventive or therapeutic purposes to prevent or reduce acute or chronic pancreatitis.
[472] KGF-2 can also be used to reduce intrinsic toxicity, a side effect of viral infection, radiation therapy, chemotherapy or other treatments. KGF-2 has cytoprotective effect on small intestinal mucus. KGF-2 is also used to prevent or alleviate mucositis due to chemotherapy, other drugs and viral infections and to stimulate the healing of its mucositis (eg oral, esophagus, intestinal, coronary, kidney and anal ulcers) It can be used for preventive or therapeutic purposes. That is, the present invention also provides a method for preventing or treating a mucosal disease or pathological condition, including ulcerative colitis, Crohn's disease, and other diseases in which mucosa is damaged, comprising administering an effective amount of KGF-2. The present invention also provides a method of preventing or treating oral (including swallowing associated with mucosal damage of the pharynx and hypopharynx), esophagus, stomach, intestinal, coronary and renal mucositis irrespective of the drug or mode of use that is the cause of such damage .
[473] KGF-2 can also be used for the treatment and / or prophylaxis of the following conditions: blisters and burns caused by chemicals, for example ovarian damage due to treatment with chemotherapeutic agents or treatment with cyclophosphamide, radiation Or cystitis due to chemotherapy, or intestinal damage due to high-dose chemotherapy. KGF-2 can be used to promote healing of incisional wounds that occur during internal healing, donor site healing, internal surgical wound healing, or cosmetic surgery.
[474] KGF-2 can promote the proliferation of endothelial cells, keratinocytes and basal keratinocytes. That is, the present invention provides a method of stimulating the proliferation of such cells, comprising contacting such cells with an effective amount of KGF-2. KGF-2 can be administered to an individual or contacted with such cells in vitro, in an amount effective to stimulate cell proliferation in vivo.
[475] The present invention also provides a method of promoting cervical epithelial healing, comprising administering an effective amount of KGF-2 to the subject. That is, the present invention provides a method for promoting healing or treatment of various pathologies associated with urinary epithelial cells (i.e., endometrial cells of the urethra tube). Tissue layers comprising such cells can be damaged by a variety of mechanisms including catheterization, surgery or bacterial infections (e. G., Infections by sexually transmitted diseases such as drugs that cause gonorrhea).
[476] The present invention also provides a method of promoting tissue healing of female reproductive tracts, comprising administering an effective amount of KGF-2. Tissue damage in the female reproductive tract can be caused by a wide range of symptoms including Candida infection, Trichomoniasis, Gardnerella, Gonorrhea, Chlamydia, Mycoplasma infection and other sexually transmitted diseases.
[477] As disclosed in Examples 10, 18 and 19, KGF-2 stimulates epidermal keratinocyte proliferation and increases epidermal cell thickness. That is, KGF-2 can be used for complete skin regeneration, complete or partial thickness recovery of skin defects including burns (i.e., regeneration of hair follicles, fine lines and conjunctival foci), and other skin defects such as psoriasis have.
[478] KGF-2 can be used to treat such lesions by promoting re-epithelialization, which is a defective adherence to the epidermis dermis, which often leads to epidermal erosion, that is, painful, open blisters. KGF-2 can be used to treat gastric ulcers and duodenal ulcers, to help heal the mucosal lining and to allow faster regeneration of the mucosal and duodenal mucosal lining. Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis can be treated by intestinal or colon Which causes destruction of mucosal surface. That is, KGF-2 can be used to accelerate resurfacing of mucosal surfaces to aid faster healing and to prevent or reduce the progression of inflammatory bowel disease. Treatment with KGF-2 is expected to have a significant effect on the production of mucosal membranes throughout the gastrointestinal tract and can be used to protect the mucosa from digestive toxicants or after surgery. As described above, KGF-2 can also be used to promote healing of intestinal or coronary anastomosis. KGF-2 can also be used to treat diseases associated with low expression of KGF-2.
[479] As disclosed in Example 32 below, KGF-2 stimulates the proliferation of lung epithelial cells. That is, KGF-2 may be administered for the purpose of prevention to prevent or prevent damage to the lung due to various pathological conditions. KGF-2 may also be administered during or after the onset of damage to promote healing. For example, KGF-2 stimulates the proliferation and differentiation of alveoli and bronchial epithelium and promotes its restoration, thereby preventing, alleviating or treating acute or chronic lung injury. Because progressive alveolar damage and inhalation disorders, which are caused by smoking inhalation and burns and causing necrosis of the bronchial epithelium and alveoli, may be due to chemotherapy, radiotherapy, lung cancer, asthma, blackout and other lung damage, KGF-2 Can be used effectively. In addition, since KGF-2 can stimulate the proliferation and differentiation of type II lung cells, it helps to treat or prevent diseases such as vitreous membrane diseases, for example, pediatric respiratory distress syndrome and pulmonary dysplasia of premature infants .
[480] Three causes of acute renal failure are systemic-related disorders (e. G., Heart failure), bowel related disorders (e. G., By chemotherapeutic agents) that cause renal tubular cell death, occlusion of the lumen of the vessel, Induced nephrotoxicity) and progression-related disorders (eg, urethral tube obstruction) [Thadhani et al. N. Engl. J. Med. 334 : 1448-1460 (1996)]. Growth factors such as insulin positive growth factor I, osteogenic protein-1, hepatocyte growth factor and epidermal growth factor have been shown to mitigate renal disease in animal models [Taub et al. Cytokine 5 : 175-179 (1993); Vakicevic et al. J. Am. Soc. Nephrol. 7 : 1867 (1996)). As disclosed in Example 31 below, KGF-2 stimulates the proliferation of renal epithelial cells and is thus useful for alleviating or treating renal diseases and etiologies such as acute and chronic renal failure and end-stage renal disease.
[481] Because KGF-2 can stimulate the proliferation and differentiation of breast tissue, it can be used to promote healing of breast tissue due to surgery, trauma, or tumors.
[482] KGF-2 can also be used to treat or prevent the onset of diabetes. In patients newly diagnosed with Type I and Type 2 diabetes, where some islet cell function remains, KGF-2 may be used to maintain the soot function to alleviate, delay or prevent persistent disease symptoms . KGF-2 can also be used as an adjuvant in islet cell transplantation to enhance or promote the function of the islet cell.
[483] In addition, the anti-inflammatory properties of KGF-2 may be beneficial in treating acute and chronic diseases in which inflammation is a major cause of diseases including psoriasis, eczema, dermatitis and / or arthritis. That is, the present invention provides a method of preventing or alleviating inflammation and inflammation-related diseases in an individual, including administering an effective amount of KGF-2.
[484] KGF-2 can be used to promote and alleviate brain tissue damage caused by trauma, surgery, or chemical hazards.
[485] In addition, since KGF-2 increases the thickness of the epidermis, the protein can be used to improve aged skin, reduce wrinkles in the skin, and reduce post-operative scarring. Scar tissue is often associated with hyperplasia of dermal fibroblasts. As described in Example 10, fibroblast proliferation is not stimulated by KGF-2. Thus, KGF-2 appears to induce wound healing, leaving minimal scar as a mitogenic promoter specific to epidermal keratinocytes. That is, the present invention provides a method of promoting wound healing while leaving minimal scarring, including administering an effective amount of KGF-2 to the subject. KGF-2 may be administered before, during, and / or after a wounding event (e.g., cosmetic surgery, sudden or intentional tissue trauma due to a shaped object).
[486] As described above, KGF-2 stimulates proliferation of keratinocytes and pores, and thus can be used to promote hair growth on hair loss surfaces and hair transplant patients. Accordingly, the present invention provides a hair growth promoting method comprising administering a sufficient amount of KGF-2 to promote the production of pores.
[487] The present invention also provides a method of protecting an individual from the effects of ionizing radiation, chemotherapy or antiviral drugs, including administering an effective amount of KGF-2. The present invention also provides a method of treating tissue damage due to exposure to ionizing radiation, chemotherapy or antiviral drugs, including administering an effective amount of KGF-2. An individual may be exposed to ionizing radiation for many reasons, including therapeutic purposes (e.g., treatment of hyperproliferative disorders), release of radioactive isotopes into an unexpected environment, or noninvasive medical diagnostic procedures (e.g., x-rays) . Also, a significant number of individuals are exposed to radioactive radon in the workplace and at home. Expected shortening lifetimes have been calculated using exposures to such an environment over a long period of time [Johnson, W. and Kearfott, K., Health Phys. 73 : 312-319 (1977)]. As disclosed in Example 23, the proteins of the present invention improve the survival rate of animals exposed to radiation. That is, KGF-2 can increase the survival rate of individuals affected by radiation, protect individuals from sub-lethal doses of radiation, and increase the rate of irradiation in the treatment of diseases such as hyperproliferative disorders.
[488] KGF-2 can also be used to protect individuals against the doses of radiation, chemotherapeutic agents or antiviral agents that are not normally resistant. For such use, or when used as described herein, KGF-2 may be administered before, after, and / or during treatment with radiation therapy / exposure, chemotherapy or antiviral agents. High dose radiation and chemotherapeutic agents may be particularly useful when treating individuals at advanced disease stages such as hyperproliferative disorders.
[489] The present invention also relates to the use of an effective amount of KGF-2 as an active ingredient in the treatment of radiation-induced oral and gastrointestinal tract damage, mucositis, intestinal fibrosis, rectalitis, radiation-induced pulmonary fibrosis, radiation- induced pneumonitis, And a method of preventing or treating a symptom such as a gonadal syndrome, radiation induced spinal cord toxicity.
[490] KGF-2 may be used alone or in combination with one or more additional drugs that protect against radiation or other drugs. A number of cytokines (e.g., IL-1, TNF, IL-6, IL-12) have been found to have such a protective effect [Neta, R. et al., J. Exp. Med. 173 : 1177 (1991)). IL-11 has also been shown to protect mucosal cells of the small intestine [Du, XX et al., Blood 83 : 33 (1994)] after radiation and chemotherapy in combination, and radiation induced chest trauma [Redlich CA et al., J. Immun. 157 : 1705-1710 (1996)). Various growth factors such as fibroblast growth factor and transforming growth factor beta-3 have also been found to protect against radiation exposure [Ding et al., Acta Oncol. 36 : 337-340 (1997); Potten, C. et al., Br. J. Cancer 75 : 1454-1459 (1997)]
[491] Hemorrhagic cystitis is a syndrome associated with exposure to drugs, viruses, and toxins as well as certain disease states. The outgrowth of the epithelium of the bladder appears to diffuse. Known therapies include intravenous, systemic and nonpharmacologic therapies [West, NJ , Pharmacotherapy 17 : 696-706 (1997)]. Some cytotoxic drugs used clinically have the side effect of causing the inhibition of proliferation of the normal epidermis of the bladder and causing destruction of the ulcer and epithelial lining which are life threatening. For example, cyclophosphamide is a cytotoxic drug that is biologically transformed mainly in the liver and actively alkylates the metabolite by the complex action of the microsomal oxidase system. Such metabolites interfere with the growth of rapidly growing malignant cells. The mechanism of action is thought to be related to the crosslinking of tumor cell DNA (Physician's Desk reference, 1997).
[492] Cyclophosphamide is an example of a cytotoxic drug that causes haemorrhagic cystitis in some patients and can lead to severe complications and, in some cases, death. Fibrosis of the urethra bladder may also progress without or with cystitis. This damage is thought to be caused by cyclophosphamide metabolites excreted in the urine. Hematuria caused by cyclophosphamide occurs for several days but may persist. In severe cases, medication or surgical treatment is needed. For severe haemorrhagic cystitis, nonconsecutive cyclophosphamide therapy should be used. In addition, deterioration of the urethral bladder generally occurs within two years after cyclophosphamide treatment and occurs in patients already with hemorrhagic cystitis [CYTOXAN (cyclophosphamide) package insert]. Cyclophosphamide is harmful to the prostate and the male reproductive system. Cyclophosphamide therapy can cause infertility and can cause testicular degeneration in some cases.
[493] As shown in FIGS. 52 and 53, whole body administration of KGF-2 to an individual stimulates proliferation of bladder and prostate epithelial cells. That is, the present invention provides a method for promoting the differentiation of bladder epithelial and prostatic epithelial cells by administering an effective amount of KGF-2 polypeptide to an individual. More particularly, as demonstrated in Figures 54 and 55, KGF-2 can be used to reduce damage by cytotoxic agents with side effects that lead to inhibition of bladder and prostate epithelial cell proliferation. In order to reduce such damage, KGF-2 may be administered before, after and / or during treatment with a cytotoxic agent or before exposure thereto. Accordingly, the present invention provides a method for reducing damage due to the inhibition of normal proliferation of bladder or prostate epithelial cells by administering an effective amount of KGF-2 to the subject. As described, inhibitors of normal proliferation of the bladder or prostate epithelium include radiation therapy (which causes acute or chronic radiation damage) and, for example, but not limited to, cyclophosphamide, Chemotherapeutic agents, or anti-neoplastic agents. In the present invention, KGF-2 is administered to alleviate or prevent fibrosis and ulceration of urinary bladder. KGF-2 is administered to relieve or prevent hemorrhagic cystitis. Appropriate dosages, formulation and administration routes are described below.
[494] As used herein, an "individual" refers to an animal, preferably a mammal (eg, an ape, cow, horse, pig, bird, sheep, rodent, goat, dog, cat, chicken, monkey, rabbit, And dolphins), with human being more preferred.
[495] The signal sequence of KGF-2 encoding amino acids 1-35 or 36 can generally be used to identify secreted proteins by hybridization and / or computer-generated algorithms.
[496] The KGF-2 nucleotide sequence can be used to isolate the 5 'sequence by hybridization. Plasmids containing the KGF-2 gene under the control of the native promoter / enhancer sequence can be used for in vitro studies to identify endogenous cells expressing the KGF-2 gene and viral transactivators.
[497] The KGF-2 protein can also be used as a positive control for experiments designed to identify peptidomimetics acting on the KGF-2 receptor.
[498] According to the present invention, such a polypeptide, or a polynucleotide encoding such a polypeptide, is used for the purpose of providing an in vitro target for scientific research, DNA synthesis, preparation of a DNA vector, and a diagnostic agent and a therapeutic agent for treating human disease And the like.
[499] A fragment of the full-length KGF-2 gene can be used as a hybridization probe for a cDNA library for isolating the full-length KGF-2 gene and isolating another gene having high sequence similarity or similar biological activity with such a gene. This kind of probe generally has more than 20 bases. However, it is preferred that the probe has more than 30 bases and not more than 50, but it may have a larger number of bases. Probes can also be used to identify genomic clone (s) containing the complete KGF-2 gene, including the cDNA clone corresponding to full-length copies and the regulatory and promoter regions, exons and introns. An example of a screen method is to synthesize an oligonucleotide probe by separating the coding region of the KGF-2 gene using a known DNA sequence. A labeled oligonucleotide having a sequence complementary to the sequence of a gene of the present invention is used to screen a library of human cDNA, genomic DNA or cDNA to determine which library element hybridizes to the probe.
[500] The present invention provides a method for identifying a receptor for a KGF-2 polypeptide. Gene encoding the receptor can be identified by a variety of methods known in the art, for example by ligand panning and FACS classification [Coligan et al., Current Protocols in Immun., 1 (2), Ch. 5 (1991). An expression cloning method is used in which polyadenylated RNA is prepared from a cell that responds to a polypeptide, and a cDNA library generated in the RNA is divided into pools to transform COS cells or other cells that do not react with the polypeptide Is preferably used. Transfected cells are grown on glass slides and exposed to labeled polypeptides. The polypeptide may be labeled by a variety of methods including iodination or insertion of the recognition site of the site-specific protein kinase. After immobilization and incubation, slides are radiographically analyzed. A positive clone is identified and a sub-pool is prepared and then re-transfected using repetitive sub-pooling and re-screening methods to obtain a single clone encoding the putative receptor.
[501] As another receptor identification method, the labeled polypeptide can be photo-affinity bound with a cell membrane or an extract preparation expressing the receptor molecule. The cross-linking material is degraded by PAGE analysis and exposed to X-ray film. The labeled complex containing the polypeptide is separated and digested into peptide fragments, and protein microarraying is performed. The amino acid sequence obtained by microsequencing is used to devise a set of modified oligonucleotide probes for cDNA library screens to identify genes encoding the putative receptor.
[502] The present invention provides methods for screening compounds to identify those that interfere with the function of KGF-2 or block the function of KGF-2. One example of such an assay involves binding mammalian keratinocytes, the compound to be screened, and ³ [H] thymidine under cell culture conditions in which keratinocytes normally proliferate. A control assay can be performed in the absence of the compound to be screened and compared to the keratinocyte expansion in the presence of the compound to determine whether the compound stimulates the proliferation of keratinocytes.
[503] To screen the antagonist, the same assay can be performed in the presence of KGF-2, measuring the activity of the compound to prevent keratinocyte proliferation and then measuring the antagonist activity. The keratinocyte expansion is measured using liquid scintillation chromatography, which measures the introduction of ³ [H] thymidine.
[504] In another method, the mammalian cell or membrane preparation expressing the KGF-2 receptor is cultured with KGF-2 labeled in the presence of the compound. The activity of the compound that enhances or blocks the interaction can then be measured. Alternatively, the response of a known second messenger system after interaction of the receptor with KGF-2 is measured and compared in the presence or absence of the compound. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channel or phosphoinositide hydrolysis.
[505] Examples of potential KGF-2 antagonists include antibodies that bind to the polypeptide, or, in some cases, oligonucleotides. In addition, a potential KGF-2 antagonist may be a mutant form of KGF-2 binding to the KGF-2 receptor, but the action of KGF-2 is effectively blocked because the second messenger response is absent.
[506] Other potential KGF-2 antagonists are antisense constructs made using antisense technology. Antisense technology can be used to control gene expression through triple helix formation or antisense DNA or RNA, both of which are based on the binding of the polynucleotide to DNA or RNA. For example, the 5 'coding portion of a polynucleotide sequence encoding a mature polypeptide of the invention is used to devise an antisense RNA oligonucleotide having a base pair length of about 10 to 40 nucleotides. DNA oligonucleotides are designed to be complementary to the region of the gene involved in transcription (triple helix - Lee et al., Nucl. Acids Res. 6 : 3073 (1979); Cooney et al., Science 241 : 456 (1988); and Dervan et al., Science 251 : 1360 (1991)] to prevent the transcription and the production of KGF-2. Antisense RNA oligonucleotides hybridize in vivo to cDNA and block translation of cDNA into KGF-2 polypeptide [Antisense - Okano, J., Neurochem. 56 : 560 (1991); Oligodeoxynucleotides as antisense inhibitors of gene expression, CRC Press, Boca Raton, Lee (1988)]. The oligonucleotides described above can also be delivered to cells so that antisense RNA or DNA can be expressed in vivo to inhibit KGF-2 production.
[507] Potential KGF-2 antagonists include small molecules that bind to the binding site of the KGF-2 receptor and occupy that site thereby forming a receptor that is inaccessible to KGF-2 to prevent normal biological activity.
[508] KGF-2 antagonists can be used to prevent the induction of new blood vessel growth or angiogenesis in the tumor. The angiogenesis stimulated by KGF-2 is also due to various etiologies including diabetic retinopathy, which can be treated by inhibiting the growth of pathogenic tissues such as antagonists and rheumatoid arthritis of the present invention .
[509] The KGF-2 antagonist can also be used to treat glomerulonephritis, which is characterized by proliferative growth of the ganglionic epithelial cells forming a mass of cells that fill the Baumann space.
[510] Antagonists can also be used to prevent fibrosis associated with fibrosis or pulmonary fibrosis and restenosis after myocardial infarction, hypertrophy of scar tissue at the time of postoperative keloid formation. KGF-2 antagonists can also be used to treat other proliferative diseases that are stimulated by KGF-2, including cancer and Kaposi sarcoma.
[511] The KGF-2 antagonist can also be used to treat keratitis, a chronic infiltration of the cornea with a uveitis inflammation characterized by epithelial cell proliferation.
[512] The antagonist can be used in the form of a composition with a pharmaceutically acceptable carrier, for example a carrier as described below.
[513] Agonists and antagonists of the polypeptides of the invention may be used in conjunction with suitable pharmaceutical carriers to constitute pharmaceutical compositions. Such compositions contain a therapeutically effective amount of an agonist or antagonist of the polypeptide and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulations should be appropriate for the mode of administration.
[514] The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. Such a container may be accompanied by a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of a pharmaceutical or biological product, and such notice shall be such as permitted by the government agency for manufacture, use or sale for human administration It reflects. Agonists and antagonists of the polypeptides of the invention may also be used in conjunction with other therapeutic compounds.
[515] Polypeptides with KGF-2 activity may also be administered with the pharmaceutical composition together with one or more pharmaceutically acceptable excipients. When administered to a patient, the total daily usage of the pharmaceutical composition of the present invention will be determined by the primary care physician within a reasonable medical judgment. The specific therapeutically effective dosage level in any particular patient will depend on the type of reaction and the degree of response to be desiccated; The specific composition of the other agent used, if any; Age, weight, general health status, sex and dietary pattern of the patient; The time of administration of the composition, the route of administration and the rate of excretion of the composition; Treatment period; The number of factors, including drugs (e.g., chemotherapeutic agents) used with the specific composition or concurrently, and similar factors known in the medical arts. Suitable formulations known in the art are described in Remington ' s Pharmaceutical Sciences (current edition), Mack Publishing Company, Easton, PA.
[516] The KGF-2 composition used in the treatment considers the clinical symptoms of the individual patient, in particular the side effects of the KGF-2 monotherapy, the delivery site of the KGF-2 composition, the mode of administration, the schedule of administration, and other factors known to the clinician And are formulated and administered in a manner compatible with good treatment. Thus, an " effective amount " of KGF-2 for the purposes of the present invention is determined by such considerations.
[517] The pharmaceutical compositions may be administered in a conventional manner, for example, orally, topically, intravenously, intraperitoneally, intramuscularly, intraarticularly, subcutaneously, intranasally, intracavitally or intradermally. The pharmaceutical composition is administered in an amount effective to treat and / or prevent a particular condition. In most cases, the dose taking into account the route of administration, symptoms, etc. is about 1 占 퐂 / kg to about 30 mg / kg per daily body weight (kg). However, the dose may be as low as about 0.001 占 퐂 / kg. However, in the specific case of topical administration, it is preferable to administer at about 0.01 / / ㎠ to 9 mg / ㎠.
[518] As a general premise, a more preferred pharmaceutically effective amount of KGF-2 at the time of parenteral administration is about 1 μg / kg to 100 mg / kg per day, which is freely determined at each treatment as described above. When administered consecutively, KGF-2 is administered at a dosage rate of about 1 μg / kg / hr to about 50 μg / kg / hr, typically by one to four injections per day or by continuous subcutaneous infusion with a minipump . Venous bag solutions or bottle solutions may also be used.
[519] The duration of KGF-2 treatment, which affects the fibrinolysis system, is considered optimal to last longer than a certain minimum number of days, and the minimum number of days is 7 days for mice. The interval after treatment and the duration of treatment required to observe the changes for the reaction to occur are judged to be different depending on the desired effect. Such treatment length is indicated in the following examples.
[520] In addition, the KGF-2 polypeptide is suitably administered by a sustained-release system. Suitable examples of an alicyclic composition include a semipermeable polymer matrix in the form of shaped articles, for example in the form of films or microcapsules. (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and -Ethyl-L-glutamate (U. Sidman et al., Biopolymers 22: 547-556 (1983)), poly (2-hydroxyethyl methacrylate (R. Langer et al . , J. Biomed. Mater. Res. 15 : 167-277 (1981) and R. Langer, Chem. Tech. 1982), ethylene vinyl acetate (see R. Langer et al., Supra) or poly-D - (-) - 3-hydroxybutyric acid (EP 133,988) The liposomes containing KGF-2 can be prepared by known methods, such as DE 3,218,121, Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688-3692 (1985) , EP 52,322, EP 36,676, EP 143,949, EP 142,641, Japanese Patent Application No. 83-118008, USP 4,485,045 and 4,544,545 (Proc. Natl. Acad. Sci. USA 77: 4030-4034 , And EP 102, 304. Usually, the liposomes are prepared by lipid components It has at least about 30 mol% cholesterol in a small (about 200~800 Å) a single-layer type, wherein the selected ratio is adjusted to the optimal KGF-2 therapy.
[521] In one embodiment, parenteral administration can be achieved by administering KGF-2 in an injectable dosage form (solution, suspension, or emulsion) that is injectable at a desired degree of purity to provide a pharmaceutically acceptable carrier, It is toxic and is formulated in admixture with other ingredients of the formulation. For example, it is preferred that the formulation does not contain oxidizing agents and other compounds known to be detrimental to the polypeptide.
[522] Typically, the preparation is prepared by uniformly and intimately contacting KGF-2 with a liquid carrier or a finely divided solid carrier or both. Then, if necessary, the product is molded into the desired formulation. The carrier is preferably a parenteral carrier, more preferably a solution which is isotonic with the blood of the recipient. Examples of such carrier excipients include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles such as non-petrol and ethyl oleate are also useful in the present invention, as are liposomes. Suitable formulations known in the art are described in Remington ' s Pharmaceutical Sciences (current edition), Mack Publishing Company, Easton, PA.
[523] It is appropriate that the carrier contains a small amount of additives such as a substance improving isotonicity and chemical stability. Such materials are non-toxic to the recipient at the dosages and concentrations employed, such as buffers such as phosphate, citrate, succinate, acetic acid and other organic acids or their salts, antioxidants such as ascorbic acid, Amino acids such as monosaccharides such as glycine glutamic acid, aspartic acid or arginine; monocarboxylic acids such as monosaccharides such as monocarboxylic acids, Chelating agents such as EDTA, sugar alcohols such as mannitol or sorbitol, counter ions such as sodium, and / or polysorbates, poloxamers, And nonionic surfactants such as PEG.
[524] KGF-2 is typically formulated in such excipients at a concentration of about 0.01 μg / ml to 100 μg / ml, preferably 0.01 μg / ml to 10 mg / ml, at a pH of about 3 to 8. The use of certain of the excipients, carriers or stabilizers described above results in the formation of KGF-2 salts.
[525] KGF-2 used for therapeutic administration should be aseptic. Sterility is easily achieved by filtration through a sterile filtration membrane (eg, 0.2 micron membrane). Therapeutic KGF-2 compositions are typically placed in a container with sterile access ports, such as intravenous solution bags or vials with plugs that can be penetrated by subcutaneous injection needles.
[526] KGF-2 is usually stored in unit or multi-dose containers, e. G. In sealed ampoules or vials, in the form of an aqueous solution or in the form of a lyophilized formulation for reconstitution. As an example of a lyophilized preparation, 10 ml vials are filled with 5 ml of sterile filtered 1% (w / v) KGF aqueous solution, and the resulting mixture is lyophilized. The injection solution is prepared by reconstituting lyophilized KGF-2 using a bacteriocidal injection.
[527] In addition, the dosage can be adjusted in a patient-specific manner such that, for example, KGF-2 activity in blood is provided at a given concentration as measured by RIA technology. Thus, the patient dose may be adjusted to be about 50-1000 ng / ml, preferably 150-500 ng / ml, as measured by the RIA.
[528] The pharmaceutical compositions of the present invention can be administered orally, rectally, parenterally, intracutally, intradermally, intravaginally, intraperitoneally, topically (in the form of powders, ointments, gels, creams, mucus or transdermal patches), buccal, . &Quot; Pharmaceutically acceptable carrier " means a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. As used herein, the term " parenteral " refers to intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
[529] A preferred KGF-2 preparation is described in U.S. Provisional Application No. 60/068493 (December 22, 1997), which is incorporated herein by reference.
[530] Agonists and antagonists of KGF-2 polypeptides that are polypeptides may also be used in accordance with the present invention by expression of such polypeptides often referred to as " gene therapy ".
[531] Thus, for example, a patient's cells can be engineered by a polynucleotide (DNA or RNA) that encodes the polypeptide in vitro, and the engineered cells are then provided to the patient to be treated with the polypeptide. Such methods are well known in the art. For example, the cells may be engineered by methods known in the art by using retroviral particles containing RNA encoding the polypeptides of the present invention. In addition, the cells may be inoculated onto a cell carrier, including biodegradable matrices (e.g., polyglycolic acid), tissue substitutes or equivalents (e.g., artificial skin), artificial organs, You may,
[532] The cells may also be engineered in vivo for expression of the polypeptide in vivo by, for example, methods known in the art. As is known in the art, a producer cell for producing an RNA-containing retroviral particle encoding a polypeptide of the present invention can be administered to a patient to engineer the cell in vivo and express the polypeptide in vivo. Other methods for administering the polypeptides of the present invention by such methods are well known to those skilled in the art from the teachings of the present invention. For example, transcription expression vehicles for engineering cells can be other than retroviruses, for example, adenoviruses that can be used to engineer cells in vivo after combination with a suitable delivery vehicle. Examples of other delivery vehicles include HSV based vector systems, adeno-associated viral vectors and inert excipients such as dextran-coated ferrite particles.
[533] Examples of retroviruses in which retroviral plasmid vectors described above can be induced include retroviruses such as Moloney Muring Leukemia virus, spleen necrosis virus, Rous Sarcoma virus, But are not limited to, Harvey Sarcoma virus, avian leukemia virus, gibbon monkey leukemia virus, human immunodeficiency virus, adenovirus, myeloproliferative sarcoma virus, and breast cancer virus.
[534] The vector comprises one or more promoters. Suitable promoters that may be used include retroviral LTR, SV40 promoter, and Miller et al., Biotechniques Vol. Such as eukaryotic promoters, including human cytomegalovirus (CMV) as described in US Pat. No. 7,198,990 (1989), or any other promoter (eg, histones, pol III and β- Promoters, but are not limited thereto). Other viral promoters that may be used include, but are not limited to, the adenovirus promoter, the thymidine kinase (TK) promoter, and the B19 paravirus promoter. Selection of suitable promoters will be apparent to those skilled in the art from the teachings herein.
[535] The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters that can be used include adenovirus promoters such as adenovirus majorire promoter or heterologous promoters such as cytomegalovirus (CMV) promoter, respiratory syncytial virus (RSV) promoter, MMT promoter, metallothionein promoter, etc. A retroviral LTR (e.g., the aforementioned denatured retrovirus LTR), a β-actin promoter such as an inducible promoter, a heat shock promoter, an albumin promoter, an apo AI promoter, a human globin promoter and a simple herpes simplex kinase promoter, An actin promoter, and a human growth hormone promoter, but are not limited thereto. In addition, the promoter may be a natural promoter that controls a gene encoding the polypeptide.
[536] The retroviral plasmid vector is used to transform a packaging cell line to form a producer cell line. Examples of packaging cell lines that can be transduced include PE501, PA317, -2, -AM, PA12 (SEQ ID NO: 2) described in Miller, Human Gene Therapy 1 : 5-14 , T19-14X, VT-19-17-H2, ΨCRE, ΨCRIP, GP + E-86, GP + envAm12 and DAN cell lines. The vector may be transfected with the packaged cells by any means known in the art. Such means include, but are electroporation, the use of liposomes, and CaPO ₄ precipitation of, but is not limited to these. As an alternative, the retroviral plasmid vector may be encapsulated in liposomes or conjugated to lipids and then administered to the host.
[537] The producer cell line produces an infectious retroviral vector comprising the polypeptide (s) with the nucleic acid sequence (s). Such retroviral vector particles can then be used to transduce eukaryotic cells in vitro or in vivo. The transduced eukaryotic cell expresses the nucleic acid sequence (s) encoding the polypeptide. Examples of eukaryotic cells that can be transduced include, but are not limited to, fetal liver cells, fetal tumor cells, hematopoietic stem cells, liver cells, fibroblasts, myoblasts, keratinocytes, endothelial cells, and bronchial endothelial cells.
[538] The present invention provides a method of treating, inhibiting and preventing the administration of a compound or pharmaceutical composition of the present invention, preferably an antibody of the present invention, to an individual. In a preferred embodiment, the compound is in a substantially purified state (e. G., Substantially free of a substance that confers its effect or produces undesired side effects). The subject is preferably an animal including, but not limited to, an animal such as a cow, a pig, a horse, a chicken, a cat, a dog, etc., more preferably a mammal, and most preferably a human.
[539] Methods and formulations of administration that may be used when the compound contains a nucleic acid or immunoglobulin have been described above, and additional suitable agents and routes of administration may be selected from those described below.
[540] Various delivery systems are known in the art for administering the compounds of the present invention, and examples thereof include liposomes, microparticles, microcapsules, recombinant intracellular encapsulation methods capable of expressing the compounds, (See, e.g., Wu and Wu, J. Biochem. 262 : 4429-4432 (1987)), constructing nucleic acids as part of a retrovirus or other vector, and the like. Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compound or composition may be administered by any convenient route, for example by infusion or infusion, by the endothelium or mucosa (e. G., Oral mucosa, rectal mucosa and intestinal mucosa) You may. Administration may be systemic or topical. In addition, the pharmaceutical compounds or compositions of the present invention may be injected into the central nervous system via any suitable route, such as intracisternal and intrathecal infusion, and intrathoracic infusion may take place, for example, Lt; RTI ID = 0.0 > Ommaya < / RTI > Also, pulmonary administration can be used, for example, using an inhaler or an injector, and a formulation containing a propellant.
[541] In a specific embodiment, it may be desirable to administer the pharmaceutical compound or composition of the present invention locally to the site in need of treatment. This may be accomplished, but not limited to, by injection with a catheter, local injection during surgery with suppositories or inserts, such as topical application with post-operative wound dressing, the implant being a sialastic membrane ) Or membranes of fibers, etc., as well as porous, non-porous or gelatinous materials. When administering a protein, including an antibody of the present invention, care should be taken to use a substance that is not absorbed by the protein.
[542] In another embodiment, the compounds or compositions can be delivered in vesicles, particularly liposomes (see Science 249 : 1527-1533 (1990); Therapy of Infectious Disease and Cancer, Lopez- Beresterin and Fidler (eds.), Liss, New York, pp. 353-365 (1989), Lopez-Berestaine, pp. 317-327).
[543] In another embodiment, the compound or composition may be delivered to a sustained release system. In one embodiment, a pump may also be used (see Langer, et al ., Ceferton, CRC Crit. Ref. Biomed. Eng. 14 : 201 (1987); Buchwald et al., Surgery 88 : 507 1980); Saudek et al ., N. Engl. J. Med. 321 : 574 (1989)). In another embodiment, polymeric materials may be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Biovailability, Drug Product Design and (Levy et al., 1983); [Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23 : 61 (1983)]; , Science 228: 190 (1985) ]; [During et al, Ann Neurol 25:... 351 (1989)]; [Howard et al, J. Neurosurg 71:.. 105 (1989)] reference). In another embodiment, the slow release system can be placed near the therapeutic target, i.e., the brain, so that only a fraction of the systemic dose is needed (e.g., Goodson, in Medical Application of Controlled Release, Vol 2, pp. 115 -138 (1984)).
[544] Other slow release systems are discussed in Langer et al., Science 249 : 1527-1533 (1990).
[545] In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid may be constructed as part of a suitable nucleic acid transcript expression vector, for example using retroviral vectors (see U.S. Patent No. 4,980,286) In combination with a homeobox-type peptide known to be introduced into the nucleus, or through the use of a particulate shock (e.g., gene gun; violystick, DuPont), or coating with a lipid or cell surface receptor or transfection agent, (See, for example, Joliot et al., Proc Natl Acad Sci USA 88: 1864-1868 (1991)), Promote. Alternatively, the nucleic acid can be introduced into cells and incorporated into the host cell DNA by homologous recombination for expression of the transcript.
[546] The present invention also provides a pharmaceutical composition. Such compositions comprise a therapeutically effective amount of a compound and a pharmaceutically acceptable carrier. In a specific embodiment, the term " pharmacologically acceptable " means that it is approved by the United States Pharmacopoeia or an animal, more specifically listed in another pharmacopeia generally recognized for use in humans, or by a federal or state regulatory agency. The term " carrier " refers to a diluent, adjuvant, excipient or vehicle to be administered with the therapeutic agent. Such pharmaceutical carriers may be sterile solutions, for example, water and oils derived from petroleum, animal, vegetable or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, water is the preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used, especially as liquid carriers for injection solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol, monostearate, talc, sodium chloride, skim milk, glycerol, , Water, and ethanol. If desired, the composition may contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, etc. The compositions may be formulated in suppository form with carriers such as typical binders and triglycerides. Oral preparations may contain standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. for pharmaceuticals. An example of a suitable pharmaceutical carrier is described in Remington ' s Pharmaceutical Sciences " by EW Martin. Such a composition may be formulated by mixing a therapeutically effective amount of the compound, preferably in purified form, The above-mentioned supplements should be appropriate for the mode of administration.
[547] In a preferred embodiment, the composition is formulated in a conventional manner as a pharmaceutical composition suitable for intravenous administration to humans. Typically, the composition for intravenous administration is a solution in a sterile isotonic aqueous buffer. If desired, the composition may also contain a solubilizing agent and a local anesthetic such as lidocaine to alleviate pain at the injection site. Typically, the ingredients are presented in unit dosage form, for example, separately or together in the form of a dry lyophilized powder or a non-aqueous concentrate in a sealed container (e.g., an ampoule or sachette indicating the amount of active ingredient). When the composition is to be administered by injection, it can be dispensed with an infusion bottle containing sterile water or salt water for the medicine. When the composition is administered by injection, an ampoule of injectable sterile water or saline can be provided so that the ingredients can be mixed prior to administration.
[548] The composition of the present invention may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, lactic acid, and the like, and those formed with anions such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, 2-ethylaminoethanol, histidine, procaine, and the like.
[549] The amount of a compound of the present invention that is effective for the treatment, inhibition and prevention of diseases or diseases related to excessive expression and / or activity of a polypeptide of the present invention can be determined by standard clinical techniques. In addition, in vitro assays can optionally be used to aid in identifying appropriate titration ranges. The exact dosage used in the formulation will depend on the route of administration and the severity of the disease or disorder and should be determined by the judgment of the clinician and the circumstances of each patient. Effective doses may also be extrapolated from dose-response curves derived from in vitro or animal model test systems.
[550] In the case of antibodies, the dose administered to the donor is typically from 0.1 mg to 100 mg per kilogram body weight of the patient. The amount of tour administered to the patient is preferably from 0.1 mg to 20 mg, more preferably from 1 mg to 10 mg, per kg of the patient's body weight. Typically, human antibodies have a longer half-life in the human body than other species antibodies due to the immune response to the external polypeptide. Thus, lower doses of human antibodies and less frequent dosing are often possible. Further, the dosage and the frequency of administration of the antibody of the present invention can be lowered by improving the absorption and the tissue infiltration (for example, penetration into the brain) of the antibody by, for example, denaturation such as lipidation.
[551] The invention also encompasses pharmaceutical packs or kits comprising one or more containers filled with one or more components of a pharmaceutical composition of the invention. Such a container may be accompanied by a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of a pharmaceutical or biological product, and such notice shall be such as permitted by the government agency for manufacture, use or sale for human administration It reflects.
[552] Antibody-Based Therapeutic Uses
[553] The invention also relates to an antibody-based therapy for administering an antibody of the invention to an animal, preferably a mammalian patient, for the treatment of one or more of the disclosed diseases, conditions or conditions. Therapeutic compounds of the invention include an antibody of the invention (including fragments, analogs and derivatives thereof as described herein) and a nucleic acid encoding the antibody of the invention (including fragments, analogs and derivatives thereof as described herein, ≪ / RTI > < RTI ID = 0.0 > and < / RTI > An antibody of the invention may be used to treat, inhibit or prevent any one or more of the diseases, diseases or conditions associated with excessive trait expression and / or activity of the polypeptide of the invention, for example, diseases, But are not limited thereto. The treatment and / or prophylaxis of a disease, disorder or condition associated with excessive expression and / or activity of a polypeptide of the present invention may include, but is not limited to, relieving symptoms associated with the disease, disorder or condition. The antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
[554] A summary of the ways in which the antibodies of the invention can be used is that the polynucleotides or polypeptides of the present invention may be administered locally or systemically, or by direct cells of, for example, complement (CDC) mediated antibodies, (ADCC). Those of skill in the art having the benefit of the teachings provided herein will know how to use the antibodies of the invention for diagnosis, observation, or treatment without undue experimentation.
[555] The antibodies of the invention may be used in combination with other monoclonal or chimeric antibodies that act to increase the number or activity of, for example, the number of activator cells that are reacted with the antibody, or with lymphokines or hematopoietic growth factors such as IL-2, IL-3 and < RTI ID = 0.0 > IL-7). ≪ / RTI >
[556] The antibodies of the present invention may be administered alone or in combination with other types of therapies (e.g., radiation therapy, chemotherapy, hormone therapy, immunotherapy, and anti-cancer agents). Typically, it is desirable to administer a product having the same species origin or species reactivity (in the case of an antibody) as the patient species. Thus, in a preferred embodiment, a human antibody, fragment derivative, analog, or nucleic acid is administered to a patient for treatment or prophylaxis.
[557] Both the treatment of polynucleotides or polypeptides of the invention and the immunological assays therewith for the treatment of diseases associated with the polynucleotides and fragments thereof include the use of the polypeptides or polynucleotides of the invention with high potency and / / Or affinity is preferably used. Such antibodies, fragments or regions preferably have affinity for the polynucleotides or polypeptides of the invention and fragments thereof. Body is a preferred binding affinity or dissociation constant ^{Kd 5 X 10 -2 M, 10} -2 M, 5 X 10 -3 M, 10 -3 M, 5 X 10 -4 M, 10 -4 M, 5 X 10 - ^{5 M, 10 -5 M, 5} X 10 -6 M, 10 -6 M, 5 X 10 -7 M, 10 -7 M, 5 X 10 -8 M, 10 -8 M, 5 X 10 -9 M ^{, 10 -9 M, 5 X 10} -10 M, 10 -10 M, 5 X 10 -11 M, 10 -11 M, 5 X 10 -12 M, 10 -12 M, 5 X 10 -13 M, 10 ^-13 M, 5 X 10 ^-14 M, 10 ^-14 M, 5 X 10 ^-15 M and 10 ^-15 M or less.
[558] Chromosome analysis
[559] In addition, the sequences of the present invention are valuable for chromosome identification. The sequence can be specifically targeted to a particular location on an individual human chromosome and crossed with it. Further, in recent years, there is a need to identify specific sites on the salt body. Chromosome display reagents based on actual sequence data (repeat polymorphism) useful for indicating chromosomal locations are prime. Mapping DNA to a saliva according to the present invention is an important first step for correlating gene-related diseases with the above-mentioned sequences.
[560] Briefly, PCR primers (preferably 15-25 bp) can be prepared from cDNA to map on chromosomes. Rapid selection of primers that do not contain one or more exons in the genomic DNA makes use of computer analysis of the 3 ' untranslated region, thus complicating the proliferation process. These primers are used in PCR searches of somatic cell hybrids containing individual human chromosomes. Only the hybrid containing the human gene corresponding to the primer is produced.
[561] PCR mapping of somatic cell hybrids is a rapid way to assign specific DNA to specific chromosomes. Utilizing the present invention with the same oligonucleotide primers can be achieved in a similar manner by full or specific chromosome fragment panels of large genomic clones. Other methods of mapping that can be used to create maps on the chromosome include a field hybrid method, a preliminary search by labeled flow classification chromosomes, and a method of constructing a chromosome-specific cDNA library through preliminary selection by hybrid have.
[562] Fluorescence in situ hybridization (FISH) of cDNA clones into meta-autosomal spreads can be used to provide accurate chromosomal location in one step. This technique can use cDNAs as short as 50 bases or 60 bases. For a review of these techniques, Verma et. al., Human Chromosomes: a Manual of Basic Techniques , Pergamon Press, New York (1988).
[563] When sequencing the sequence at the correct chromosomal location, the physical location of the sequence on the chromosome may be correlated with the genetic map data. Such data can be found, for example, in V. McKusick, Mendelian Inheritance in Man, online at Johns Hopkins University Welch Medical Library. Next, the relationship between the genes that map on the same chromosomal region and the disease is identified through binding analysis (co-inheritance of physically adjacent genes).
[564] It is then necessary to determine the difference in cDNA or genomic sequence between the infected and uninfected individuals. If a mutation is observed in some or all of the infected individuals, excluding any normal individuals, the mutation is likely to be a disease predisposition.
[565] With the recent resolution of physical mapping and genetic mapping techniques, precisely localized cDNAs in the chromosomal region associated with the disease can be one of 50 to 500 potential markers (1 gene per 20 kb and 1 It is presumed to have a megabase mapping resolution).
[566] The present invention will be described with reference to the following examples. However, the present invention is not limited to such an embodiment. Unless otherwise specified, all parts or amounts are by weight.
[567] Hereinafter, frequently used methods and / or terms will be described in order to facilitate understanding of the embodiments.
[568] A " plasmid " refers to p when uppercase letters and / or numbers are preceded / followed or lower than the following. As used herein, starting plasmids may be commercially available, commercially available or commercially available, or may be constructed from commercially available plasmids according to published methods. In addition, plasmids equivalent to those described above are known in the art and will be apparent to those skilled in the art.
[569] &Quot; Cleavage " of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only in certain sequences in the DNA. The various restriction enzymes used in the present invention are commercially available, and their reaction conditions, cofactors and other requirements are used as known to those skilled in the art. For analysis, typically 1 μg of plasmid or DNA fragment is used with about 20 units of enzyme in about 20 μl of buffer. For isolation of the DNA fragment for plasmid construction, 25 to 250 units of enzyme in a larger volume were used to usually cut 5 [mu] g to 50 [mu] g of DNA. The amount of buffer and substrate suitable for the specific restriction enzyme is specified by the manufacturer. Usually, the incubation time of about 1 hour at 37 ° C is used, but may be changed according to the instructions of the supplier. After cleavage, direct reaction is electroporated on polyacrylamide gel to isolate the desired fragments.
[570] The size separation of the segmented fragments is carried out using 8% polyacrylamide gels described in Goeldel, D. et al . [ Nucleic Acids Res., 8: 4057 (1980)].
[571] &Quot; Oligonucleotide " refers to a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands that can be chemically synthesized. Such synthetic oligonucleotides do not have a 5 ' phosphate and thus ligate to another oligonucleotide without adding phosphate with ATP in the presence of kinase. The synthetic oligonucleotide is ligated to the dephosphorylated fragment.
[572] &Quot; Ligation " refers to the process of forming a phosphodiester bond between two double stranded nucleic acid fragments (see p. 146 of Maniatis, T., et al.). Unless otherwise indicated, the ligation may be performed using known buffers and conditions, along with 10 units of T4 DNA ligase (" ligase ") per 0.5 μg, which is an approximate equimolar amount of the DNA fragment to be ligated have.
[573] When such external DNA was introduced into the cell membrane, the cells were " transformed " by foreign humans. External DNA may not be integrated (covalently) or integrated into the chromosomal DNA, thereby forming the cell genome. Prokaryotes and enzymes, such as extrinsic DNA, may be retained on episomal elements. Regarding eukaryotic cells, stably transfected or transfected cells are those where external DNA is integrated into the chromosome and bound to the daughter cells through the chromosome strand. This ability is evidenced by the ability of eukaryotic cells to establish cell lines or clones composed of daughter cells that contain external DNA. Examples of transformations are described in Graham, F. and Van Dear, A., Virology, 52 : 456-457 (1973).
[574] &Quot; Transduced " or " transduced " refers to the process by which cells absorb external DNA and integrate the foreign DNA into their chromosomes. Transduction can be accomplished, for example, by transfection, which refers to various techniques by which cells absorb DNA, or by infection using viruses to transfer DNA into cells.
[575] Gene therapy
[576] Another aspect of the present invention relates to a gene therapy method for treating diseases, diseases and symptoms. The gene therapy method relates to transfection of the KGF-2 polypeptide of the present invention by introducing nucleic acid (DNA, RNA and antisense DNA or RNA) into an animal. The method requires polynucleotides encoding the KGF-2 polypeptide that bind effectively to the promoter and other genetic elements necessary for expression of the polypeptide by the target tissue. Such gene therapy and delivery techniques are well known in the art. See, for example, WO 90/11092, which is incorporated herein by reference.
[577] Thus, for example, a cell obtained from a patient is manipulated using a polynucleotide (DNA or RNA) containing a promoter that can be bound to the KGF-2 polynucleotide by in vitro manipulation, The cells may be provided to a patient to be treated with the polypeptide. Such methods are well known in the art. See, for example, the references incorporated herein by reference [Belldegrum, A., et al., J. Natl. Cancer Inst. 85 : 207-216 (1993); Ferrantini, M. et al., Cancer Research 53 : 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153 : 4604-4615 (1994); Kaido, T., et al. Int. J. Cancer 60 : 221-229 (1995); Ogura, H., et al., Cancer Research 50 : 5102-5106 (1990); Santodonato, L., et al., Human Gene Therapy 7 : 1-10 (1996); Santodonato, L., et al., Gene Therapy 4 : 1246-1255 (1997); And Zhang, JF et al., Cancer Gene Therapy 3 : 31-38 (1996). In one embodiment, the cells to be manipulated are arterial cells. The arterial cells can be injected directly into the artery, injected into tissue around the artery, or reintroduced into the patient through a catheter injection or the like.
[578] As will be described in more detail below, KGF-2 polynucleotide constructs can be used in any method of delivering injectable material to animal cells, for example, by injection into tissue (heart, muscle, skin, lung, liver, etc.) And the like. KGF-2 polynucleotide constructs may be delivered using a pharmaceutically acceptable liquid or aqueous carrier.
[579] In one embodiment, the KGF-2 polynucleotide is delivered as a polynucleotide naked. Polynucleotide, DNA or RNA The term " naked " is intended to include any delivery vehicle that acts to assist, facilitate or facilitate intracellular introduction of viral sequences, viral particles, liposomal preparations, lipofectin, ≪ / RTI > However, KGF-2 polynucleotides may also be delivered with liposomal preparations and lipofectin preparations which may be prepared according to methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466 and 5,580,859, which are incorporated herein by reference.
[580] The KGF-2 polynucleotide vector construct used in the gene therapy method is preferably a construct that is not integrated into the host genome and does not contain a sequence that they can replicate. Suitable vectors include pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; PSVK3, pBPV, pMSG and pSVL available from Pharmacia; And pEF1 / V5, pcDNA3.1 and pRc / CMV2 available from Invitrogen. Other suitable vectors are readily apparent to those skilled in the art.
[581] Any strong promoter known in the art can be used to induce the expression of KGF-2 DNA. Suitable promoters include adenovirus promoters such as the adenoviral major late promoter; Or a heterologous promoter, e. G., A cell enlargement virus (CMV) promoter; Respiratory syncytial virus (RSV) promoter; Inducible promoters such as MMT promoter, metallothionein promoter; Heat shock promoter; Albumin promoter; Apo AI (ApoAI) promoter; Human globin promoter; A viral thymidine kinase promoter, for example, a herpes simplex thymidine kinase promoter; Retroviral LTRs; b-actin promoter; And human growth hormone promoters. The promoter may be a native promoter for KGF-2.
[582] Unlike other gene therapy methods, one major advantage in introducing the nucleic acid sequence itself into target cells is the transiotic nature of the polynucleotide synthesis of the cells. Studies have shown that by introducing a non-replicating DNA sequence into a cell, it is possible to provide for generation of a polypeptide of interest for a period of less than six months.
[583] The constructs of KGF-2 polynucleotides are useful for the treatment and / or prophylaxis of organs, such as muscle, skin, brain, lung, liver, spleen, thymus, heart, lymph, blood, bone, cartilage, pancreas, , The intestine, the testes, the ovaries, the uterus, the rectum, the nervous system, the eyes, the endocrine glands and connective tissues. The space between the tissues can be made up of an intracellular fluid, a point polysaccharide matrix in the reticular fibers of the organ tissue, elastic fibers of the blood vessel or chamber walls, collagen fibers of the fibrous tissue, or the same matrix in the connective tissue covering the bone cells or muscle cells . It is a similarly occupied space by the lymphatic fluid of circulating plasma and lymphatic channels. It is preferable to deliver the tissue to the space between the muscle tissues for the following reasons. They can be conveniently delivered by injection into tissues containing these cells. Although delivery and expression can be accomplished in stem cells of undifferentiated or less fully differentiated cells, e. G., Blood or skin fibroblasts, they are preferably delivered and continuously expressed into differentiated, non-dividing cells. Muscle cells in vivo are particularly excellent in their ability to take and express polynucleotides.
[584] When injecting an acid sequence intact, the effective dose of DNA or RNA will range from about 0.05 mg / kg body weight to about 50 mg / kg body weight. A preferred dosage is about 0.005 mg / kg to about 20 mg / kg, and a more preferred dosage is about 0.05 mg / kg to about 5 mg / kg. Of course, those skilled in the art will appreciate that such dosages will vary depending on the tissue site being injected. The appropriate effective dose of the nucleic acid sequence will be readily determined by those skilled in the art and will vary depending upon the symptom to be treated and the route of administration.
[585] A preferred route of administration is a parenteral route of administration that injects into the space between tissues. However, other methods of inhalation of aerosol formulations may be used for delivery to other parenteral routes, for example to the mucous membranes of the lung or bronchial tissues, throat or nose. In addition, KGF-2 DNA constructs can be delivered to the arteries during angioplasty by the catheter used in the procedure.
[586] The intact polynucleotide can be delivered by any method known in the art, including direct needle injection at the delivery site, intravenous injection, topical administration, catheter injection, and so-called "quot; gene gun " method, but is not limited thereto. These delivery methods are well known in the art.
[587] The constructs may be delivered using a delivery vehicle such as, for example, viral sequences, viral particles, liposomal preparations, lipofectin, precipitants, and the like. Such a method of delivery is known in the art.
[588] In certain embodiments, the KGF-2 polynucleotide construct is complexed with a liposomal formulation. Liposomal preparations for use in the present invention include cationic, anionic and neutral preparations. However, cationic liposomes are particularly preferred. This is because a rigid charged complex can form between the cationic liposome and the polyanionic nucleic acid. Cationic liposomes, in their functional form, contain plasmid DNA [Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84: 7413-7416, which is incorporated herein by reference; mRNA [Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86: 6077-6081, which is incorporated herein by reference; And purified transcription factors [Debs et al., J. Biol. Chem. (1990) 265: 10189-10192, which is incorporated herein by reference).
[589] Cationic liposomes are readily available. N, N, N-triethylammonium (DOTMA) liposomes are particularly useful, for example, in GIBCO BRL (Grand Island, NY) under the trade name Lipofectin . [Felgner et al., Proc. Natl Acad. Sci. USA (1987) 84: 7413-7416. Which is incorporated herein by reference). Other commercially available liposomes include transfectace (DDAB / DOPE) and DOTAP / DOPE (Boehringer).
[590] Other cationic liposomes can be prepared from materials that are readily available using techniques well known in the art. For example, PCT Publication No. WO 90/11092, which describes a method for the synthesis of DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonio) propane) liposome, ). Methods for preparing DOTMA liposomes are described in the literature [P. Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417. Similar methods can be used to prepare liposomes from other cationic lipid materials.
[591] Similarly, anionic liposomes and neutral liposomes are readily available from Avanti Polar Lipids (Birmingham, Ala.) And can be readily prepared from readily available materials. Such materials include phosphatidyl, choline, cholesterol, phosphatidyl, ethanolamine, dioloylphosphatidylcholine (DOPC), dioloylphosphatidylglycerol (DOPG), and dioloylphosphatidylethanolamine (DOPE). These materials may also be mixed with DOTMA and DOTAP starting materials in suitable proportions. Methods for making liposomes using these materials are well known in the art.
[592] For example, using various combinations of commercially available dioleo-phosphatidylcholine (DOPC), dioloylphosphatidylglycerol (DOPG), and dioloylphosphatidylethanolamine (DOPE) (with or without the addition of cholesterol) Of liposomes can be prepared. Thus, for example, a DOPG / DOPC vehicle can be prepared by drying 50 mg each of DOPG and DOPC under a stream of nitrogen gas in a sonication vial. The sample is vacuum pumped overnight and hydrated with deionized water the next day. Thereafter, using a heating system Model 350 350 sonicator equipped with an inverted cup (bath type) probe at the highest setting while circulating the bath at 15 EC, Samples are sonicated for 2 hours. Alternatively, the negative charge vesicles can be produced without ultrasonic treatment to produce multilamellar vesicles. Alternatively, uniamellar vesicles of different sizes can be prepared by extruding negative charge vesicles through a nucleopore membrane. Other methods are well known in the art and are available to those skilled in the art.
[593] Liposomes may include multilamellar vesicles (MLVs), small unit vesicles (SUVs), or large unit vesicles (LUVs), and preferably include SUVs. Various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al., Methods of Immunology (1983), 101: 512-527, which is incorporated herein by reference. For example, MLVs containing nucleic acids can be prepared by attaching a thin film of phospholipid to the wall of a glass tube followed by hydration with a solution of the material to be encapsulated. SUVs are produced by prolonged sonication of MLVs to produce a homogeneous population of unitary liposomes. The material to be taken is added to a suspension of preformed MLVs and sonicated. In the case of using liposomes containing cationic lipids, the dry lipid membrane is resuspended in a suitable solution such as sterile water or in an isotonic buffer such as 10 mM Tris / NaCl, sonicated, and the preformed liposome is mixed directly with the DNA do. Because positively charged liposomes bind to cationic DNA, liposomes and DNA form very stable complexes. SUVs are used with small nucleic acid fragments. LUVs are prepared by a number of methods known in the art. Commonly used methods include the Ca ²⁺ -EDTA chelation method [Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394: 483; Wilson et al., Cell (1979) 17: 77); Ether injection method [Deamer, D. and Bangham, A., Biochim. Biophys. Acta (1976) 443: 629; Ostro et al., Biochem. Biophys. Res. Commun . (1977) 76: 836; Fraley et al., Proc. Natl. Acad. Sci. USA (1979) 76: 3348; Detergent dialysis [Enoch, H. and Strittmatter, P., Proc. Natl. Acad. Sci. USA (1979) 76: 145; And reverse-phase evaporation (REV) [Fraley et al., J. Biol. Chem. (1980) 255: 10431; Szoka, F. and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75: 145; Schaefer-Ridder et al., Science (1982) 215: 166). All of the above-mentioned documents are incorporated herein by reference.
[594] Generally, the ratio of DNA to liposome will be from about 10: 1 to about 1:10. Preferably, the ratio is from about 5: 1 to about 1: 5. More preferably, the ratio is from about 3: 1 to about 1: 3. It is even more preferred that the ratio is about 1: 1.
[595] U.S. Patent No. 5,676,954 (incorporated herein by reference) describes the injection of a genetic material complexed with a cationic liposome carrier into a mouse. U.S. Patent Nos. 4,897,355; 4,946,787; 5,049,386; 5,459,127; 5,589,466; 5,693,622; 5,580,859; 5,703,055; WO 94/29469, which is incorporated herein by reference, discloses cationic lipids for use in transforming DNA into cells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859, 5,703,055, and International Publication No. WO 94/29469, which is incorporated herein by reference, provides a method of delivering a DNA-cationic lipid complex to a mammal.
[596] In certain embodiments, retroviral particles comprising RNA comprising a sequence encoding KGF-2 are used to manipulate cells in vivo or ex vivo. Examples of retroviruses from which retroviral plasmid vectors can be derived include Moloney Murine Leukemia virus, spleen necrosis virus, Rous sarcoma virus, Harvey Sacoma virus, But are not limited to, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma virus and mammalian tumor virus, no.
[597] A producer cell line is formed by transfecting a packaging cell line with a retroviral plasmid vector. Examples of packaging cell lines that can be transformed include, but are not limited to, PE501, PA317, R-2, R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP + E-86, GP + envAm12, But are not limited to, the DAN cell line described in Miller, Human Gene Therapy 1: 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may be used to transfect the packaging cells by any method known in the art. Such means include, but are not limited to, electroporation, liposome usage, and CaPO ₄ precipitation. In alternative embodiments, the retroviral plasmid vector may be encapsulated into liposomes, coupled with lipids, and then administered to the host.
[598] The producer cell line produces infectious Retrovirus vector particles containing a polynucleotide encoding KGF-2. Such retroviral vector particles can then be used to transduce eukaryotic cells in vivo or in vitro. Transduced eukaryotic cells will express KGF-2.
[599] In certain other embodiments, cells are manipulated in vivo or ex vivo with KGF-2 polynucleotides contained within adenovirus vectors. The adenovirus will be engineered to encode and express KGF-2 while at the same time its replication ability in the normal lytic virus survival cycle is inactivated. By expressing the adenovirus without integrating the viral DNA into the host cell chromosome, it reduces the concerns about embryonic development. In addition, adenovirus has been used as a live enteric vaccine with excellent stability profiles for many years (Schwartz, AR et al., (1974) Am. Rev. Respir. Dis . 109: 233-238]. Finally, adenovirus-mediated gene transduction has been demonstrated in a number of cases, including transforming alpha-1-antitrypsin and CFTR into the lungs of cotton rats (Rosenfeld, MA et al., 1991 ) Science 252 : 431-434; Rosenfeld et al., (1992) Cell 68: 143-155]. In addition, extensive studies of attempts to evaluate adenovirus as a causative agent of human cancer have been consistently negative (Green, M. et al., (1979) Proc. Natl. Acad. Sci. USA 76 : 6606].
[600] Suitable adenoviral vectors that can be used in the present invention are described in the literature (e.g., Kozarsky and Wilson, Curr. Opin. Genet. Devel . 3 : 499-503 (1993); Rosenfeld et al. , Cell 68 : 143-155 (1992); Engelhardt et al ., Human Genet. Ther. 4: 759-769 (1993); Yang et al., Nature Genet. 7 : 362-369 (1994); Wilson et al . , Nature 365 : 691-692 (1993); And U.S. Patent No. 5,652,224, which are incorporated herein by reference). For example, the adenoviral vector Ad2 is useful and can be grown in human 293 cells. These cells contain the E1 region of the adenovirus and constitutively express Ela and Elb complementing the deficient adenovirus by providing the deleted gene product in the vector. In addition to Ad2, other adenovirus variants (e.g. Ad3, Ad5 and Ad7) can also be used in the present invention.
[601] The adenovirus used in the present invention is preferably replication deficient. Replication deficient adenoviruses require the help of helper viruses and / or packaging cell lines to form infectious particles. The resulting virus can infect cells and express the polynucleotide operatively linked to the promoter, but it can not be replicated in most cells. Replication-deficient adenoviruses will delete one or more of the following genes in whole or in part: Ela, Elb, E3, E4, E2a or L1 to L5.
[602] In certain other embodiments, adeno-associated virus (AAV) is used to manipulate cells in vitro or in vivo. AAV is a naturally occurring deficient virus that requires helper virus to generate infectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol. 158 : 97 (1992). It is also one of several viruses that can integrate his DNA into non-dividing cells. A vector containing as few as 300 base pairs of AAV can be packaged and integrated, but the space for exogenous DNA is limited to about 4.5 kb. Methods for making and using such AAV are known in the art. See, for example, U.S. Patent Nos. 5,139,941, 5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745 and 5,589,377.
[603] For example, suitable AAV vectors for use in the present invention will include all sequences necessary for DNA replication, encapsulation, and host-cell integration. KGF-2 polynucleotide constructs can be prepared by standard cloning methods, for example, Sambrook (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989). The recombinant AAV vector is then transformed into packaging cells infected with helper virus using any standard technique, for example, lipofection, electroporation, calcium phosphate precipitation, and the like. Suitable helper viruses include adenovirus, cytomegalovirus, vaccinia virus or herpes virus. Once the packaging cells have been transfected and infected, they will produce infectious AAV virus particles comprising a KGF-2 polynucleotide construct. These virus particles are then used to transduce eukaryotic cells in vivo or ex vivo. The transduced cells will contain KGF-2 polynucleotide constructs integrated into the genome and will express METH1 and / or KGF-2.
[604] Other methods of gene therapy involve the feasible association of endogenous polynucleotide sequences (e. G., Encoding KGF-2) and heterologous control regions through homologous recombination (see, e. U. S. Patent No. 5,641, 670; 1996. 9. 26. Published International Publication No. WO 96/29411; Published International Publication No. WO 94/12650; Koller et al ., Proc. Natl. Acad. Sci. USA 86 : 8932-8935 (1989); And Zijlstra et al. , Nature 342 : 435-438 (1989)]. This method involves the activation of genes that are present in the target cell but are not normally expressed in the cell or are expressed at a level lower than the predetermined level.
[605] Polynucleotide constructs containing a promoter having a target sequence next to the promoter are prepared using standard techniques in the art. Suitable promoters are described herein. The target sequence is sufficiently complementary to the endogenous sequence to permit homologous recombination of the promoter-target sequence with the endogenous sequence. The target sequence is sufficiently close to the 5 ' end of the KGF-2 endogenous polynucleotide sequence so that the promoter will be operably linked to the endogenous sequence at homologous recombination.
[606] Promoters and target sequences can be amplified using PCR. Preferably, the amplified promoter comprises a restriction enzyme site separated at the 5 'and 3' ends. The 3 'end of the first target sequence has the same restriction enzyme site as the 5' end of the amplified promoter and the 5 'end of the second target sequence has the same restriction enzyme site as the 3' end of the amplified promoter. The amplified promoter and target sequence are digested and linked to each other.
[607] Promoter-target sequence constructs may be used in conjunction with the polynucleotides themselves or with transformation-facilitating agents, such as liposomes, viral sequences, viral particles, whole viruses, lipofection, precipitants, etc. Lt; / RTI > The promoter-target sequence may be delivered using any method, including direct injection, intravenous injection, topical administration, catheter injection, particle stimulation, and the like. The method is described in more detail below.
[608] The promoter-target sequence construct is taken by the cell. The homologous recombination between the construct and the endogenous sequence occurs under the control of the promoter so that the endogenous KGF-2 sequence is located. The promoter then induces the expression of the endogenous KGF-2 sequence.
[609] The polynucleotide encoding KGF-2 may be co-administered with other polynucleotides encoding other proteins. Such proteins include but are not limited to acid and basic fibroblast growth factors, VEGF-1, epithelial growth factor alpha and beta, platelet-derived endothelial cell growth factor, platelet-derived growth factor alpha and beta, Growth factors, colony stimulating factors, macrophage colony stimulating factors, granulocyte / macrophage colony stimulating factors, and nitric oxide synthase, but are not limited thereto.
[610] The polynucleotide encoding KGF-2 preferably comprises secretory signal sequences that facilitate secretion of the protein. Typically, the signal sequence is located at the 5 'end of the coding region or at the coding region of the polynucleotide that is expressed at the 5' end. The signal sequence may be homologous or heterologous to the polynucleotide of interest, and may be homologous or heterologous to the cell being transformed. In addition, the signal sequence may be chemically synthesized using methods known in the art.
[611] The mode of administration of any of the above-described polynucleotide constructs may be used due to a mode of expressing one or more molecules in an amount sufficient to provide a therapeutic effect. These include direct injection, whole body injection, catheter injection, biolistic injector, particle acceleration (ie, "gene gun" method), gelfoam sponge depot method (Eg, Alza minipump, oral or suppository solid pharmaceutical formulation (tablets or pills), decanting during surgery, or topical application. For example, foreign genes were expressed in the liver of rats by direct injection of the calcium phosphate-precipitated plasmid itself into the liver and rat females of rats, or by direct injection of protein-coated plasmids into the portal vein (Kaneda et al . Science 243: 375 (1989)).
[612] The preferred local administration method is direct injection. It is preferred to administer the recombinant molecule of the present invention complexed with the delivery vehicle by injection, or by topical administration in the area of the artery. Topically administering the composition within the arterial region means injecting the composition in centimeters, preferably millimeters, in the artery.
[613] Other topical administration methods involve contacting the polynucleotide construct of the invention at or around the surgical wound. For example, the patient may be operated on, the polynucleotide construct coated on the surface of the wound tissue, or the construct injected into the area of the wound tissue.
[614] Therapeutic compositions useful for systemic administration include the recombinant molecules of the invention complexed to a target delivery vehicle of the present invention. Suitable delivery vehicles for applications using systemic administration include liposomes comprising a ligand for targeting the vehicle to a particular site.
[615] Preferred systemic administration methods include intravenous injection, aerosol, oral and skin (topical) delivery, and the like. Intravenous injection can be carried out using standard methods in the art. Aerosol delivery can also be carried out using standard methods in the art (see, for example, Stribling et al ., Proc. Natl. Acad. Sci. USA 189 : 11277-11281 (1992), incorporated herein by reference). Oral delivery can be accomplished by complexing the polynucleotide construct of the present invention to a carrier capable of withstanding digestion by the digestive enzymes in the intestine of an animal. Examples of such carriers include plastic capsules or tablets known in the art. Local delivery can be accomplished by mixing a polynucleotide construct of the invention with a lipophilic reagent (e.g., DMSO) that can be passed into the skin.
[616] The determination of an effective amount of a substance to be delivered may depend on a number of factors including, for example, the chemical structure and biological activity of the substance, the age and weight of the animal, and the exact condition and its severity and the route of administration requiring treatment. The frequency of treatment will depend on a number of factors, such as the amount of polynucleotide construct administered per dose, and the health and medical history of the subject. The exact amount, number of doses, and time of administration may be determined by the physician or veterinarian.
[617] The therapeutic compositions of the present invention may be administered to any animal, preferably a mammal and an avian. Preferred animals include humans, dogs, cats, mice, rats, rabbits, sheep, cows, horses and pigs, and humans are particularly preferred.
[618] In certain embodiments, a nucleic acid comprising a sequence encoding an antibody or a functional derivative thereof is administered to treat, inhibit or prevent a disease or disorder associated with abnormal expression and / or activity of a polypeptide of the invention by gene therapy . Gene therapy refers to a therapy performed by administering an expressed or expressible nucleic acid to a subject. In this embodiment of the invention, the nucleic acid produces its cryptic protein mediating the therapeutic effect.
[619] Any method for gene therapy available in the art may be used in accordance with the present invention. Such a method is illustrated below.
[620] For a general review of gene therapy, see the literature (see Goldspiel et al. , Clinical Pharmacy 12 : 488-505 (1993); Wu and Wu, Biotherapy 3 : 87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32 : 573-596 (1993); Mulligan, Science 260 : 926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62 : 191-217 (1993); May, TIBTECH11 (5) : 155-215 ( 1993). Methods known in the art as available recombinant DNA techniques are described in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); And Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).
[621] In a preferred embodiment, the compound comprises a nucleic acid sequence encoding an antibody, said nucleic acid sequence being part of an expression vector that expresses the antibody or fragment thereof or a chimeric protein or heavy or light chain in a suitable host. In particular, such nucleic acid sequences have promoters that are operably linked to the antibody coding region, such promoters being inducible or constitutive, and optionally tissue-specific. In other specific embodiments of the invention, it is possible to provide a chromosomal expression of the antibody encoding the nucleic acid by using a nucleic acid molecule located next to the region in which the antibody coding sequence and other predetermined sequences promote homologous recombination at a site in the genome (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86 : 8932-8935 (1989); Zijlstra et al. , Nature 342 : 435-438 (1989)]. In certain embodiments, the expressed antibody molecule is a single chain antibody; Alternatively, the nucleic acid sequence comprises a sequence encoding both the heavy and light chains of the antibody, or fragments thereof.
[622] Delivery of the nucleic acid into the patient may be indirect, if the patient is directly exposed to the nucleic acid or nucleic acid-carrying vector, or directly if the cell is injected into the patient after first being transformed into a nucleic acid in vitro. These two approaches are respectively known as in vivo or in vitro gene therapy methods.
[623] In certain embodiments, the nucleic acid sequence is administered directly in vivo, where it is expressed to produce an encoded product. This can be accomplished by any of a number of methods known in the art, for example by constructing them as part of a suitable nucleic acid expression vector, administering it into cells, using a defective or attenuated retrovirus or other viral vector (See, for example, gene gun, biolistic, duopont), lipid or cell-surface receptor (see, for example, U.S. Patent No. 4,980,286), or by direct injection of DNA itself, Or coating with a transforming agent, a method of encapsulation in liposomes, microparticles or microcapsules, a method of introducing them bound to known peptides for nuclear transfer, or a method of administering them which are bound to receptor-mediated cellular foreign body absorbed ligands Methods [see, for example, Wu and Wu, J. Biol., Chem. 262 : 4429-4432 (1987)], which may be used to target cell types that specifically express the receptor. In another embodiment, lysosomal denaturation of the nucleic acid can be avoided by forming a nucleic acid-ligand complex comprising a fusogenic viral peptide wherein the ligand disrupts the endosomal. In another embodiment, nucleic acids can be targeted in vivo for cell-specific uptake and expression by targeting specific receptors (see, for example, PCT Publication No. WO 92/06180; WO 92/22635; WO 92 / WO 93/14188, WO 93/20221). Alternatively, the nucleic acid can be introduced into cells by homologous recombination and incorporated into host cell DNA for expression (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86 : 8932-8935 (1989); Zijlstra et al., Nature 342 : 435-438 (1989)).
[624] In certain embodiments, a viral vector containing a nucleic acid sequence encoding an antibody of the invention is used. For example, retroviral vectors can be used [Miller et al., Meth. Enzymol. 217 : 581-599 (1993)). These retroviral vectors contain the necessary components to accurately package the viral genome and integrate into the host cell DNA. The nucleic acid sequence encoding the antibody used in the gene therapy is cloned into one or more vectors that facilitate delivery of the gene into the patient. A more detailed description of retroviral vectors can be found in Boesen et al., Biotherapy 6 : 291-302 (1994), which teaches that hematopoietic stem cells are more resistant to chemotherapy Describes the use of retroviral vectors that deliver the mdr1 gene to stem cells. Other references which illustrate the use of retroviral vectors in gene therapy include Clowes et al., J. Clin. Invest. 93 : 644-651 (1994); Kiem et al., Blood 83 : 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4 : 129-141 (1993); And Grossman and Wilson, Curr. Opin. in Genetics and Devel. 3 : 110-114 (1993).
[625] Adenoviruses are other viral vectors that can be used for gene therapy. Adenovirus is a particularly effective carrier for delivering genes to the respiratory epithelium. Adenoviruses inherently infect respiratory epithelia that cause mild illness. Other targets for the adenovirus-based delivery system are liver, central nervous system, endothelial cells and muscle. Adenovirus has the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3 : 499-503 (1993) provides an introduction to adenoviral-based gene therapy. Bout et al., Human Gene Therapy 5 : 3-10 (1994) described the use of adenoviral vectors that transfer genes to the respiratory epithelium of rhesus monkeys. Other examples of methods of using adenovirus in gene therapy are described in Rosenfeld et al., Science 252 : 431-434 (1991); Rosenfeld et al., Cell 68 : 143-155 (1992); Mastrangeli et al., J. Clin. Invest. 91 : 225-234 (1993); PCT Publication WO 94/12649; And Wang, et al. , Gene Therapy 2 : 775-783 (1995). In a preferred embodiment, an adenovirus vector is used.
[626] Adeno-associated virus (AAV) has also been proposed for use in gene therapy [Walsh et al., Proc. Soc. Exp. Biol. Med. 204 : 289-300 (1993); U.S. Patent No. 5,436,146].
[627] Another approach to gene therapy involves introducing genes into cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transformation, or viral infection. Typically, the transformation method involves introducing a selectable marker into the cell. The cells are then taken and placed under selection to isolate the cells expressing the transgene. These cells are then delivered to the patient.
[628] In this embodiment, the nucleic acid is introduced into the cell prior to in vivo administration of the resulting recombinant cell. Such introduction may be carried out, for example, by transformation, electroporation, microinjection, infection using a bacteriophage or viral vector containing a nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, Gene transfer, gene transfer, spore fusion, and the like. Various techniques for introducing foreign genes into cells are known in the art {see, e.g., Loeffler and Behr, Meth. Enzymol. 217 : 599-618 (1993); Cohen et al., Meth. Enzymol. 217 : 618-644 (1993); Cline, Pharmac. Ther. 29 : 69-92m (1985)], which can be used according to the present invention, but the necessary development and physiological functions of the recipient cells are not destroyed. The above technique should allow the nucleic acid to be stably expressed in the cell by stably introducing the nucleic acid into the cell, and preferably to be inherently expressed in the progeny of the cell.
[629] The resulting recombinant cells can be delivered to the patient using a variety of methods known in the art. Recombinant blood cells (e. G., Hematopoietic stem or progenitor cells) are preferably administered intravenously. The expected amount of cell usage will depend on the desired effect, the condition of the patient, and can be determined by one of ordinary skill in the art.
[630] Cells into which the nucleic acid can be introduced for gene therapy purposes include any desired cell type, including epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; Blood cells such as T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, macrophages, granulocytes; But are not limited to, those obtained from various stem cells or progenitor cells, particularly hematopoietic stem cells or progenitor cells such as bone marrow, umbilical cord blood, peripheral blood, and fetal liver.
[631] In a preferred embodiment, the cells used in gene therapy are derived from the patient's autologous tissue.
[632] In embodiments where recombinant cells are used in gene therapy, nucleic acid sequences encoding the antibody may be introduced into the cells, thereby expressing them with the cells or their progeny, and then introducing the recombinant cells into vivo for therapeutic purposes . In certain embodiments, stem or progenitor cells are used. Any stem and / or progenitor cells that can be maintained in vitro in isolation can be usefully employed in accordance with this embodiment of the invention (see, for example, PCT Publication No. WO 94/08598; Stemple and Anderson, Cell 71 : 973-985 (1992); Rheinwald, Meth. Cell Bio. 21A : 229 (1980); And Pittelkow and Scott, Mayo Clinic Proc. 61 : 771 (1986)).
[633] In certain embodiments, the nucleic acid introduced for gene therapy purposes comprises an inducible promoter operably linked to the coding region so that the expression of said nucleic acid can be controlled by the control of the presence or absence of a suitable transcription inductor Demonstration of Therapeutic or Prophylatic Activity).
[634] The compounds or pharmaceutical compositions of the present invention are preferably tested in vitro before being used in humans and then tested in vivo for the desired therapeutic or prophylactic activity. For example, in vitro assays for demonstrating therapeutic or prophylactic effects of a compound or pharmaceutical composition include the effect of a compound on a patient tissue sample or cell line. The effect of a compound or composition on a cell line and / or tissue sample may be determined using techniques known to those skilled in the art, including, but not limited to, rosette formation assays and cell lysis assays . In accordance with the present invention, in vitro assays that can be used to determine whether administration of a particular compound is required include a procedure in which a patient tissue sample is grown in culture, exposed to a dosing compound, and monitored for the effect of such compound in a tissue sample There is an in vitro cell culture assay.
[635] Immune activity
[636] Agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, may be useful for treating disorders or deficiencies of the immune system by activating or inhibiting the proliferation, differentiation or mobilization (chemotaxis) of immune cells . Immune cells are developed through a hematopoietic process that produces lymphoid cells (B and T lymphocytes) and myeloid cells (platelets, red blood cells, neutrophils and macrophages) from pluripotent stem cells. The pathogenesis of the immunodeficiency or disorder may be a genetic cause, a physical cause, for example, some autoimmune disorder or cancer, or infection, obtained by chemotherapy or toxicity. In addition, agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, can be used as markers or detectors of certain immune system diseases or disorders.
[637] KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, may be useful for detecting or treating a deficiency or disorder of hematopoietic cells. KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, are useful for the differentiation and proliferation of hematopoietic cells including pluripotent stem cells in treating disorders associated with certain (or many) types of hematopoietic cell depletion Can be used to increase. Examples of immunodeficiency syndromes include, but are not limited to, blood protein disorders (e.g., non-gammaglobulinemia, abnormal gammaglobulinemia), capillary vasomotor ataxia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphocytopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency syndrome (SCID), Wiskott-Aldrich Disorder, , Anemia, thrombocytopenia, or hemoglobinuria, but are not limited thereto.
[638] In addition, KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, may also be used to modulate hemostatic activity (to stop bleeding) or thrombolytic activity (clot formation). For example, by increasing hemostasis or thrombolytic activity, agonists or agonists of KGF-2 polynucleotides or polypeptides, or KGF-2, can be used for the treatment or prevention of blood clotting disorders (e.g., fibrinogenemia, factor deficiency), blood platelet disorders Eg, thrombocytopenia), or trauma, external surgery, or other causes. Alternatively, a KGF-2 polynucleotide or polypeptide, or agonist or antagonist of KGF-2, can reduce hemostatic or thrombolytic activity and inhibit blood coagulation that is important in the treatment of heart attacks (infarcts), seizures or scars Or dissolve.
[639] In addition, agonists or agonists of KGF-2 polynucleotides or polypeptides, or KGF-2, may also be useful in treating or detecting autoimmune disorders. Many autoimmune disorders are caused by improper recognition of immune cells as foreign substances. Such inappropriateness of recognition leads to an immune response that leads to the destruction of the host tissue. Therefore, administration of a KGF-2 polynucleotide or polypeptide, or an agonist or antagonist of KGF-2, which is capable of inhibiting the proliferation, differentiation or chemotaxis of an immune response, particularly T-cells, is effective in the treatment of autoimmune disorders .
[640] Examples of autoimmune disorders that may be treated or detected include but are not limited to Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Reiter's Disease, Stiff-Man Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Onychomycosis, Swollen Pemphis, , Autoimmune thyroiditis, systemic lupus erythematosus, autoimmune pulmonary inflammation, Guillain-Barre syndrome, insulin-dependent diabetes mellitus and autoimmune inflammatory eye diseases.
[641] Similarly, allergic reactions and symptoms, such as asthma (especially allergic asthma) or other respiratory diseases, can also be treated with agents or antagonists of KGF-2 polynucleotide or polypeptide, or KGF-2. In addition, these molecules can be used to treat anapalacia, hypersensitivity to antigenic molecules, or blood group incompatibility.
[642] In addition, agonists or antagonists of KGF-2 polynucleotide or polypeptide, or KGF-2, can be used to treat and / or prevent organ rejection or graft-versus-host disease (GVHD) . The organ rejection is caused by host immune cell destruction of the graft through the immune response. Similarly, immune response is involved in GVHD, but in this case the foreign transplantation immune cells destroy host tissue. Administration of a KGF-2 polynucleotide or polypeptide, or an agonist or antagonist of KGF-2, to inhibit an immune response, particularly T-cell proliferation, differentiation or chemotaxis, is an effective treatment method for organ rejection or GVHD prevention .
[643] Similarly, agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, can also be used to control inflammation. For example, a KGF-2 polynucleotide or polypeptide, or an agonist or antagonist of KGF-2, may inhibit proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat inflammatory conditions in both chronic and acute conditions. Examples of such inflammatory conditions include infection related inflammation (e.g., sepsis shock, sepsis or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury Inflammatory bowel disease, Crohn's disease or cytokine (eg, TNF or IL-1), and inflammatory bowel disease, inflammatory bowel disease, Crohn's disease or cytokine Symptoms, and the like.
[644] Hyperproliferative Disorders
[645] KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, can be used to treat or detect hyperproliferative disorders, including tumors. Agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, may inhibit the hyperproliferation of the disorder through direct or indirect interactions. Alternatively, agonists or agonists of KGF-2 polynucleotides or polypeptides, or KGF-2, can proliferate other cells capable of inhibiting hyperproliferative disorders.
[646] For example, hyperproliferative disorders can be treated by increasing the immune response of hyperproliferative disorders, in particular by increasing antigenic quality, or by proliferating, differentiating or mobilizing T-cells. Such an immune response can be enhanced by enhancing the existing immune response or by initiating a new immune response. Alternatively, reducing the immune response may be a treatment for hyperproliferative disorders such as chemotherapeutic agents.
[647] Examples of hyperproliferative disorders that can be treated or detected with agents or antagonists of KGF-2 polynucleotides or polypeptides or KGF-2 include but are not limited to abdominal, bones, chest, digestive tract, liver, pancreas, peritoneum, , Pituitary gland, testis, ovary, thymus, thyroid gland), eyes, head and neck, nerves (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, It is not.
[648] Similarly, other hyperproliferative disorders may also be treated or detected with KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2. Examples of such hyperproliferative disorders include, but are not limited to, tumors located in the above-described systemic system, such as, but not limited to, gamma globulinemia, lymphoproliferative disorders, dysoproteinemia, purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia ), Gaucher's Disease, histiocytosis, and any other hyperproliferative disease.
[649] Cardiovascular disorder
[650] A KGF-2 polynucleotide or polypeptide, or an agonist or antagonist of KGF-2, encoding KGF-2 can be used to treat cardiovascular disorders, including peripheral arterial disease, such as, for example,
[651] Cardiovascular disorders include cardiovascular disorders such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformation, congenital heart defects, pulmonary atresia, and scimitar syndrome. Congenital heart defects include aortic aneurysm, tremor, coronary artery anomalies, crisscross heart, right ventricle, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, Syndrome, left ventricular hypertrophy, tetralogy of fallot, transpositon of great vessel, double outlet right ventricle, tricuspid atresia, arterial continence, and septic shock, For example, aortic pulmonary arterial septal defect, endomyoclonus defect, Lutembacher's syndrome, Palo's syndrome, and ventricular heart septal defect.
[652] Cardiovascular disorders also include cardiovascular diseases such as arrhythmia, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial) The risk of cardiac arrest, cardiac arrest, congestive heart failure, congestive cardiomyopathy, seizure dyspnea, cardiogenic edema, cardiac hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, cardiac valve disease, myocardial disease, myocardial ischemia, pericardial effusion, pericarditis (including stricture and constriction), pneumopericardium, postcardioversion syndrome, Cardiovascular complications, scimitar syndrome, cardiovascular syphilis, and cardiovascular tuberculosis), cardiovascular complications, cardiovascular complications, culosis).
[653] Arrhythmias include atrial fibrillation, atrial fibrillation, atrial fibrillation, bradycardia, exophthalmia, Adams-Stokes Syndrome, bundle-branch block, Oriental block, long QT syndrome, Parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome ), Sick sinus syndrome, tachycardias, and ventricular fibrillation. Emptysmophilia is characterized by ataxia, atrial fibrillation, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ejaculatory anomalies, Sinus tachycardia, Torsades de Pointes, and ventricular insufficiency.
[654] Heart valve diseases include aortic valve regurgitation, aortic valve stenosis, cardiac murmur, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral insufficiency, mitral stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, Tricuspid atresia, tricuspid insufficiency, and tricuspid stenosis.
[655] Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic valve stenosis, pulmonary hypoplasia, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibrosis, myocardial infarction fibrosis, Kearns syndrome Syndrome, myocardial reperfusion injury, and myocarditis.
[656] Myocardial ischemia includes coronary diseases such as angina, coronary artery aneurysms, coronary atherosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
[657] In addition, cardiovascular diseases include vascular diseases such as aneurysms, angiogenesis disorders, hemangiomas, bacterial hemangiomas, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, , Sturge-Weber Syndrome, angioenteric edema, aortic disease, Takayasu's Arteritis, aortitis, Leriche's Syndrome, aortic obstructive disease, arteritis, enarteritis, Hypertension, hypotension, ischemia, peripheral vascular disease, phlebitis, pulmonary vein, pulmonary vein thrombosis, pulmonary vein thrombosis, pulmonary arterial hypertension, pulmonary venous insufficiency, (CRA) syndrome, retinal vein occlusion, scimitar syndrome, superior vena cava syndrome, telangiectaemia, cerebrovascular disease, ), Atacia telangiectasia, hereditary hemorrhagic telangiectasia, varicocele, varicose veins, varicose veins, vasculitis and venous insufficiency.
[658] Aneurysms include dissecting aneurysms, pseudoaneurysms, infectious aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, cardiac aneurysms, and iliac aneurysms.
[659] Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid artery stenosis, fibrous dysplasia, mesenteric vascular occlusion, Moyamoya disease, renal artery occlusion, retinal artery occlusion and occlusive thromboangiitis.
[660] Cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery disease, cerebral artery embolism and thrombosis, carotid thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, cerebral hemorrhage, cerebral hemorrhage, , Epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), clavicular submandibular gland syndrome, periventricular leukomalacia, vascular headache, Vocal headache, migraine, and ulnar basal artery circulation failure.
[661] Embolism includes air embolism, amniotic embolism, cholesterol embolism, blue toe syndrome, fat embolism, pulmonary embolism, and thromboembolism. Examples of thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid thrombosis, venous thrombosis, Wallenberg's syndrome, and hemorrhagic phlebitis.
[662] Ischemia includes cerebral ischemia, ischemic colitis, compartment syndrome, anterior compartment syndrome, myocardial ischemia, reperfusion injury and peripheral limb ischemia. The vasculitis may include, but is not limited to, aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocellular lymphadenopathy syndrome, obstructive thrombotic inflammation, hypersensitive vasculitis, Schoenlein- purpura, allergic dermatitis, and Wegener's granulomatosis.
[663] Agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, are particularly effective in the treatment of severe limb ischemia and coronary diseases. Administration of KGF-2 polynucleotides and polypeptides to the experimentally induced ischemic rabbit hindlimb, as shown by the examples, can restore blood pressure ratio, blood flow, angiographic score, and capillary density.
[664] The KGF-2 polypeptide may be administered directly to the delivery site, by intravenous injection, topical administration, catheter injection, biolistic injector, particle accelerator, gelfoam sponge depot, May be administered using any method known in the art including, but not limited to, osmotic pumps, oral or suppository solid pharmaceutical preparations, decanting during surgery or topical application, spray delivery, and the like have. Such methods are well known in the art. The KGF-2 polypeptide may be administered as part of a pharmaceutical composition as described in more detail below. Methods for delivering KGF-2 polynucleotides are described in more detail herein.
[665] Anti-angiogenic activity
[666] The spontaneous balance between endogenous stimulants and inhibitors of angiogenesis lies in the predominant inhibitory effect. See Rastinejad et al., Cell 56 : 345-355 (1989). In rare cases where angiogenesis occurs in normal physiological conditions such as wound healing, organ regeneration, fetal development and female reproductive processes, angiogenesis is tightly regulated and spatially and temporally restricted. These regulatory functions do not play a role under the conditions of pathological angiogenesis characterized by solid tumor growth and the like. Uncontrolled angiogenesis is pathologic and leads to the progression of many tumor and non-tumor diseases. A number of serious diseases, including solid tumor growth and metastasis, arthritis, some types of eye diseases, and psoriasis, are affected by abnormal angiogenesis. See Moses et al ., Biotech. 9 : 630-634 (1991), Folkman et al ., N. Engl. J. Med. 333 : 1757-1763 (1995)], Auerbach et al ., J. Microvasc. Res . 29 : 401-411 (1985)], Folkman et al., Advances in Cancer Research , Klein and Weinhouse, ed., Academic Press, New York, pp. 175-203 (1985), Patz et al ., Am. J. Opthalmol. 94 : 715-743 (1982) and Folkman et al. Science 221 : 719-725 (1983). The angiogenic process contributes to the disease state in many pathological conditions. For example, many data have been accumulated suggesting that the growth of solid tumors depends on angiogenesis. See, for example, Folkman and Klagsbrun, Science 235 : 442-447 (1987).
[667] The present invention provides a method of treating an angiogenesis-related disease or disorder by administering a KGF-2 polynucleotide and / or polypeptide of the present invention, as well as an agonist or antagonist of KGF-2. Malignant and metastatic conditions that may be treated with the polynucleotides and polypeptides of the invention, agonists or antagonists include, but are not limited to, those described herein and other malignancies, solid tumors and cancers known in the art (See Fishman et al. [Medicine 2 Edition, JB Lippincott Co., Philadelphia (1985)] for these diseases).
[668] Ocular diseases associated with angiogenesis that can be treated with KGF-2 polynucleotides and polypeptides of the present invention (including KGF-2 agonists and / or antagonists) include angiogenesis glaucoma, diabetic retinopathy, retinoblastoma, But are not limited to, uveitis, premature aging, corneal graft angiogenesis as well as other inflammatory eye diseases, ocular tumors, and ocular diseases associated with choroidal or iris angiogenesis. See Waltman et al ., Am. J. Ophthal. 85 : 704-710 (1978) and Gartner et al ., Surv. Ophthal . 22 : 291-312 (1978).
[669] Additional diseases that can be treated with the KGF-2 polynucleotides and polypeptides of the invention, including KGF-2 agonists and / or antagonists, include hemangiomas, arthritis, psoriasis, angiofibrosis, atherosclerotic plaque, But are not limited to, pain, hemorrhagic joints, hypertrophic scarring, adhesion-deficient fractures, Osler-Weber syndrome, pyogenic granulomas, sclerotic edema, trachoma, and vascular adhesions.
[670] In addition, diseases and / or conditions that can be treated with the KGF-2 polynucleotides and polypeptides of the invention, including KGF-2 agonists and / or antagonists, include solid tumors, hematologic tumors (e.g., leukemia) (Eg, diabetic retinopathy, premature retinopathy, complete obsessive compromise, corneal transplant rejection, diabetic retinopathy, diabetic retinopathy, diabetic retinopathy, Endometritis, angiogenesis, granulation, hypertrophic scarring (keloid), adhesion-deficient fracture, sclerotic edema, trachoma, vasodilatation, retinopathy, retinopathy of prematurity, retinopathy of prematurity , Ischemic preconditioning angiogenesis, Isler-Weber's syndrome, plaque angiogenesis, capillary vasculopathy, hippocampal joint, (Rochele minalia quintosa), a feline scleroderma (Rochele minalia quintosa), a follicular dermatosis (Rochele minalia quintosa), a follicular dermatosis, a follicular dysgenesis, a wound granulosis, Crohn's disease, atherosclerosis, Ulcer (Helicobacter pylori), Barthonell's rash and Bacillus angiomatosis, and the like.
[671] Digestive disease
[672] KGF-2 was shown to promote the proliferation of GI tract cells. Thus, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or detect peptic diseases.
[673] Examples of peptic diseases that can be treated or detected include, but are not limited to, biliary diseases (e.g., biliary tract tumors, biliary obstruction, biliary tract diseases including gallbladder disease, cholangitis, total biliary cirrhosis such as total biliary cysts, total gut duct stones and total gut tumors; Cholestasis, biliary obstruction, allagile syndrome and liver cirrhosis, gallbladder disease, gallstone disease, gallbladder cancer, gallbladder cancer, gallbladder cancer, gallbladder cancer, Gallbladder disease, Hirschsprung's disease, intestinal atresia, Meckel's diverticulum), gastrointestinal disorders (e.g., gallbladder disease such as cancer, (E.g., fistulae of the biliary tree and esophagus fistula [e.g. esophageal fistulae], canker, intestinal fistula [e.g., fistulae of the rectum]), digestive tract fistula (E.g., bowel fistula such as pancreatic fistula), gastrointestinal tumors (e.g., total squamous cell carcinoma, cholangiocarcinoma including gallbladder tumor), esophageal tumors, gastric tumors (e. G., Colon polyps such as adenomatous polyposis coli) Colon tumors, duodenal tumors, ileal tumors, bowel polyps [e.g., colon polyps such as adenomatous polyposis coli, Gardner ' s syndrome, poisons, , Gastrointestinal tumors (rectal tumors such as rectal tumors, such as rectal tumors, stomach tumors, pancreatic tumors, and peritoneal tumors, including anal tumors and anal gland tumors) Esophageal disease such as Barrett's esophagus, esophagus and gastric varices, esophageal atresia, esophageal cysts, esophageal diverticulum such as gencurricular dystrophy, esophageal autoimmune disorders such as CREST syndrome, Esophageal perforation such as rheumatoid arthritis, esophageal stenosis such as Malaray-Weiss syndrome, esophagus such as digestive esophagitis, traumatic diaphragmatic hernia, such as rheumatoid arthritis, rheumatoid arthritis, rheumatoid arthritis, Diaphragmatic hernia, and hilar hernia).
[674] Examples of gastrointestinal diseases that can be treated or detected include, but are not limited to, gastroenteritis, such as cholera disease, gastrointestinal bleeding (e. G., Leukemia, hematochezia and peptic ulcer), hernias (e.g., diaphragmatic hernias including traumatic diaphragmatic hernia, (Eg, appendicitis, cecal disease including cecum tumors such as appendicitis, colonic disease [ischemic colitis, ulcerative colitis such as toxic megaloblastic disease (eg, hypertrophic hernia, , Rectal colitis such as pseudomembranous small intestine colitis, rectal colitis, functional colorectal diseases such as colonic pseudo-obstruction, colon tumors such as colon polyps such as adenomatous polyposis coli, rectal colorectal tumors such as hereditary colorectal tumors and non polyposis , Sigmoid colon tumor, colonic diverticulitis, colonic polyposis, Hirschsprung's disease and toxic megacolonosis, rectal colitis, and S-phase colon tumor (Eg, duodenal tumors, duodenal obstruction (eg, upper rectal arteriovenous syndrome), diabetic nephropathy, diabetic nephropathy, diabetic nephropathy, diabetic nephropathy, , Duodenal ulcer (e.g., curling ulcer and duodenitis)], enteritis (e.g., small bowel colitis including membranous enterocolitis), ileus disease [e.g., ileal tumors and ileitis], immunoproliferative small bowel disease, inflammatory bowel disease Atherosclerosis, vesicular infection, cryptosporidia, bilateral amoebiasis, amoebic dysplasia, and giardiasis [e.g., ulcerative colitis and Crohn's disease], bowel obstruction, parasitic bowel disease , Rectal polyps (including adenomatous polyposis coli), colon rectal tumors [for example, rectal fistula (including rectal fasciitis)], glandular tumors such as cecal tumors (including appendicitis) For example, Bowel syndrome, bowel-palsy syndrome), bowel obstruction, bowel obstruction, bowel polyps (e.g., colon polyps (e.g., adenomatous polyposis coli), Gardner's syndrome, (Including, for example, anorexia nervosa syndrome, duodenal obstruction), adherent stools, intestinal pseudo-obstruction [e.g., colonic pseudo-obstruction], intussusception, intestinal perforation, (Eg, plant tumors), absorptive disorder syndrome [eg, Blunt Loop Syndrome], celiac disease, lactose intolerance, long fat dystrophy, glandular syndrome, tropical sprue, obstructive mesenteric vessels, Rectal diseases such as anal fissure (anal tumors such as anal canal tumors, anal fever, anal fever, bowel incontinence, hemorrhoids), rectal [e.g. rectal colitis], rectal Rectal fistulas such as rectal fistula, rectal tumors such as rheumatoid arthritis, rectal tumors such as rheumatoid arthritis, rectal tumors such as rheumatoid arthritis, rectal tumors such as rectal tumors, rectal diseases such as rectal prolapse, peptic ulcer, peptic ulcer, peptic ulcer bleeding, peptic ulcer perforation, gastric ulcer, Eurythmia syndrome, posttraumatic syndrome [e.g., rapid migraine syndrome], gastrointestinal [e.g., nonalcoholic acidosis], duodenal gastroeshty [e.g., biliary atresia], gastric fistula, gastric mucosal prolapse, gastric outlet obstruction [e.g. pyloric stenosis] Gastritis [e.g., atrophic gastritis and hypertrophic gastritis], gastritis, gastritis, gastric ulcer, gastric tumor, gastric rupture, gastric ulcer and gastrointestinal torsion, gastrointestinal tuberculosis, Pancreatic insufficiency, pancreatic tumor, and pancreatitis), peritoneal disease (e. G., Peritoneal papilloma, intraperitoneal hemorrhage, intestinal metaplasia), pancreatic diseases such as cystic fibrosis, pancreatic cysts such as pancreatic cysts, pancreatic fistula, Mesenteric lymphadenitis, mesenteric vascular occlusion, peritoneal fat layer disease, peritoneal tumor, peritonitis, celiac disease, subdiaphragmatic abscess and peritoneal tuberculosis).
[675] Peptic diseases that can be treated or detected include liver disease. Liver diseases include, but are not limited to, acute yellow hepatic atrophy, intrahepatic cholestasis (such as Allergic Syndrome and biliary cirrhosis), fatty liver (such as alcoholic fatty liver and Ray syndrome), hepatic vein thrombosis, , Hepatitis A, hepatitis A, hepatitis C, hepatitis C, hepatitis C, hepatitis C, hepatitis B, hepatitis B, (E.g., chronic active hepatitis and hepatitis E), liver lobular degeneration, hepatoblastoma, hepatic dyssynergia, portal hypertension (such as, for example, , Liver cirrhosis (e.g., alcoholic liver cirrhosis, biliary cirrhosis and experimental liver cirrhosis), alcoholic liver disease (such as alcoholic fatty liver, alcoholic hepatitis and alcoholic liver cirrhosis), parasitic liver disease Physealgia, Hepatosplenomegaly and acute hepatic insufficiency), liver tumors, parenchymal hepatitis, erythropoietic porphyria and hepatic porphyria (such as acute intermittent porphyria and full bodily porphyria), hepatic tuberculosis (e. G. And Zellweger syndrome.
[676] Examples of oral dental diseases that can be treated or detected include, but are not limited to, lowered diseases (e.g., Crohn's disease, giant cell granuloma, mandibular disorders such as palate, pharyngeal, Pierre Robin syndrome, maxillary prodromal and mandibular withdrawal, Dental cysts, dentin-forming cysts, calcified dental cysts, dermoid cysts [e.g., muscular cysts], dementia-induced cysts [e.g., partial dentin-like cysts (Including temporomandibular joint disorders such as temporomandibular joint syndromes, mandibular tumors, maxillary prodrome, and mandibular retraction), submandibular gliomas such as mandibular tumors (e.g., mandibular tumors), maxillary tumors and palatal tumors, (E.g., Maxill's disease [e.g., maxillary tumor]), oral disease (e.g., Bevet's syndrome, Burning's Mouth syndrome, oral candidiasis, dry sick, lesional epithelial hyperplasia, , Oral squamous cell carcinoma, oral mucositis syndrome, oral malformations [e.g., lenticulitis, palatal adenopathy, gingival fibrosis, dagger desiccation, codulitis, Oral gum tumors such as gingival tumors (gum tumors), oral gum disease (e.g., hair flaccid tumors), oral tumors (such as dermatomyosarcoma) Oral tumors, salivary gland tumors such as parotid tumors, sublingual tumors, mandibular dermatomas and tongue tumors, hydrocephalus, oral fistulas [eg, fistulas, oral maxillary sinus fistulas and salivary glands], oral bleeding (Eg, periapical abscess and periapical periapical granuloma) and dermoid cysts), dental porphyria (eg, alveolar bone loss, root gut lobe [eg, gingival bleeding], gingival hyperplasia Gingival hyperplasia, gingival hyperplasia, gingival hyperplasia, gingivitis [eg, gingival saliva, gingival pockets, necrotizing ulcerative gingivitis, giant cell granuloma and crown circumflex), dentinal adhesions loss, dermoid cysts, dermoid fasciitis [ (E.g., parotid pockets and kyphosis), tooth deprivation, tooth loss, tooth movement (e.g., mesial migration of teeth and tooth mobility), hippocampus, salivary gland disease Salivary gland stones], salivary gland fistula, salivary gland tumors [e.g., parotid tumors, sublingual tumors and mandibular dorsal tumors]), salivary glands, necrotizing salivary glands burns, salivary gland secretions, mandible (E.g., Stevens-Johnson syndrome, aphthous stomatitis, denture stomatitis, and herpetic stomatitis)), osteoarthritis (e.g., (E.g., tongue, tongue, glaucoma, and oral tuberculosis), pharyngeal disease (e.g., pharyngitis, such as non-pharyngeal disease) Malignant tumors, oral tumors (including tonsil tumors, pharyngitis, post-pharyngeal abscesses, tonsillitis, and pelvic dysplasia), tumors of the stomach, (E.g., temporomandibular joint syndromes), dental diseases (e.g., Alzheimer's disease, dental deposits [including dental calculi and dental plaques], tooth leaks, dental pulp diseases Impaired dentin, dental lesions, hypercalcinaemia, occlusive dysfunction [eg, traumatic tooth closure, interdental canine, angle class I obstruction (including occlusion, fissure, tooth pulp calcification, dental pulp exposure, dental pulp gangrene, second dentin and dental calculus) Deficiency Dental plaque dysplasia, dental plaque insufficiency, dental plaque dysplasia, dental plaque dysplasia, dental plaque dysplasia, dental plaque dysplasia, dental plaque dysplasia, Teeth erosion, tooth erosion, eruption of an entire tooth, tooth impaction, tooth impairment (for example, dental caries, dental caries, and dental caries) Tooth fracture (dental fracture syndrome and tooth dislocation), tooth loss, tooth reabsorption [e.g., root absorption and tooth eruption], and toothache).
[677] Eye disease
[678] KGF-2 was shown to promote proliferation of ocular cells. Thus, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used in the treatment and / or detection of eye diseases.
[679] Examples of eye diseases that can be treated or detected include, but are not limited to, stable fatigue, conjunctival diseases, conjunctival neoplasms, conjunctivitis (allergic, bacterial, inclusive, neonatal eye, trachoma, viral, acute bleeding), conjunctivitis (infectious or dry) Corneal edema, corneal angiogenesis, corneal opacity, corneal opacity, keratitis, acanthamoeba keratitis, corneal ulcer, herpetic keratitis, keratitis, keratoconjunctivitis, keratoconjunctivitis, keratoconjunctivitis (hereditary) (Acanthosis, retinopathy, retinopathy), hereditary ocular disease (albinism, retinopathy, retinopathy, retinopathy, retinopathy, Hereditary corneal dystrophy, gyrate atrophy, hereditary ophthalmia, retinopathy of prematurity, pigmented retinitis), ocular hemorrhage (choroidal hemorrhage, hyphema, (Bacterial conjunctivitis, encapsulated conjunctivitis, neonatal conjunctivitis, trachoma colitis, infectious keratoconjunctivitis, intrauterine tuberculosis), fungal eye infections, parasitic eye infections (including acute amoeba keratitis , Viral eye infections (viral conjunctivitis, acute hemorrhagic conjunctivitis, cytomegaloviral retinitis, herpes zoster anititis, herpetic keratitis, keratitis), purulent uveitis (Conjunctival tumor, eyelid tumors, orbital tumors, uveal tumors (choroidal tumors, iris tumors), eyelid diseases (blepharitis, ophthalmologic tumors) (Dacryocystitis, dry eye sundromes, dry conjunctiva, conjunctivitis, scleroderma), pseudoaneurysms (scleroderma, scleroderma, ptosis, Lens disease (aphthalmia, phosgene aneurysm, cataract, lens subunit, lens lithotripsy, eye hypertension, glaucoma (visual-occlusive angiogenesis, visual-obstructive angiogenesis, ocular hypertension), sicca syndrome, (Esotropia), optic nerve palsy (protozoa), optic nerve palsy (ocular hypertension), ocular hypertension, ocular mobility disorder (amblyopia, nystagmus, nerve paralysis, ocular paralysis, dorsal syndrome, Horner syndrome, chronic progressive external ophthalmoplegia, (Ocular hypertrophy, ocular hypertrophy, Grave disease, orbital plasma cell granuloma, orbital tumor), abnormal pore function (pupillary thickening, Retinal pigment epithelium, diabetic retinopathy, retinal artery occlusion, retinal pigment epithelium, retinal pigment epithelium, retinal pigment epithelium, retinal pigment epithelium, retinal pigment epithelium, Retina Retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, retinitis pigmentosa, Choroidal hemorrhage, choroidal tumor, choroidal defect, choroiditis, choroiditis, choroidal retinitis, retinal vein occlusion), scleroderma (scleritis), uveitis (Irregular atrophy), iris disease (deprivation syndrome, iridocyclitis, iridocytoma), uveitis (anterior uveitis, sympathicotomy, anterior vesicet syndrome, iridocyclitis, iritis, posterior uveitis, choroiditis, (Mild to moderate uveitis, localized planitis, purulent uveitis (internal organs), uveitic meningoencephalitis syndrome), visual impairment (amblyopia, blindness, Double vision, night blindness, dark spot, under-normal vision) and proliferative vitreoretinopathy.
[680] Skin and connective tissue disorders
[681] KGF-2 promotes cell proliferation in skin and connective tissue. Thus, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used in the treatment and / or detection of skin and / or connective tissue.
[682] Examples of connective tissue diseases include cartilage diseases such as recurrent chondroitinitis and tatea syndrome; Cellulitis; Collagen diseases such as Elardian's syndrome, keloids (including acne keloid), mucopolysaccharidosis I, necrotizing necrosis (including annular granuloma, nevus nevus), and incomplete osteogenesis; Skin relaxation; Dermatomyositis; Dufuq Trang construction; Homocystinuria; Lupus erythematosus (including skin, plaques, fatty deposits, whole body and nephritis); Marfan's syndrome; Mixed connective tissue disease; Mucinosis (including vesicles, mucopolysaccharidosis [I, II, UU, IV, IV and VII]), mucinous adenomatous sclerosis and synovial cysts; Connective tissue tumor; Noan sind romel pelvic pain; (Including cystic erythema, small nodular non-pyogenic and peritoneal); Penile hardening; Pseudo-amyloid elastase; Rheumatic diseases (including arthritis [including rheumatoid arthritis, childhood rheumatoid arthritis, Kaplan's syndrome, Pelt's syndrome, rheumatoid nodule, obstructive vertebral and steer disease], hyperlipidemia, multiple myalgia rheumatism); (CREST syndrome), and systemic lupus erythematosus (CREST syndrome).
[683] Examples of skin diseases include proliferating vascular lymphatic vessels with eosinophilia; Scars (including hypertrophic scars); Skin fistula, cuis laxative; Dermatitis (including dermatitis), atopic dermatitis, contact dermatitis (allergic contact, allergic toxicodendron), irritant dermatitis (phototoxicity, diaper rash), occupational dermatitis; (Including, for example, toxic epidermal necrolysis, nodular erythema, serosal), eczema (including sweating disorders, neuropsychiatric, neurodermatitis, and radiation dermatitis); seborrheic dermatitis, herpes dermatitis, seborrheic dermatitis, Dermatomyositis; Erythema (including acute rash), facial dermatosis (acne-shaped rash [keloid, lavage, normal and pavilion-lacoux), erythema (Including skin soreness, cholesterol species [including middle ear]), glandular (congenital glandular erythema, epidermal erythematosus, epidermal erythematosus, Atherosclerosis, atherosclerosis, atherosclerosis, and atherosclerosis, including, but not limited to, lupus erythematosus, laminar lupus, imaginary lupus, X-ray associated lupus, and Sogren-Larsson syndrome) (Necrosis), necrosis (necrosis), necrosis (necrosis), photodynamic disorders (photoallergic or phototoxic dermatitis, bacillary thickening, sunburn and pigmented sclerosis), pigmentation (Acanthosis nigricans), sunspots, Poets-elimination syndrome, hypochromatosis, albinism, pibaldism, albinism, pigmental ataxia, pigmentation ≪ / RTI > hives and pigmented skin).
[684] Examples of skin diseases include; Pruritus (including anal and vulva); Cicatricialis (including gibbous gonorrhea and gibberellicosis); Sclap skin disease; Adult acne vulgaris, neonatal skin sclerosis; Skin diseases such as hair diseases (alopecia, folliculitis, hyperhidrosis, Kinky hair syndrome), crude diseases (crude-patellar syndrome, intestinal obstruction or early onset, Tumors), marine diseases (Hansen's disease, hyperhidrosis, bovine hepatitis, Hanjin, Fox-Poditis, tumors); (S), genetic skin diseases (such as, for example, alfynism, skin laxity, benign familial pemphigus, porphyria, dermatophytosis, ectodermal dysplasia, Ellis van der Klee syndrome, lesional dysplasia, Augmentation, young line); Infectious skin diseases such as dermatomyositis, infantile fungus, candidiasis, pigment fungus, Madura mycosis, paracoxidioid fungi, sporotracum vulgaris, Yusen; Bacterial skin diseases such as cervical fasciculatominosis, bacillary multiple hemangiomas, infertility, solitary, chronic diabetic rubella, scarlet fever, inguinal granuloma, purulent sinusitis, Madura mycosis, Dermatitis, nondiagnosis, staphylococcal skin infections [franglichia, carnellan, impetigo, vaginal hot skin syndrome], skin syphilis, skin tuberculosis, yose); Parasitic dermatoses (such as, for example, larval migraine, respiratory < / RTI > Viral skin diseases (e.g., infectious erythema, sporadic rash, simple hypoglycem, moolusum contagiosum, and warts).
[685] Other examples of skin diseases include metabolic dermatoses such as painful alopecia, fatty dystrophy, lethargic necrosis, porphyria, burning yellow pustules, amyopathies (ulcers); Papulosquamus skin disease (including tachyarrhagic rash), parpasoriasis, intranasal and psoriasis; Vascular dermatosis, such as Bartz's syndrome, mucocutaneous lymphadenitis syndrome, polyarteritis nodosa, gingival fibrosis, Takayasu's arteritis; Hypertrophic dermatosis (including extracellular septicemia), blisters, herpetic pregnancy, bullous pox, bullous pemphigoid, pemphigus vulgaris; Skin tumor; Skin ulcers, such as gastric ulcers, leg ulcers, foot ulcers, diabetic foot ulcers, varicose ulcers, and gangrenous hypertrophy.
[686] Urinary tract diseases and disorders
[687] KGF-2 can promote the proliferation of the non-genital phase. Thus, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and detect male and female genital diseases and / or disorders and pregnancy complications.
[688] Examples of urological and male genital diseases that may be treated or detected include epididymitis, male genital tumors, penile tumors, prostate tumors, testicular tumors, scrotal hematomas, genital herpes, scrotal species, male infertility, schizophrenia, penile diseases (Eg, hypertrophic, urethral hypothermia, penile hardening, penile tumors, whiplash, jawbone whiplash, penile ankle), prostate diseases such as hypertrophy, tumors and prostatitis, sexual dysfunctions such as erectile dysfunction and vasculogenesis erectile dysfunction, (Menstrual, kidney), urinary tract abnormalities, bladder exstrophy, latent testis, urethral glaucoma, urethral glaucoma, polycystic ovary, polycystic kidney disease, (Including autosomal dominant and autosomal recessive), hereditary nephritis, sexual dysfunction, genital dysplasia, mixed penile dysplasia, hemispheric, caustic leukemia, Kalman syndrome, Clepelter syndrome, (Bladder, kidney, ureter, urethra), bladder disease (stones, exstrophy, fistula, bladder sore throat, neck closure, tumors) Renal hypertension, renal hypertension, renal hypertension, renal hypertension, renal hypertension, renal hypertension, renal hypertension, renal hypertension, renal hypertension, renal hypertension, renal insufficiency, Renal failure, kidney stones, renal stones, renal stone necrosis, cystic tension, polycystic kidney, polycystic kidney disease (autosomal dominant, autosomal recessive), spongy kidney, renal failure (renal primary idiopathic diabetes, acute renal failure, Nephritis, glomerulonephritis, glomerulonephritis, glomerulonephritis, glomerulonephritis, lupus nephritis, hereditary nephritis, intestinal nephritis, balkan nephropathy, pyelonephritis, yellow granuloma, glomerulonephritis, nephrolithiasis, , Shin , Renal vein occlusion, renal osteodystrophy, congenital defects in tubular transport, tubulointerstitial disease, renal amino acidosis, cystinuria, cystic fibrosis, renal pelvic inflammatory disease, Neurodegenerative syndrome, hypogonadism, renal tuberculosis, uremia, hemoglobinuria syndrome, Wegener granulomatosis, gelberger syndrome, proteinuria, diabetic nephropathy, diabetic nephropathy, (Urinary tract infection), urethritis (urinary tract obstruction, urethral stricture), urethritis (lighter syndrome), urinary stones (bladder, urinary tract, urinary tract) Urinary incontinence, urinary incontinence, stress urinary incontinence, urinary retention), urinary tract infection (bacteriuria, pyuria, bilateral schistosomiasis) .
[689] Examples of female reproductive diseases and complications of pregnancy that can be treated or detected include adnexal diseases (such as adnomatitis, ovarian inflammation, ovarian cirrhosis), tubal diseases (such as tubal tumors and tuberculosis), ovarian diseases (including ovarian, Ovarian cysts, ovarian cysts, ovarian cysts, endometriosis, female genital tumors, ovarian tumors, uterine tumors, cervical tumors, endometrial tumors, vaginal tumors, ovarian cysts, ovarian cysts, ovarian cysts, polycystic ovary syndrome, immature ovarian failure, ovarian hyperstimulation syndrome, (Such as amenorrhea, menstrual irregularities, excessive menstruation, menorrhagia and premenstrual syndrome), sexual pregnancy, sexual dysfunction (such as sexual intercourse), sexual dysfunction Urinary tract hyperplasia, polycystic kidney [autosomal dominant and autosomal recessive], hereditary nephropathy, malignant tuberculosis, female genital tuberculosis, urinary tract diseases (e.g., (Bladder, ureter, urethra), uterine disease (including bladder, urethra, urethra), sexual dysfunction (such as genital organs [46 XY, mixed], Turner syndrome, (Such as cervical disease [cervical cancer, cervical erosion, cervical hypertrophy, arthritis, cervical cancer, endometrial hyperplasia, endometritis, uterine bleeding, menorrhagia, Uterine perforation, vaginal diseases such as external auditory candidiasis, sexual dyspnea, vaginal ailments, white fever, gonorrhea, rectal fasciitis, vaginal tumors, vaginal tumors, vaginitis [trichomoniasis and uterine endometrial tumors), uterine prolapse, uterine rupture, Embryo transfer, embryo transfer, embryo transfer, embryo transfer, embryo abortion, abortion abortion, vaginal miscarriage, fetal lethal transmission, embryo absorption, embryo absorption, fetal disease (Chorioamnionitis, Fetal growth retardation, fetal hyperglycemia and meconium aspiration), gestational herpes, delivery complications (for example, placental excretion, dysmenorrhea, uterine atresia, fetal membranes, fetal distress syndrome, fetal distress syndrome, Placental adherence, placenta retention, placenta retention, placenta retention, placental adhesion, placental adhesion, placenta previa, postpartum hemorrhage, uterine rupture, prematurity, amniotic fluid depression, Placental abortion), amniotic fluid hyperplasia, cardiovascular pregnancy complication, amniotic fluid embolism, blood pregnancy complication, infectious complication (sepsis complication, parasitic pregnancy complication, postpartum complication), tumor pregnancy complication (trophoblastic tumor, Gestational diabetes, pregnancy outcome, pregnancy outcome, pregnancy outcome, pregnancy outcome, pregnancy outcome, pregnancy outcome, pregnancy outcome, Postpartum hemorrhage and postpartum infections (eclampsia, HELLP syndrome, anterior-eclampsia, EPH pregnancy intoxication, pregnancy outbreaks), obesity disorders, disorders of milk secretion (e.g., Chiari-Promomel syndrome, milk leakage and mastitis) .
[690] sterility
[691] As described above, KGF-2 polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists can be used to treat male or female infertility. Thus, in one embodiment of the invention, there is provided a method of using KGF-2 polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists to treat and / or prevent male infertility. In another embodiment, there is provided a method of using KGF-2 polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists to treat and / or prevent female infertility. Preferred KGF-2 polypeptides for treating infertility include KGF-2 33, ample lengths and mature KGF-2, KGF-2 28, and amino acids 77-208, 80-208 and 93-208 of KGF- Polypeptides as well as any of the KGF-2 mutations described herein. Polynucleotides encoding these polypeptides are also preferable.
[692] Preferred methods of administration of KGF-2 for treating or preventing infertility include oral, rectal, parenteral, intracisternally, intradermal, intravaginal, intraperitoneal, topical (including powders, ointments, gels, creams, Patches), buccal, or oral or nasal sprays. Other modes of administration are described herein. The KGF-2 polynucleotide, polypeptide, variant, antibody, agonist and / or antagonist is preferably administered together with the pharmaceutical carrier as part of a pharmaceutical composition. Suitable carriers are described herein.
[693] The KGF-2 polynucleotides, polypeptides, variants, antibodies, agonists and / or antagonists can be used for the treatment of environmental causes such as coffee, MSG, plastic surgery, Nutrasweet, alcohol, food additives, Exhaust and pollution; age; Congenital infertility; Low modulus sperm; Infectious diseases such as mumps, tuberculosis, influenza, small pox, cytomegalovirus (CMV) infection, chlamydia, mycoplasma, gonorrhea, syphilis and other sexual transmission diseases; Endocrine diseases such as diabetes; Neurological diseases such as hemiplegia; High fever; Endometriosis; Toxins, such as those found in lead, ethylene oxide, chemical and materials industries, such as the paper industry, contained in paints, varnishes and automotive manufacturing reagents; Chemotherapy; Low or overweight; Obesity or overweight gain; stress; Ovulation disorder; Hormone imbalance, Cushings Syndrome; Tubular collapse; Pelvic infection; Surgical adhesions; Intrauterine device (IUD); Cervical disorders such as anatomical problems, cervical infection and mucus quality; Cervical stenosis; Uterine disorders such as intrauterine adhesion, endometriosis and / or endometrial infection; Asherman's Syndrome, uterine fibrosis; Ovarian scar tissue; Ovarian cysts including chocolate cysts; Asthenospermia; Mature suspension; Hypospermia; Sertoli Cell-syndrome; Gonadotropin deficiency, including those resulting from an extended pituitary tumor that damages LH and FSH secretion, resulting from a surgical trauma injury, a trauma that damages the portal blood supply; Anabolic steroids; nicotine; Forbidden drugs such as marijuana, heroin and cocaine; Habitual exposure to alkaline drugs, procarbazine, some halogenated hydrocarbons used in pesticides and large amounts of alcohol; Pelvic inflammatory disease (PID); Epididymitis; Exposure to toxic substances or risks such as lead, cadmium, mercury, ethylene oxide, vinyl chloride, radiation and x-rays; Prescription drugs for ulcers or psoriasis; Intrauterine DES exposure; Elevated temperature - male penile exposure to hot water, vortex, and steam; Healing; Undisturbed testicles; Vitamin deficiency; Prior abortions; And cyclophosphamide. ≪ Desc / Clms Page number 7 >
[694] KGF-2 polynucleotides, polyprepides, modifications, antibodies, agonists and / or antagonists may also be used to treat or prevent primary infertility or secondary infertility. KGF-2 can also be used to treat transient infertility or permanent infertility.
[695] The KGF-2 polynucleotide, the peptidic peptide, the variant, the antibody, the agonist and / or the antagonist may be administered in combination with other fertilizing agents such as chlorimitin citrate (clomid, verticalpin), progesterone and / or 17 beta -estradiol can do.
[696] KGF-2 can be used to treat female infertility during natural pregnancy or during assisted reproduction. Assistive reproductive techniques include IVF, embryo transfer (ET), gonadotropin transfer (GIFT), embryo transfer (ZIFT), donor oocytes, donor sperm and embryo donor embryos, in vitro fertilization (IVF) And micro-manipulation of the oocyte and embryo. In IVF-ET, oocytes are surgically removed, modified in vitro, and placed in the uterus or fallopian tube of the same woman. In the oocyte donation, as in ET, the oocyte is recovered from the donor, modified in vitro, and transferred to infertility recipients. This procedure requires concurrency between the provider and the recipient, which is generally achieved by administering a steroid hormone to the recipient. In standard IVF-ET, treatment performed to induce multiple follicular growth often leads to insufficient luteal function. Therefore, implantation can not be performed without using a supplemental treatment using molecules such as KGF-2.
[697] One preferred delivery method of KGF-2 for the treatment or prevention of female infertility is disclosed in U.S. Patent No. 5,869,081, the disclosure of which is incorporated herein by reference, Type emission system.
[698] Polysiloxane carriers have been used as delivery methods of progesterone as a contraceptive method for lactating women (Croxatto et al., 1991, in "Female Contraception and Male Fertility Regulation. Advances in Gynecological and Obstetric Research Series," Reinnebaum et al. (Simon et al., 1986, Fertility and Sterility, 46: 619), which is functionally equivalent to the function of estradiol in women [Stumpf et al., 1982, J. Clin. Endocrinol. Metab., 58: 17β-estradiol and / or progesterone-saturated polysiloxane rings and cylinders for endometrial priming are disclosed in women of agonadal. This ring and cylinder system was used to achieve serum levels of 17 [beta] -estradiol and progesterone that fall within the normal range for the entire menstrual cycle. U.S. Patent No. 4,816,257 discloses the use of a polysiloxane ring containing 17 -Estradiol or 17 -Estradiol and progesterone to mimic normal steroid hormone levels in functionally non-gonadal human women.
[699] The present invention provides a method of administering KGF-2 for the achievement and maintenance of pregnancy. The method of the present invention comprises the steps of inserting a carrier containing KGF-2 into the vagina of a woman and maintaining said carrier in the vagina for about 1 to 28 days. In a preferred embodiment, the carrier is a polysiloxane ring having an ex vivo release rate of from about 1 [mu] l / day to 1000 mg / day, whilst this amount is at a therapeutic discretion.
[700] The method of the present invention can also be used to treat or prevent infertility in women undergoing assisted reproduction. The method comprises the steps of inserting a carrier containing KGF-2 into the vagina of a woman and maintaining the carrier in vagina for 7 to 12 weeks of gestation. In a preferred embodiment, the carrier is a polysiloxane ring wherein the rate of ex vivo release is from about 1 [mu] l / day to 1000 mg / day KGF-2.
[701] The present invention relates to a method for administering KGF-2 to a female having a functioning ovary and functionally to a non-gonadal female. A woman with a functioning ovary that can not be contraindicated because the infertile woman or partner is infertile can become pregnant through assisted reproduction techniques. However, hormone therapy used to induce multiple follicular growth causes insufficient production of progesterone by the corpus luteum. Thus, the initiation and maintenance of implantation is impaired. Functionally, non-gonadal females are infertile, resulting from undeveloped or improperly developed ovaries, surgical removal of the ovaries, or other ovarian failure or dysfunction. Assisted reproductive techniques such as OD, IVF and ET functionally allow women with non-gonads to become pregnant. However, this assisted reproductive technique requires a hormone replacement technique to achieve the endometrium to achieve and maintain the pregnancy.
[702] Thus, in the present invention, KGF-2 can be used to treat or prevent infertility, in particular through the promotion of embryo implantation. The present invention provides a method of administering KGF-2 for the attainment and maintenance of pregnancy by assisted reproductive techniques in a human female of a normal gonad and a female of a functionally non-gonadal. The method comprises the steps of inserting a KGF-2 containing carrier into the vagina of a normal gonadal human or a functionally non-gonadal female, and maintaining the carrier in the vagina for at least about 28 days.
[703] The present invention also provides a method for hormone replacement therapy for a human female undergoing assisted reproductive therapy. The method comprises the steps of inserting a carrier containing KGF-2 into the vagina of a woman undergoing assisted reproductive therapy and maintaining the carrier in vagina for about 7 to 12 weeks in pregnancy.
[704] The physiologically acceptable carrier containing KGF-2 useful in the method of the present invention is preferably a ring-shaped solid carrier made of silicone rubber (or alternatively referred to herein as polysiloxane) or other suitable material. The delivery of steroid hormones by polysiloxane rings is well known in the art. The rate of release of KGF-2 from the polysiloxane ring depends on factors including the surface area of the ring. Thus, the amount of KGF-2 in the ring is readily described in terms of the in vitro release rate of KGF-2 released from the ring. In vivo release rates are commonly used in the art to characterize hormone containing polysiloxanes. KGF-2 containing polysiloxane rings having an in vitro release rate of from about 0.001 mg to about 1000 mg per day are considered useful in the methods of the present invention. In a preferred embodiment, the polysiloxane ring has an in vitro release rate of from about 0.1 mg to about 100 mg of KGF-2 per day. In a most preferred embodiment, the polysiloxane ring has an in vitro release rate of from about 0.1 mg to about 10 mg of KGF-2 per day.
[705] The KGF-2 containing polysiloxane carrier is administered by inserting into the vagina. This ring is inserted in the vagina and located near the cervix. The ring can be inserted and removed in a manner similar to that of a diaphragm conventionally used by female patients, thus providing another advantage of the present invention.
[706] The carrier containing KGF-2 may be administered about 2 to 7 days, preferably 3 days, before embryonic transfer, and may be supplemented by other hormone administration, for example, oral administration of 17 [beta] -estradiol or progesterone. In a preferred embodiment, the carrier is a ring and the embryo transfer is inserted on the third day prior. The carrier is removed after about 28 days and replaced with another carrier. If pregnancy occurs, the carrier should have enough KGF-2 to maintain pregnancy until luteal placental transfer, where dosing can be done discontinuously. In a preferred embodiment, the ring is kept in the vagina continually, and the administration is stopped at about 12 weeks of gestation.
[707] Injury, occupational disease
[708] KGF-2 was shown to stimulate the proliferation of various tissues. Thus, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat injuries or occupational diseases.
[709] Examples of treatable and diagnosed injuries, occupational diseases and addictions include: occupational diseases such as farmer's disease, farmer's lungs and silo filler's disease, avian lovers' lungs, occupational dermatitis, Silicosis such as gas coma, laboratory infection, asbestosis, beryllium poisoning, cholinergic disease, Kaplan's syndrome, iron deposits, silicosis such as silicosis and silica tuberculosis, addiction such as alcoholism, for example alcoholism such as alcoholic cardiomyopathy Alcoholic diarrhea, Alcoholic withdrawal delirium, Silver poisoning, Scrubs and stab wounds, such as spider gonorrhea, insect cramps and stab wounds, snake saccharides, Tick poisoning, for example tick tick paralysis, cadmium poisoning, carbon tetrachloride poisoning, drug poisoning, for example Water-induced sudden onset, drug-induced rash, e.g., toxic epidermal melasma, sputum erythematosus and seropositivity, drug-induced dyskinesias and neurotic malignant syndromes, erectile dysfunction, fluorosis, food poisoning such as botulism, Mushroom poisoning, Salmonella food poisoning and staphylococcus food poisoning, gas poisoning, such as carbon monoxide poisoning, inert gas anesthesia, toxic hepatitis, lead poisoning, mercury poisoning, fungal poisoning, for example, Induced neuropathy, wounds and injuries such as abdominal injuries such as traumatic diaphragmatic hernias, splenic rupture, such as non-necrosis, gastrointestinal rupture, Traumatic amputations, arm injuries such as frontal limb injuries such as radial and vertebral fractures, correlated fractures, shoulder dislocations, shoulder fractures, tennis elbow and wrist injuries, asphyxiation, Such as, for example, chemical burns, electrical burns, inhalation burns, burns, burns, burns, burns, Such as smoke inhalation injury, eye burns and sunburn, left upper limb, dislocation such as buttocks and shoulder dislocation, drowning, eg drowning, electrical burns and light injury, esophagus perforation, leakage of diagnostic and therapeutic substances, For example, femoral fracture such as hip fracture, for example, femoral neck fracture, fracture fracture, fracture fracture, unilateral fracture, Fractures such as open fractures, spontaneous fractures, stress fractures, unconventional fractures such as fractures of the upper arm of the elbow, radial fractures such as call fractures, rib fractures, shoulder fractures, skull fractures such as jaw fractures, The mandible Fracture of the maxilla, orbital fracture and orbital fracture, vertebral fracture, tibial fracture, fracture of the femur, for example, fracture of the montegia, frostbite, eg alumnae, injury such as finger injury, head injury, Such as concussions, cerebrospinal fluid, CSF rupture, occlusive head injuries, facial injuries such as facial injuries such as eye injuries such as eye burns, eye extrinsic and perforative eye injuries, jaw fractures, For example, mandibular and maxillary fractures, mandibular injuries such as mandibular and maxillary fractures, maxillary fractures, maxillary fractures, brainstem, skull fractures such as mandibular and maxillary fractures such as mandibular and maxillary fractures, For example, sunstroke, leg injuries such as ankle injuries, femoral fractures such as hip fractures such as femoral neck fractures, foot injuries, hip dislocations, knee injuries and tibial fractures, bottle, Multiple scarring, radiation injury such as radiation-induced abnormalities, radiation-induced leukemia, radiation-induced tumors, radiation necrosis, laboratory radiation injury, radiation pneumonitis and radiation dermatitis, Such as fractures, splenic rupture such as nonspecific, gastrointestinal rupture, uterine rupture such as uterine perforation, magnetic cleavage, traumatic shock such as fracture syndrome, soft tissue injury, spinal injury such as spinal compression, spinal injury, For example, vertebral fractures and knee injuries, sprains and strains, for example, repetitive strain injuries, dry cell injuries, chest injuries such as aneurysms, heart injuries and rib fractures, For example, fracture syndromes, tooth dislocations, vaginal perforation, injury, non-perforating injuries such as concussion and occlusive head injuries and perforation injuries such as perforating eye injuries, gunshot wounds, If it includes a needle injury.
[710] Blood and lymphatic diseases
[711] KGF-2 polynucleotides, polypeptides, agonists and / or antagonists may be used to treat and / or screen blood and lymphatic diseases.
[712] Examples of blood and lymphatic diseases that can be treated and retrieved include: anemia (e.g., anemia of the fanconi), hemolytic anemia (e.g., autoimmune hemolytic anemia and congenital hemolytic anemia such as congenital erythropoietic anemia, Congenital non-hemolytic hemolytic anemia, sickle-cell anemia, such as hemoglobin SC disease and sickle cell trait, hereditary elliptical constitution and glucose phosphate dehydrogenase deficiency, such as hypersensitivity, hemoglobin C disease, hereditary spherocytosis, Hematopoietic anemia (e. G., Iron-thalassemia, e. G., Hematopoietic < / RTI > (Eg, malignant anemia), myelogenous anemia, neonatal anemia (eg, fetal-fetal anemia) (Eg, transfusion and fetal-maternal transfusion), refractory anemia (eg, refractory anemia), iron amyloid anemia, erythrocyte amyloidosis, fetal wilting (eg, fetal hydrops and nuclear jaundice), Rh immunization, (E. G., Amyloidosis, e. G., Amyloidosis, e. G., Leukemia), hyperglycemia Amyloid neuropathy and cerebral amyloid angiography, chronic glomerulonephritis, heavy chain diseases such as immunoproliferative small bowel disease, multiple myeloma, POEMS syndrome, valgendroma to globulinemia), protein S deficiency.
[713] Other examples of treatable and searchable blood and lymphatic diseases include: bone marrow diseases such as ankylosing spondylitis, myelodysplastic syndromes (e.g., refractory anemia, such as refractory anemia, Anemia, seizure hemoglobinuria and myelogenous leukemia), myeloproliferative diseases (e.g., myelopathic anemia, acute myelogenous leukemia, hematopoietic response, myelofibrosis, myelogenous leukemia, intrinsic erythropoiesis, hemorrhagic thrombocythemia and thrombocytosis) Hemoglobinopathies such as sickle cell anemia (e.g. hemoglobin SC disease and sickle cell trait), hemoglobin SC disease, thalassemia (including alpha-thalassemia, such as fetal hydration and beta Thalassemia), hemorrhagic ischemia, such as, for example, Abrinino cinemia, Christmas illness, disseminated intravascular coagulation, factor VII deficiency, factor XI deficiency, factor XII deficiency, factor XIII (Including factor V deficiency and factor X deficiency), Swartzmann syndrome, Bernard-Suller syndrome, hemolytic uremic syndrome, platelet storage pool deficiency, thrombocytopenia, hemolytic thrombocytopenia (thrombocytopenia purpura (Including, for example, idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura and Wisscott-Aldrich syndrome), globulin-forming purpura, 쇤line-henoch-jaune, thrombocytopenic purpura (idiopathic thrombocytopenic purpura) Thrombocytopenic purpura, thrombocytopenic purpura, Thoracic-Aldrich syndrome, hereditary thrombotic capillary dilatation, vitamin K deficiency (including neonatal hemorrhagic disease) and Bonnbrand disease, leukocyte diseases such as eosinophilia (eosinophilic hyperplasia, Eosinophilia - myalgia syndrome, eosinophilic granuloma and hypereosinophilic syndrome, such as increased lung eosinophilia), infectious mononucleosis, Leukocytopenia (including neutropenia and lymphocytopenia, including idiopathic CD4-positive T-lymphocytopenia), Pelgere-posterior hyperplasia, phagocytic bacterial function (Including Kidyak-Higashi syndrome, chronic granulomatous disease, Job Syndrome), methemoglobinemia, pancytopenia, polycythemia vera, hematogenous leukemia and sulphated hemoglobinemia.
[714] Further examples of treatable and diagnostible blood and lymphatic diseases include: lymphadenopathy (including cat-scratch disease and mesenteric lymphadenitis), lymphangiectasia, lymphatic tuberculosis, lymphatic edema (epitheliopathy and filariasis), limpocell, Lymphoid proliferative diseases (non-gamma globulinemia, amyloidosis, such as amyloid neuropathy and cerebral amyloid angiopathy, giant lymph node hyperplasias, heavy chain diseases such as immunoproliferative small intestine disease, immunoblastic lymphadenopathy, infectious mononuclear species, hair detail Lymphoma (including Hodgkin's disease, Hodgkin's lymphoma, such as B-cell lymphoma, Burkitt's lymphoma, leukemia, leukemia, leukemia, myeloma leukemia including acute myeloid leukemia and acute myeloma leukemia) Kidney lymphoma, AIDS-related lymphoma, mucosal-associated lymphoid tissue lymphoma and small cell lymphoma, disseminated lymphoma, such as disseminated large lymphoma , Small cell lymphoma, small non-associated cell lymphoma, follicular lymphoma, e.g., follicular large-cell lymphoma, follicular < RTI ID = 0.0 > Lymphoma, small cell lung lymphoma, small cell lymphoma, small cell lymphoma, follicular small cell lymphoma, follicular small cell lymphoma, follicular small cell lymphoma, mixed cell lymphoma and follicular small cell lymphoma, high grade lymphoma such as immunoblastic large cell lymphoma, lymphoblastic lymphoma, Cell lymphomas, disseminated small cut-cell lymphomas, large lymphomas such as disseminated large lymphoma, follicular large-cell lymphoma, follicular large-cell lymphoma, follicular large-cell lymphoma, Giant large cell lymphoma, Ki-1 large cell lymphoma and immunoblastic large cell lymphoma, low grade lymphoma, e. G., Follicular mixed cell lymphoma , Mucosal-associated lymphoid tissue, follicular small cut cell lymphoma, and small lymphocytic lymphoma, mixed cell lymphoma such as diffuse mixed cell lymphoma and follicular mixed cell lymphoma, follicular small cut Cell lymphoma, small lymphocytic lymphoma, and small non-cleaved cell lymphoma, t-cell lymphoma, such as lymphoblastic lymphoma, dermal T-cell lymphoma such as Ki-1 large-cell lymphoma, Sezary syndrome and peripheral T-cell lymphoma, undifferentiated lymphoma such as disseminated large-cell lymphoma and small non-excised cell lymphoma such as Burkitt's lymphoma, lymphomatous papillomatosis, , Sarcoidosis (including pulmonary sarcoidosis and subepithelial adenocarcinoma), tumor dissolution syndrome, mucocutaneous lymphadenopathy syndrome, cutaneous endometriosis (goshiosis, histiocytosis, e.g., malignant tissue diseases, Such as eosinophilic granulocytes, Had-Scholler-Cyclin syndrome and Letterer-Siebe disease, non-Langerhans cell lines, such as acute monocytic leukemia, large lymphoma such as Ki-1 large cell lymphoma, Langerhans- - histiocytosis, such as senegal histiocytosis, nigro-Picky disease, psoriatic blue tissue syndrome and inflammatory yellow granuloma, mast cell sarcoma), non-respiratory disease (hypervascular disease, myelogenous leukemia, , Splenic rupture such as nonspecific, non-tubal and spleen tuberculosis), thymic hyperplasia, thymoma, lymph node tuberculosis, for example, Kings Evil.
[715] Tumor-like diseases and abnormalities
[716] KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat, prevent and / or detect a neoplastic disease and / or an abnormality.
[717] Examples of hereditary diseases and disorders that can be treated or searched include: drug-induced dyskinesia, multiple dyskinesias such as Alagill's syndrome, Angelman syndrome, basal cell nevus syndrome, Beckweed-Bydeman syndrome, Bloom syndrome Dysentery syndrome, Down syndrome, ectodermal dysplasia, e. G. Ellis-van Klewd syndrome and local dermatogenesis deficiency, Gardner ' s syndrome, frontal encephalopathy, Prader-Willi syndrome, Prunella syndrome, Prunella syndrome, Congenital rubella syndrome, Rubenstein-Taibe syndrome, Parkinson's disease, Parkinson's disease, Syndrome, short rib-polydactylil syndrome, and Denver's syndrome, Wolfman syndrome, gelbeather syndrome, radiation-induced abnormalities, chromosomal anomalies, Pseudo-Willy syndrome, sex chromosomal abnormalities such as Bonnevie-Ulrich syndrome, dermatophyte deficiency, e.g., dermatomyositis, e.g., dermatomyositis, 46, XY gonadal dysgenesis, mixed gonadal dysfunction, Kalman syndrome, Klepelter syndrome, eye thalassic syndrome, Turner syndrome, and XYY karoni type and digestive system abnormalities.
[718] Respiratory diseases
[719] KGF-2 stimulated cell proliferation in respiratory tract. Thus, KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or detect respiratory diseases.
[720] Examples of treatable or detectable respiratory tract diseases include, but are not limited to, bronchial disorders such as asthma (including exercise-induced asthma and asthma persistence), bronchial fistula, bronchial hyperreactivity, bronchial tumor, bronchospasm, bronchiolitis, bronchitis Laryngeal carcinoma, laryngeal tumor, laryngeal tumor, laryngoconjunctivitis, laryngospasm, laryngitis, such as Crohn's disease, laryngeal stenosis (including laryngeal pneumonia, bronchopneumonia, and bronchopneumonia), ciliary motility diseases such as Cartagener syndrome, , Laryngeal tuberculosis, speech paralysis, speech disorders such as schizophrenia and depression), lung diseases such as atelectasis such as mesenchymal syndrome, bronchopulmonary syndrome, congenital cystic fibrosis of the lung, cystic fibrosis , Pulmonary transforming cell sarcoma, hemoptysis, lung abscess, fungal lung disease such as allergic bronchial aspergillosis and neomycostasis carinii pneumonia, interstitial lung disease, For example asphyxia, berylliosis, internal < RTI ID = 0.0 > cholinergic < / RTI > inflammation, Kaplan disease, Crohn ' s disease, Alzheimer's disease, Lung fibrosis, Wegener's granuloma), obstructive pulmonary disease, viral bronchitis, emphysema, parasitic lung disease such as alveolar sclerosis, pulmonary arthropathy, for example, arthritis, Bronchopneumonia, aspiration pneumonia, such as bronchopneumonia, bacterial pneumonia, such as tuberculous laryngeal pneumonia, mycoplasma pneumonia, bronchopneumonia, Plasma pneumonia, Rickettsia pneumoniae and Staphylococcus pneumoniae, Newmastisis carinii pneumonia, Virus pneumonia), pulmonary alveolar proteinosis, pulmonary edema, pulmonary eosinophilia, pulmonary vein occlusion, respiratory distress syndrome , Such as glaucomatous block disease, adult respiratory distress syndrome, Schizmita syndrome, Shilopillar disease, pulmonary tuberculosis, e.g. Nasal polyps, such as nasal polyps, sinus tumors such as maxillary sinus tumors, ethmoid sinusitis, frontal sinusitis, oesophageal carcinoma, , Sinusitis such as facial and sphenoid sinusitis, fever fever, perennial allergic rhinitis, atrophic rhinitis and vasomotor rhinitis, rhinitis such as non-sclerosis).
[721] Respiratory diseases that can be treated and / or diagnosed include: pleural diseases such as chylothorax, pleural sinusitis (e.g., tuberculous sinusitis), hemopneumothorax, hemothorax, hypopyon, pleurisy, pleural effusion such as malignant pleural effusion , Pleural tumors such as malignant pleural effusion, pleurisy such as pleural pneumonia, thoracic tuberculosis, pleural tuberculosis such as tuberculous sinusitis, respiratory disorders such as apnea such as sleep apnea, , Chest-stroking breathing, coughing, dyspnea such as paroxysmal dyspnea, wheezing, hyperventilation such as respiratory alkalosis, laryngospasm, moxibustion, oral respiration, respiratory distress syndrome, Respiratory distress syndrome, low respiration, endogenous positive-pressure respiratory and respiratory paralysis, respiratory distress syndrome, respiratory distress syndrome, respiratory distress syndrome, respiratory distress syndrome, respiratory acidosis, For example Exogenous allergic alveolitis, such as those of avian enthusiasts and farmers, allergic bronchitis aspergillosis, asthma, such as exercise-induced asthma and asthma persistence, fever fever, allergic rhinitis , Bronchial cysts such as bronchial cysts, bronchopulmonary sequestration, posterior occlusion, congenital cysts of the lungs, malformations of the ciliary body, Cartagener's disease, schizmitter's disease, tracheobronchial enlargement, respiratory tract rupture, Bronchitis such as bronchitis such as viral bronchitis, general cold, pleural sinusitis, such as tuberculous sinusitis, influenza, laryngitis, such as epiglottitis, Legionella disease, e. G. For example, leukodystrophy, pleurisy, pleural effusion such as pleural pneumonia, pneumonia such as bronchopneumonia, pleural pneumonia, respiratory pneumonia such as lipopneumonia, bacterial pneumonia, Mycoplasma pneumonia, Mycoplasma pneumonia, Rickettsia pneumoniae and Staphylococcus pneumoniae, Newstomy stiffness pneumonia, viral pneumonia, rhinitis, non-sclerosis, sinusitis, such as ethocelitis, frontotemporal, maxillary sinusitis and sphenoiditis, tonsillitis, Such as bronchial tuberculosis, tuberculosis, pertussis, pertussis, respiratory tract tumors, such as bronchial tumors, laryngeal tumors, pulmonary tumors, for example, Bronchogenic carcinomas such as maxillary sinus tumors, pleural tumors such as malignant pleural effusion, tracheal tumors, organ diseases, eg, bronchogenic carcinomas, bronchogenic carcinomas, For tracheal tumors, tracheal stenosis, tracheitis, bronchopulmonary hypertrophy, and tracheobronchial leak.
[722] Examples of treatable and diagnosable otorhinolaryngological disorders include: ciliary dysmotility disorders such as, for example, car- tagener's disease, ear infections such as middle ear cholesteatoma, acquired acquired dysplasia, heterozygous, migraine, hearing disorders, Such as hearing loss, sudden hearing loss, partial hearing loss, such as bilateral hearing loss, inductive hearing loss, functional hearing loss, high frequency hearing loss, sensory nerve impairment such as hearing loss, noise-induced hearing loss and senile hearing loss, For example, a herpes zoster, a maze disease such as cochlea, an endolymphatic species such as menorrhagia, myosinitis, vestibulosis, such as movement disorders such as space dysentery, dizziness, For example, neurofibromatosis such as neurofibromatosis 2, central auditory disease, neuropathy, neurofibromatosis, neurofibromatosis, for example, neurofibromatosis, e.g. neurofibromatosis, For example, , Laryngeal perforation), posterior laryngeal disease such as laryngeal sarcoma, occipital larynx, laryngeal tumor, laryngeal periostitis, laryngospasm, laryngitis such as cough, laryngeal stenosis, laryngeal tuberculosis, speech paralysis, Nasal polyps, nasal polyps, sinus tumors, such as maxillary sinus tumors, nasal polyps, nasal polyps, nasal polyps, nasal polyps, Rhinitis, such as sinusitis, sinus tumors such as sinusitis, sinusitis such as sinusitis, sinusitis, facial sinus and sphenoid sinus, fever fever, perennial allergic rhinitis, atrophic rhinitis and vasomotor rhinitis, A neurofibromatosis 2, a nasal tumor, such as a nasal polyp, a sinus tumor, such as a maxillary sinus tumor, a pharyngeal tumor, such as a hypopharyngeal tumor, a neuroendocrine tumor, for example, Nontoxic Amount, job Doosung tumor, for example, one-way tumor, throat, pharynx station perch, tonsillitis and tongue bind deficiency.
[723] Neuropathy
[724] KGF-2 polynucleotides, polypeptides, agonists and / or antagonists can be used to treat and / or detect neuropathy.
[725] Examples of neuropathy that can be treated and / or diagnosed include: brain disorders (including metabolic brain diseases such as phenylketonuria, such as maternal phenylketonuria, pyruvate carboxylase deficiency, pyruvate Neurogenic tumors, such as subpleural tumors, ventricular tumors such as choroidal neurotic tumors, hypothalamic tumors, tumoral tumors, cannabis diseases, cerebellar diseases, brain tumors, For example, atrophic ataxia, such as spinal cerebellar degeneration, e.g., vasodilatatory ataxia, cerebellar ataxia, pre-ederior ataxia, macado-Joseph's disease, olivary neuroblastoma, (Eg, carotid artery disease such as carotid artery thrombosis, carotid artery stenosis and Miyamoto's disease, cerebral vascular disease, cerebral vasculopathy, Cerebral arteriosclerosis, cerebral arteriovenous dysplasia, cerebral artery aneurysm, cerebral embolism and thrombosis such as carotid artery thrombosis, thrombosis and valenberg disease, cerebral hemorrhage, such as epidural hematoma, dura mater Cerebral palsy and subarachnoid hemorrhage, brain fracture, cerebral ischemia such as transient cerebral ischemia, clavicular submandibular gland syndrome and posterior vertebral artery hypertrophy, vascular dementia such as dementia, periventricular white matter aphasia, coronary headache, Migraine headaches, migraine headaches, dementia such as AIDS dementia complexes, senile dementia such as Alzheimer's disease and Kruszfeld-Jakob disease, senile dementia such as Alzheimer's disease and progressive nuclear paralysis, vascular dementia, Such as dementia, encephalitis such as axonal encephalitis, viral encephalitis such as encephalitis, Japanese encephalitis, St. Louis encephalitis, tick-borne encephalitis and West Nile fever, acute disseminated encephalopathy Such as, for example, asthma, such as asthma, meningoencephalitis, such as, for example, uveal meningoencephalitis, Parkinson's disease and subacute sclerosing panencephalitis, subacute sclerosing panencephalitis, cerebral palsy such as periventricular softening, epilepsy such as general epilepsy such as infantile seizures, Partial seizures such as complex partial epilepsy, frontal lobe epilepsy and trunk lobe epilepsy, post traumatic epilepsy, epileptic seizure status such as persistent partial epilepsy, Haller-Boders-Spars syndrome, hydrocephalus, e.g., Dendy-Walker syndrome and general pressure hydrocephalus, hypothalamic diseases such as hypothalamic tumors, brain malaria, sleeping seizures such as deafness seizures, septic grayish myelitis, cerebral tumors, Rett's syndrome, Reye's syndrome, Disease, brain toxoplasmosis, intracranial tuberculosis and gelbeerger syndrome, central nervous system infection, such as AIDS dementia complex, brain abscess, subdural hypoplasia, A bacterial meningitis such as a bacterial meningitis such as, for example, a bacterial meningitis, such as a bacteremia, a bacteremia, a bacteremia, a bacteremia, Hemophilus meningitis, listerioma meningitis, meningococcal meningitis such as Waterhouse-Friedrichs's syndrome, pneumococcal meningitis and meningitis tuberculosis, fungal meningitis such as cryptococcal meningitis, subdural effusion, meningococcal meningitis Encephalitis, e.g., uveal meningoencephalitis syndrome, myelitis such as transverse myelitis, nerve syphilis such as spinal cord, poliomyelitis, such as auricular poliomyelitis and posterior poliomyelitis syndrome, prion diseases - Jacob disease, bovine spongiform encephalopathy, Jestman-Straussler syndrome, crew, scrapie), brain toxoplasmosis Neuroendocrine tumors such as brain tumors such as subendothelial tumors, ventricular tumors such as choroidal neurotic tumors, hypothalamic and perianal tumors, meningiomas, spinal tumors such as epidural tumors, Neurodegenerative diseases such as canker dystrophy, generalized brain sclerosis such as adrenal leukodystrophy, axonal satellite encephalitis, globular cell leukodystrophy, generalized brain sclerosis such as dysentery leukodystrophy, allergic encephalomyelitis, necrotizing hemorrhage Transverse myelitis, ophthalmoplegia, scrape, swayback, chronic fatigue syndrome, visceral, hypertensive neuropathy, meningiomas, spinal cord diseases such as congenital anomalies, such as cerebrospinal fluid, progressive multi-point leukodystrophy, multiple sclerosis, Myasthenia gravis, spinal cord compression, spinal cord compression, spinal cord tumors such as epidural tumors, spinal cord convulsion, spinal cord, striae, myasthenia gravis, Gangliosidosis G (M1), Sandhoff's disease, Thai-Sachs syndrome, Down syndrome, Down syndrome, Down syndrome, Gangliosidosis, Parkinson's disease, Parkinson's disease, Parkinson's disease, Parkinson's disease, Parkinson's disease, Parkinson's disease, Parkinson's disease, Parkinson's disease, Neurological disorders such as complete neurotoxicity, neurological deficits, neurological deficits, neurological deficits, neuropsychiatric disorders, neuropsychiatric disorders, neuropsychiatric disorders, Deficits, such as aphasia, such as, for example, dysmenorrhea, Arnold-Karyi formation disorder, brain disorders, meninges, meningocervical spinal cord, spinal fusion deficiencies such as cystic vertebral and latent vertebral arthritis, For example, Hereditary sensory and autonomic neuropathy such as congenital ankle and familial autonomic dysfunction, neurological symptoms (eg, cognitive impairment such as Gustman's syndrome, Communication disorders such as forgetfulness, such as retrograde amnesia, aphasia, neurogenic bladder, depressive episodes, auditory disorders such as hearing loss, partial hearing loss, voice mobilization and tinnitus, speech disorders such as aphasia, insomnia For example aphasia, such as aphasia, such as aphasia, Broca's aphasia and Venickian aphasia, dysphonia, aphasia, aphasia, such as aphasia, such as acquired dyslexia, Dementia, anorexia, ankylosing spondylitis, speech disturbances such as aphasia, and depression, cerebellar rejection, delirium, Such as atherosclerosis, ataxia, hallucination, menstrual pain, movement disorders, such as Angelman's syndrome, ataxia, anorexia, chorea, dystonia, hypotonia, muscle relaxation, Such as Stevens-Man syndrome, paresthesias, paralysis, facial paralysis, such as ear shingles, gastroparesis, hemiplegia, ophthalmoplegia such as diplopia, Duan syndrome, Horner's syndrome, chronic progression Paranephritis, e.g. Brown-Sikhard syndrome, limb paralysis, respiratory paralysis and speech paralysis, paralysis, ghost limb, taste disorders, such as paranasal sinus paralysis, such as Kors syndrome, training paralysis, Sleep disorders, such as, for example, hyperplasia, crenin-Levin syndrome, insomnia and sleepwalking, convulsions, such as tinnitus, tinnitus and taste disorders, visual disturbances such as amblyopia, obtusight, color blindness, diplopia, For example, the opening disorder, numbness, Such as, for example, facial laxity, congestion, persistent plant conditions and syncope and dizziness, neuromuscular disorders such as congenital working conditions, amyotrophic sclerosis, Lambert-Eaton myasthenia, motor neuropathy, such as spinal muscular atrophy, Myocardial infarction syndrome, peripheral nerve palsy, stiff-man syndrome, peripheral nerve palsy, peripheral nerve palsy, stiff-man syndrome, peripheral nerve palsy, Niger-Hoff Manifest, posterior poliomyelitis syndrome, muscle dystrophy, grave myasthenia, atrophic muscular atrophy, congenital dystonia, Idiopathic neuropathy, schizophrenia, schizophrenia, schizophrenia, schizophrenia, schizophrenia, schizophrenia, schizophrenia, schizophrenia, For example, neuropathy, such as aural neuropathy, for example neuro-neuropathy 2, facial neuropathy such as facial nerve pain, Postural, visual motion disorders such as amblyopia, nystagmus, nerve paralysis, ophthalmoplegia such as Duan syndrome, Horner's syndrome, chronic progressive external ophthalmoplegia such as Kors syndrome, strabismus such as esotropia and exotropia, Such as neuropathy, ocular neuropathy, for example, ataxia, for example, hereditary atrophy, ocular disc drains, ocular neuropathies such as optic nerve sheaths, papillary edema, triple neuralgia, speech paralysis, Diabetic neuropathy such as diabetic foot, neuropsychiatric syndrome such as carpal tunnel syndrome, arousal syndrome, cervical exit syndrome, ulnar nerve compression syndrome, neuralgia, neuropathy, Neuralgia and acute neuralgia, neuritis, such as experimental allergic neuritis, optic neuritis, multiple neuritis, multiple neuromyositis and neuromyositis such as multiple nerves Hereditary sensory and autonomic nervous disorders such as congenital hyperalgesia and familial disorders such as familial hypercholesterolemia and familial hyperalgesia, , POEMS syndrome, sciatica, unexplained sweating and tetany).
[726] Metabolic and endocrine diseases
[727] KGF-2 polynucleotides, polypeptides, agonists and / or antagonists may be useful for treating and / or diagnosing metabolic or endocrine diseases.
[728] Examples of nutritional and metabolic diseases that can be treated or diagnosed are: acid deficiency, acid-base imbalance, acidosis (including lactic acid, syndrome or respiratory), diabetic ketoacidosis, ketosis, alkalosis, respiratory alkalosis, calcium It has been reported that metabolic sisorders, calcification, resistant calcium formation, CREST syndrome, nephrolithiasis, pathological decalcification, hypocalcemia, hypocalcemia, tetany, osteomalacia, parathyroid hyperplasia, (Including laboratory and insulin-dependent, lipodynamic, non-insulin-dependent), diabetic vascular disease, diabetic foot, diabetic gastrointestinal tract, fetal diabetes mellitus, diabetes mellitus Hyperglycemia, hypoglycemia, hyperlipidemia, hyperlipidemia, hypoglycemia, hypoglycemia, hypoglycemia, hyperlipoproteinemia, hyperglycemia, hyperglycemia, hyperglia (Including blind loop syndrome, celiac disease, lactose intolerance, intestinal lipid disorders, tropic sprue), congenital disorders of metabolism (congenital disorders of amino acid metabolism, ocular albinosis, Amyloid neuropathy, brain amyloid angiopathy, amyloid angiopathy, amyloid angiopathy, amyloid angiopathy, amyloid angiopathy, amyotrophic lateral sclerosis, amyloid angiopathy, amyloid angiopathy, (Type I, II, III, III, IV, III, IV), congenital disorders of the metabolism of hydrocarbons, such as congenital disorders of fructose metabolism (fructose-1,6-diphosphatase deficiency, fructose resistance), galactosemia, glucose tolerance, IV, V, VI, VII, VIII), oxathiosis, primary and oxathiosis, mannososis, mucopolysaccharide (I, II, III, VI, VII), polycarboxylase deficiency, Ambassador Bait Diabetes mellitus syndrome, Gilbert's disease, chronic idiopathic jaundice, lipid metabolism, diabetic nephropathy, diabetic nephropathy, diabetic nephropathy, diabetic nephropathy, diabetic neuropathy, (Type III, IV, V), familial lipoprotein lipase deficiency, low lipoproteinemia (including non-beta-lipoproteinemia, low beta-lipoproteinemia, lecithin acyltransferase deficiency, Lepidoptera, Sogren-Larson syndrome, sphingolipidosis (adrenal leukodystrophy, Fabry's disease, gangliosidosis, diabetic retinopathy), hyperlipidemia (cholesterol ester storage disease, Sandhoff's disease, Thai-Sachs disease, Gossyp disease, globular cell leukodystrophy, migratory leukodystrophy, nematicopic disease, The present invention provides a method of treating a disease selected from the group consisting of mucositis, mitochondrial proliferative disease, mitochondrial myocarditis, MELAS syndrome, MERRF syndrome, external chronic progressive ophthalmoplegia, lysosomal storage diseases such as cholesterol ester storage disease, mannososis, (I, II, III, IV, VI and VII), congenital malformations of metal metabolism, hemochromatosis, hepatotenic tick hypersensitivity, hypophosphatasia, familial hypophosphatemia, Periodic paralysis, and congenital disorders of purine-pyrimidine metabolism, gingival hyperparathyroidism, mucoripidosis, fucoside accumulation, poppyria (erythropoietic, erythropoietic, hepatic, acute intermittent, For example, gout, gouty arthritis and Lewis-Nehon syndrome, congenital disorders of syncope, such as, for example, nephrotic syndrome, nephrotic syndrome, cystinuria, heart trophoblastic, cystinuria, Electrolyte depletion, dehydration, hypocalcemia, hypereosinophilia, hypophosphatemia, protein-deficient bowel disease, intestinal lymphangiectasia, and water-electrolyte imbalance (dehydration, hypercalcemia, hyperglycemia, hypoglycemic syndrome, Diabetes mellitus, hypernatremia, hypothyroidism, calamities, hyponatremia, hypocalcemia, hyponatremia, inappropriate adhd syndrome, water toxicity), amyloidosis, ulcer disease, infantile malnutrition such as childhood malnutrition, , Vitamin B ₁₂ deficiency (anemia, malignancy), vitamins A, vitamin B deficiency, cholin deficiency, folate deficiency, pellagra, pyridoxine deficiency, riboflavin deficiency, thiamine deficiency, berryberry, D deficiency, (osteomalacia, fatty tissue), vitamin E deficiency (fatty tissue disease), vitamin K deficiency, magnesium deficiency, potassium deficiency, protein deficiency (protein- ), Swayback, diabetic obesity, morbid obesity, Pickwickian syndrome, Prader-Willi Syndrome, and starvation.
[729] Examples of endocrine diseases that can be treated or diagnosed are: adrenal disease (cortical disease, northertic tumor), adrenal and function (Cushing's syndrome, Aladosteronemia, Barter's disease), adrenal insufficiency Adult adrenal hyperplasia, Waterhouse-Friedrichsson syndrome, Chest tumors, Male chest tumors, Chest fibrocystic disease, Gynecomastia, Lactation disorders, eg, Diabetic (diabetic, insulin-dependent, wolfram syndrome, liposomal, and noninsulin-dependent), diabetic vascular disease, diabetic foot, diabetes mellitus Diabetic nephropathy associated with diabetic foot, diabetic obesity, gastrointestinal diabetes mellitus, fetal diabetes mellitus, dwarfism (coccaccia syndrome, pituitary syndrome, pituitary hyperplasia, , Endocrine tumors such as adrenal cortical tumors, multiple endocrine tumors (type 1, 2a, 2b), endocrine-like syndrome syndrome, ACTH syndrome (global), Solinger-Ellison syndrome, ovarian tumors Thyroid gland, thyroid gland, gonadal disorders such as adrenal hyperplasia (congenital), feminization, testicular feminization, hyperandrogenemia, hypogonadism, hypothyroidism, Osteoporosis, ovarian cysts, ovarian cysts, multiple cystic ovary syndromes, non-mature ovarian failure, ovarian hyperstimulation syndrome, ovarian tumors, Mayg Syndrome, delayed maturation and osteoporosis. (46, XY, mixed type) and Turner syndrome, halitosis, leukodystrophy, Galmal syndrome, Clepelton syndrome, testicular feminization, testicular disease, eg, potential (Exotropia) and Sollinger-Ellison syndrome, parathyroid disease, e.g., hyperthyroidism, hyperthyroidism, testicular hypertrophy, testicular tumors, malignancies, hypercholesterolemia, hyperinsulinemia, teratogenic endocrine type syndromes such as ACTH syndrome Pituitary gland, pituitary gland, thyroid gland, pituitary syndrome, pituitary gland, hypertrophy, hypertrophy, hypogastric hypoplasia (diabetic insipidus, tumor diabetic insipidus, Pheochromocytomas, pituitary tumors, Nelson's syndrome, autoimmune multiple endocrinology, progesterone, vener syndrome, thymic hyperplasia, thymic diseases, such as uteroid disease, pituitary adenomatosis, Thyrotoxicosis, hyperthyroidism (cretinism and muscles), thyroid hormone resistance syndrome, hyperthyroidism, hyperthyroidism, hyperthyroidism, hyperthyroidism, Malignant tumor, thyroid gland, thyroiditis (autoimmune, subacute, purulent), thyrotoxicosis, thyrotoxicosis and endocrine pneumonia.
[730] Disease at a cellular level
[731] Diseases associated with inhibition of cell apoptosis or increased cell survival that can be treated or diagnosed by agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 include, but are not limited to, cancer (e.g., vesicular lymphoma, p53 mutation And cancer such as colon cancer, heart tumor, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, adenocarcinoma, testicular cancer, gastric cancer, neurocytoma, myxoma, myoma, But are not limited to, lymphoma, endothelial cell, osteoblastoma, osteoclastoma, osteogenic sarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer; Autoimmune disorders (e. G., Multiple sclerosis, Sjogren ' syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, (Eg, herpes viruses, poxviruses and adenoviruses), inflammation, graft disease to the host, acute graft rejection and chronic graft rejection, and the like . In a preferred embodiment, KGF-2 polynucleotides or polypeptides of the invention, and / or antagonists are used to inhibit the growth, progression and / or metastasis of cancer in the above-listed diseases.
[732] Additional diseases or conditions associated with increased cell survival include diseases associated with the progression and / or metastasis of malignant tumors and related disorders, such as, for example, an autoimmune disease or condition associated with increased cell survival, such as a KGF-2 polynucleotide or polypeptide or an agent or antagonist of KGF- For example, leukemia (e.g., acute leukemia such as acute lymphocytic leukemia, acute myelogenous leukemia (myeloblastic, zygotic cell, bone morphogenetic, monocytic and acute leukemia etc.) and chronic leukemia, (E.g., chronic spinal cord cysts (granulocytic leukemia and chronic lymphocytic leukemia), intrinsic erythropoiesis, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Such as Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors, such as sarcomas and carcinomas, such as fibrosarcoma, The present invention relates to a method for the treatment of sarcoma, sarcoma, sarcoma, sarcoma, sarcoma, sarcoma, sarcoma, sarcoma, sarcoma, sarcoma, sarcoma, sarcoma, sarcoma, sarcoma sarcoma, sarcoma sarcoma, lymphangioma sarcoma, lymphatic endothelial sarcoma, synovial sarcoma, mesothelioma , Breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary carcinoma, cystadenocarcinoma, follicular carcinoma, bronchus carcinoma, renal cell carcinoma, liver carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder cancer, epithelial carcinoma, glioma, astrocytoma, malignant neoplasm, craniopharyngioma But are not limited to, adenocarcinoma, pineal gland, angioblastoma, auditory neoplasm, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.
[733] A disease which can be detected or treated by a KGF-2 polynucleotide or polypeptide, and an agonist or antagonist of KGF-2, wherein the disease associated with an increase in the number of cells inducing AIDS; Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration, and brain tumors or diseases such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Related tumors]; Autoimmune disorders (e.g., multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, multiple myositis, systemic inflammation Myelodysplastic syndrome (e. G., Aplastic anemia), graft versus host disease, ischemic injury (e. G., Myocardial infarction, stroke and reperfusion injury) Hepatitis related liver damage, ischemia / reperfusion injury, cholestosis (bile duct injury), and liver cancer]; Toxic-induced liver diseases (such as those caused by alcohol), pulmonary shock, cachexia and anorexia.
[734] Wound treatment and epithelial cell proliferation
[735] In accordance with another aspect of the present invention there is provided a pharmaceutical composition comprising KGF-2 polynucleotides, such as KGF-2 polynucleotides, to stimulate epithelial cell proliferation and basal keratinocytes for wound healing, and to stimulate hair vesicle production and dermal wound healing, Or using an agonist or antagonist of the polypeptide and KGF-2. Agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 can be used for the treatment of wound healing, incisional wounds, deep wounds associated with dermal and epidermal damage, eye tissue wounds, tooth tissue wounds, oral wounds, diabetic ulcers, Abnormalities such as ulcers, elbow ulcers, arterial ulcers, venous congestion ulcers, burns due to heat exposure or chemicals, and other complaints related to uremia, malnutrition, vitamin deficiencies and steroids, radiation therapy and systemic treatment of tumor suppressors and antimetabolites And may be clinically useful for stimulating wound healing, including wound healing conditions. KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 may be used to enhance dermis re-establishment following dermal loss.
[736] Agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 can be used to increase adhesion of skin grafts to wound beds and stimulate re-epithelization from wound beds. The following are the types of implants that can be used to increase the attachment of KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 to wound beds: autografts, artificial skin, allografts, Bone grafts, Bone grafts, Bone grafts, Dermal grafts, Delayed grafts, Apples, Epidermoid grafts, Fascia grafts, Whole-thickness skin grafts, Heterologous grafts, Heterologous grafts, Homologous grafts A graft, a hyperplastic graft, a surface graft, a mesh graft, a mucosal graft, an Oer-Thierry graft, a long-term graft, a patch graft, a transplant graft, a whole graft graft, a partial graft graft, and a thick ruptured graft. KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 can also be used to enhance skin strength and improve the appearance of aging skin.
[737] Agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 will also cause hepatocyte proliferation and changes in epithelial cell proliferation in the lung, heart, pancreas, stomach, small intestine, and large intestine. KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 can be used to treat epithelial cells such as sebocytes, hair cysts, hepatocytes, type II lung cells, mucin producing germ cells and other epithelial cells, , Proliferation of the progenitor cells contained within the liver and gastrointestinal tract can be promoted. KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 may promote the proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
[738] Agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 may also be used to reduce the side effects of digestive tract toxicity due to radiation, chemotherapy or viral infection. KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 may have cytoprotective effects on the small intestine mucosa. KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 may also promote the healing of mucositis (mouth ulcers) caused by chemotherapy and virus infection.
[739] Agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 can further be used for the complete regeneration of the skin (e.g., repopulation of hair vesicles, glands and sebaceous glands) in whole and partial skin defects, including burns, And can be used for the treatment of other skin defects such as skin. Agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 can be treated by accelerating the re-epithelialization of lesions of vesiculopathic epilepsy, which causes frequent open and painful blisters due to poor adhesion of the epidermis to underlying dermis . Agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 also help treat gastric and duodenal ulcers and accelerate the scar formation of mucosal walls and the regeneration of the mucosal and duodenal walls of the duodenum. Inflammatory bowel diseases such as Crohn's disease and ulcerative colitis are diseases that result in destruction of the mucosal surface of the small intestine or colon, respectively. Thus, KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 can be used to aid faster healing and promote resurfacing of mucosal surfaces to prevent the progression of inflammatory bowel disease. Treatment with a KGF-2 polynucleotide or polypeptide and an agonist or antagonist of KGF-2 is expected to have an important effect on the production of mucosa throughout the gastrointestinal tract and to protect the intestinal mucosa from digestive or post- Can be used. Agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 can be used to treat KGF-2 low expression related diseases.
[740] Moreover, KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 can be used to prevent and treat lung damage due to various pathological conditions. Growth factors, such as KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2, promote proliferation and differentiation, promote the repair of damage to the repertoire and bronchial epithelium, thereby preventing or treating acute or chronic lung injury. For example, a model resulting in a gradual loss of reward, and inhalation damage resulting from smoke inhalation and burns resulting in necrosis of the bronchioles epithelium and reptile, may be caused by KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 Lt; / RTI > can be effectively treated. In addition, agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 stimulate proliferation and differentiation of type II lung cells, leading to the development of glaucoma diseases (e.g., infant respiratory distress syndrome and bronchopulmonary dysplasia) in premature infants, Lt; RTI ID = 0.0 > and / or < / RTI >
[741] Agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 stimulate the proliferation and differentiation of hepatocytes, causing acute liver failure, viral hepatitis and toxins (including acetaminophen, carbon tetrachloride and other hepatotoxins ), ≪ / RTI > and liver diseases, such as liver damage caused by < RTI ID = 0.0 >
[742] Moreover, KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 can be used to treat or prevent diabetic neuropathy. In patients with newly diagnosed Type I and II diabetes (in which case some islet cell function remains), agonists or antagonists of KGF-2 polynucleotides or polypeptides and KGF-2 maintain sodal function, May be used to alleviate, delay, or prevent the symptoms. In addition, KGF-2 polynucleotides or polypeptides and agonists or antagonists of KGF-2 may be used as adjuvants in islet cell transplantation to improve or enhance islet cell function.
[743] Infectious disease
[744] Agonists or antagonists of KGF-2 polynucleotides or polypeptides or KGF-2 can be used to treat or detect infectious agents. For example, infectious diseases may be treated by increasing the immune response, particularly by increasing the proliferation and differentiation of B and / or T cells. The immune response can be increased by increasing the existing immune response or by starting a new immune response. On the other hand, KGF-2 polynucleotides or polypeptides or agonists or antagonists of KGF-2 may inhibit direct infectious agents without the need to cause an immune response.
[745] The virus is an example of an infectious agent that can cause a disease or condition that can be treated or detected by a KGF-2 polynucleotide or polypeptide or an agonist or antagonist of KGF-2. Examples of viruses include, but are not limited to, arboviruses, adenoviruses, arenaviruses, alteriviruses, birnaviruses, vanniaviruses, caliciviruses, sarcoviruses, coronaviruses, (Eg, paramyxoviridae, morvilivirus, rabodoviridae), oropharyngeal viruses (eg, hepatoviruses, hepatitis viruses, hepatitis viruses), herpes viruses (cytomegalovirus, herpes simplex, (Eg, influenza), papovavirus, parvovirus, picornavirus, poxvirus (small fox or vaccinia), reovirus (eg rotavirus), retrovirus (HTLV-I, HTLV- Lentivirus), and toviruses (e. G., Ruby viruses). Viruses belonging to these groups can cause a variety of diseases or conditions including but not limited to arthritis, bronchitis, encephalitis, eye infections (eg, conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, Cerebrospinal Flu viruses, Chicken Pox, Chicken Pox, Hemorrhagic fever, measles, mumps, parainfluenza, rabies, cold, folio, leukemia, rubella, Sexually transmitted diseases, skin diseases (eg, Kaposi, warts), and viremia. Agonists or antagonists of KGF-2 polynucleotides or polypeptides or KGF-2 may be used to treat or detect these conditions or diseases.
[746] Similarly, bacterial or fungal pathogens which may cause disease or symptom and which can be treated or detected by KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2 include, but are not limited to, the following gram- - include positive bacteria and fungi: actinomycetes (eg, corynebacterium, mycobacterium, Norcadia), aspergillosis, basilaceae (eg, anthrax, clostridium), bacteriocidae, Bacterial strains such as Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Kosidiumiomycosis, Cryptococcosis, Dermatosi Kosensee, Enterobacteriaceae (Clevesiella, Salmonella, Serratia, Yersinia), Erysipelotrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Maiko Plasmathales, Pseudomonas spp., Pseudomonas spp., Pseudomonas spp., Pseudomonas spp., Pseudomonas spp., Pseudomonas spp., Pseudomonas spp. Coccal. These bacterial or fungal infections can cause, but are not limited to, bacterial hematomas, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (eg, AIDS related infections) Inflammatory bowel disease, peripheral inflammation, peritonitis, associated infections, Reiter's Disease, airway infections such as pertussis or sinusitis, sepsis, Lyme Disease, Cat-Scratch Disease, Tuberculosis, botulism, tuberculosis, botulism, botulism, gangrene, tetanus, rheumatic fever, rheumatic fever, scarlet fever, sexual dyspepsia, skin, pancreatitis, typhus, food poisoning, typhoid, pneumonia, gonorrhea, meningitis, chlamydia, syphilis, diphtheria Diseases [eg, cellulitis, dermatocycoses], intoxication, urinary tract infection, wound infection. Agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, can be used to detect or treat any of these conditions or diseases.
[747] In addition, parasitic pathogenic agents that cause diseases or conditions that can be detected or treated by KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, include Amebiasis, Babesiosis, Coccidiosis, Cryptosporia But are not limited to, diacylglycerol, dexamethasone, dianthamoebiasis, duralin, ectoparasitic, guaradiasis, helminthiasis, reismaniasis, tylerialis, toxoplasmosis, tripopaminomic acid and trichomonas no. These parasites include a variety of parasites including, but not limited to, ophthalmoplegia, sickle cell disease, eye infections, intestinal diseases (eg, dysentery, dysmenorrhoea), liver disease, lung disease, opportunistic infections (eg, AIDS related), malaria, Disease, or symptom. ≪ / RTI > Agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, can be used to detect or treat any of these conditions or diseases.
[748] Treatment with a KGF-2 polynucleotide or polypeptide, or an agonist or antagonist of KGF-2, may comprise administering to the patient an effective amount of a KGF-2 polypeptide, or removing the cells from the patient and administering KGF-2 polynucleotide , Then sending the engineered cells back to the patient (e.g., in vivo treatment) would be desirable. In addition, the use of a KGF-2 polypeptide or polynucleotide as an antigen in a vaccine can induce an immune response to infection.
[749] play
[750] KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2 can be used to differentiate, proliferate, and attract cells to induce tissue regeneration (see Science 276: 59-87 (1997)). Tissue regeneration can be used to treat a variety of conditions such as congenital defects, trauma (wound, burn, incision or ulcer), aging, diseases (eg osteoporosis, asteocarthritis, periodontal disease, liver failure) Can repair, replace or protect tissue damaged by fibrosis, reperfusion injury, or systemic cytokine injury.
[751] Examples of tissues that can be regenerated using the present invention include tissues (including pancreas, liver, intestine, kidney, skin, endothelium), muscle tissue (smooth muscle, skeletal muscle or myocardium) , Hematopoietic tissues and skeletal tissues (bone, cartilage, tendons and ligaments). Regeneration preferably occurs without scarring or scarring. Regeneration may also involve angiogenesis.
[752] In addition, agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, can improve tissue regeneration which is difficult to treat. For example, by improving tendon / ligament regeneration, the regeneration time after injury can be shortened. The KGF-2 polynucleotides or polypeptides of the invention, or agonists or antagonists of KGF-2, can also be used for preventative purposes to avoid damage. Certain diseases that can be treated include tendinitis, carpal tunnel syndrome, other tendons or ligamentous defects. Other examples of tissue regeneration of non-healing wounds include ulcers, ulcers associated with cardiovascular dysfunction, surgical wounds and traumatic wounds.
[753] Similarly, neural tissue and brain tissue can be regenerated by proliferating and differentiating neurons using KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2. Diseases that can be treated using this method include central nervous system and peripheral nervous system diseases, neuropathies or mechanical and traumatic disorders [eg, spinal cord disorders, head trauma, cerebrovascular disease and stoke]. Alzheimer ' s disease, Parkinson ' s disease, Huntington ' s disease (e. G., Neuropathy, disease, amyotrophic lateral sclerosis and Shy-Drager syndrome] can all be treated using KGF-2 polynucleotides or polypeptides, agonists or antagonists of KGF-2.
[754] Chemistry
[755] Agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, may have chemotactic activity. The chemotactic molecule may be attached to a specific body site, e. G., An inflammatory site, an infectious site, or an area of interest, such as a cell, e. G., Monocytes, fibroblasts, neutrophils, T- cells, mast cells, eosinophils, epithelium and / Attracts or activates the site of hyperplasia. The mobilized cells can then fight / fight or treat certain traumas or abnormalities.
[756] Agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, may enhance the chemotactic activity of certain cells. These chemoattractant molecules can then be used to treat inflammation, infection, hyperproliferative disorders or other immune system disorders by increasing the number of targeted cells at specific locations in the body. For example, chemoattractant molecules can be used to treat other trauma to the wound and tissue by inducing immune cells to the site of injury. As chemoattractant molecules, KGF-2 can also induce fibroblasts that can be used to treat wound healing.
[757] It is also contemplated that KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, may inhibit chemotactic activity. These molecules can also be used to treat disorders. Thus, KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, can be used as chemotaxis inhibitors.
[758] Binding activity
[759] KGF-2 polypeptides can be used to screen molecules that bind to KGF-2 or molecules that bind KGF-2. KGF-2 and the molecule may activate (agonist), augment, inhibit (antagonize), or decrease the activity of KGF-2 or its molecular binding. Examples of such molecules include antibodies, oligonucleotides, proteins (e.g., receptors) or small molecules.
[760] The molecule is preferably intimately associated with a natural ligand of KGF-2, such as a fragment of a ligand, or a natural substrate, ligand, structure or functional mimetic. [Coligan et al. , Current Protocols in Immunology 1 (2): Chapter 5 (1991)]. Similarly, the molecule may be intimately associated with a natural receptor to which KGF-2 binds, or a fragment of the receptor to which at least KGF-2 (e.g., an active site) may be bound. In either case, the molecule can be rationally designed using known techniques.
[761] Screening for these molecules preferably includes generating appropriate cells expressing KGF-2 either as a secreted protein or on the cell membrane. Preferred cells include mammals, yeast, Drosophila or E. coli. And the like. The cell (or cell membrane comprising the expression polypeptide) expressing KGF-2 is then preferably contacted with a test compound potentially comprising said molecule to inhibit, stimulate or bind KGF-2 or the activity of said molecule .
[762] The assay may be a simple conjugation test of a candidate compound for KGF-2 in which the binding is detected in an assay involving competition with the labeled competitor or by labeling. In addition, the assay can test whether a candidate compound results in a signal produced by binding to KGF-2.
[763] Alternatively, the assay can be performed using a cell-free preparation, a polypeptide / molecule attached to a solid support, a chemical library, or a naturally occurring mixture. The assay may simply include mixing the candidate compound with a solution containing KGF-2, measuring KGF-2 molecular activity or binding, and comparing the KGF-2 activity or binding to a reference material .
[764] Preferably, ELISA assays can be used to measure KGF-2 concentration or activity in a sample (e.g., a biological sample) using a monoclonal or polyclonal antibody. The antibody can measure KGF-2 concentration or activity by binding directly or indirectly to KGF-2, or by competing with KGF-2 for a substrate.
[765] Alternatively, the receptor to which KGF-2 binds can be identified by a number of methods known to those skilled in the art. Examples of such methods include ligand panning and FACS sorting [Coligan, et al . Current Protocols in Immun., 1 (2), Chapter 5, (1991)]. For example, polyadenylated RNA can be prepared from cells that are reactive to the polypeptide, such as NIH3T3 cells and SC-3 cells known to contain multiple receptors for FGF-based proteins, RTI ID = 0.0 > cDNA < / RTI > library into pools to transform COS cells or other cells that are not reactive with the polypeptide. The transformed cells grown on the glass slide are labeled and then exposed to the polypeptide of the invention. The polypeptide may be labeled by a variety of means, such as by inclusion or iodination of the recognition site for the site-specific protein kinase.
[766] After immobilization and incubation, the slides were analyzed radiographically. A positive pool is identified and a sub-pool is generated and re-transfected using repeated sub-pooling and re-screening methods to obtain a single clone encoding the putative receptor.
[767] As an alternative approach to receptor identification, the labeled polypeptide may be photoaffinity binding to an extract formulation or cell membrane that expresses the receptor molecule. The crosslinked material is analyzed by PAGE analysis and exposed to X-ray film. The labeled complex containing the receptor of the polypeptide is excised to digest into peptide fragments and protein microsequencing is performed. A gene encoding the putative receptor can be identified by designing a dextran oligonucleotide probe set using the amino acid sequence obtained from microsequencing and screening the cDNA library.
[768] Also, by modulating the activity of KGF-2 using gene-shuffling, motif-shuffling, exon-shuffling, and / or codon-shuffling (collectively referred to as "DNA shuffling" , Agonists and antagonists of KGF-2. Generally, U.S. Patent Nos. 5,605,793; 5,811, 238; 5,830, 721; 5,834,252 and 5,837,458 and Patten et al., Curr. Oppion Biotechnol. 8: 724-33 (1997); Harayama, S., Trends Biotechnol. 16 (2): 76-82 (1998); Hansson, L.O. et al., J. Mol. Biol. 287: 265-76 (1999); And Lorenzo, M.M. and Blasco, R., Biotechniques 24 (2): 308-13 (1998) (each of these patents and documents cited herein by reference). In one embodiment, the alteration of the KGF-2 polynucleotide and the corresponding polypeptide may be by DNA shuffling. DNA shuffling involves the combination of two or more DNA fragments by homology or site-specific recombination. In other embodiments, the KGF-2 polynucleotide or the polypeptide of interest may be altered by performing a random mutation prior to recombination by error-prone PCR, random nucleotide insertion or other methods. In another embodiment, one or more components, motifs, sections, fragments, domains, fragments, etc. of KGF-2 of the present invention are combined with one or more components, motifs, sections, fragments, domains, fragments, etc. of one or more heterologous molecules . In a preferred embodiment, the heterologous molecule is a member of the fibroblast proliferative factor family. In a further preferred embodiment the heterologous molecule is selected from the group consisting of growth factors such as platelet-derived growth factor (PDGF), insulin-like growth factor (IGF-I), transforming growth factor BMP-4, BMP-5, BMP-6, BMP-7, Actin A (EGF), fibroblast growth factor (FGF), TGF-beta, bone morphogenetic protein And B, decapentaplegic, 60A, OP-2, dorsalin, growth differentiation factors (GDFs), nodal, MIS, inhibin-alpha, TGF - beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 5, and glial-derived neurotrophic factor (GDNF).
[769] Other preferred fragments are biologically active KGF-2 fragments. Biologically active fragments exhibit similar activities to those of KGF-2 polypeptide, but are not necessarily the same. The biological activity of the fragment may include an improved predetermined activity, or a reduced undesired activity.
[770] The present invention also provides a screening method for a compound for identifying a compound that modulates the polypeptide action of the present invention. Examples of such assays include mammalian fibroblasts, the polypeptides of the invention, the compounds to be screened and the combination of ³ [H] thymidine under cell culture conditions, where the fibroblasts will normally proliferate. After performing a control analysis in the absence of the compound to be screened, the amount of fibroblast proliferation in the presence of the compound is compared by measuring the uptake of ³ [H] thymidine in each case to determine whether the compound stimulates proliferation . The amount of fibroblast proliferation is determined by liquid scintillation chromatography, which measures the incorporation of ³ [H] thymidine. Both the agonist compound and the antagonist compound can be identified by this process.
[771] In another method, a mammalian cell or membrane preparation expressing a receptor for a polypeptide of the invention is incubated with the labeled polypeptide of the invention in the presence of the compound. The ability of the compound to enhance or block such interaction can then be measured. Alternatively, the ability of the compound to measure the response of a known second messenger system after interaction of the KGF-2 receptor with the compound to be screened, and to bind to the receptor and induce a second messenger response, A potential agonist or antagonist. Such second messenger systems include, but are not limited to, cAMP guanylate cyclase, ion channel or phosphoinositide hydrolyzate.
[772] All of the above methods can be used as diagnostic or prognostic markers. The molecules found using these assays can be used to activate or inhibit KGF-2 / molecule to induce a particular outcome (eg, angiogenesis) in the patient or to treat the disease. In addition, agents capable of inhibiting or enhancing the production of KGF-2 from moderately engineered cells or tissues can be found using this assay.
[773] Thus, the present invention provides a method of treating cancer comprising: (a) incubating KGF-2 and a candidate binding compound; And (b) determining whether the binding has taken place. The present invention also relates to a method for the determination of the biological activity of KGF-2 comprising the steps of (a) incubating KGF-2 and a candidate compound, (b) analyzing the biological activity, and (c) Lt; RTI ID = 0.0 > antagonists. ≪ / RTI >
[774] In addition, a beta-pleated sheet region disclosed in FIG. 4 and Table 1 can be used to identify molecules that bind to KGF-2 experimentally. Particular embodiments of the invention thus relate to polynucleotides encoding or encoding polypeptides comprising, or consisting of, the amino acid sequences of each beta-planar structural region shown in Figure 4 / A further embodiment of the present invention relates to a polynucleotide encoding a KGF-2 polypeptide comprising, or consisting of, all of the beta screening structural regions shown in Figure 4 / Table 1, or any combination thereof. A further preferred embodiment of the present invention relates to a polypeptide comprising, or consisting of, the KGF-2 amino acid sequence of each of the beta screening structural regions shown in Figure 4 / A further embodiment of the present invention relates to a KGF-2 polypeptide comprising, or consisting of, all of the beta screen structures disclosed in Figure 4 / Table 1, or any combination thereof.
[775] Antisense and ribozyme (antagonist)
[776] In certain embodiments, the antagonist according to the present invention is a nucleic acid corresponding to the sequence contained in SEQ ID NO: 1, or a complementary strand thereof, and / or the nucleotide sequence contained in the depository clone 75977. In one embodiment, the antisense sequence is generated internally by the organism, and in other embodiments, the antisense sequence is administered separately (see, for example, O'Connor, J., Neurochem. 56 : 560 (1991), Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)]. Antisense technology can be used to control gene expression through antisense DNA or RNA, or through tri-helical formation. Antisense technology is described, for example, in Okano, J. Neurochem. 56 : 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1998). Triple helical structure formation is described, for example, in Lee et al., Nucleic Acids Research 6 : 3073 (1979); Cooney et al., Science 241 : 456 (1988); And Dervan et al., Science 251: 1300 (1991). These methods are based on binding of polynucleotides to complementary DNA or RNA.
[777] For example, a 5 'coding portion of a polynucleotide encoding the mature polypeptide of the invention can be used to devise an antisense RNA oligonucleotide of about 10 to 40 base pairs in length. By designing the DNA oligonucleotide to be complementary to the gene region involved in transcription, transcription can be blocked and a receptor can be generated. Antisense RNA oligonucleotides are hybridized in vivo to mRNA and block translation of mRNA molecules into receptor polypeptides.
[778] In one embodiment, the KGF-2 antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or portion thereof that produces the antisense nucleic acid (RNA) of the present invention is transcribed. Such a vector comprises a sequence encoding a KGF-2 antisense nucleic acid. Such a vector may remain in the episome or be chromosomally integrated as long as it can be transcribed to produce the desired antisense RNA. Such vectors may be constructed using standard recombinant DNA technology methods in the art. The vector may be a plasmid, virus, or other vector known in the art used for replication and expression in vertebrate cells. Expression of a sequence encoding KGF-2, or a fragment thereof, can be by any promoter known in the art to function in vertebrate, preferably human, cells. Such promoters may be inducible or constitutive. Such promoters include the SV40 early promoter region [Bernoist and Chambon, Nature 29: 304-310 (1981)], the promoter contained in the 3 'long terminal repeating unit of Rous sarcoma virus [Yamamoto et al ., Cell 22: 787-797 (1980)], the herpes thymidine promoter [Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981)] and the regulatory sequence of the metallothionein gene [Brinster, et al ., Nature 296: 39-42 (1982)].
[779] The antisense nucleic acid of the present invention contains a sequence complementary to at least a part of the RNA transcript of the KGF-2 gene. However, although complete complementarity is desirable, it is not required. As used herein, the term " complementary to at least a portion of the RNA " means a sequence that has sufficient complementarity to be capable of hybridizing to RNA and forms a stable duplex. Thus, in the case of double-stranded KGF-2 antisense nucleic acid, one strand of duplex DNA will be tested, or triple strand formation will be analyzed. The ability to hybridize will depend on both the length of the antinense nucleic acid and the degree of complementarity. Generally, the larger the hybridized nucleic acid, the more stable the duplex (which may be a triplex as the case may be) and the greater the mismatch with the KGF-2 RNA that can form it. Those skilled in the art will be able to identify tolerance mismatches using standard procedures to determine the melting point of hybridized composites.
[780] Oligonucleotides that are complementary to the 5 ' end of the message (e.g., including the 5 ' untranslational sequence and including the AUG start codon) should work most efficiently at the translational inhibition. However, sequences complementary to the 3 'untranslated sequence of mRNA were found to be effective at inhibiting mRNA translation. See generally Wagner, R., 1994, Nature 372 : 333-335. Thus, oligonucleotides complementary to the 5'- or 3'-untranslated unencrypted regions of KGF-2 shown in Figures 1A-1B can be used in an antisense approach to inhibit the translation of endogenous KGF-2 mRNA. Oligonucleotides complementary to the 5 'untranslated region of the mRNA should contain the complement of the AUG start codon. Antisense oligonucleotides that are complementary to the coding region of mRNA are less effective at inhibiting translation, but can be used in accordance with the present invention. Regardless of whether the antisense nucleic acid is designed to be hybridized to the 5'-, 3- or coding region of KGF-2 mRNA, the antisense nucleic acid should be at least six nucleotides in length and an oligonucleotide ranging in length from 6 to about 50 nucleotides desirable. In a particular aspect, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.
[781] Polynucleotides of the present invention can be DNA or RNA or chimeric mixtures or derivatives or variants thereof, single-stranded or double-stranded. Oligonucleotides can be modified at the base, sugar or phosphate backbone to improve, for example, molecular stability, hybridization, and the like. Oligonucleotides may be used in conjunction with other additional groups, e. G., To target peptides (e. G., To target host cell receptors in vivo), or agents that facilitate cross-cell traversal (see, e.g., Letsinger et al . Natl. Acad Sci. USA 86: 6553-6556; Lemaitre et al ., 1987, Proc. Natl Acad Sci 84: 648-652, PCT Publication No. WO 88/09810, ), Or hematopoietic barrier (see, for example, PCT Publication No. WO 89/10134, published on April 25, 1988), a hybridization-inducing cleavage agent (see, for example, Krol et al . , 1988, BioTechniques 6: 958-976) or an intercalating agent (see, for example, Zon, 1988, Pharm. Res. 5 : 539-549). To this end, oligonucleotides may be coupled to other molecules, such as peptides, hybridization-inducing crosslinkers, transporters, hybridization-inducing cleansers, and the like.
[782] The antisense oligonucleotides are selected from the group consisting of 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5- (carboxyhydroxymethyl) Carboxymethylaminomethyl uracil, dihydrouracil, beta-D-galactosylcuechin, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methyl 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyamino Methyl-2-thiouracil, beta-D-mannosylquioine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid (v), wybutoxysin, pseudouracil, cuedosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N- 2-carboxypropyl) uracil, at least one modified base moiety selected from the group consisting of (acp3) w, and 2,6-diaminopurine.
[783] Antisense oligonucleotides may include one or more modified sugar moieties selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, cucurros, and hexose.
[784] In yet another embodiment, the antisense oligonucleotide is selected from the group consisting of phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphodiamidate, methylphosphonate, alkylphosphotriester, And one or more modified phosphate backbones selected from the group consisting of (including, but not limited to) formal acetals or analogs thereof.
[785] In yet another embodiment, the antisense oligonucleotide is an a-anomeric oligonucleotide. Non-anomeric oligonucleotides, unlike conventional b-units, form specific double-strand hybrids with complementary RNAs in which the strands run parallel to each other (Gautier et al., 1987, Nucl. Acids Res . 15: 6625 -6641). The oligonucleotides can be used to amplify the nucleotide sequence of 2'-O-methyl ribonucleotide (Inoue et al., 1987, Nucl. Acids Res. 15 : 6131-6148) or chimeric RNA-DNA analogue (Inoue et al., 1987, FEBS Lett. 215 : 327-330).
[786] The polynucleotides of the present invention can be synthesized using standard methods known in the art, for example, automatic DNA synthesizers (e.g., commercially available from Biosearch, Applied Biosystems, etc.). For example, phosphorothioate oligonucleotides can be prepared by the method of Stein et al. [1988, Nucl. Acids Res. 16: 3209), and methylphosphonate oligonucleotides can be synthesized by controlled pore glass polymer supports (Sarin et al ., 1988, Proc. Natl. Acad. Sci. USA 85 : 7448 -7451).
[787] Although antisense nucleotides complementary to the KGF-2 encoding region sequence can be used, it is most preferred that they are complementary to the transcript untranslated region.
[788] Latent antagonists according to the present invention also include catalytic RNA or ribozymes (see, for example, PCT International Publication No. WO 90/11364, published on October 10, 1990; Sarver et al., Science 247 : 1222-1225 (1990)]. A ribozyme that cleaves mRNA in site-specific recognition sequences can be used to destroy KGF-2 mRNA, but it is preferred to use a hammerhead ribozyme. The hammerhead ribozyme cleaves the mRNA at the side position of the region forming a complementary base pair with the target mRNA. The only condition is that the target mRNA has two base sequences: 5'-UG-3 '. Hammerhead lyophilized constructs and production are well known in the art and are described in more detail in Haseloff and Gerlach Nature 334 : 585-591 (1988). Within the nucleotide sequence of KGF-2 there are a number of potential hammerhead ribozyme cleavage sites (FIG. 1A-1B). In order to increase efficiency and minimize intracellular accumulation of non-functional mRNA transcripts, it is desirable to manipulate the ribozyme so that the cleavage recognition site is located near the 5 'end of KGF-2 mRNA.
[789] As in the antisense approach, the ribozymes of the present invention can be composed of oligonucleotides that have been modified (e.g., improved stability, targeting, etc.) and must be delivered to cells expressing KGF-2 in vivo . The DNA construct encoding the ribozyme can be introduced into the cell in the same manner as described above in connection with the introduction of the antisense encoding DNA. A preferred method of delivery involves the use of a DNA construct that "encodes" the ribozyme under the control of a strong constitutive promoter, for example, a pol III or pol II promoter, through which the transformed cells express an endogenous KGF-2 message Will create a sufficient amount of ribozyme to destroy the translation and inhibit translation. Unlike antisense molecules, since ribozymes are catalysts, lower intracellular concentrations are required for efficiency.
[790] Antagonist / agonist compounds can be used to inhibit the proliferative effect and cellular growth (i. E., Angiogenic stimulation of the tumor) of the polypeptides of the invention on tumor cells and tissues, and thus, Delay or inhibit cell growth and proliferation.
[791] In addition, antagonists / agonists can be used to prevent hypervascular diseases and proliferation of epithelial lens cells can be prevented after extracapsular cataract surgery. In addition, prevention of the mitogenic activity of the polypeptides of the present invention may be desirable in the case of restenosis following balloon angioplasty.
[792] In addition, antagonists / agonists can be used to prevent scar tissue growth during wound healing.
[793] In addition, antagonists / agonists can be used to treat the diseases described herein.
[794] Other activity
[795] Since the polypeptide of the present invention has an ability to stimulate vascular endothelial cell proliferation, the polypeptide of the present invention can be used for stimulating re-angiogenesis of ischemic tissue due to various diseases such as thrombosis, arteriosclerosis and other cardiovascular diseases. These polypeptides can be used to stimulate angiogenesis and limb regeneration as described below.
[796] Polypeptides are a source of division to various cells of different origin (e.g., fibroblasts and skeletal muscle cells), and are thus susceptible to damage or injury due to injury, burns, postoperative tissue damage and ulceration These polypeptides can be used for wound healing.
[797] The polypeptides of the present invention can be used to stimulate neural growth and to treat and prevent some neuronal or neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, and neuronal damage resulting from AIDS related syndromes. KGF-2 has the ability to stimulate chondrocyte proliferation, which can be used to increase bone and rhizome regeneration and assist tissue grafts or bone grafts.
[798] The stimulation of keratinocyte growth using the polypeptide of the present invention can prevent skin aging caused by sunburn.
[799] KGF-2 polypeptides can be used to prevent hair loss because FGF family members activate hair-forming cells and promote melanocyte growth. In addition, the polypeptide of the present invention can promote the growth and differentiation of hematopoietic cells and bone marrow cells when used in combination with other cytokines.
[800] KGF-2 polypeptides may be used to maintain the organ prior to transplantation, or to support cell culture of the primary tissue.
[801] The polypeptides of the present invention can be used to induce mesenchymal origin to differentiate early embryos.
[802] KGF-2 polynucleotides or polypeptides, or agonists or antagonists of KGF-2, can increase or decrease the differentiation or proliferation of embryonic stem cells in addition to the hematopoietic system, as described above.
[803] Agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, may be used to identify mammalian characteristics such as kidney, body weight, hair color, eye color, skin, percentage of adipose tissue, pigmentation, For example, plastic surgery). Similarly, a KGF-2 polynucleotide or polypeptide, or an agonist or antagonist of KGF-2, may be used to modulate mammalian metabolism that affects catabolism, assimilation, energy treatment, utilization and storage.
[804] Agonists or antagonists of KGF-2 polynucleotides or polypeptides, or KGF-2, may be used in the treatment of angiogenesis, bradycardia, periodic rhythm, depression (including depression), violence, resistance to pain, Activity), hormone or endocrine levels, appetite, sexual desire, memory, stress, or other cognitive abilities, to alter the mental or physical state of a mammal.
[805] An agonist or antagonist of KGF-2 polynucleotide or polypeptide, or an agonist or antagonist of KGF-2 can be used, for example, as a food additive or preservative, a fat inclusion, a lipid, a protein, a carbohydrate, a vitamin, Other nutritional ingredients may also be used.
[806] The above-mentioned applications are available in a variety of hosts. Such hosts include humans, rats, rabbits, goats, guinea pigs, camels, horses, mice, rats, hamsters, pigs, micro-pigs, chickens, And the like, but the present invention is not limited thereto. In certain embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In a preferred embodiment, the host is a mammal. In a most preferred embodiment, the host is a human.
[807] Diagnosis and Imaging
[808] Labeled antibodies, and derivatives and homologues thereof, that specifically bind to a polypeptide of interest may be used for diagnostic purposes to detect, diagnose, or monitor diseases, disorders, and / or conditions associated with abnormal expression and / Lt; / RTI > The present invention relates to a method of determining the level of expression of a polypeptide of interest by (a) analyzing the expression of a polypeptide of interest in a cell or body fluid of an individual using one or more antibodies specific for the polypeptide of interest, and (b) Wherein the decrease or increase in the level of polypeptide gene expression analyzed as compared to the expression of the abnormal expression of the polypeptide is indicative of abnormal expression.
[809] The present invention relates to a method of determining the level of expression of a polypeptide of interest by (a) analyzing the expression of a polypeptide of interest in an individual's cells or body fluids using one or more antibodies specific for the polypeptide of interest, and (b) Wherein a decrease or increase in the level of polypeptide gene expression analyzed as compared to the expression level of the polypeptide is indicative of a particular disease. In the context of cancer, the presence of a relatively high amount of transcript in the autopsied tissue from an individual may represent a precursor for the development of the disease, or provide a means for detecting the disease prior to the appearance of substantial clinical symptoms It is possible. This type of more definitive diagnosis allows medical professionals to use preventive measures or aggressive therapies earlier to prevent the development or further development of cancer.
[810] The antibodies of the present invention can be used to analyze protein concentrations in biological samples using classical immunohistological methods known to those skilled in the art (Jalkanen, et al., J. Cell. Biol. 101: 976-985 1985); J. Cell. Biol. 105: 3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays such as enzyme linked immunosorbant assay (ELISA) and radioimmunoassay (RIA). Suitable antibody assay labels are known and include glucose oxidase; Radioisotopes, iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In), and technetium ( ⁹⁹ Tc); Luminescent labels such as luminol; And fluorescent labels such as fluorescein and rhodamine and biotin.
[811] One aspect of the invention is the detection and diagnosis of a disease or condition associated with an abnormal expression of a polypeptide of interest in an animal, preferably a mammal, and most preferably a human. In one embodiment, the diagnosis is a) administering (e.g., parenterally, subcutaneously, or intraperitoneally) a labeled molecule-effective amount specifically binding to a polypeptide of interest; b) waiting for a predetermined time interval after administration so that the labeled molecule is preferentially concentrated at the location in the subject where the polypeptide is expressed (and the unbound labeled molecule is removed at the background level); c) determining the background level; And d) detecting the labeled molecule in the subject to detect that the labeled molecule has a certain disease or disorder associated with abnormal expression of the polypeptide of interest. Background levels can be determined by a variety of methods, including comparing the amount of the detected marker molecule to a predetermined standard value for a particular system.
[812] Those skilled in the art will appreciate that the size of the subject and the imaging system used will determine the amount of imaging required to produce a diagnostic image. For a radioactive isotope portion, for human subjects, the amount of radioactive isotope to be injected will generally be in the range of about 5 to about 20 milli-squares of ⁹⁹ mTc. The labeled antibody or antibody fragment will then preferentially accumulate at the cellular site containing the particular protein. In vivo tumor imaging is described in SW Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibiotics and Their Fragment" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, SW Burchiel and BARhodes, eds, Masson Publishing Inc. (1982)). .
[813] Depending on several variables, including the type of label used and the mode of administration, the time interval after administration is such that the labeled molecule is preferentially concentrated at the site of the subject and that unbound labeled molecules are removed at the background level is between 6 and 48 hours Or 6 to 24 hours or 6 to 12 hours. In other embodiments, the time interval after administration is 5 to 20 days or 5 to 10 days.
[814] In one embodiment, the monitoring of the disease or disorder is performed, for example, by repeating the method for diagnosing the disease or disorder one month after the initial diagnosis, six months after the initial diagnosis, and one year after the initial diagnosis.
[815] The presence of the labeled molecule can be detected in the patient using known methods for in vivo scanning. These methods depend on the type of label used. One skilled in the art will be able to determine the appropriate method for detection of a particular label. Methods and apparatus that can be used in the diagnostic methods of the present invention include, but are not limited to, computer tomography (CT), whole body scanning such as positional emission tomography, magnetic resonance imaging (MRI), and sonography.
[816] In certain embodiments, the molecule is labeled with a radioactive isotope and is detected in a patient using a radiation-sensitive surgical instrument (Thurston et al., U.S. Patent No. 5,441,050). In another embodiment, the molecule is labeled with a fluorescent compound And is detected in the patient using a reactive scanning device. In another embodiment, the molecule is labeled with a positron emitting metal and detected in a patient using positron emission-tomography. In another embodiment, the molecule is labeled with a paramagnetic label and is detected in the patient using magnetic resonance imaging (MRI).
[817] Kit
[818] The present invention provides kits that can be used in the methods described above. In one embodiment, the kit comprises an antibody of the invention, preferably a purified antibody, in one or more containers. In certain embodiments, a kit of the invention comprises a substantially separate polypeptide comprising an epitope that specifically immunoreacts with an antibody contained in the kit. The kit of the present invention preferably further comprises a regulatory antibody that does not react with the polypeptide. In another specific embodiment, a kit of the invention comprises means for detecting binding of an antibody to the polypeptide (e. G., The antibody comprises a detectable substrate, e. G., A fluorescent compound, an enzyme substrate, A radioactive compound or a luminescent compound, or a second antibody recognizing the first antibody may be combined with a detectable substrate).
[819] In another particular embodiment of the invention, the kit is a diagnostic kit for use in screening serum containing proliferating and / or cancer polynucleotides and antibodies specific for the polypeptide. Such kits may comprise regulatory antibodies that do not react with the polypeptide of interest. Such a kit may comprise a substantially separate polypeptide antigen comprising an epitope that specifically immunoreacts with one or more anti-polypeptide antigen antibodies. Such kits also include means for detecting binding of the antibody to the antigen (e. G., The antibody can be conjugated to a fluorescent compound such as rhodamine or fluorescein, which can be detected by flow cytometry have). In certain embodiments, the kit may comprise recombinantly produced or chemically synthesized polypeptide antigens. The polypeptide antigen of the kit may be attached to a solid support.
[820] In a more specific embodiment, the detection means of the kit described above comprise a solid support to which the polypeptide antigen is attached. Such kits may include non-attached reporter-labeled anti-human antibodies. In such embodiments, binding of the antibody to the polypeptide antigen can be detected by binding of the reporter-labeled antibody.
[821] In a further embodiment, the invention includes a diagnostic kit for use in screening sera comprising an antigen of a polypeptide of the invention. The diagnostic kit comprises a substantially separate antibody that specifically immunoreacts with a polypeptide or polynucleotide antigen, and means for detecting binding of the polynucleotide or polypeptide antigen to the antibody. In one embodiment, the antibody is attached to a solid support. In certain embodiments, the antibody may be a monoclonal antibody. The means of detection of the kit may comprise a second labeled monoclonal antibody. Alternatively or additionally, the detection means may comprise a labeled competitive antigen.
[822] In one diagnostic configuration, the test serum is reacted with the solid phase reagent having the surface-binding antigen obtained by the method of the present invention. The reagent is bound to a specific antigen antibody, the unbound serum component is removed by washing, and the reagent is reacted with a reporter-labeled anti-human antibody, Combine the reagent with the reporter. The reagent is washed again to remove unbound labeled antibody and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme (Sigma, St. Louis, MO) that is detected by incubating the solid phase in the presence of a suitable fluorimetric, luminescent, or colorimetric substrate.
[823] The solid surface reagents in this assay can be prepared using known techniques for attaching proteinaceous materials to solid support materials, such as, for example, polymer beads, dip sticks, 96-well plates or filter materials do. These attachment methods generally involve covalently bonding proteins to a chemical reactor on a solid support such as an active carboxyl, hydroxyl or aldehyde group, usually via a free amine group, or non-specifically adsorbing the protein to a support . Alternatively, a streptavidin-coated plate can be used with the biotinylated antigen (s).
[824] Thus, the present invention provides a kit or assay system for performing such diagnostic methods. The kit generally comprises a support having a reporter-labeled anti-human antibody and a surface-binding recombinant antigen for detecting a surface-bound anti-antigen antibody.
[825] The present invention has been generally described, and the present invention will be more readily understood by reference to the following examples. However, the following examples are provided for illustrative purposes and are not intended to limit the present invention.
[826] Example 1
[827] Bacterial expression and purification of KGF-2
[828] Initially, a DNA sequence encoding KGF-2, ATCC # 75977 is amplified using a PCR oligonucleotide primer (including a signal peptide sequence) corresponding to the 5 'and 3' end sequences of the processed KGF-2 cDNA. The 5 'oligonucleotide primer is represented by the following sequence: 5'CCCCACATGTGGAAATGGATACTGACACATTGTGCC 3' (SEQ ID NO: 3) sequentially contains 30 nucleotide sequences of the KGF-2 coding sequence starting from the putative initiation codon with Af1III restriction enzyme . The 3 'sequence is 5' CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3 '(SEQ ID NO: 4) contains sequence complementary to the HindIII site and 26 nucleotides of KGF-2 in sequence. Restriction enzyme sites are associated with restriction enzyme sites on the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, Calif.). pQE-60 encodes antibiotic resistance (Amp ^r ), the bacterial origin of replication, the IPTG-regulatable promoter operator (P / O), the ribosome binding site (RBS), the 6-His tag and the restriction enzyme site. Subsequently, pQE-60 is digested with NcoI and HindIII. The amplified sequence is bound to pQE-60 and inserted into the frame. Then , E. coli strain M15 / rep4 (Qiagen, Inc) is transformed using a binding mixture according to the method described in Sambrook, J., et al., Molecular Cloning, A Laboratory Manual, Cold Spring Laboratory Press . M15 / rep 4 contains multiple copies of the plasmid pREP4, which expresses the lacI replicas and confer kanamycin resistance (Kan ^r ). Identification of the transformants is accomplished by their ability to proliferate on LB plates, and ampicillin / kanamycin resistant colonies are selected. Plasmid DNA is isolated and confirmed by inhibition assay. Clones containing the desired construct are grown overnight (O / N) in a liquid culture of LB medium supplemented with both Amp (100 ug / ml) and Kan (25 ug / ml). The overnight cultures are used to inoculate large-scale cultures at a ratio of 1: 100 to 1: 250. Cells are grown until optical density 600 (OD ⁶⁰⁰ ) is between 0.4 and 0.6. IPTG ("isopropyl-BD-thiogalactopyranoside") is added to give a final concentration of 1 mM. The IPTG reacts with the lacI repressor and separates from the operator, thereby directly transcribing the promoter. Cells are grown for an additional 3 or 4 hours. The cells are then harvested by centrifugation. The cell pellet is solubilized in 6 moles of guanidine HCl, which is a chaotropic agent. After purification, the solubilized KGF-2 is purified from the solution by chromatography on heparin affinity under conditions which allow protein to bind intimately (Hochuli, E et al., J. Chromatography 411 : 177-184 (1984)). KGF-2 (75% purity) is eluted from the column with high salt buffer.
[829] Example 2
[830] Expression and purification of truncated KGF-2 bacteria
[831] Initially, a DNA sequence encoding KGF-2, ATCC # 75977 is amplified using a PCR oligonucleotide primer corresponding to the 5 ' and 3 ' end sequences of the truncated KGF-2 polypeptide. The truncated form is a peptide without 36 amino acid signal sequences, in which the methionine and alanine residues are added in front of the cysteine residues to contain 37 amino acids of the full-length protein. The 5 'oligonucleotide primer is represented by the following sequence: 5'CATGCCATGGCGTGCCAAGCCCTTGGTCAGGACATG 3' (SEQ ID NO: 5) sequentially contains the NcoI restriction enzyme site and the 24 nucleotide sequence of the KGF-2 coding sequence. 3 'sequence is 5' CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3 '(SEQ ID NO: 6) contains sequence complementary to HindIII site and 26 nucleotides of KGF-2 gene. Restriction enzymes are associated with restriction enzyme sites on the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth, Calif.). pQE-60 encodes antibiotic resistance (Amp ^r ), bacterial origin of replication, or IPTG-regulatable promoter operator (P / O), ribosome binding site (RBS), 6-His tag and restriction enzyme sites. Next, pQE-60 is digested with NcoI and HindIII. The amplified sequence is ligated into pQE-60 and inserted into the frame. Then, E. coli strain M15 / rep4 (Qiagen, Inc) is transformed using a binding mixture according to the method described in Sambrook, J., et al., Molecular Cloning, A Laboratory Manual , Cold Spring Laboratory Press . M15 / rep 4 contains multiple copies of the plasmid pREP4, which expresses the lacI replicas and confer kanamycin resistance (Kan ^r ). Identification of the transformant is accomplished by its ability to proliferate on the LB plate, and an ampicillin / kanamycin resistant colony is selected. Plasmid DNA is isolated and confirmed by inhibition assay. Clones containing the desired construct are grown overnight (O / N) in a liquid culture of LB medium supplemented with both Amp (100 ug / ml) and Kan (25 ug / ml). The overnight cultures are used to inoculate large-scale cultures at a ratio of 1: 100 to 1: 250. Cells are grown until the optical density 600 (OD ⁶⁰⁰ ) is 0.4 to 0.6. IPTG (" isopropyl-BD-thiogalactopyranoside ") is added to a final concentration of 1 mM. Inactivation of the lacI repressor induces IPTG, resulting in elimination of the promoter and increased gene expression. The cells are further proliferated for 3 or 4 hours. The cells are then harvested by centrifugation. The cell pellet is solubilized in 6 moles of guanidine HCl as a chaotropic agent. After purification, the solubilized KGF-2 is purified from the solution by chromatography on heparin affinity under conditions which allow protein to bind intimately (Hochuli, E et al., J. Chromatography 411 : 177-184 (1984)). The KGF-2 protein is eluted from the column with high salt buffer.
[832] Example 3
[833] Cloning and Expression of KGF-2 Using Baculovirus Expression System
[834] The DNA sequence encoding the full length KGF-2 protein, ATCC # 75977 is amplified using a PCR oligonucleotide primer corresponding to the 5 'and 3' sequences of the gene sequences described below:
[835] 5 'primer 5'GCG GGATCC GCCATC TGGAAATGGATACTCAC ATG 3' (SEQ ID NO: 7) appears as a sequence, BamHI restriction enzyme site (bold portion), 6-nucleotide similar to the effective signal in translation initiation in eukaryotic cells (Kozak, M. , J. Mol. Biol., 196 : 947-950 (1987)) and the first 17 nucleotides of the KGF-2 gene immediately following it (the translation "ATG" initiation codon is underlined in the sequence) .
[836] The 3 'primer is shown as 5'GCGCGGTACCACAAACGTTGCCTTCCT 3' (SEQ ID NO: 8) and contains 19 nucleotides complementary to the cleavage site of the restriction endonuclease Asp 718 and the 3 'non-translation sequence of the KGF-2 gene. The amplified sequence is isolated from 1% agarose gel using a kit available from Qiagen, Inc., Chatsworth, Calif. The fragment is then digested with endonucleases BamHI and Asp718 and then purified on 1% agarose gel. This fragment is named F2.
[837] The vector pA2 (a modification of the pVL941 vector, described below) is used to express KGF-2 protein using a baculovirus expression system (Summers, MD & Smith, GE, A manual of methods for baculovirus vectors and insect cell culture procedures , Texas Agricultural Experimental Station Bulletin No. 1555 (1987)). This expression vector sequentially contains the strong polyhidrin promoter of the Autographa californica neuritic polyhydrosis virus (AcMNPV), and the recognition site of the restriction endonucleases BamHI and Asp 718. Efficient polyadenylation is carried out using the polyadenylation site of simian virus (SV) 40. To facilitate selection of the recombinant virus, the beta-galactosidase gene is inserted from Escherichia coli in the same orientation as the polyhydrin promoter and the polyadenylation signal of the polyhydrin gene thereafter. The polyhydrin sequences are adjacent to the viral sequences of cell-mediated homologous recombination that are co-infected with wild-type viral DNA on both sides. As many other baculovirus vectors, pAc373, pVL941, and pAcIMI (Luckow, VA & Summers, M., Virology, 170 : 31-39) can be used.
[838] The plasmid is digested with restriction enzymes BamHI and Asp 718. The DNA is then isolated from the 1% agarose gel using a commercially available kit (Qiagen, Inc., Chatworth, Calif.). This vector DNA is named V2.
[839] Fragment F2 and plasmid V2 bind to T4 DNA ligase. Escherichia coli HB101 cells are transformed and PCR and oligonucleotide cloning are used together to identify a plasmid containing the KGF-2 gene (pBACKGF-2). The cloned fragment sequence is confirmed by DNA sequencing.
[840] 1.0 [mu] g of linearized baculovirus commercially available from 5 [mu] g of the plasmid pBacKGF-2 using the lipofection method (Felgner, et al., Proc. Natl. Acad. Sci. USA , 84: 7413-7417 (&Quot; BaculoGold ^TM baculovirus DNA ").
[841] 1 μg of BaculoGold ^™ virus DNA and 5 μg of plasmid pBacKGF-2 are mixed in a sterile well of a microtiter plate containing 50 μl of Grace's medium of serum glass (Life Technologies Inc., Gaithersburg, Md.). Add 10 μl of Lipofectin and 90 μl of Grace medium, mix and incubate at room temperature for 15 min. Subsequently, the transfection mixture is added dropwise to Sf9 insect cells (ATCC CRL 1711). This insect cell was seeded on a 35 mm tissue culture plate with 1 ml of Grace medium without serum. Mix the newly applied solution while moving the plate back and forth. The plate is then incubated at 27 ° C for 5 hours. After 5 hours, the transfection solution is removed from the plate and 1 ml of Grace insect medium is added. This insect medium was supplemented with 10% fetal calf serum. The plate is put back into the incubator and incubation is continued at 27 ° C for 4 days.
[842] Four days later, the supernatant is collected and a flake assay is performed, which assay is performed in a manner similar to that provided by Summers and Smith (see above). As a variation of this assay, agarose gels with "Blue Gal" (Life Technologies Inc., Gaithersburg) are used, which readily isolate blue-stained flakes (a detailed description of "flake analysis" (See Life Technologies Inc., Gaithersburg, page 9-10), as described in the user's instructions for culture and bacteriology.
[843] Four days after the sequential dilution, the virus is added to the cells and the blue-stained flakes are picked up using an Eppendorf pipette tip. The agar containing the recombinant virus is re-suspended in an Eppendorf tube containing 200 mu l of Grace's medium. The agar is briefly centrifuged off and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. These supernatants of the culture supernatant are collected after 4 days and stored at 4 ° C.
[844] Sf9 cells are proliferated in Grace medium supplemented with 10% heat-activated FBS. Cells are infected with recombinant baculovirus V-KGF-2, causing the multiplicity of infection (MOI) to be 2. After 6 hours the medium is removed and replaced with SF900 II medium (Life Technologies Inc., Gaithesburg) with methionine and cysteine removed. After 42 hours, 5 μCi of ³⁵ S methionine and 5 μCi of ³⁵ S cysteine (Amersham) are added. Afterwards, the cells are incubated for 16 hours, and the labeled proteins are collected by centrifugation and recorded with SDS-PAGE and automatic emission recorder.
[845] Example 4
[846] Most vectors used to transiently express KGF-2 protein gene sequences in mammalian cells should carry the SV40 origin of replication. This is because the vector is replicated in high numbers of cells (eg, COS cells), resulting in the expression of the T antigen necessary for initiation of viral DNA synthesis. Any other mammalian cell line may be used for this purpose.
[847] Since a general mammalian expression vector has a promoter element, it initiates transcription of mRNA and mediates signals necessary for transcription termination and polyadenylation of protein coding sequences and transcripts. Additional elements include an enhancer, a Kozak sequence, and a donor-mediated interference site and a receptor site for RNA splicing. High-efficiency transcripts are produced by the early and late promoters of SV 40, the long terminal repeat (LTR) of retroviruses (e.g. RSV, HTLVI, HIVI) and the cytomegalovirus (CMV) Lt; / RTI > However, cellular signals may also be used (e. G., The human actin promoter). Suitable vectors for use in practicing the invention include vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109) . Usable mammalian host cells include human Hela, 283, H9 and Jurkart cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7, and CVl, African green monkey cells, quail QC1-3 cells, 293T cells, Chinese hamster ovary cells.
[848] Alternatively, the gene can be expressed in a stable cell line containing the gene integrated into the chromosome. Co-infection with selectable markers such as dhfr, gpt, neomycin, and hygromycin allows the identification and isolation of transfected cells.
[849] The transfected gene can be amplified to express a large amount of the encoded protein. DHFR (dihydrofolate reductase) is a marker useful for forming cell lines that carry hundreds or even thousands of copies of the gene of interest. Another useful selectable marker is the enzyme glutamine synthetase (GS) (Murphy et al ., Biochem J. 227 : 277-279 (1991); Bebbington et al., Bio / Technology 10 : 169-175 The animal cells are grown in a selective medium and the most resistant cells are selected.The cell lines contain amplified genes integrated into the chromosome.The Chinese hamster ovary (CHO) cells are frequently used for protein production.
[850] Expression vectors pC1 and pC4 were transfected with the strong promoter (LTR) of Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447 (March, 1985)) and CMV-inferase fragments (Boshart et al., Cell 41 : 521-530 (1985)). A number of cloning sites, such as the restriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate cloning of the gene of interest. The vector contains a polyadenyl and a termination signal in addition to the 3 ' intron of the rat pre-insulin gene.
[851] A. Expression of Recombinant KGF-2 in COS Cells
[852] Expression of the plasmid KGF-2 HA was confirmed by 1) SV 40 replication origin, 2) ampicillin resistance gene, 3) E. coli replication origin, 4) CMV promoter and its polylinker site, SV 40 intron and polyadenylation site derived from pcDNAI / Amp (Invitrogen). The HA tag corresponds to an epitope derived from the < RTI ID = 0.0 > influenza hemagglutinin < / RTI > protein as described above (Wilson, I., et al., Cell 37: 767, (1984)). Injecting HA tags into target proteins facilitates detection of recombinant proteins with antibodies recognizing HA epitopes. Since the DNA fragment encoding the entire KGF-2 precursor HA tag is fused within the frame with the HA tag, the expression of the recombinant protein occurs under the CMV promoter.
[853] Plasmid constructs are as follows:
[854] The DNA sequence encoding KGF-2, ATCC # 75977 is constructed by PCR using two primers: the 5 'primer, 5'TAACGAGGATCCGCCATCATGTGGAAATGGATACTGACAC 3' (SEQ ID NO: 9) contains the BamHI site, 2 < / RTI > of the nucleotide sequence encoding KFG-2. The 3 'sequence 5' TAAGCACTCGAGTGAGTGTACCACCATTGGAAGAAATG3 '(SEQ ID NO: 10) contains the sequence complementary to the XhoI site, the HA tag, and the last 26 nucleotides of the coding sequence of KGF-2 (without stop codon). Therefore, the PCR product contains the BamHI site, the KGF-2 coding sequence, the XhoI site, the HA tag fused in the frame, and the translation termination stop codon next to the HA tag. PCR amplified DNA fragment and vector pcDNA-3'HA were digested with BamHI and XhoI restriction enzyme and bound to produce pcDNA-3'HA-KGF-2. The binding mixture is transformed in E. coli strain XL1 blue (Stratagene Cloning Systems, La Jolla, Calif.) And the transformed cultures are plated on an ampicillin medium plate and resistant colonies are selected. Plasmid DNA is isolated from the transformants and examined for PCR and inhibition assays for the presence of the correct fragments. For expression of recombinant KGF-2, COS cells are transfected with the expression vector by the DEAE-DEXTRAN method (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual , Cold Spring Laboratory Press (1989)). Expression of KGF-2 HA protein is detected by radiolabeling and immunoprecipitation methods (Harlow, E, & Lane, D., Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, (1988)). Cells were transfected with ³⁵ S-cysteine for 2 h after transfection for 8 h. The culture medium was then harvested and the cells were washed with detergent (RIPA buffer-150 mL NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5 , I., et al., Id, 37 : 767 (1984)). Both cell culture medium and culture medium were precipitated with HA-specific monoclonal antibody. The precipitated proteins were analyzed on a 15% SDS-PAGE gel.
[855] B. Expression and purification of human KGF-2 protein using CHO expression system
[856] The vector pC1 is used to express the KGF-2 protein. Plasmid pC1 is a derivative of the plasmid pSV 2-dhfr (ATCC No. 37146). These two plasmids contain the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary cells or other cells are deficient in dihydrofololate activity and they are transfected with the plasmid and then proliferated in selective media supplemented with the chemotherapeutic agent methotrexate (alpha minus MEM, Life Technologies) . The amplification process of the DHFR gene in cells resistant to methotrexate (MTX) is well documented (Alt, FW, Kellems, R., M., Bertino, JR, and Schimke, RT, 1978, J. Biol. Chem . 253: 13357-1370, Hamlin, JL and Ma, C. 1990, Biochem, et Biophys Acta 1097:... 107-143, Page, MJ and Sydenham, MA 1991, Biotechology Vol.9; 64-68)) . Cells that proliferate with increasing concentrations of MTX exhibit resistance to the drug by overproduction of the target enzyme, DHFR, as a result of DHFR gene propagation. If the second gene binds to the DHFR gene it co-propagates and overexpresses. Developing a cell line that carries more than 1000 copies of a gene is a state-of-the-art technology. Subsequently, when mettrexate is recovered, the cell line contains an amplified gene integrated into the chromosome.
[857] Plasmid pC1 is a strong promoter of the long terminal repeat (LTR) of Rouse Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, March 1985: 438-4470) and human cytomegalovirus (CMV) , Cell 41 : 521-530, 1985). The lower promoters are the following single restriction endonuclease sites that allow integration of the taxa: BamHI, Pvull, and Nrul. The plasmid behind these cloning sites sequentially contains the transcription termination codon, the 3 'intron and the polyadenylation site in all three reading frames of the rat preproinsulin gene. Other high efficiency promoters can be used for expression, examples of which include the human [beta] -actin promoter, the SV40 early or late promoter or the LTR from other retroviruses (e.g., HIV and HTLVI). In the case of polyadenylation of mRNA, other signals, such as globin genes or other signals derived from human growth hormone, may also be used.
[858] Stable cell lines carrying the gene of interest integrated into the chromosome are also selectable at co-transfection with selectable markers such as gpt, G418, or hygromycin. It is also advantageous to use one or more selectable markers, such as G418 and Metrotreksite, at the beginning of the first time.
[859] Plasmid pC1 is digested with restriction enzyme BamHI and then diphosphorylated using calf intestinal phosphate according to procedures known in the art. The vector is then separated from the 1% agarose gel.
[860] The DNA sequence encoding KFG-2, ATCC # 75977 is amplified using a PCR oligonucleotide primer corresponding to the 5 'and 3' sequences of the gene:
[861] 5 'primer for the underlined BamHI restriction enzyme, and FIG. 1 KFG-2 sequence (SEQ ID NO: 1) of the 21 nucleotide sequence 5 which is contained in the sequence of the "represented by TAACGA GGATCC GCCATCATGTGGAAATGGATACTGACAC 3' (SEQ ID NO: 9) . Providing the 5 'end of the amplified fragment encoding human KFG-2 in the expression vector as described below provides an efficient signal peptide. Kozak, M., J. Effective signals for translational initiation in eukaryotic cells, as described in Mol. Biol., 196 : 974-950 (1987), are located at appropriate locations in the vector region of the construct.
[862] The 3 'primer contained a BamHI restriction enzyme and a 5' TAAGCA (SEQ ID NO: 1) sequence containing the nucleotides corresponding to the last 26 nucleotides of the KFG-2 coding sequence represented by GGATCC TGAGTGTACCACCATTGGAAGAAATG 3 '(SEQ ID NO: 10).
[863] The amplified fragments were separated from 1% agarose gel as described above and then digested with endonuclease BamHI and again purified on 1% agarose gel.
[864] The isolated fragment and the dephosphorylated vector are then ligated to the T4 DNA ligase. E. coli HB101 cells are transformed and the bacteria containing the plasmid pC1 are identified. The sequence and orientation of the inserted gene is confirmed by DNA sequencing.
[865] Transfection of CHO-DHFR- cells
[866] Chinese hamster ovary cells deficient in active DHFR enzyme are used for transfection. 5 μg of the expression plasmid C1 is co-transfected with 0.5 μg of the plasmid pSV neo using the lipofecting method (see Felgenr et al., Supra). The plasmid pSV2-neo contains a dominant selectivity marker, wherein the gene neo originates from Tn5, which encodes an enzyme that confers resistance to antibiotics, including G418. The cells are seeded in alpha minus MEM supplemented with 1 mg / ml G418. Two days later, cells are added with trypsin and seeded in a hybridoma cloning plate (Greiner, Germany) and cultured for 10-14 days. After this period, the monoclon is added to trypsin and seeded in a 6-well Petri dish using various concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). The clones growing in the highest concentration of methotrexate are transferred to a new 6-well plate containing even higher concentrations of methotrexate (500 nM, 1 μM, 2 μM, 5 μM). The same procedure is repeated until the clone is grown at a concentration of 100 [mu] M.
[867] Expression of the desired gene product is analyzed by Western blot analysis and SDS-PAGE.
[868] Example 5
[869] Transcription and translation of recombinant KFG-2 in vitro
[870] The PCR product is derived from the cloned cDNA in the pA2 vector used for insect cell expression of KFG-2. The primers used in this PCR are: 5 'ATTAACCCTCACTAAAGGGAGGCCATGTGGAAATGGATACTGACACATTGTGCC 3' (SEQ ID NO: 11) and 5 'CCCAAGCTTCCACAAACGTTGCCTTCCTCTATGAG 3' (SEQ ID NO: 12).
[871] First, the primer contains the sequence of the T3 promoter 5 'at the ATG start codon. The second primer encodes the inverted complement of the stop codon complementary to the 3 'end of the KFG-2 open reading frame.
[872] The resulting PCR product is purified using a commercially available kit from Qiagen. 0.5 [mu] g of the DNA is used as a template for the transcription-translation reaction in vitro. This reaction is carried out using a kit available from Promega under the name TNT. The assay was carried out as described in the kit's instructions, using the radiolabeled methionine as substrate, but using only half the amount of reagent indicated and the reaction was carried out at 33 占 for 1.5 hours do.
[873] 5 [mu] l of the reaction product is electrophoretically separated into 10 to 15% of polyacrylamide gel denaturing. The gel is allowed to stand in a mixture of water: methanol: acetic acid having a volume ratio of 6: 3: 1 for 30 minutes. The gel is dried under heat and vacuum and exposed to X-ray film for 16 hours. When the film is developed, a band of radiolabeled proteins corresponding in size to the conceptually translated KFG-2 appears. This strongly suggests that the clone cDNA of KFG-2 contains an open reading frame encoding the expected size of the protein.
[874] Example 6
[875] Expression through gene therapy
[876] Fibroblasts are obtained from skin tissue sections. Tissue the tissue produced in the tissue culture medium and cut into small pieces. Place the small piece of tissue on the wetted surface of the tissue culture flask and add about 10 pieces to each flask. The flask is turned upside down and allowed to stand overnight at room temperature. After standing at room temperature for 24 hours, flip the flask again and fix the tissue piece to the bottom of the flask and add fresh medium (eg Ham's F12 medium, 10% FBS, penicillin and streptomycin). This is incubated at 37 ° C for about one week. At this time, fresh medium is added and changed every 2-3 days. Fibroblast monolayers appear when the cells are maintained for an additional 2 weeks in the culture medium. The monolayer is added with trypsin and scaled into a large flask.
[877] PMV-7 (Kirschmeier, PT et al., DNA, 7 : 219-25 (1988)) adjacent to the LTR of Moloney murine sarcoma virus is digested with EcoRI and HindIII. And then treated with calf intestinal phosphate. Using a glass bead, the linear vector is fractionated on an agarose gel and purified.
[878] The cDNA encoding the polypeptide of the present invention is amplified using PCR primers corresponding to the 5 'and 3' terminal sequences, respectively. The 5 'primer and the 3' primer containing the EcoRI site further contain HindIII. The Moloney murine sarcoma virus linear backbone is added together with the amplified EcoRI and HindIII fragment equivalents, which are carried out in the presence of T4 DNA ligase. The resulting mixture is maintained under suitable conditions to bind the two fragments. The binding mixture is used to transform the bacterial HB 101, which is then plated onto an agar-containing kanamycin to confirm that the vector has inserted the gene of interest properly.
[879] Amphoteric pA317 or GP + am12 packaging cells are grown in tissue culture at confluence density of DMEM (Modified Eagle Medium of Dulbecco) to proliferate with 10% calf serum (CS), penicillin and streptomycin. The MSV vector containing the gene is added to the medium and the packaging cells are transduced with the vector. As a result, packaging cells produce infectious viral particles containing the gene (packaging cells are also now referred to as producer cells).
[880] Fresh medium is added to the transfected producer cells and the medium is collected from 10 cm plates of confluent producer cells. The used medium containing the infectious viral particles is filtered through a microporous filter to remove the separated producer cells and the fibroblasts are infected with this medium. The medium is removed from the semi-confluent plate of fibroblasts and replaced immediately with the medium of the producer cells. Remove this medium and replace it with fresh medium. If the viral titer is high, virtually all fibroblasts are infected, so no selection is necessary. If the titer is very low, then it is necessary to use a retroviral vector with a selectable marker such as neo or his .
[881] The processed fibroblasts are then inserted into the host, either alone or after propagation to the confluence point on the cytodex 3 microcarrier beads. As a result, fibroblasts produce protein products.
[882] Example 7
[883] How to Treat Wounds with KFG-2 Stimulation in a Mouse Model with Diabetes
[884] To demonstrate that the healing process is promoted by KFG-2, we use a genetically diabetic mouse model that underwent a wound healing process. A model that is being treated for full thickness wounds in db + / db + mice is a well characterized, clinically relevant, and reproducible model of the wound healing process. The healing of diabetic wounds can depend on and play left back, rather than contraction on whether the epithelial cells form the granulation tissue (Gartner, MH, etc., J. Surg, Res 52:. 389 (1992); Greenhalgh, DG , etc. Am, J. Pathol , 136: 1235 (1990).
[885] Animals with diabetes have many features observed in type II diabetes melitus. Homozygous (db + / db +) mice are obese when compared with normal heterozygotes (db + / + m). Mutated diabetic mice (db + / db +) have a single autosomal recession variation on chromosome 4 (db +) (Coleman et al., Proc Natl Acad Sci USA 77 : 283-293 (1982)). The animals exhibit bulimia, alternation, and polyuria. Mutated diabetic mice (db + / db +) increase blood glucose levels, exhibit normal or increased insulin levels and inhibit cell-regulated immune responses (Mandel et al . , J. Immunol. 120 : 1375 (1978); Debray-Sachs, Immunol 51 (1) : 1-7 (1983); Leiter et al . , Am. J. of Pathol. 114 : 46-55 (1985)). Peripheral neuropathy, cardiovascular complications, cardiovascular lesions, basement membrane hypertrophy and glomerular filtration abnormalities are seen in these animals (Norido, F. et al . , Exp. Neurol, 83 (2) : 221-232 (1984); Robertson et al., Diabetes29 1): 60-67 (1980); Giacomelli , etc. d, Lab Invest, 40 (4 ):. 460-473 (1979); Coleman, DL, diabetes 31 (Suppl): 1-6 (1982) these homozygous diabetic Mice promote hyperglycemia resistant to insulin analogous to human type II diabetes (Mandel et al. , J. Immunol. 120 : 1375-1377 (1978)).
[886] The character observed in these animals suggests that the healing in this model may be similar to the healing observed in human diabetes (Greenhalgh et al . , Am. J. of Pathol. 136 : 1235-1246 (1990)). This study demonstrates that KFG-2 has a potent stimulating effect in treating pre-wound thickness in one-year-olds with heterozygous diabetes mellitus and one-year olds with diabetes mellitus. The remarkable effect of collagen fibers, increase in granular tissue in the dermis and reconstruction of the epithelial cells, appeared in animals treated with KFG-2. Exogenous administration of growth factors can trigger the formation of granular tissue by attracting inflammatory cells to the wound site.
[887] animal
[888] Genetically diabetic female C57BL / KsJ (db + / db +) mice and non-diabetic (db + / + m) heterozygotes were used in this study (Jackson Laboratories). These animals were purchased at 6 weeks of age and started studying at 8 weeks of age. Animals were individually housed in us and given food, water and ribitum. All procedures were performed using aseptic techniques. The experiments were carried out in accordance with the guidelines and guidelines of the Animal Handling and Use Committee of the Human Genome Society, Ansys Corporation, and the handling and use of laboratory animals.
[889] KGF-2
[890] The recombinant human KGF-2 used in wound healing studies was over-expressed and purified from pQE60-Cys37, an E. coli expression vector system (pQE-9, Qiagen). The protein expressed from this construct is KGF-2 from cysteine at position 37 to serine at position 208 with the 6X (His) tag attached to the N-terminus of the protein (SEQ ID NO: 29-30 ) (Fig. 15). Fractions containing more than 95% pure recombinant material were used in the experiments. Keratinocyte growth factor-2 was formulated in a vehicle containing 100 mM Tris, 8.0 and 600 mM NaCl. Final concentrations were 80 ug / ml stock solution and 8 ug / ml stock solution. Diluted from the mother liquor using the same vehicle.
[891] Surgical wound
[892] The previously reported method [Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172: 245-251 (1990)]. Briefly, animals were injected intraperitoneally with a wound-forming blade, avertin (0.01 mg / ml), 2,2,2-tribromoethanol and 2-methyl-2-butanol dissolved in deionized water Lt; / RTI > The hairs on the back of the animal were removed and the skin was washed with 70% ethanol solution and iodine. Prior to wounding, the surgical site was dried with sterile gauze. Using a Keyes tissue punch, the wound was covered with a sufficient thickness of 8 mm. Immediately after applying the wound, the surrounding skin was softened and the wound expansion was suppressed. The wound area was left open during the experiment. Topical treatment was applied for 5 consecutive days starting from the day of wounding. Prior to treatment, the wound area was gently rinsed with sterile saline and a gauze sponge.
[893] The wounds were visually observed at two days intervals on and after the operation, and pictures were taken at a fixed distance. The wound closure was measured on day 1 and day 8 on days 1-5. Wound measurements were taken horizontally and vertically using graduated Jameson calipers. The wound was considered healed if the granular tissue was no longer visible and the wound was covered with continuous epithelial tissue.
[894] Using two different doses of KGF-2, i.e., 50 쨉 l of vehicle, one dose was administered for 8 days at 4 쨉 g per wound per day and the other was given at 40 ug per wound per day for 8 days. Respectively. The vehicle control was administered with 50 μl of the vehicle solution.
[895] Animals were euthanized by intraperitoneal injection of sodium pentobarbital (300 mg / kg) on day 8. Wounds and surrounding skin were collected for histological and immunohistochemical studies. Tissue samples were placed in 10% neutral buffered formalin in tissue cassettes between biopsied sponges for further processing.
[896] Experiment plan
[897] Three groups of 10 animals (5 diabetic and 5 non-diabetic controls) were evaluated:
[898] 1) vehicle placebo control
[899] 2) < RTI ID = 0.0 > KGF-2 &
[900] 3) KGF-2 40 μg / day
[901] The study was planned as follows:
[902] N group process N = 5 db + / db + N = 5 db + / + mVehicle Vehicle50 μl 50 μl N = 5 db + / db + N = 5 db + / + mKGF-2KGF-24 / / 50 4 4 / / 50 ㎕ N = 5 db + / db + N = 5 db + / + mKGF-2KGF-240 mu g / 50 mu l 40 mu g / 50 mu l
[903] Measurement of wound area and occlusion
[904] The area was measured on the vertical and horizontal axes to obtain the total square area of the wound area and the wound closure was analyzed. Thereafter, the shrinkage was evaluated by measuring the difference between the initial wound area (day 0) and the wound area after treatment (day 8). The wound area on the first day was 64 mm2, which was consistent with the skin punch size. Calculated using the following equation:
[905] [Open area on the eighth day] - [Open area on the first day] / [Open area on the first day]
[906] histology
[907] The specimens were fixed in 10% buffered formalin and the paraffin-containing block was sectioned perpendicular to the wound surface (5 mu m) and incised using a Reichert-Jung microtome. Normal hematoxylin-eosin (H & E) staining was performed on the cross-sections of the divided wounds. Histological examination of the wound was used to assess whether the healing process and the morphological appearance of the treated skin were altered by treatment with KGF-2. This assessment includes the identification of the presence of cell accumulation, inflammatory cells, capillaries, fibroblasts, re-epithelialization and epidermal maturation [Greenhalgh, DG et al., Am. J. Pathol. 136 : 1235 (1990)) (Table 1). A lens micrometer with a graduated blinded observer was used.
[908] Immunohistochemistry
[909] Re-epithelization
[910] Tissue sections were immunohistochemically stained with polyclonal anti-human keratin antibodies using an ABC Elite detection system. Human skin was used as a positive control, while non-immunized IgG was used as negative control. Keratinocyte growth was determined by evaluating re-epithelialization of the wound using a calibrated lens micrometer.
[911] Cell proliferation marker
[912] The proliferating cell nuclear antigen / cyclin (PCNA) of the skin specimen was expressed as an ABC elite detection system using an anti-PCNA antibody (1:50). Human clone cancer was used as a positive tissue control group, and human brain tissue was used as a negative tissue control group. Each specimen contained sections that were missing primary antibodies and were replaced by non-immunized mouse IgG. The proliferation of these fragments is represented by 0-8 steps from the side showing low proliferation to the side showing high proliferation to indicate the degree of proliferation.
[913] Statistical analysis
[914] Experimental data were analyzed using the unpaired t test. A p value of < 0.05 was considered significant. The data are expressed as mean ± SEM.
[915] result
[916] Effect of KGF-2 on wound closure
[917] Diabetic mice showed a healing process that was impaired compared to heterozygous normal mice. A dose of 4 ug of KGF-2 per site appeared to produce the maximal response in diabetic and non-diabetic animals (Figures 5 and 6). These results were statistically significant (p = 0.002 and p <0.0001) when compared to the cushion control. Treatment with KGF-2 resulted in a final mean closure of 60.6% in the 4 μg / day group and 34.5% in the 40 μg / day group. In the cushion control group, only 3.8% of the wound closure occurred by day 8. The wound area measurement of db + / db + mice treated with KGF-2 was repeated on days 2 to 5 and on day 8 of wound emergence, and after 3 days of wounding, the total wound area (mm 2) Improvement has been demonstrated. Improvement was continued and statistically significant results were observed at the end of the experiment (Fig. 7). Repeated measurements of the db / + m group of animals receiving KGF-2 showed that the wound area was significantly reduced compared to the buffered control (Fig. 8). These results demonstrate the fast wound closure rate of KGF-2 treated animals.
[918] Effect of KGF-2 on histological scores
[919] A histologic evaluation of KGF-2 in the diabetic (db + / db +) model on day 8 demonstrated a statistically significant improvement in wound score when compared to the cushion control (p <0.0001). The pharmacological effects observed in both 4 과 and 40 용량 of the dose of KGF-2 were not statistically different from each other. The buffered control group showed minimal cell accumulation without granulation tissue or epithelial migration while the 4 ㎍ and 40 용량 doses of KGF-2 (p <0.0001 & p = 0.06, respectively) showed wound epithelial, neovascularization, And fibroblast and collagen deposition (Figure 9).
[920] Histological evaluation of skin wounds was performed on hematoxylin-eosin stained samples. The scoring standard included from 1 to 12 steps, i.e., from step 1 showing minimal cell accumulation with little or no granulation, to the presence of 12 fibroblasts rich in collagen deposition and wound epithelium covering the wound (Table 1) .
[921] [Table 1] Score in histology
[922] Score Standard 1-3No minimal cell accumulation. No granulation tissue or epithelium running. 4-6Thin, immature granules dominated by inflammatory cells but with some fibroblasts, capillaries or collagen deposits. Minimal epithelial migration. 7-9Moderately thick granular tissue that can contain up to more fibroblasts and collagen deposition areas from areas dominated by inflammatory cells. Extensive neovascularization. The epithelium may range from a minimum to an appropriate range. 10-12Thick vascular granulation tissue dominated by fibroblasts and extensive collagen deposition. The epithelium covers partially or completely wound.
[923] After dosing with both KGF-2 doses, the evaluation of non-diabetic littermates showed no significant activity when compared to the buffered control for all assessed measures (Figure 10). Buffered controls showed immature granular tissue, inflammatory cells and capillaries. The mean score was higher than in the diabetic group, which represents the impaired healing process of diabetes (db + / db +) mice.
[924] Effect of KGF-2 on re-epithelization
[925] The extent of re-epithelialization was measured using cytokeratin immunostaining. Scores were displayed based on the degree of occlusion in steps of 0 (not closed) to 8 (complete closure). There was a statistically significant improvement (p &lt; 0.001) in the score of re-epithelization compared to the buffer control group in the 4 / / day administration group (Fig. 11). In this group, keratinocytes were observed to be ubiquitous in the newly formed epithelium overlying the wound. In addition, the capacity of both KGF-2 showed a mitotic figure at various stages. The re-epithelialization level was significantly improved (p = 0.006 and 0.01, respectively) in the non-diabetic group in both doses of KGF-2 (Fig. 12).
[926] Effect of KGF-2 on cell proliferation
[927] Proliferative nuclear antigen immunostaining was demonstrated to have significant proliferation in both 4 및 and 40 군 groups (Fig. 13). The non-diabetic group showed similar results as the two dose-administered groups of KGF-2, which showed a significantly significant score when compared to the buffered control (Fig. 14). Especially epidermal proliferation was observed in the basal layer of epidermis. In addition, high density PCNA-labeled cells were observed in dermis, especially in hair follicles.
[928] conclusion
[929] The results demonstrate that KGF-2 specifically stimulates the growth of major epidermal keratinocytes. In addition, this experiment demonstrates that locally applied recombinant human KGF-2 significantly promotes the healing rate of incised cuticle wounds to the full thickness of diabetic mice. Histological evaluation shows that KGF-2 induces keratinocyte proliferation that thickens the epidermis. This proliferation is localized in the basal layer of the epidermis, as evidenced by proliferating nuclear antigen (PCNA). At the dermis level, collagen deposition, fibroblast proliferation and neovascularization reshaped the normal structure of the skin.
[930] High-density PCNA-labeled cells in KGF-2 treated animals show stimulation of keratinocytes at the hair follicle, fibroblast, and dermal-epidermal levels, in contrast to the buffer group with little PCNA-labeled cells. Improvement of the healing process by KGF-2 was continuously observed in this experiment. This effect was statistically significant at the estimated parameters (re-epithelialization% and wound closure%). It is important that PCNA-labeled keratinocytes are mainly observed in the lower basal layer of the epidermis. The dermis exhibited normalized tissue with fibroblasts, collagen and granulated tissue.
[931] The observed activity in non-diabetic animals indicates that KGF-2 shows a significant pharmacological response in the percentage of wound closure on day 8 as well as during the course of the experiment based on daily measurements. Although the histopathological evaluation was not significantly different compared to the cushion control, keratinocyte growth and PCNA score demonstrated significant effects.
[932] In summary, these results demonstrate that KGF-2 exhibits significant activity in both damaged incision wound and normal incision wound using the db + / db + mouse model and, therefore, has no effect on surgical wound, diabetic ulcer, venous congestion ulcer, And would be useful in the treatment of wounds, including other skin conditions.
[933] Example 8
[934] KGF-2 mediated wound healing in steroid-impaired rat model
[935] Inhibition of wound healing by steroids has been well documented in a variety of in vitro and in vivo systems [Wahl, SM Glucocorticoids and Wound healing. In Anti-Inflammatory Steroid Action: Basic and Clinical Aspects. 280-302 (1989); Wahl, SM et al., J. Immunol. 115 : 476-481 (1975); Werb, Z. et al., J. Exp. Med. 147 : 1684-1694 (1978)). Glucocorticoids inhibit angiogenesis and decrease vascular permeability [Ebert, RH, et al., An. Intern. Med. 37: 701-705 (1952)], reducing fibroblast proliferation and reducing collagen synthesis [Beck, LS et al., Growth Factors. 5 : 295-304 (1991); Haynes, BF, et al., J. Clin. Invest. 61 : 703-797 (1978)], induces a temporary decrease in circulating monocytes [Haynes, BF, et al., J. Clin. Invest. 61 : 703-797 (1978); Wahl, SM Glucocorticoids and wound healing. In Antiinflammatory Steroid Action: Basic and Clinical Aspects. Academic Press. New York. pp. 280-302 (1989)]. It is well known that systemic administration of steroids impairs wound healing as observed in rats (Beck, LS et al., Growth Factors. 5 : 295-304 (1991); Haynes, BF, et al., J. Clin. Invest. 61 : 703-797 (1978); Wahl, SM Glucocorticoids and wound healing. In Antiinflammatory Steroid Action: Basic and Clinical Aspects. Academic Press. New York. pp. 280-302 (1989); Pierce, GF, et al., Proc. Natl. Acad. Sci. USA. 86 : 2229-2233 (1989)].
[936] To demonstrate that KGF-2 promotes healing, the effects of multi-local application of KGF-2 on skin wounds cut to full thickness in rats healed by systemic administration of methylprednisolone were evaluated. In vitro studies have demonstrated that KGF-2 specifically stimulates the growth of key human epidermal keratinocytes. This example demonstrates that topically applied recombinant human KGF-2 promotes wound healing of the incised wound to the full thickness of the rat, by wound interval measurement using graduated Jameson calipers and by tissue morphology and immunohistochemistry do. Histological evaluation demonstrates that KGF-2 promotes re-epithelization, thereby promoting wound healing.
[937] animal
[938] In this example, young and mature male Sprague Dawley rats (Charles River Laboratories) weighing 250-300 g were used. The animals purchased eight weeks and were nine weeks old at the start of the study. The healing response of the rats was impaired by systemic administration of methylprednisolone (17 mg / kg / rat, intramuscular administration) at the time of wounding. Animals were housed individually, and food and water were given unlimited. All manipulations were performed using aseptic techniques. These studies were conducted in accordance with the guidelines and guidelines of the Human Genome Sciences, Inc. Institutional Animal Care and Use Committee and the Guidelines for the Care and Use of Laboratory Animals.
[939] KGF-2
[940] The recombinant human KGF-2 was over-expressed and purified from pQE60-Cys37, an E. coli expression vector system (pQE-9, Qiagen). The protein expressed from this construct is KGF-2 from cysteine at position 37 to serine at position 208 with the 6X (His) tag attached to the N-terminus of the protein (SEQ ID NO: 29-30) 15). Fractions containing more than 95% pure recombinant material were used in the experiments. KGF-2 was formulated in a vehicle containing 1X PBS. The final concentrations were 20 μg / ml and 80 μg / ml of the mother liquor. Diluted from the mother liquor using the same vehicle.
[941] KGF-2 28 was over-expressed and purified from the E. coli expression vector system. Fractions containing more than 95% pure recombinant material were used in the experiments. KGF-2 was formulated in a vehicle containing 1X PBS. The final concentrations were 20 μg / ml and 80 μg / ml of the mother liquor. Diluted from the mother liquor using the same vehicle.
[942] Surgical wound
[943] A wound-up protocol was performed according to the method of Example 7 above. Animals were anesthetized by intramuscular injection of ketamine (50 mg / kg) and xylazine (5 mg / kg). The hairs on the back of the animal were removed and the skin was washed with 70% ethanol solution and iodine. Prior to wounding, the surgical site was dried with sterile gauze. Using a Keyes tissue punch, the wound was covered with a sufficient thickness of 8 mm. The wound area was left open during the experiment. Test material was topically applied continuously for 7 consecutive days starting from the wounded day, followed by methylprednisolone. Prior to treatment, the wound area was gently rinsed with sterile saline and a gauze sponge.
[944] The wounds were visually observed at the time of wounding and at the end of treatment, and pictures were taken at a fixed distance. Wound closure was measured on days 1-5 and on day 8. The wound was measured horizontally and vertically using a graduated Jameson caliper. The wound was considered healed if the granular tissue was no longer visible and the wound was covered with continuous epithelial tissue.
[945] Using two different doses of KGF-2, ie, 50 μl of vehicle, one dose of KGF-2 was administered for 5 days at 1 μg per wound per day for 5 days and the other at 4 μg per wound per day Respectively. Vehicle controls received 50 ㎕ of 1X PBS.
[946] Animals were euthanized by intraperitoneal injection of sodium pentobarbital (300 mg / kg) on day 8. Wounds and surrounding skin were collected for histological study. Tissue samples were placed in 10% neutral buffered formalin in tissue cassettes between biopsied sponges for further processing.
[947] Experiment plan
[948] Four groups of 10 animals (5 methylprednisolone treated groups and 5 non-glucocorticoid treated groups) were evaluated:
[949] 1) Untreated group
[950] 2) vehicle placebo control
[951] 3) KGF-2 &lt; RTI ID = 0.0 &gt;
[952] 4) KGF-2 4 μg / day.
[953] The study was planned as follows:
[954] n group processing
[955] · Glucocorticoid-treatment
[956] N = 5 untreated -
[957] N = 5 vehicle 50 μl
[958] 50 μl of N = 5KGF-2 (1 μg)
[959] N = 5 KGF-2 (4)) 50 ㎕
[960] · No glucocorticoids
[961] N = 5 untreated -
[962] N = 5 vehicle 50 μl
[963] 50 μl of N = 5KGF-2 (1 μg)
[964] N = 5 KGF-2 (4)) 50 ㎕
[965] Measurement of wound area and occlusion
[966] The area was measured on the vertical and horizontal axes to obtain the total square area of the wound area and the wound closure was analyzed. Thereafter, the closure was evaluated by measuring the difference between the initial wound area (day 0) and the wound area after treatment (day 8). The wound area on the first day was 64 mm2, which was consistent with the skin punch size. Calculated using the following equation:
[967] [Open area on the eighth day] - [Open area on the first day] / [Open area on the first day]
[968] histology
[969] The specimens were fixed in 10% buffered formalin and the paraffin-containing block was divided vertically into the wound surface (5 mu m) and dissected using an Olympus microtome. Normal hematoxylin-eosin (H & E) staining was performed on the cross-sections of the divided wounds. Whether the healing process and the morphological appearance of the treated skin was improved by KGF-2 treatment was assessed by histological examination of the wound. Blind observers used a calibrated lens micrometer to measure the distance of the wound space.
[970] Statistical analysis
[971] The experimental data were analyzed using the unpaired t test. A p value of &lt; 0.05 was considered significant. The data are expressed as mean ± SEM.
[972] result
[973] Comparison of the wound closure of methylprednisolone treated and untreated untreated control demonstrated that methylprednisolone-treated rats had significant damage to the wounding process 8 days after wounding compared to normal mice. Total wound area was measured as 58.4 mm 2 in the methylprednisolone injected group and 22.4 mm 2 in the untreated glucocorticoid group (Fig. 16).
[974] Effect of KGF-2 on wound closure
[975] Systemic administration of methylprednisolone to rats at wounding formation delayed the wound closure of normal rats (p = 0.002). At the end of the 8th day, the wound closure of the methylprednisolone-damaged group at the end of the experiment was statistically significantly higher than that of the untreated group when treated with KGF-2 (1 μg p = 0.002 & 4 μg p = 0.005) (Fig. 16). The percentage of wound closure in the group receiving 1 μg KGF-2 was 60.2% (p = 0.002) and the percentage of wound closure in the group receiving 4 μg KGF-2 was 73% (p = 0.0008). On the other hand, the wound closure of the untreated group was 12.5% and the vehicle placebo group was 28.6% (Fig. 17).
[976] On days 1 to 8, longitudinal analysis of wound closure in the glucocorticoid group revealed a significant reduction in wound size on wound days 3 to 8 at both doses of the KGF-2 treated group (Figure 18) .
[977] The results demonstrate that the group treated with 4 ug of KGF-2 promoted wound closure to a statistically significant level when compared to the untreated group (p = 0.05) (Fig. 19A). Although it is difficult to assess the ability of proteins or other compounds to promote normal animal wound healing (because the recovery is rapid), KGK-2 has been shown to promote wound healing in these models.
[978] Histopathological evaluation of KGF-2 treated wounds
[979] Histological morphology of the wound space showed that the wound distance of the KGF-2 treated group was reduced. The wound interval observed in the untreated group was 5336μ, whereas in the group treated with KGF-2 1μg, the wound interval was reduced to 2972μ. In the group treated with KGF-2 4μg, the wound interval was reduced to 3086μ (p = 0.04) 20).
[980] Effect of KGF-2 △ 28 on wound healing
[981] The effects of KGF-2Δ28 and PDGF-BB in wound healing of methylprednisolone-impaired rats were also investigated. This experiment was performed under the same conditions as for the KGF-2 protein except that the His tag was not attached to the KGF-2Δ28 protein and the wound healing process was measured on days 2, 4, 6, 8 and 10 . The buffer vehicle for this protein was 40 mM NaOAc and 150 mM NaCl, pH 6.5 for all but the " E2 " formulation of the full length KGF-2. The buffer vehicle for the " E2 " formulation was 20 mM NaOAc and 400 mM NaCl, pH 6.4.
[982] The results shown in FIG. 19B demonstrate that KGF-2Δ28 promotes wound closure to a statistically significant level and reverses the effect of methylprednisolone on wound healing compared to the untreated group.
[983] conclusion
[984] This example demonstrates that KGF-2 reversed the effect of methylprednisolone on wound healing. Exogenous application of growth factors can promote granulation tissue formation by attracting inflammatory cells into the wound. Similar activity was also observed in KGF-2 based on daily measurements in animals that did not receive methylprednisolone, indicating that KGF-2 showed a significant pharmacological response in wound closure percentage on day 5. The clinico-corticoid-damaged wound healing rat model has been shown to be a suitable and reproducible model for the measurement efficiency of KGF-2 and other compounds in the wound healing area.
[985] In summary, the results demonstrate that KGF-2 exhibits significant activity in both the glucocorticoid-impaired incision wound model and the normal incision wound model. Thus, KGF-2 has been implicated in systemic treatment with surgical wounds, diabetic ulcers, venous congestion ulcers, burns and other abnormal wound healing symptoms, such as uremia, malnutrition, vitamin deficiencies and steroids and anti- It will be clinically useful in stimulating wound healing, including treatment.
[986] Example 9
[987] Tissue distribution of KGF-2 mRNA expression
[988] To investigate the expression levels of genes coding for the KGF-2 protein in human tissues, Sambrook et al. Northern blot analysis is performed using the method described in &lt; RTI ID = 0.0 &gt; Probe corresponding to the entire open reading frame of KGF-2 of the present invention (SEQ ID NO: 1) to obtain by PCR, using the redi prime ^TM DNA labeling system (Amersham Life Science) with ³² P according to the manufacturer's instructions Respectively. After labeling, the probe was purified according to the manufacturer's protocol number PT1200-1 using a CHROMA SPIN-100 ^TM column (Clontech Laboratories, Inc.). Thereafter, various human tissues were examined for the expression of genes encoding KGF-2 using purified and labeled probes.
[989] Multi-tissue Northern (MTN) blots containing poly A RNA derived from various human tissues (H) or human immune system tissues (IM) were obtained from Clontech, and ExpressHyb ^TM hybrid formation was performed according to manufacturer's protocol number PT1190-1 Solution (clontech) and a labeled probe. After hybrid formation and washing, the blot was mounted and exposed to film overnight at -70 DEG C, and the films were developed according to standard procedures.
[990] Approximately 4.2 kb of the main mRNA species was observed in most human tissues. KGF-2 mRNA was relatively abundant in the heart, pancreas, placenta and ovary. Small mRNA species of about 5.2 kb were also found to be ubiquitous. The identity of these 5.2 mRNA species was not clear. There is a possibility that the 5.2 kb transcript could alternatively encode a linked form of KGF-2 or a third member of the KGF system. KGF-2 cDNA was 4.1 kb consistent with the size of 4.2 kb mRNA.
[991] Example 10
[992] Keratinocyte proliferation assay
[993] Dermal keratinocytes are cells in the epidermis of the skin. The growth and spread of keratinocytes in the skin is an important process in wound healing. Thus, proliferation assay of keratinocytes is a useful indicator of protein activity in stimulating keratinocyte growth and consequently healing wounds.
[994] However, keratinocytes are difficult to grow in vitro. A small number of keratinocyte cell lines are present. These cell lines have different cells and genetic defects. Primary dermal keratinocytes are selected for this assay to avoid the complexity of the assay by cell defects, such as critical growth factor receptor or dependence of critical growth factors for growth. These major keratinocytes are purchased from Cronetics, Inc. (San Diego, Calif.).
[995] Keratinocyte proliferation assay using alamarBlue
[996] Alarama blue is a viable blue dye that is metabolized by mitochondria when added to the medium. The dye then turns red in the tissue culture supernatant. The amount of the red dye can be directly quantified by measuring the difference in optical density from 570 nm to 600 nm. These measurements reflect cell activity and cell number.
[997] Normal major dermal keratinocytes (CC-0255, NHEK-Neo pooled) are purchased from CloNetix, Inc. (San Diego, CA). These cells are passage 2. The keratinocytes are grown in complete keratinocyte growth medium (CC-3001, KGM; Cronetics, Inc.) until they reach 80% confluency. The cells are trypsinized according to the manufacturer's specifications. In summary, cells were washed twice with Hank's balanced salt solution. 2-3 ml of trypsin was added to the cells for about 3-5 minutes at room temperature. Trypsin neutralization solution was added and cells were harvested. Cells were spun at 600 x g for 5 minutes at room temperature and plated on fresh flasks at 3,000 cells per cm 2 using preheated media.
[998] For proliferation analysis. The keratinocytes are plated at 1,000 to 2,000 keratocytes per well of a Corning flat bottom 96-well plate in complete medium, except for the outermost column. The outer wells are filled with 200 [mu] l of sterile water. This helps to keep the well temperature and humidity changes to a minimum. Cells are grown overnight at 37 ° C under 5% CO ₂ . Cells are washed twice with keratinocyte basal medium (CC-3101, KBM, Cronetics, Inc.) And 100 쨉 l of KBM is added to each well. Incubate for 24 hours. Dilute the growth factors in KBM to make a series of dilutions, and add 100 μl to each well. KGM is used as a positive control and KBM is used as a negative control. Six wells are used for each concentration point. Incubate for 2 to 3 days. At the end of the incubation period, the cells are washed once with KBM and 100 μl of 10% v / v AlamarBlue and KBM premixed in the medium are added. In the KGM positive control, incubate for 6 to 16 hours until the color of the medium begins to turn red. Measure OD 570nm minus OD 600nm by placing the plates directly in a plate reader.
[999] result
[1000] Stimulation of keratinocyte proliferation by KGF-2
[1001] KGF-2 (i.e., starting from the amino acid Cys37 as described in Examples 7 and 8 above) and the N-terminal deletion mutants KGF-2Δ33 and KGF-2Δ28 were found to be active in stimulating epidermal keratinocyte growth (SEQ ID NO: 2), KGF-2 33 (batch number E1) and KGF-2 protein (batch number E3) -2 &gt; 28 (batch number E2). The KGF-2 protein stimulated the growth of epidermal keratinocytes with an EC50 of about 5ng / ml corresponding to that of FGF7 / KGF-1 (Figure 21A). On the other hand, other FGFs such as FGF-1 and FGF-2 did not stimulate the growth of major keratinocytes. The EC50 for KGF-2 33 was 0.2 ng / ml and the EC50 for KGF-2 28 was 2 ng / ml (Figures 21B and 21C). Thus, KGF-2 appeared to be as effective as FGF7 / KGF in stimulating proliferation of major epidermal keratinocytes. However, KGF-2 33 is more potent in stimulating keratinocyte proliferation than " Cys (37) &quot; KGF-2 and KGF-2 28 described in Examples 7 and 8 described above.
[1002] Scar formation of the wound tissue includes hyperplasia of dermal fibroblasts. To determine whether the stimulatory effect of KGF-2 was specific only to keratinocytes and not to fibroblasts, mouse Balb.c.3T3 fibroblasts and human lung fibroblasts were tested. No fibroblasts of any type responded to KGF-2 in the proliferation assay. Thus, KGF-2 appears to be a specific mitogen for epidermal keratinocytes but not for mesenchymal cells such as fibroblasts. This suggests that KGF-2 is unlikely to cause scar formation in the wound tissue.
[1003] Example 11
[1004] A. Mitogen Effect of KGF-2 on Cells Transformed with Specific FGF Receptors
[1005] In order to determine whether the FGF receptor isoform mediates the proliferative effect of KGF-2, the effect of KGF-2 on cells expressing the specific FGF receptor isoform was assessed by Santos-Ocampo et al. J. Biol. Chem. 271 : 1726-1731 (1996). FGF7 / KGF is known to induce mitogenesis of epithelial cells by specific activation by binding to FGFR2iiib form [Miki et al. Science 251: 72-75 (1991). Thus, in the mitogenesis assay, the proliferative effect of KGF-2 was tested using cells expressing one of the following FGF receptor isoforms: FGFR1iiib, FGFR2iiib, FGFR3iiib and FGFR4.
[1006] Mice genetic analysis of cells expressing FGF receptor
[1007] Santos-Ocampo et al. J. Biol. Chem. 271 : 1726-1731 (1996), thymidine incorporation of BaF3 cells expressing specific FGF receptors was performed. Briefly, BaF3 cells expressing the specific FGF receptor were washed and resuspended in Dulbecco's modified Eagel's medium, 10% neonatal bovine serum, L-glutamine. About 22,500 cells per well were plated in 96-well assay plates in media containing 2 ug / ml heparin. Reagents were added to each well to give a total volume of 200 [mu] l per well. Cells were incubated at 37 [deg.] C for 2 days. Thereafter, ¹ mu Ci of 3H-thymidine was added to each well in a volume of 50 mu l. Cells were harvested after 4 to 5 hours by filtration through glass fiber paper. The incorporated ³ H-thymidine was counted with a Wallac beta plate scintillation counter.
[1008] result
[1009] The results, KGF-2 protein [there the N- terminal Met FIG Thr (36) of the first with the addition to -Ser (208) (SEQ ID NO: 2) is, as indicated by the ³ H- thymidine incorporation Similarly, it was shown to strongly stimulate proliferation of Baf3 cells expressing the KGF receptor, FGFR2iiib isoform (Fig. 22A). Interestingly, a weak stimulatory effect of KGF-2 on the proliferation of Baf3 cells expressing FGFR1iiib isoform was observed. KGF-2 did not show any effect on the cells expressing FGFR3iiib or FGFR4 of the receptor.
[1010] FGF7 / KGF stimulated the proliferation of cells expressing the KGF receptor, FGFR2iiib, but did not stimulate FGFR1iiib isoform. The difference between KGF-2 and FGF7 / KGF was interesting. In a control experiment, aFGF stimulated its receptors FGFR1iiib and iiic, and bFGF stimulated its receptor FGFR2iiic. Thus, these results suggested that KGF-2 binds to FGFR2iiib isoform and stimulates mitogenesis. Unlike FGF7 / KGF, KGF-2 also binds to FGFR1iiib isoforms to stimulate mitogenesis.
[1011] B. Mitogen Effect of KGF-2 33 on Cells Transformed with Specific FGF Receptors
[1012] As demonstrated above, both FGFs or both KGF-1 and KGF-2 bind to and activate the FGF2iiib receptor (FGFR2iiib). The proliferative effect of KGF-2 33 in mitochondrial assays was examined using cells expressing one of the following FGF receptor isoforms: FGFR2iiib or FGFR2iiic (2iiic receptor-transfected cells are included as negative control).
[1013] As described in Part A of this embodiment, this experiment was performed. Briefly, 10% conditioned medium derived from the culture of 10% calf serum (not BCS-fetal serum), WEHI3 cells (grown in RPMI containing 5% BCS), 50 nM -Mercaptoethanol, L-Glu (2% of 100X stock solution) and pen / strep (1% of 100X stock solution).
[1014] For analysis, BaF3 cells were washed twice in RPMI medium containing 10% BCS and 1 [mu] g / ml heparin. BaF3 cells (22,000 / well) were plated in 96-well plates in 150 占 퐇 of RPMI medium containing 10% BCS and 1 占 퐂 / ml heparin. Acidic FGF, basic FGF, KGF-1 (HG15400) or KGF-2 protein (HG03400, 03401, 03410 or 03411) was added at a concentration of about 0-10 nM. The cells were incubated at 37 [deg.] C for 48 hours in a final volume of 200 [mu] l. All analyzes were performed three times. Tritiated thymidine (0.5 μCi) was added to each well at 37 ° C. for 4 hours and cells were harvested by filtration through glass fiber paper. Thereafter, the total amount of radioactivity incorporated by the liquid scintillation counting method was measured. The following positive controls were used: basic FGF and acidic FGF for FGFR2iiic cells; Acidic FGF and KGF-1 for FGFR2iiib. The following negative controls were used: basal medium (RPMI medium containing 10% BCS and 1 ug / ml heparin).
[1015] result
[1016] The results, as the [Thr (36) -Ser (208 ) having an addition of an N- terminal Met], KGF2 △ 33 and △ 28 KGF2 protein KGF2, indicated by ³ H- thymidine incorporation, And strongly stimulated the proliferation of Baf3 cells expressing KGF receptor FGFR2iiib isoform (Fig. 22A-C). The KGF-2 protein had no effect on cells expressing the FGFR2iiic form of the receptor. These results suggested that KGF-2 protein binds to FGFR2iiib isoform and stimulates mitogenesis. Furthermore, KGF-2 33 was able to stimulate BaF3 cell proliferation better than KGF-2 [Thr (36) -Ser (208)].
[1017] Example 12
[1018] A. Construction of KGF-2 optimized E. coli
[1019] In order to increase the expression level of the full-length KGF-2 in the E. coli expression system, the codon of the amino terminal portion of the gene was optimized with the E. coli codon for the use. For the synthesis of the optimized region of KGF-2, a series of six oligonucleotides were synthesized: number 1 to 6 (sequences are mentioned below). These overlap oligos were used for PCR reactions in 7 rounds under the following conditions:
[1020] Degeneration 95 degrees 20 seconds
[1021] Annealing 58 degrees 20 seconds
[1022] Height 72 degrees 60 seconds
[1023] Using 1 μl of the first PCR reaction using KGF-2 synthetic 5 'BamHI as the KFG-2 synthetic primer 6 and 5' primer as the 3 'primer and using the same conditions as described above, PCR reaction was designed. The product prepared by the final reaction was digested with AvaII and BamHI. KGF-2 constructs of Example 1 were digested with AvaII and HindIII, and the fragments were isolated. These two fragments were cloned into pQE-9 digested with BamHI and HindIII in three fragment ligations.
[1024] Primers were used to construct optimized synthetic KGF-1 1/208:
[1025] KGF-2 synthetic primer 1:
[1026] ATGTGGAAATGGATACTGACCCACTGCGCTTCTGCTTTCCCGCACCTGCCGGGTTGCTGCTGCTGCTGCTTCCTGCTGCTGTTC (SEQ ID NO: 31)
[1027] KGF-2 synthetic primer 2:
[1028] CCGGAGAAACCATGTCCTGACCCAGAGCCTGGCAGGTAACCGGAACAGAAGAAACCAGGAACAGCAGCAGGAAGCAGCAGCA (SEQ ID NO: 32)
[1029] KGF-2 synthetic primer 3:
[1030] GGGTCAGGACATGGTTTCTCCGGAAGCTACCAACTCTTCTTCTTCTTCTTTCTCTTCTCCGTCTTCTGCTGGTCGTCACG (SEQ ID NO: 33)
[1031] KGF-2 synthetic primer 4:
[1032] GGTGAAAGAGAACAGTTTACGCCAACGAACGTCACCCTGCAGGTGGTTGTAAGAACGAACGTGACGACCAGCAGAAGACGG (SEQ ID NO: 34)
[1033] KGF-2 synthetic primer: 5
[1034] CGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAACGGTAAAGTTTCTGGGACCAAA (SEQ ID NO: 35)
[1035] KGF-2 synthetic primer 6:
[1036] TTTGGTCCCAGAAACTTTACCGTTTTTTTCGATTTTCAG (SEQ ID NO: 36)
[1037] KGF-2 synthetic 5'BamHI
[1038] AAAGGATCCATGTGGAAATGGATACTGACCCACTGC (SEQ ID NO: 37)
[1039] The resulting clones are shown in Figure 23 (SEQ ID NOS: 38 and 39).
[1040] B. Construction of E. coli optimized mature KGF-2
[1041] In order to further increase the expression level of the mature form of KGF-2 in the E. coli expression system, the codon of the amino terminal portion of the gene was optimized with the E. coli codon used. The truncated form of KGF-2 was constructed starting from threonine 36, corresponding to the maturation type of KGF-1. In a PCR reaction using BspHI 5 'KGF-2 (the sequence is given below) as the 5' primer and HindIII 3 'KGF-2 (given in the sequence below) as the 3' primer, the E. coli synthetic KGF from Example 12A -2 as a template. The amplification procedure was performed for 25 cycles using the standard conditions given in Example 12A above. The resulting product was digested with BspHI and HindII and cloned into E. coli expression vector pQE60 digested with NcoI and HindIII.
[1042] BspHI 5 'KGF-2 primer:
[1043] TTTCATGACTTGTCAAGCTCTGGGTCAAGATATGGTTC (SEQ ID NO: 40)
[1044] HindIII 3 ' KGF-2 primer:
[1045] GCCCAAGCTTCCACAAACGTTGCCTTCC (SEQ ID NO: 41)
[1046] The resulting clone is shown in Figure 24A (SEQ ID NOS: 42 and 43).
[1047] C. Construction of alternative E. coli optimized maturation KGF-2
[1048] To further increase the expression level of KGF-2 mature form in the E. coli expression system, a codon of 53 amino acids was changed to the E. coli codon for the amino terminal portion of the E. coli optimized gene. For the synthesis of the optimized region of KGF-2, a series of six oligonucleotides were synthesized: Nos. 18062, 18061, 18058, 18064, 18059 and 18063 (the sequences are mentioned below). These overlap oligos were used for PCR reactions in 7 rounds under the following conditions:
[1049] Degeneration 95 degrees 20 seconds
[1050] Annealing 58 degrees 20 seconds
[1051] Height 72 degrees 60 seconds
[1052] After 7 rounds of synthesis, 5 'primer 18169 for the above region and 3' primer 18060 for the entire region were added to the PCR reaction containing 1 mu l derived from the initiation reaction of 6 oligonucleotides. The product was amplified for 30 revolutions using the following conditions:
[1053] Degeneration 95 degrees 20 seconds
[1054] Annealing 55 degrees 20 seconds
[1055] Height 72 degrees 60 seconds
[1056] The second PCR reaction was designed to amplify the 3 'region of the gene using primers 18066 and 18065 under the same conditions as described above for 25 revolutions. The product was separated from the agarose gel. The gel piece containing the product was diluted in 10 mM Tris, 1 mM EDTA, pH 7.5. 1 [mu] l of each taken from each diluted gel piece was used in an additional PCR reaction in which primer 18169 was used as the 5 'primer and primer 18065 as the 3' primer. The product was amplified for 25 cycles using the same conditions as described above. The product produced by the final reaction was digested with Eco R1 and Hind III and cloned into pQE60 (now pQE6) which was also cut with Eco R1 and HindIII.
[1057] Sequence of 5 'synthetic primer:
[1058] 18169 KGF2 5'EcoRI / RBS:
[1059] TCAGTGAATTCATTAAAGAGGAGAAATTAATCATGACTTGCCAGG [SEQ ID NO: 44]
[1060] 18062 KGF2 Synthetic New R1 sense:
[1061] TCATGACTTGCCAGGCACTGGGTCAAGACATGGTTTCCCCGGAAGCTA [SEQ ID NO: 45]
[1062] 18061 KGF2 Synthetic R2 Sense:
[1063] GCTTCAGCAGCCCATCTAGCGCAGGTCGTCACGTTCGCTCTTACAACC [SEQ ID NO: 46]
[1064] 18058 KGF2 Synthetic R3 Sense:
[1065] GTTCGTTGGCGCAAACTGTTCAGCTTTACCAAGTACTTCCTGAAAATC [SEQ ID NO: 47]
[1066] 18066 KGF 2 20bp Ava II Sense:
[1067] TCGAAAAAAACGGTAAAGTTTCTGGGAC [SEQ ID NO: 48]
[1068] 18064 KGF2 Synthetic F1 antisense:
[1069] GATGGGCTGCTGAAGCTAGAGCTGGAGCTGTTGGTAGCTTCCGGGGAA [SEQ ID NO: 49]
[1070] 18059 KGF2 Synthetic F2 antisense:
[1071] AACAGTTTGCGCCAACGAACATCACCCTGTAAGTGGTTGTAAGAG [SEQ ID NO: 50]
[1072] 18063 KGF2 Synthetic F3 antisense:
[1073] Gt;
[1074] 18060 KGF 2 AvaII antisense:
[1075] TTCTTGGTCCCAGAAACTTTACCG [SEQ ID NO: 52]
[1076] 18065 KGF2 HindIII 3 'stop:
[1077] AGATCAGGCTTCTATTATTATGAGTGTACCACCATTGGAAGAAAG [SEQ ID NO: 53]
[1078] The synthetic KGF-2 gene and the corresponding amino acid sequence are shown in Figure 24B (SEQ ID NOS: 54 and 55).
[1079] Example 13
[1080] Construction of KGF-2 deletion mutants
[1081] A deletion mutant was constructed from the 5 ' and 3 ' terminals of the KGF-2 gene using the optimized KGF-2 construct from Example 12A as the template. E. coli. Lt; RTI ID = 0.0 &gt; gene &lt; / RTI &gt; For 5 'deletion, the primers listed below were used as 5' primers. These primers include the ATG coding for the initiation methionine and the indicated restriction sites. KGF-2 (FGF-12) 208 amino acid 3 'HindIII primer was used as the 3' primer. PCR amplification was performed for 25 revolutions using standard conditions as mentioned in Example 12. The product for KGF-2 36aa / 208aa deletion mutants was cut into BspHI for the 5 'position, HindIII for the 3' position, and cloned into pQE60 digested with BspHI and HindIII. All other products were digested with NcoI for the 5 'restriction enzyme, digested with HindIII at the 3' position and cloned into pQE60 digested with NcoI and HindIII. 36aa / 153aa and 128aa 3 'HindIII were used as 3' primers and FGF-12 36aa / 208aa was used as 5 'primers for KGF-2 (FGF-12). For FGF-12 62aa / 153aa, 128aa 3'HindIII was used as the 3 'primer and FGF-12 62aa / 208aa was used as the 5' primer. The name of the resulting clone indicates the first and last amino acid of the polypeptide resulting from deletion. For example, KGF-2 36aa / 153aa means that the first amino acid of the deletion mutant is 36 and the last amino acid is the amino acid 153 of KGF-2. In addition, as shown in Figures 25 to 33, each mutant has an N-terminal Met added thereto.
[1082] Sequence of deletion primer:
[1083] FGF12 36aa / 208aa:
[1084] 5 'BsphI GGACCCTCATGACCTGCCAGGCTCTGGGTCAGGAC [SEQ ID NO: 56]
[1085] FGF12 63aa / 208aa:
[1086] 5 'NcoI GGACAGCCATGGCTGGTCGTCACGTTCG [SEQ ID NO: 57]
[1087] FGF12 77aa / 208aa:
[1088] 5 'NcoI GGACAGCCATGGTTCGTTGGCGTAAACTG [SEQ ID NO: 58]
[1089] FGF12 93aa / 208aa:
[1090] 5 'NcoI GGACAGCCATGGAAAAAAACGGTAAAGTTTC [SEQ ID NO: 59]
[1091] FGF12 104aa / 208aa:
[1092] 5 'NcoI GGACCCCCATGGAGAACTGCCCGTAGAGC [SEQ ID NO: 60]
[1093] FGF12 123aa / 208aa:
[1094] 5 'NcoI GGACCCCCATGGTCAAAGCCATTAACAGCAAC [SEQ ID NO: 61]
[1095] FGF12 138aa / 208aa:
[1096] 5 'NcoI GGACCCCCATGGGGAAACTCTATGGCTCAAAAG [SEQ ID NO: 62]
[1097] FGF12 3 'HindIII: (used for all of the deletion clones above)
[1098] CTGCCCAAGCTTATTATGAGTGTACCACCATTGGAAG [SEQ ID NO: 63]
[1099] FGF12 36aa / 153aa:
[1100] 5 ' BsphI (as described above)
[1101] 3 'HindIII CTGCCCAAGCTTATTACTTCAGCTTACAGTCATTGT [SEQ ID NO: 64]
[1102] FGF12 63aa / 153aa:
[1103] 5 'NcoI and 3' HindIII, as described above.
[1104] Sequences for the resulting deletion mutants are shown in Figures 25 to 33 (SEQ ID NOS: 65 to 82).
[1105] Proteinase inhibitors, such as guanidine hydrochloride (Gu-HCl), are used to inhibit protein denaturation when expressing KGF-2Δ28 (amino acids 63-208) in E. coli. For example, the E. coli paste is resuspended in 50 mM Tris-acetate, 10 mM EDTA-NA ₂ , pH 7.7 ± 0.2 and lysed. The lytic suspension is treated with the same volume of 1.0 M Gu-HCl solution and gently stirred at 2-8 [deg.] C for 2-4 hours. Thereafter, the suspension is centrifuged, filtered, and then dropped onto the first column for purification. The initial purified product is placed on an SP-Sepharose FF column eluting the bound KGF-2 with a salt gradient. The resulting SP-Sepharose eluting pool was diluted, 0.2 μm filtered, and loaded onto the Fructose gel COO- (S) column. Elution was carried out through a salt gradient, and elution pools were dialyzed and concentrated in buffer.
[1106] Example 14
[1107] Construction of cysteine mutants of KGF-2
[1108] The KGF-2 (FGF-12) template from Example 12A was amplified using constructs of the C-37 mutant primer 5457 5 'BsphI and 5258 173aa3' HindIII. Primer 5457 5 'BsphI changes cysteine 37 to serine. Amplification was performed for 25 cycles using the standard conditions described above in Example 12A. The resulting product was digested with BspHI and HindIII and cloned into E. coli expression vector pQE60 digested with BspHI and HindIII (Fig. 34) (SEQ ID NO: 83).
[1109] For the mutation of cysteine 106 to serine, two PCR reactions were designed for oligonucleotide site induction mutagenesis of the cysteine. In the first reaction, 5453 Bsphl was used as the 5 'primer and 5455 was used as the 3' primer. In the second reaction, 5456 was used as the 5 'primer and 5258 HindIII was used as the 3' primer. An amplification reaction was performed for 25 revolutions under the standard conditions described in Example 12. One μl of each PCR reaction was used as a template in a sequential reaction using 5453 BspHI as a 5 'primer and 5258 HindIII as a 3' primer. Amplification was carried out for 25 revolutions using the standard conditions described in Example 12. The product was digested with BspHI and HindIII and cloned into E. coli expression vector pQE60 digested with NcoI and HindIII.
[1110] Two PCR reactions were required to prepare C-37 / C-106 mutants. Primers 5457 Bsph1 and 5455 were used to generate mutant 5 'regions in which cysteine 37 was substituted with serine and primers 5456 and 5258 HindIII were used to generate 3' regions of the mutant in which cysteine 106 was replaced with serine. In the second reaction, a C-37 / C-106 mutant was prepared using the 5457 Bsphl primer as the 5 'primer, the 5258 HindIII primer as the 3' primer, Respectively. The PCR product was digested with BsphI and HindIII and cloned into pQE60 digested with NcoI and HindIII. The resulting clone is shown in Figure 35 (SEQ ID NO: 84).
[1111] Sequence of cysteine mutant primers:
[1112] 5457 BspHI:
[1113] GGACCCTCATGACCTCTCAGGCTCTGGGT (SEQ ID NO: 85)
[1114] 5456:
[1115] AAGGAGAACTCTCCGTACAGC (SEQ ID NO: 86)
[1116] 5455:
[1117] GCTGTACGGTCTGTTCTCCTT (SEQ ID NO: 87)
[1118] 5453 BspHI:
[1119] GGACCCTCATGACCTGCCAGGCTCTGGGTCAGGAC (SEQ ID NO: 88)
[1120] 5258 HindIII:
[1121] CTGCCCAAGCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO: 89)
[1122] Example 15
[1123] Preparation and purification of KGF-2 (FGF-12)
[1124] The DNA sequence coding for the optimized mature protein described in Example 12B (i.e., amino acids T36 to S208 of KGF-2) was cloned into plasmid pQE-9 (Qiagen). E. coli (M15 / rep4; Qiagen) was grown in stationary phase overnight at 37 占 폚 in LB containing 100 占 퐂 / ml ampicillin and 25 占 퐂 / ml kanamycin. Using the culture, fresh LB medium containing 100 μg / ml ampicillin and 25 μg / ml kanamycin was inoculated with 1:50 dilution. Cells were grown to 0.7 OD ₅₉₅ at 37 ° C and induced to a final concentration of 1 mM by the addition of isopropyl 1-thio-bD-galactopyranoside (IPTG). 3 to 4 hours later, the cells were collected by centrifugation, 60mM NaPO _4, and 5 volumes buffer to buffer containing 360mM NaCl: resuspended at a ratio of 1 volume of cell paste. After rupture in Mautin Gaulin, the extract was adjusted to pH 8.0 by the addition of NaOH and purified by centrifugation.
[1125] The purified soluble extract was applied to a porous HS-50 column (2.0 x 10.0 cm; Perceptive Biosystems, Inc.) and the bound protein was eluted with 50 mM NaPO ₄ pH 8 containing 0.5 M, 1.0 M and 1.5 M NaCl 0.0 &gt; 0. &Lt; / RTI &gt; After the KGF-2 eluted in the 1.5M salt fraction, it was diluted 5-fold with the salt fraction in 50mM NaPO ₄ pH6.5 made to a final concentration of 300mM. The fractions containing KGF-2 were then sequentially passed through a porous HQ-20 column (2.0 X 7.0 cm; Perceptive Biosystems, Inc.), and then a porous CM-20 column (2.0 X 9.0 cm; Perceptive Biosystems , Inc.). The KGF-2 (FGF-12) -containing fractions eluted with about 500 mM to about 750 mM NaCl were collected, diluted and then applied again to the CM-20 column and concentrated. Finally, the protein was dialyzed against 40 mM NaOAC pH 6.5; And separated on a gel filtration column (S-75; Pharmacia) in 150 mM NaCl (batch E-5). Alternatively, the gel filtration column was run in phosphate buffered saline (PBS, batch E-4). KGF-2-containing fractions were collected and protein concentration was measured by Bio-Rad Protein Assay. The protein was judged to be> 90% pure by SDS-PAGE. Finally, the endotoxin level measured by Limulus Amebocyte Lysate Assay (Cape Cod Associates) was found to be 1Eμ / mg . Proteins prepared by this method could bind to heparin, a hallmark of FGF system members.
[1126] Example 16
[1127] A. Construction of the N-terminal deletion mutant KGF-2 33
[1128] In order to increase the expression level of KGF-2 in E. coli and to improve the solubility and stability of E. coli expressing KGF2, the deletion mutant KGF-2 33 ( KGF-2 aa 69-208) (SEQ ID NO: 96). The reason for producing such deletion mutants was based on the following observations. First, mature KGF2 (KGF-2 aa 36-208) contains an odd number (3) of cysteine residues that can be aggregated due to internal molecules forming disulfide bridges. The deletion mutants of KGF 33 only contain only two cysteine residues, which cause the internal molecules to form disulfide bridges and thereby reduce the potential for aggregation. Reduction of aggregation should be induced to increase the yield of active KGF2 protein. Second, KGF 33 deletion mutants are absent from KGF-1 and do not appear to be important for activity, but E. coli. Lt; RTI ID = 0.0 &gt; poly-serine &lt; / RTI &gt; Thus, removal of poly-serine stretch can increase the level of expression of the active KGF-2 protein. Third, E. coli. Gt; 33 &lt; / RTI &gt; results in a natural cleavage of KGF-2 between residues 68 and 69. Thus, KGF2 33 can be effectively processed and is expected to be stable in E. coli.
[1129] Construction of KGF2 33 in pQE6
[1130] Polymerase Chain Reaction Induction Amplification and E. coli Protein Expression To subclone KGF2Δ33 into vector pQE6, two oligonucleotide primers (5952 and 19138) complementary to a given region of KGF2 were synthesized as follows:
[1131] Primer 5952:
[1132] 5'GCGGCACATGTCTTACAACCACCTGCAGGGTG3 '(SEQ ID NO: 91)
[1133] Primer 19138:
[1134] 5 'GGGCCCAAGCTTATGAGTGTACCACCAT3' (SEQ ID NO: 92)
[1135] Terminus restriction site in the case of N-terminal primer 5952, while a HindIII restriction site was included in the case of C-terminal primer 19138. In addition, primer 5952 was obtained from E. coli. While the primer 19138 contains an adjacent ATG sequence in a frame with a KGF2 coding region that allows translation of the cloned fragment. (Preferably used in E. coli) in a frame with a KGF2 coding region ensuring correct translation termination.
[1136] Polymerase chain reaction was performed using the nucleotide sequences for mature KGF-2 (aa 36-208) (working example in Example 12C) as standard conditions known in the art. The resulting amplicon was digested with AflIII and HindIII and cut and subcloned into the NcoI / HindIII digested pQE6 protein expression vector.
[1137] Construction of KGF2 33 in pHE1
[1138] Two subunit oligonucleotide primers (6153 and 6150) complementary to a given region of KGF2 were synthesized in the following base sequences to subclone KGF2 33 into the polymerase chain reaction induced amplification and E. coli expression vector pHE1.
[1139] Primer 6153:
[1140] 5 'CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG3' (SEQ ID NO: 93)
[1141] Primer 6150:
[1142] 5 'CCGGCGGTACCTTATTATGAGTGTACCACCATTGG3' (SEQ ID NO: 94)
[1143] Included is the NdeI restriction site in the case of N-terminal primer 6153 while the Asp718 restriction site in the case of C-terminal primer 6150. In addition, primer 6153 was obtained from E. coli. Lt; / RTI &gt; contains the adjacent ATG sequence in the frame with the KGF2 coding region which allows translation of the cloned fragment in E. coli. (Preferably used in E. coli) in a frame with a KGF2 coding region ensuring correct translation termination.
[1144] Polymerase chain reaction was performed using the nucleotide sequences for mature KGF-2 (aa 36-208) (working example in Example 12C) as standard conditions known in the art. The resulting amplicon was digested with NdeI and Asp718 and cleaved and subcloned into the NdeI / Asp718 digested pHE1 protein expression vector.
[1145] Nucleotide sequence of KGF2 33:
[1146] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAA (SEQ ID NO: 95)
[1147] Amino acid sequence of KGF 33:
[1148] MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAN1NKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMWALNGKGAPRRGQKTRRKNTSAHFLPMVVHS (SEQ ID NO: 96)
[1149] B. Configuration of optimized KGF-2 Δ33
[1150] In order to increase the expression level of E. coli KGF2 [Delta] 33, the codons of the complete gene were optimized so that they could be best used for E. coli. Since the template used to generate KGF2 [Delta] 33 is codon-optimized within the N-terminal region, the C-terminal amino acid (84-208) requires optimization.
[1151] First, the amino acid 172-208 was codon optimized to generate KGF2 33 (s172-208). This was accomplished by a nested PCR strategy. The oligonucleotides PM07 and PM08 (corresponding to amino acids 172-208) were combined and annealed by heating them to 70 占 and cooling to 37 占 폚. The annealed oligonucleotides were then used as templates for standard PCR reactions performed by primers PM09 and PM10. In a separate PCR reaction following standard conditions well known to those skilled in the art and using a KGF2 [Delta] 33 as template, oligonucleotides PM05 (overlapping the Pstl site in the coding region of KGF2) and PM11 were used to amplify amino acids 84-172 The corresponding KGF2 region was amplified. In the third PCR reaction, the product of the first PCR reaction (corresponding to the codon optimized amino acid 172-208) and the product of the second PCR reaction (corresponding to codon non-optimized amino acids 84-172) were combined and oligonucleotides PM05 and Lt; RTI ID = 0.0 &gt; PM10. &Lt; / RTI &gt; The resulting amplicon is digested with Pst1 / HindIII and subcloned into pQE6KGF2Δ33 digested with Pst1 / HindIII to effectively replace the corresponding noncodon-optimized region to generate pQE6KGF2Δ33 (s172-208).
[1152] In order to complete the codon optimization of KGF2, a synthetic gene codon optimized for the region of KGF2 corresponding to amino acids 84-172 was generated using oligonucleotide overlap. First, four oligonucleotides (PM31, PM32, PM33 and PM34) were combined and subjected to seven cycles of the following PCR: 94 占 폚, 30 seconds; 46.5 DEG C, 30 seconds; And 72 ° C for 30 seconds.
[1153] The second PCR reaction, followed by primers PM35 and PM36, was performed according to standard procedures using 1 l of the first PCR reaction as template. The resulting codon-optimized gene fragments were then digested with Pst1 / Sall and subcloned into Pst1 / Sall digested pQE6KGF2Δ33 (s172-208) to generate a fully optimized KGF2 encoding gene, pQE6KGF2Δ33.
[1154] To generate an alternative E. coli protein expression vector, KGF2 33 was PCR amplified using primers PM102 and PM130 on pQE6KGF 33. The resulting amplicon was digested with NdeI and EcoRV and subcloned into the pHE1 expression vector digested with NdeI and Asp718 (blunt ended) to generate pHE1Δ33.
[1155] Oligonucleotide sequence used in the construction of codon-optimized KGF2 A33:
[1156] PM05: CAACCACCTGCAGGGTGACG (SEQ ID NO: 97)
[1157] PM07:
[1158] AACGGTCGACAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCACGTCGTGGTCAGAAAACCCGTCGTAAAAACACC (SEQ ID NO: 98)
[1159] PM08:
[1160] GGGCCCAAGCTTAAGAGTGTACCACCATTGGCAGAAAGTGAGCAGAGGTGnTTTACGACGGGTTTTCTGACCACG (SEQ ID NO: 99)
[1161] PM09: GCCACATACATTTGTCGACCGTT (SEQ ID NO: 100)
[1162] PM10: GGGCCCAAGCTTAAGAGTG (SEQ ID NO: 101)
[1163] PM11: GCCACATACATTTGTCGACCGTT (SEQ ID NO: 102)
[1164] PM31:
[1165] CTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCCTTCACCAAATACTTCCTGAAAATCGAAAAAACGGTAAAGTTTCTGGTACCAAG (SEQ ID NO: 103)
[1166] PM32:
[1167] AGCTTTAACAGCAACAACACCGATTTCAACGGAGGTGATTTCCAGGATGGAGTACGGGCAGTTTTCTTTCTTGGTAC CAGAAACTTTACC (SEQ ID NO: 104)
[1168] PM33:
[1169] GGTGTTGTTGCTGTTAAAGCTATCAACTCCAACTACTACCTGGCTATGAACAAGAAAGGTAAACTGTACGGTTCCAAAGAATTTAACAAC (SEQ ID NO: 105)
[1170] PM34:
[1171] GTCGACCGTTGTGCTGCCAGTTGAAGGAAGCGTAGGTGTTGTAACCGTTTTCTTCGATACGTTCTTTCAGTTTACAGTCGTTGTTAAATTCTTTGGAACC (SEQ ID NO: 106)
[1172] PM35: GCGGCGTCGACCGUGTGCTGCCAG (SEQ ID NO: 107)
[1173] PM36: GCGGCCTGCAGGGTGACGTTCGTTGG (SEQ ID NO: 108)
[1174] PM102: CCGGCGGATCCCATATGTCTTACAACCACCTGCAGG (SEQ ID NO: 109)
[1175] PM130: CGCGCGATATCTTATTAAGAGTGTACCACCATTG (SEQ ID NO: 110)
[1176] Nucleotide sequence of KGF2 33 (s172-208):
[1177] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCCTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGTACCAAGAAAGAAAACTGCCCGTACTCCATCCTGGAAATCACCTCCGTTGAAATCGGTGTTGTTGCTGTTAAAGCTATCAACTCCAACTACTACCTGGCTATGAACAAGAAAGGTAAACTGTACGGTTCCAAA GAATTTAACAACGACTGTAAACTGAAAGAACGTATCGAAGAAAACGGTTACAACACCTACGCTTCCTTCAACTGGCAGCACAACGGTCGACAAATGTATGTGGCACTGAACGGTAAAGGTGCTCCACGTCGTGGTCAGAAAACCCGTCGTAAAAACACCTCTGCTCACTTTCTGC CAATGGTGGTACACTCTTAA (SEQ ID NO: 111)
[1178] Amino acid sequence of KGF2 33 (s172-208):
[1179] MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSlLEITSVEIGVVAVKAINSNYYLANINKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMWALNGKGAPRRGQKTRRKNTSAHFLPMVVHS (SEQ ID NO: 112)
[1180] C. Composition of the N-terminal deletion mutant KGF-2 4
[1181] To increase the expression level of KGF2 in E. coli and improve the stability and solubility of KGF2 expressed in E. coli, the deletion mutant KGF2Δ4 (amino acids 39-208) from which the first 38 amino acids of the pretreated KGF2 had been removed was replaced with cysteine at position 37 Respectively. Since the resulting KGF2 deletion molecule contains an even number of cysteines, it is necessary to improve the expression level of the active protein by reducing the problems caused by aggregation due to intramolecular disulfide bridge formation.
[1182] Two oligonucleotide primers (PM61 and 19138) were synthesized with the following base sequences in order to enable the polymerase chain reaction performed by amplification and subcloning of KGF2 [Delta] 4 into the E. coli protein expression vector, pQE6.
[1183] PM61: CGCGGCCATGGCTCTGGGTCAGGACATG (SEQ ID NO: 113)
[1184] 19138: GGGCCCAAGCTTATGAGTGTACCACCAT (SEQ ID NO: 114)
[1185] For the N-terminal primer (PM61), the NcoI restriction site was integrated, whereas for the C-terminal primer (19138) the HindIII restriction site was integrated. In addition, PM61 contains a frame with the KGF2 coding region and an adjacent ATG sequence to allow translation of the cloned fragments in E. coli, whereas 19138 is a frame with a KGF2 coding region which ensures correct translation in E. coli, (Preferentially used for Escherichia coli).
[1186] The polymerase chain reaction is carried out using the full length KGF2 (aa 36-208) as a template and template (constructed in Example 12C), which is well known to those skilled in the art. The resulting amplicon is restricted to NcoI and HindIII and subcloned into the NcoI / HindIII digested pQE6 protein expression vector.
[1187] Nucleotide sequence of KGF2 4:
[1188] ATGGCTCTGGGTCAAGATATGGTTTCTCCGGAAGCTACCAACTCTTCCTCTTCCTCTTTCTCTTCCCCGTCTTCCGCTGGTCGTCACGTTCGTTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAA (SEQ ID NO: 115)
[1189] Amino acid sequence of KGF2 4:
[1190] MALGQDMVSPEATNSSSSSFSSPSSAGRHVRSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQKTRRKNTSAHFLPMVVHS (SEQ ID NO: 116)
[1191] Example 17
[1192] KGF-2Δ33 stimulated wound healing in normal rats
[1193] To demonstrate that KGF-2Δ33 promotes the healing process, wound healing at the incisional wound site was tested using the following model.
[1194] A 6 mm cut wound was formed in the back of Sprague Dawley rats (n = 5) with a Keyes skin perforator. (40 mM NaOAc and 150 mM NaCl, pH 6.5 buffer) and buffer (40 mM NaOAc and 150 mM NaCl, pH &lt; RTI ID = 0.0 &gt; 6.5). The wound area was measured daily using a calibrated Jameson caliper. The wound size was expressed in square millimeters. At the last day, the wound area was measured and collected for further analysis. Statistical analysis was performed using an unpaired t test (mean 占 SE). The evaluation parameters were wound closure rate (%), histological score (1-3 minimal cell accumulation, no new skin formation, 4-6 immature skin formation, inflammatory cell, capillary; Cells, mature skin with new epithelium 10-12 fibroblasts, collagen, epithelium), reperfusion and immunohistochemistry.
[1195] Treatment with KGF-2 Δ33 at 3 days post-wound showed reduced wound size (30.4 mm 2 at 4 μg, p = 0.006, 33.6 mm 2 at 1 μg, p = 0.0007) when compared to the 38.9 mm 2 buffer control . On day 4 after wounding, treatment with KGF-2 Δ33 resulted in a reduction in wound size (27.2 mm 2 at 0.1 μg, 27.9 mm 2 at 27 μg, p = 0.002, 0.4 μg, p = 0.04) when compared to the 33.8 mm 2 buffer control . Treatment with KGF-2 Δ33 at 5 days post-wound showed a decrease in wound size (18.1 mm 2 at 4 μg, p = 0.02) when compared to the 25.1 ㎟ buffer control. See FIG.
[1196] After wound collection on day 5, additional parameters were measured. KGF-2 Δ33 showed an increase in wound closure rate (71.2%, p = 0.02) at 4 μg compared to the buffer control 60.2%. Also, the administration of KGF-2 33 showed an improvement in the histological scores (1 ug to 8.4, p = 0.005, 4 ㎍ to 8.5, p = 0.04) at 1 and 4 占 compared to the buffer control of 6.4. In addition, re-epithelization was improved (1 ug at 1389 ㎛, p = 0.007 at 4 ㎍, 1220 ㎛ at 4 ㎍, p = 0.02) in 1 ㎍ and 4 ㎍ KGF-2 Δ33 as compared to the 923 ㎛ buffer control. See FIG.
[1197] This study demonstrates that daily treatment with KGF-2 [Delta] 33 promotes wound healing rates in normal animals as indicated by a reduction in total wound area. In addition, evaluation of histological evaluation and re-epithelization of wound samples also demonstrates that KGF-2 [Delta] 33 improves the healing rate in this normal rat model.
[1198] Example 18
[1199] KGF-2 Δ33 effect on tensile strength and epidermal thickness in normal rats
[1200] The following experiment was conducted to demonstrate that KGF-2 Δ33 increases the tensile strength and skin thickness of wound area.
[1201] A 2.5 cm full length midline incision wound site was formed on the back of male Sprague Dawley rats (n = 8 or 9). Skin incision was closed using equidistant metal skin staples. Buffer (40 mM NaOAc and 150 mM NaCl, pH 6.5) or KGF-2 33 (40 mM NaOAc and 150 mM NaCl, pH 6.5 buffer) was topically applied at wound formation. Four wound strips 0.5 cm wide were excised on day 5. This sample was used for the study of fracture strength, hydroxyproline measurement and histological evaluation using an Instron ^TM skin tension meter. The fracture strength was defined as the maximum force sustained by each wound site before rupture. Statistical analysis was performed using the adjunct t test (mean ± SE).
[1202] In an incised skin rat model, topically applied KGF-2 33 showed a statistically significant increase in fracture strength, tensile strength and epidermal thickness as a result of intrathecal application after wounding. In one study, the fracture strength of wound sites treated with KGF-2 at 1, 4, and 10 μg was significantly higher (107.3 g at 1 μg, p = 0.0006, 126.4 g at 4 μg, p < 0.0001, 123.8 g at 10 [mu] g, p < 0.0001). See FIG.
[1203] Epidermal thickness was measured under a light microscope on a Masson Trichrome section. KGF-2 Δ33 treated wound areas showed increased skin thickness (1 μg at 60.5 μg, 4 μg at 66.51 μg, p = 0.01, at 10 μg at 59.6 μg) in contrast to the 54.8 μ buffer control. See FIG.
[1204] These studies demonstrate that the single incisional application of KGF-2 is characterized by increasing and promoting the wound healing process to increase the fracture strength and epidermal thickness of the incision wound site.
[1205] Example 19
[1206] KGF-2 Δ33 effect on normal rat skin
[1207] The following experiment was conducted to measure the effect of KGF-2 33 on normal rat skin after intradermal injection.
[1208] Six intradermal injections of placebo or KGF-2 [Delta] 33 (40 mM NaOAc and 150 mM NaCl, pH 6.5 buffer) at a concentration of 1 and 4 [mu] g / 50 μl in male adult SD rats (n = 3) Respectively. At 24 and 48 hours, animals were sacrificed 2 hours after injecting 5-2'-bromodeoxyuridine (BrdU) (100 mg / kg intraperitoneal). The epidermal thickness was measured from the skin forming layer to the base layer bottom. Approximately 20 measurements were made along the injection site and the average thickness was determined. This measurement was done using a microscope corrected on a mason trichrome staining section under a light microscope. BrdU scoring was performed by two blind observers under a light microscope using the following scoring system: 0-3 no to minimal BrdU labeled cells; 4-6 intermediate labeling; 7-10 Strongly labeled cells. Animals were sacrificed at 24 and 48 hours after injection. Statistical analysis was performed using a subtype test (mean ± SE).
[1209] The skin treated with KGF-2 Δ33 at 24 hours showed increased skin thickness (1 μg to 32.2 μ, p <0.001, 4 μg to 35.4 μ, p <0.0001) when compared to the 27.1 μ buffer control. The skin treated with KGF-2 Δ33 at 48 hours showed an increased skin thickness (34.0 μ at 1 μg, p = 0.0003, 42.4 μ at 4 μg, p <0.0001) compared to the 27.8 μ buffer control. See FIG. In addition, KGF-2 Δ33 treated skin showed increased BrdU immunostaining (4.73, p = 0.07, 4 ㎍DPTJ 6.85, p <0.0001) at 48 hours compared with 3.33 of the buffer control group. See FIG.
[1210] These studies demonstrate that intradermal injection of KGF-2 increases and promotes epidermal thickness. Thus, KGF-2 will have applications in preventing or alleviating wrinkles, improving skin aging, reducing scar formation or improving healing from cosmetic surgery. KGF-2 may also be used prophylactically to prevent or reduce oral mucositis (oral ulcers), intestinal inflammation associated with chemotherapy or other drugs.
[1211] Example 20
[1212] Anti-inflammatory effect of KGF-2 on PAF-induced foot edema
[1213] To demonstrate the anti-inflammatory effects of KGF-2, the following experiments were performed using the PAF-induced paw edema inflammation model.
[1214] Groups of four Lewis rats (190-210 gm) were incubated with 120 μl of a solution containing 2.5 nMol of PAF, 125 μg Ckb-10 (B5), 24 μg of LPS, 73 μg of KGF-2 {N - Thr (36) -Ser (208) of Fig. 1 (SEQ ID: 2) with terminal Met or subcutaneously injected into the sole of the right hind paw with a reagent not containing the protein. The left hind paw was used as a parallel control group by providing the same amount of buffer. Foot volume was quantified immediately prior to PAF injection, or 30 and 90 minutes, using a hematology system. And the change rate (%) of the foot volume was calculated.
[1215] [table]
[1216] The test reagents in Experiments Nos. 1 and 2 Group (N = 4)PAF (R.) 2.5 nMolCk -10 (R) 1.04 mg / mlLPS (R.) 200 占 퐂 / mlKGF-2 (R.) 0.73 mg / mlBuffer One20 μl---100 μl 220 μl100 μl--- 320 μl-100 μl-- 420 μl--100 μl-
[1217] As shown in Figure 42, the right hind paw scanned with PAF alone had a significant increase in foot volume (75% or 100% for experiment number 1 or 2, respectively) at 0.5 hour post-injection, as expected , The left hind paw that received the buffer, or the right hind paw that received LPS or SEB alone exhibited mild symptoms of edema (data not shown). However, when KGF-2 was provided topically with PAF, there was a substantial reduction (25% or 50% for Experiment No. 1 or 2, respectively) when compared to PAF alone attacked foot. Decreased foot edema was not observed in animals receiving PAF with Ckb-10 (a different protein), LPS or SEB (two inflammatory mediators). These results suggest that the anti-inflammatory effect of KGF-2 is specific and not due to the specific non-specific nature of the protein.
[1218] Effect of KGF-2 Δ33 on PAF-induced foot edema in rats
[1219] After the above experiment with KGF-2 33 to confirm in vivo biological activity and stimulation of wound healing in order to stimulate keratinocyte proliferation, KGF-2 33 was further evaluated in the PAF induced foot edema model of rats . Groups of four Lewis rats (190-210 gm) were subcutaneously injected into the soles of the right hind paw with 120 μl of solution containing 210 μg of KGF-2 Δ33 or albumin along with 2.5 nMol of PAF. The left hind paw was used as a parallel control by providing the same amount of buffer, albumin or KGF-2 33 alone. Foot volume was quantified at various intervals after PAF injection using a blood flow system. The percentage change in foot volume was measured.
[1220] As shown in Figure 43, the right hind paw injected with PAF and albumin had a significant increase in foot volume (75%) at 0.5 hour post-injection, while buffer, albumin or KGF-2 Δ33 alone The accommodated left hind paw showed some edema symptoms. However, when KGF-2 Δ33 was provided locally with PAF, there was a substantial reduction in foot volume (mean 20%) when compared to PAF and albumin-attacked feet throughout the entire experiment, which was completed within 4 hours. These results support the anti-inflammatory properties of KGF-2 33.
[1221] [table]
[1222] Test reagent Group (N = 4)PAF 2.5 nMolAlbumin 2.1 mg / mlKGF-2 331 mg / mlBuffer One20 μl100 μl-- 220 μl-100 μl- 3-120 μl-- 4--120 μl- 5---120 μl
[1223] Thus, KGF-2 is useful for treating acute and chronic conditions in which inflammation is a key pathogenesis, including but not limited to psoriasis, eczema, dermatitis and / or arthritis.
[1224] Example 21
[1225] Effect of KGF-2 Δ33 on end-to-end colonic aortic rat model
[1226] This example demonstrates that KGF-2 [Delta] 33 increases the intestinal recovery rate in bowel or colonic anastomosis of Wistar or Sprague Dawley rats. The use of rats in experimental anastomosis is a well characterized, appropriate and reproducible model of surgical wound healing. This model can also be extended to study the effects of chronic steroid therapy or various chemotherapy regimens on the quality and speed of surgical wound healing in the colon and small intestine {Mastboom WJB et al . Br. J. Surg. 78 : 54-56 (1991), Salm R. et al . , J. Surg. Oncol. 47 : 5-11, (1991), Weiber S. et al . Eur. Surg. Res. 26 : 173-178 (1994). The healing of union is similar to that of wound healing in any part of the body. The initial state of healing is characterized by acute inflammation, followed by fibroblast proliferation and the synthesis of collagen. Collagen is gradually modeled, and the wound is strengthened as new collagen is synthesized {KorudaM.J. And Rolandelli RH J. Surg. Res. 48 : 504-515 (1990). Most postoperative surgical complications, such as anastomotic leakage, occur during the first few days after surgery, and the strength of the intestine during that period is largely ensured by the ability to maintain a suture on the wound margin. The suture maintenance capacity of the gastrointestinal tract has been reported to decrease to about 80% during first postoperative days (Hogstrom H and Haglund U. Acta Chri Scand 151 : 533-535 (1985), Jonsson K, et al . Am J. Surg. 145 : 800-803 (1983).
[1227] Male adult SD rats (n = 5) were intramuscularly anesthetized with a combination of ketamine (50 mg / kg) and xylazine (5 mg / kg). The abdominal cavity was opened with a midline incision of 4 cm in length. A 1 cm wide segment of the left colon was abdominally inferior to the abdominal inversion while preserving the marginal vessels. Single-layer end-to-end anastomosis was performed with an 8-10 blocked 5-0 cricil inversion suture to restore the intestinal continuum. Thereafter, the anastomosis site was topically treated with a buffer of 1 and 4 μg of buffer or KGF-2 Δ33 by syringe. The incision wound was closed with a 3-0 running silk suture for the muscle layer and a surgical staple for the skin. The treatment was then administered daily, consisting of buffer or KGF-2 Δ33, and subcutaneously administered at 1 and 5 mg / kg. Weighed on the day of surgery and every day thereafter. Animals were anesthetized 24 hours after the final treatment (5 days). The animals were anesthetized, received barium enema and then x-rayed at a fixed distance. Radiation analysis by two blinded observers showed that the group treated with KGF-2 Δ33 had 1) a reduced rate of barium leakage at the surgical site, 2) a lower degree of contraction at the surgical site, and 3) And increased the presence of distant feces pellets.
[1228] [table]
[1229] Colon anastomosis radiography groupPresence rate of fecesContraction rateApical expansion rateAbdominal leak rate No treatment (N = 5)20%80%80%60% Buffer (N = 5)40%60%80%75% KGF-2 33 [1 mg / kg] (N = 5)60%20%100%20% KGF-2 33 [5 mg / kg] (N = 4)100%0%75%25%
[1230] Example 22
[1231] Structure of carboxy terminal mutation of KGF-2
[1232] The carboxyl terminal of KGF-2 is greatly charged. The density of these charged residues can affect the stability of the protein and thus its solubility. A series of mutations were generated in this region of the genome to generate muteins that could stabilize proteins in solution. In order to produce the point mutants 194 R / E, 194 R / Q, 191 K / E, 191 K / Q, 188 R / E, 188 R / Q, In a PCR reaction using well-known standard conditions, 5952 KGF [Delta] 335 'Afl III 5' primers were used with the indicated 3 'primers containing appropriate point mutations for KGF-2. The resulting products were restricted to AflIII and HindIII and cloned into E. coli expression vector pQE60 restricted to NcoI and HindIII.
[1233] KGF2 33,194 R / E Structure:
[1234] The following primers were used:
[1235] 5952 KGFA 33 5 'Afl III:
[1236] 5 'GCGGCACATGTCTTACAACCACCTGCAGGGTG3' (SEQ ID NO: 117)
[1237] KGF2 3'HindIII 194aa R / E:
[1238] 5'CTGCCC AAGCTT TTATGAGTGTACCACCATTGGAAGAAAGTGAGCAGAGGTGTTTTT TTC TCGTGTTTTCTGTCC3 '(SEQ ID NO: 118)
[1239] KGF2 33,194 R / E Nucleotide sequence:
[1240] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGMCAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAGAAAACACGA AAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG GAA (SEQ ID NO: 119)
[1241] KGF2 33,194 R / E Amino acid sequence:
[1242] MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMWALNGKGAPRRGQKTR E KNTSAHFLPMVVHS (SEQ ID NO: 120)
[1243] KGF2 33,194 R / Q Structure:
[1244] The following primers were used:
[1245] 5952 KGF Δ33 5 'Afl III:
[1246] 5'GCGGCACATGTCTTACAACCACCTGCAGGGTG 3 '(SEQ ID NO: 121)
[1247] KGF2 33 HindIII 194aa R / Q:
[1248] 5 'CTGCCC AAGCTT TTATGAGTGTACCACCATTGGAAGAAAGTGAGCAGAGGTGTTTTT CTG TCGTGTTTTCTGTCC3' (SEQ ID NO: 122)
[1249] KGF2 33,194 R / Q Nucleotide sequence:
[1250] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGA AAAACACGACAGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG CAG (SEQ ID NO: 123)
[1251] KGF2 33,194 R / Q Amino acid sequence:
[1252] MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMWALNGKGAPRRGQKTR Q KNTSAHFLPMVVHS (SEQ ID NO: 124)
[1253] KGF2 33,191 K / E Structure:
[1254] The following primers were used:
[1255] 5952 KGF Δ33 5 'Afl III:
[1256] 5'GCGGCACATGTCTTACAACCACCTGCAGGGTG3 '(SEQ ID NO: 125)
[1257] KGF2 3 'HindIII 191aa K / E
[1258] 5 'CTGCCC AAGCTT TTATGAGTGTACCACCATTGGAAGAAAGTGAGCAGAGGTGTTTTTCCTTCGTGT TTC CTGTCCTCTCCTTGG3' (SEQ ID NO: 126)
[1259] KGF2 33,191 K / E nucleotide sequence:
[1260] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAG ACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG GAA (SEQ ID NO: 127)
[1261] KGF2 33,191 K / E Amino acid sequence:
[1262] MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLANlNKKGKLYGS KEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPRRGQ E TRRKNTSAHFLPMVVHS (SEQ ID NO: 128)
[1263] KGF2 33, 191 K / Q Structure:
[1264] The following primers were used:
[1265] 5952 KGF 33 5 'Afl III:
[1266] 5'GCGGCACATGTCTTACAACCACCTGCAGGGTG3 '(SEQ ID NO: 129)
[1267] KGF2 3 'HindIII 191 aa K / Q
[1268] 5 'CTGCCC AAGCTT TTATGAGTGTACCACCATTGGAAGAAAGTGAGCAGAGGTGTTTTTCCTTCGTGT CTG CTGTCCTCTCCTTGG3' (SEQ ID NO: 130)
[1269] KGF2 33, 191 K / Q nucleotide sequence:
[1270] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGAGAGGACAG ACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG CAG (SEQ ID NO: 131)
[1271] KGF2 33, 191 K / Q Amino acid sequence:
[1272] MSYNHLQGDVRWRKLFSFTKYFLKlEKNGKVSGTKKENCPYSILElTSVElGVVAVKAINSNYYLANINKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMWALNGKGAPRRGQ TRRKNTSAHFLPMVVHS Q (SEQ ID NO: l32)
[1273] KGF2 33, 188 R / E Structure:
[1274] The following primers were used:
[1275] 5952 KGF 33 5 'Afl III:
[1276] 5'GCGGCACATGTCTTACAACCACCTGCAGGGTG3 '(SEQ ID NO: 133)
[1277] KGF2 3 'HindIII 188aa R / E:
[1278] 5 'CTGCCC AAGCTT TTATGAGTGTACCACCATTGGAAGAAAGTGAGCAGAGGTGTTTTTCCTTCGTGTTTTCTGTCC TTC CCTTGGAGCTCCTTT3' (SEQ ID NO: 134)
[1279] KGF2 33, 188R / E Nucleotide sequence:
[1280] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGGGAAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG (SEQ ID NO: 135)
[1281] KGF2 33, 188R / E Amino acid sequence:
[1282] MYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNGKGAPR E GQKTRRKNTSAHFLPMVVHS (SEQ ID NO: 136)
[1283] KGF2 33, 188 R / Q Structure:
[1284] The following primers were used:
[1285] 5952 KGF Δ33 5 'Afl III:
[1286] 5'GCGGCACATGTCTTACAACCACCTGCAGGGTG3 '(SEQ ID NO: 137)
[1287] KGF2 3 'HindIII 188aa R / Q:
[1288] 5 'CTGCCC AAGCTT TTATGAGTGTACCACCATTGGAAGAAAGTGAGCAGAGGTGTTTTTCCTTCGTGTTTTCTGTCC CTG CCTTGGAGCTCCTTT3' (SEQ ID NO: 138)
[1289] KGF2 33, 188 R / Q nucleotide sequence:
[1290] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGAAAAGGAGCTCCAAGG GGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG CAG (SEQ ID NO: 139)
[1291] KGF2 33, 188 R / Q Amino acid sequence:
[1292] MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERlEENGYNTYASFNWQHNGRQMYVALJNGKGAPR GQKTRRKNTSAHFLPMVVHS Q (SEQ ID NO: 140)
[1293] KGF2 A33, 183K / E Structure:
[1294] For the mutant 183K / E, two PCR reactions were set up for oligonucleotide directed mutagenesis of the lysine. In one reaction, 5952 KGFΔ33 5 'AflIII was used as the 5' primer and the KGF2 183aa K / E antisense was used as the 3 'primer in the reaction. In the second reaction, the KGF2 5 '183aa K / E sense was used as the 5' primer and the KGF2 3 'HindIII TAA stop was used as the 3' primer. KGF-2 33 was used as the template for this reaction. The reaction was amplified under standard conditions well known to those skilled in the art. One microliter from each of these PCR reactions was used as a template in subsequent reactions using 5453 Bsphl as the 5 'primer and 5258 HindIII as the 3' primer. Amplification was performed using standard conditions well known to those skilled in the art. The resulting products were restricted to AflII and HindIII and cloned into E. coli expression vector pQE60 restricted to NcoI and HindIII.
[1295] The following primers were used:
[1296] 5952 KGF Δ33 5 'Afl III:
[1297] 5'GCGGCACATGTCTTACAACCACCTGCAGGGTG3 '(SEQ ID NO: 141)
[1298] KGF2 5 '183aa K / E sense:
[1299] 5 'TTGAATGGAGAA GGA GCTCCA3' (SEQ ID NO: 142)
[1300] KGF2 183aa K / E Antisense:
[1301] 5 'TGGAGCTCC TTC TCCATTCAA3' (SEQ ID NO: 143)
[1302] KGF2 3 'HindIII TAA Stop:
[1303] 5 'CTGCCC AAGCTT TTA TGAGTGTACCACCATTGG3' (SEQ ID NO: 144)
[1304] KGF2 33, 183K / E nucleotide sequence:
[1305] ATGTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTGCCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTTGCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGAAGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCTGMGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATTTAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGGA GGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACACCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAG GAA (SEQ ID NO: 145)
[1306] KGF2 33, 183K / E Amino acid sequence:
[1307] MSYNHLQGDVRWRKLFSFTKYFLKIEKNGKVSGTKKENCPYSILEITSVEIGVVAVKAINSNYYLAMNKKGKLYGSKEFNNDCKLKERIEENGYNTYASFNWQHNGRQMYVALNG E GAPRRGQKTRRKNTSAHFLPMVVHS (SEQ ID NO: 146)
[1308] Example 23
[1309] Effect of KGF-2 on survival after total body irradiation of Balb / c mice
[1310] Ionizing radiation is commonly used to treat many malignant tumors including lung and breast, lymphoma and pelvic tumors (Ward, WF et al., CRC Handbook of Animal Models of Pulmonary Disease , CRC Press, pp. 165-195 }. However, radiation-induced injuries (lung, bowel, etc.) have limited strength and continuity of radiation therapy {Morgan, GW et al. , Int. J. Radiat. Onco1. Biol. Phys. 31 : 361 (1995). Gastric mucosa has a rapid cell cycle and is particularly sensitive to cytotoxic agents {Potten, CS, et al., In: Cytotoxic Insult to Tissue, Churchill Livingstone , pp. 105-152 (1983). Some of the manifestations of intestinal radiation injury include acute rectalitis, intestinal fibrosis, stenosis and fistula formation (Anseline, DF et al . Ann. Surg. 194 : 716-724 (1981). Treatment to protect normal structures from radiation without altering the radiation sensitivity of the tumor is advantageous for the management of such disorders. Regardless of the area to be irradiated, the dose of radiation is limited by the radiation sensitivity of normal tissue. Complications following systemic or partial body irradiation include pneumonia, fibrosis, gastrointestinal injury, and bone marrow disorders.
[1311] Some cytokines, including IL-1, TNF, IL-6 and IL-12, show post-TBI radiation protection effects {Neta, R. et al . , J. Exp. Med. 173 : 1177 (1991)}. IL-11 has been implicated in the treatment of small intestinal mucosal cells {Du, XX et al., Blood 83 : 33 (1994)} and radiation induced chest injuries {Redlich, CA et al., The Journal of Immunology 157: 1705-1710 )}.
[1312] animal
[1313] All experiments were performed using BALB / c mice. Animals were purchased at 6 weeks of age and 7 weeks of age at the start of the study. All manipulations were performed using the Mouton technique. This study was conducted in accordance with the guidelines described by Human Genome Science Inc., Institutional Animal Care and Use Commissions, which examined and approved the experimental protocol.
[1314] KGF-2
[1315] The protein was composed of a 141 amino acid human protein called KGF-2 33. This protein is the truncated isomer of KGF-2 lacking the first 33 amino-terminal residues of the mature protein. The gene encoding this protein was cloned into an E. coli expression vector. Fractions containing 95% or more pure recombinant material were used in the experiments. KGF-2 was prepared in a vehicle containing 40 mM Na acetate + 150 mM NaCl, pH 6.5. The same vehicle was used to make a dilution from the stock solution.
[1316] Whole body investigation and experiment design
[1317] The mice were irradiated with 519 RADS (5.19 Gy) using a 68 Mark I Shepherd cesium applicator. KGF-2 33 was started two days before irradiation and subcutaneously injected continuously for 7 days after the irradiation. We got daily weight from all mice. Mice were randomized to receive one of three treatments: whole body irradiation (TBI) and buffers, TBI and KGF-2 33 (1 mg / kg sq), TBI and KGF-2 33 (5 mg / kg sq) . Two independent experiments were performed.
[1318] result
[1319] Two studies were conducted using the investigated animals. In the first study, animals were irradiated with 519 RADS (5.19 Gy). The animals were treated with buffer or KGF-2 33 daily for 1 day and 5 mg / kg s.q. for 2 days prior to irradiation and then daily for 7 days thereafter. On the 25th day after systemic irradiation, 1/5 of the animals survived in the buffer group. In contrast, KGF-2 treated group survived 5/5 animals at 1 mg / kg and 4/5 animals at 5 mg / kg (FIG. 44).
[1320] In addition, KGF-2 treated animals showed 0.9% and 5.3% body weight gain at 20 days after TBI. By contrast, the buffer treatment group had a 4.2% weight loss on day 20. The control animals corresponding to the normal non-irradiated ages showed a 6.7% weight gain at the same time (FIG. 45).
[1321] Animals in the second study were also surveyed with 519 RADS (5.19 Gy). These animals were treated with buffer or KGF-2 33 daily for 1 day and 5 mg / kg sq. For 2 days prior to irradiation and then daily for 7 days thereafter. On day 15 after systemic irradiation, all animals in the buffer group died. The survival rate of KGF-2 at 1 mg / kg was 30% and the survival rate was 60% at 5 mg / kg. On the 25th day after TBI, the 1 mg / kg group showed 20% survival rate and the 5 mg / kg group showed 50% survival rate (FIG. 46).
[1322] conclusion
[1323] In summary, these results demonstrate that KGF-2 has a protective effect after TBI. The ability of KGF-2 to increase the survival rate of TBI-treated animals is useful for radiation-induced injury and is also useful for increasing the rate of irradiation in the treatment of malignant tumors.
[1324] Example 24
[1325] Evaluation of KGF-2 in TPA model of skin inflammation in mice
[1326] To demonstrate that KGF-2 alleviates the progression of contact dermatitis, the mouse Tetradecanoyl Phorbol Acetate (TPA) induced skin inflammation model was used. The use of female BALB / c and male Swiss Webster mice in skin irritation experiments is a well-characterized, appropriate and reproducible model of contact dermatitis. These varieties of mice have been shown to develop long-term inflammatory responses after topical application of TPA, consisting of leukocyte hemodynamics, vascular permeability and local migration, and these human changes are similar to those of human dermatitis {Rao et al., 1993 , Inflammation 17 (6): 723; Rao et al., 1994, J. Kipid Mediators Cell Signaling 10 : 213}.
[1327] Mice were injected intraperitoneally or subcutaneously with vehicle or KGF-2 at 60 minutes after topical application of TPA (4 [mu] g / ear) applied with a solution in acetone (200 [mu] g / Or intravenously. The control received 20 [mu] l of acetone as topical application. After 4 hours of TPA application, an increase in ear thickness was measured and the ear was incised for histology. In order to measure the vascular permeability according to TPA, mice were sacrificed after 15 minutes by intravenous injection into mice via tail vein with Evans blue (300 mg / kg) at predetermined time after topical application of TPA. Ear was dissected out, extracted with dimethylformamide, and centrifuged. Absorbance readings were measured spectrophotometrically at 590 nm.
[1328] Example 25
[1329] Effect of KGF-2 Δ33 on wound healing
[1330] The biological effects of KGF-2 [Delta] 33 on skin are based on initial in vitro data indicating the ability of KGF-2 to stimulate primary human epidermal keratinocytes as well as rat and pro-B BaF3 cells transfected with FGFR isoform 2 iiib Respectively. Initial experiments were performed to determine the biological effects of KGF-2 33 after intradermal administration. After intradermal study, KGF-2 33 was explored in various wound healing models (e.g., full thickness perforated biopsy wound and incision wound) to determine its efficacy as a wound healing agent.
[1331] Effect of KGF-2 Δ33 on wound healing in glucocorticoid-impaired rat model
[1332] Damaged wound healing is an important clinical problem associated with various pathological conditions, such as diabetes, and is a complication of systemic administration of steroids or antidepressants. Treatment with systemic glucocorticoids has been shown to impair wound healing in human and animal models of tissue regeneration. Reduction of single cell levels and inhibition of procollagen synthesis were observed after glucocorticoid administration. Therefore, inflammatory conditions and matrix synthesis of healing are important factors involved in the complex process of tissue repair. In the current study, the effect of multiple topical application of KGF-2 was assessed for total thickness incision skin wounds of the rats where healing was impaired by systemic administration of methylprednisolone.
[1333] Sprague Dawley rats (n = 5 / treatment group) were given 8 mm back wound to compromise healing. The wound sites were topically treated daily with buffer or KGF-2 at doses of 0.1, 0.5 and 1.5 占 퐂 and volume of 50 占 퐇. The wound sites were measured on days 2, 4, 6 and 8 using calibrated Jameson calipers. On day 6 (data not shown) and on day 8 (FIG. 47), the KGF-2 treated group showed a statistically significant decrease in wound closure when compared to the buffer control group.
[1334] Effect of KGF-2 Δ33 on wound healing of diabetic mouse model
[1335] Genetically diabetic homozygous females ( db + / db +) mice weighing 30-35 g at 6 weeks of age were provided with a full-thickness back wound with a 6 mm biopsy perforator. The wound was left open and treated daily with 0.1, 0.5 and 1.5 μg of placebo or KGF-2. Wound closure was measured using a Jameson caliper. On day 10, the animals were euthanized and wound sites were harvested for histology.
[1336] KGF-2 showed significant improvement in wound closure rate at 0.1 μg (p = 0.02) compared with placebo or untreated group.
[1337] In addition, the administration of KGF-2 showed an improvement in histological score at 0.1 ㎍ (p = 0.03) compared to the placebo or untreated group (p = 0.01) and at 1.5 ㎍ compared to the untreated group .
[1338] conclusion
[1339] Based on the above results, KGF-2 shows significant activity in the damaged state such as glucocorticoid administration and diabetes. Therefore, KGF-2 can be used for post-operative wound, diabetes or poor circulation (e.g., venous insufficiency and vein ulceration), burns and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and steroid and anti- Of the total body weight.
[1340] Example 26
[1341] Effect of KGF-2 Δ33 on oral mucosa
[1342] Clinically used cytotoxic agents have the disadvantageous effect of inhibiting normal epithelial proliferation in certain local areas, such as oral mucosa, resulting in life-threatening disorders in mucosal barriers. The present inventors have constructed a study investigating the efficacy of KGF-2 in this clinical field. Data supports the therapeutic effect of KGF-2 in a model of mucositis.
[1343] Effect of KGF-2 Δ33 on hamster oral mucosa
[1344] We sought to determine whether KGF-2 could induce normal oral mucosal epithelial proliferation. The effect of KGF-2 on the oral mucosa was assessed by male Golden Syrian hamsters. The hamster's bladder was treated daily with topical application to a hamster buccal anesthetized with a buffer or KGF-2 33 (0.1, 1 and 10 占 퐂 / min) in a volume of 100 占 퐇 per nerve. The compound was contacted with buccal bones for a minimum of 60 seconds and then swallowed. Seven days after treatment, animals were injected with BrdU and sacrificed as described above. Proliferating cells were labeled using anti-BrdU antibodies. Figure 48 shows that there was a significant increase (cell proliferation) of BrdU labeling when animals were treated with 1 ug and 10 ug of KGF-2 [Delta] 33 (as compared to buffered treatment).
[1345] Topical treatment with KGF-2 causes normal epithelial cell proliferation. Based on these results, KGF-2 is clinically useful for the prevention of oral mucositis caused by any chemotherapeutic agent (or other toxic drug regimen), radiotherapy, or any combination chemotherapy-radiotherapy regimen . In addition, KGF-2 may be useful as a therapeutic agent by reducing the severity of damage to the oral mucosa resulting from toxic drugs (chemotherapy) or radiation therapy.
[1346] Example 27
[1347] Effect of KGF-2 Δ33 on ischemic wound healing in rats
[1348] The purpose of the experiment provided in this example is to determine the efficacy of KGF-2 in wound healing using an ischemic wound healing model.
[1349] Blood supply to the topical skin was partially halted by raising the short-axis total thickness random muscle flap (3 x 4 cm). The entire thickness wound is made up of localized skin and consists of a muscle flap. Six adult adult Sprague Dawley rats were randomly assigned to this study (5 rats / group / time point) with the KGF-2 33 treatment group and the placebo group. The wound sites were collected on days 1, 3, 5, 7, 10 and 15 after wound formation.
[1350] Wound fracture strength did not show a significant difference between KGF-2 and buffered treatment groups at the early days by 10 and 15 days after wound formation.
[1351] The results showed that KGF-2 significantly improved wound healing strength in ischemic wound healing after 10 days of wound formation. These results also suggest that ischemia delays the healing process in both groups when compared to data already obtained from normal wound healing studies.
[1352] This muscle flap model provides data and information in ischemic situations obtained from venous reflux. These results suggest that KGF-2 may be used for the treatment of chronic venous leg ulcers caused by impaired venous regurgitation and / or dysfunction.
[1353] Example 28
[1354] Assessment of KGF-2 in healing colon
[1355] The results of this experiment demonstrate that KGF-2 [Delta] 33 increases the rate of intestinal recovery in models of bowel or colon adduction of Wistar or Sprague Dawley rats. In addition, this model can be used to illustrate that KGF-2 and its isomers increase the ability of the stomach or colon to join sutures.
[1356] Using rats in experimental anastomosis is a well-characterized, appropriate, and reproducible model of surgical wound healing. This model can also be extended to study the effects of chronic steroid therapy or various chemotherapy regimens on the quality and speed of surgical wound healing in the colon and small intestine {Mastboom WJB et al . Br. J. Surg. 78 : 54-56 (1991), Salm R. et al . , J. Surg. Oncol. 47 : 5-11, (1991), Weiber S. et al . Eur. Surg. Res. 26 : 173-178 (1994). The healing of union is similar to that of wound healing in any part of the body. The initial state of healing is characterized by acute inflammation, followed by fibroblast proliferation and the synthesis of collagen. Collagen is gradually modeled, and the wound is strengthened as new collagen is synthesized {Koruda MJ and Rolandelli RH J. Surg. Res. 48 : 504-515 (1990). Most postoperative surgical complications, such as anastomotic leakage, occur during the first few days after surgery, and the strength of the intestine during that period is largely ensured by the ability to maintain a suture on the wound margin. The suture maintenance capacity of the gastrointestinal tract has been reported to decrease to about 80% during first postoperative days (Hogstrom H and Haglund U. Acta Chri Scand 151 : 533--535 (1985), Jonsson K, et al . Am J. Surg. 145 : 800-803 (1983).
[1357] The rats were anesthetized intramuscularly with a combination of ketamine (50 mg / kg) and xylazine (5 mg / kg). Animals were placed on healing pads during skin disinfection, surgery and post-surgery. The abdominal cavity was opened with a midline incision of 4 cm in length. A 1 cm wide segment of the left colon was abdominally inferior to the abdominal inversion while preserving the marginal vessels. The end-to-end anastomosis of the monolayer was performed with an 8-10 blocked 8-0 prolene inversion suture to restore the intestinal continuum. The incision wound was closed with a 3-0 running silk suture for the muscle layer and a surgical staple for the skin. A daily clinical evaluation consisting of individual weight, body temperature and food consumption pattern was performed for each animal.
[1358] KGF-2 [Delta] 33 and placebo treatments were continued daily post-operatively daily, subcutaneously, topically, intraperitoneally, intramuscularly, intragastrically or intracolonally, until sacrifice on day 7. There was an untreated control, placebo and KGF-2 Δ33 groups. Two hours prior to euthanasia, the animals were intraperitoneally injected with 100 mg / kg BrdU. Animals were euthanized 24 hours after final treatment (5 days). A midline incision was made in the medial abdominal wall and 1 cm long colon segments including anastomosis were removed. A third segment at the surgical site was taken for total collagen analysis.
[1359] In a series of two experiments, male adult SD rats (n = 5) were anesthetized and a single-layer end-to-end anastomosis of the prostate colon was accommodated with an 8-10 blocked 6-0 prolactin inversion suture. Thereafter, the anastomosis site was topically treated with a buffer of 1 and 4 μg of buffer or KGF-2 Δ33 by syringe. Animals were then treated intraperitoneally with buffer or KGF-2 33 at a concentration of 1 mg / kg or 5 mg / kg daily thereafter. On day 5, animals were euthanized, colon excised, frozen immediately in liquid nitrogen, lyophilized, and collagen counted. Collagen concentrations were expressed as ug collagen / mg dry weight tissue. Statistical analysis was performed using an adjunct t test (mean ± SE). On the fifth day, the rats were anesthetized, received the barium enema, and then radiographically analyzed. From two experiments, the barium enema drug assessment of end-to-end left colon cancer showed a consistent decrease in abdominal leakage to animals treated with KGF-2 Δ33 at 1 and 5 mg / kg. This data is shown in the following table. The fracture strength of the surgical site was also measured using a tension meter. There was no significant difference between the buffer group and KGF-2 33. As shown in FIG. 49, a significant increase in collagen content at the surgical site was observed at 1 mg / kg of KGF-2 33 (p = 0.02) and 5 mg / kg (p = 0.004) of the buffer control group.
[1360] [table]
[1361] Colon anastomosis radiography groupPresence rate of fecesAnastomosis contraction ^* Abdominal leak rate In the non-treated group (N = 8)50%2.075% Buffer (N = 8)57%1.050% KGF-2 33 [1 mg / kg] (N = 8)50%1.337% KGF-2 33 [1 mg / kg] (N = 9)77%1.611%
[1362] ^* Anastomotic contraction scoring: 0 - no contraction; 1-5 - Minimal to Severe Shrinkage
[1363] Male adult SD rats (n = 5) were intramuscularly anesthetized with a combination of ketamine (50 mg / kg) and xylazine (5 mg / kg). The abdominal cavity was opened with a midline incision of 4 cm in length. A 1 cm wide segment of the left colon was abdominally inferior to the abdominal inversion while preserving the marginal vessels. A single-layer end-to-end anastomosis was performed with an 8-10 blocked 6-0 prolene inversion suture to restore the intestinal continuum. Thereafter, the anastomosis site was topically treated with a buffer of 1 and 4 μg of buffer or KGF-2 Δ33 by syringe. The incision wound was closed with a 3-0 running silk suture for the muscle layer and a surgical staple for the skin. The treatment was then administered daily, consisting of buffer or KGF-2 Δ33, and subcutaneously administered at 1 and 5 mg / kg. Weighed on the day of surgery and every day thereafter. Animals were anesthetized 24 hours after the final treatment (5 days). The animals were anesthetized, received barium enema and then x-rayed at a fixed distance. The anastomosis site was then resected for histopathology and biomechanical analysis.
[1364] Example 29
[1365] Evaluation of KGF-2 Δ33 in Inflammatory Bowel Disease Model
[1366] KGF-2 is a protein that induces keratinocyte proliferation in vitro and is active in various wound healing models in vivo. The aim of this study was to determine whether KGF-2 is efficacious in a rat and colitis model induced by random exposure to dextran sodium sulfate in drinking water.
[1367] Six to eight weeks old female Swiss Wistar mice (20-25 g, Charles River, Rayleigh, NC) were treated with sodium sulfate (DSS, 36,000-44,000 MW, American International Chemistries, ) Was used in a model of inflammatory bowel disease induced by randomly administered 4% solution of the inflammatory bowel disease. Three parameters were used for efficacy determination: 1) a clinical score based on the evaluation of stool; 2) a histological score based on an evaluation of the colon; And 3) weight change. The clinical score consisted of two parts, summing up a maximum score of four. Stool tenacity is 0 = short; 1 = dryness; 2 = rated with diarrhea. Blood in stool 0 = no blood; 1 = blood not visible; And 2 = a score of 0 to 2 for large intestinal bleeding. An average group score of 3 or more indicates a likely lethal rate and the disease progressing past the treatable stage. Clinical scores were taken at 0, 4, 5, 6 and 7 days. Elevated, crossing and descending colon slides were evaluated in a blinded fashion based on an inflammation score (0-3) and a line and score (0-4) to reach a histological score. Body weight was measured daily. Data were expressed as mean + SEM. Was by using an unpaired Student's t-test determined a significant difference when compared to the disease control ^{(* p <0.05; ** p} <0.01; *** p <0.001).
[1368] When DSS-treated mice were given daily intraperitoneal injection of KGF-2 [Delta] 33 at a dose of 1, 5 or 10 mg / kg for 7 days, KGF-2 reduced the clinical scores by 28%, 38% Respectively. Histologic evaluations corresponded closely with dose-dependent inhibition of the clinical score, and doses of 1, 5, and 10 mg / kg reduced the histological scores by 26%, 48%, and 51%, respectively. KGF-2 also significantly reduced weight loss associated with DSS-induced colitis.
[1369] In a second study, the relative efficacy of KGF-2 33 (10 mg / kg) in the case of daily intraperitoneal or subcutaneous administration was compared. The animals injected intraperitoneally with KGF-2 at the end of the 7th day of experiment had a significant 34% reduction in clinical score, whereas the subcutaneously injected KGF-2 had a significant 46% reduction. In addition, subcutaneous administration significantly reduced weight loss compared to DSS control. Based on clinical scores and body weight measurements, subcutaneous administration of KGF-2 was at least as efficacious as intraperitoneal administration.
[1370] Example 30
[1371] Effect of KGF-2 33 on normal bladder and prostate, and in hematopoietic cystitis induced by cyclophosphamide in rats
[1372] The purpose of this example is to show that KGF-2 33 is able to stimulate bladder proliferation in normal rats and that there is a therapeutic effect of KGF-2 33 in a rat model with cyclophosphamide-induced hemorrhagic cystitis To show.
[1373] Some clinically used cytotoxic agents inhibit normal epithelial proliferation in the bladder, potentially life-threatening ulceration and breakdown within the epithelium of the bladder. For example, cyclophosphamide may cause haemorrhagic cystitis in some patients, and serious and potentially fatal complications. Fibrillization of the bladder may also proceed with or without cystitis. This damage is thought to be caused by the cyclophosphamide metabolites secreted in urine. Severe hemorrhagic cystitis results in discontinuation of cyclophosphamide treatment. In addition, malignant tumors of the bladder usually occur within two years after treatment with cyclophosphamide and occur in patients who had previously had hemorrhagic cystitis (written on the CYTOXAN (cyclophosphamide) packaging). Cyclophosphamide has toxicity to the prostate and the male reproductive system. Cyclophosphamide therapy can lead to infertility, which can lead to some testicular atrophy.
[1374] Effect of KGF-2 △ 33 on normal bladder, testis and prostate
[1375] Experimental Design
[1376] Male Sprague-Dawley rats (160-220 g) (n = 4-6 / treatment group) were used in this study. KGF-2 33 was administered at a dose of 5 mg / kg / day. Recombinant KGF-2 33 or buffer (40 mM sodium acetate + 150 mM NaCl at pH 6.5) was administered daily intraperitoneally or subcutaneously for 1-7 days and the next day the rat was sacrificed. In order to investigate the reversibility of the effect induced by KGF-2 33, additional animals were dosed daily into the peritoneal cavity with KGF-2 33 for a period of 7 days without treatment and then sacrificed.
[1377] On the day of lethality, rats were intravenously administered BrdU 100 mg / kg. Two hours later, the rats were given an overdose of ether and the removed organs were selected. Tissue samples were fixed in 10% neutral buffered formalin for 24 h and paraffin-embedded. To detect the incorporation of BrdU into cloned cells, immunohistochemical procedures were performed on 5-micron fragments using mouse anti-BrdU monoclonal antibody and ABC elite detection system. This fragment was counterstained slightly with hematoxylin.
[1378] Fragments were read by a blind observer. The number of proliferating cells was counted in 10 random fields per animal at 10 times magnification on the prostate. To evaluate the effect of KGF-2 33 in the bladder, the sections of these tissues were prepared and the number of proliferating and non-proliferating cells were counted in 10 random fields at 20x magnification. Results are expressed as a percentage of labeled cells for unlabeled cells. Data are presented as mean + SEM. Statistical analysis (unpaired t-test with two tails) was performed using the Star View software package and statistical significance was defined as P < 0.05.
[1379] result
[1380] bladder
[1381] The intraperitoneal injection of KGF-2 33 induced proliferation of bladder epithelial cells over a 7 day trial period, but this did not affect organ weights (black squares, Figure 52). Subcutaneous administration induced a small increase in proliferation, but failed to obtain statistical significance (black circle, FIG. 52).
[1382] Prostate and testicles
[1383] Both subcutaneous and intraperitoneal administration of KGF-2 33 induced significant proliferation of the prostate gland (Fig. 53), but after two doses this was normalized. Intraperitoneal treatment with long-term KGF-2 △ 33 did not increase the weight of the prostate and testes.
[1384] Effect of KGF-2 △ 33 on Hemophilic Cystitis Induced by Cyclophosphamide
[1385] Experimental Design
[1386] Male Sprague Dawley rats (300-400 g) (n = 5 / group) were intravenously injected with KGF-2 33 at a concentration of 1 to 5 mg / kg via the tail vein and after 24 hours, cyclophosphamide 200 mg / kg was intraperitoneally injected. On the last day, 48 hours after cyclophosphamide administration, rats were intraperitoneally injected with 100 mg / kg of BrdU. Two hours later, the rats were sacrificed by CO 2 administration. Fixation of the bladder was achieved by direct injection of 10% formalin into the intracavity of the bladder and by washing the outside of the bladder with formalin. After 5 minutes, the bladder and prostate were removed. The bladder and prostate were paraffin embedded, section cut, stained with H & E, and stained with mouse anti-BrdU monoclonal antibody. The extent of urinary epithelium damage was assessed using the following scoring system: bladder categorization by two different observers, and the degree of injury of the cortex was recorded. (Uretery injuries were scored with 0, 25%, 50%, 75%, and 100% loss of cortical blood). In addition, the thickness of the bladder wall was measured at 10 random sites per fragment and was expressed in [mu] m.
[1387] result
[1388] Visual observation
[1389] In rats treated with placebo and cyclophosphamide, the bladder was thick and hard. After injecting 10% formalin, a slight swelling of the bladder was observed. However, in the group pretreated with KGF-2 33, greater flexibility was observed after direct injection of formalin, which meant a smaller degree of fibrosis.
[1390] Microscopic observation
[1391] Figure 54 shows the results of KGF-2 33 pretreatment on ulcer sites in the bladder. In normal rats (saline control) treated with intraperitoneal saline, bladder was histologically normal and ulceration was not observed in the uroepithelium. Cyclophosphamide intraperitoneal injection at 200 mg / kg formed ulcers in the bladder epithelium, 25-50% (mean 37%) of the total epithelial area. When KGF-2 33 was administered 24 hours before the administration of cyclophosphamide, the area of the ulcer was significantly reduced (1 mg / kg 0.4% p = 0.0128, And 5 mg / kg 5%, p = 0.0338%).
[1392] Figure 55 shows the effect of KGF-2 33 on the thickness of the bladder wall, including the epithelium, the smooth muscle layer and the serosal surface. In the population treated with buffer alone, the thickness of the bladder wall was about 40 μm. Treatment with cyclophosphamide resulted in a 5-fold increase in the thickness of the bladder wall to 210 μm. Preliminary treatment with cyclophosphamide-treated animals with KGF-2 33 significantly inhibited cyclophosphamide expansion of the bladder wall compared with cyclophosphamide alone (1 mg / kg 98.6 占 퐉 (p = 0.007) and 5 mg / kg 52.3 占 퐉 (p <0.0001).
[1393] Visual observation
[1394] Prostate: In rats treated with buffer and cyclophosphamide, a significant dilatation of the prostate (acini) and significant dilatation of the gap was observed compared to normal. In addition, it has been observed that the prostate inside the epithelium is shorter and more dense than the normal prostate tissue. The pretreatment of KGF-2 33 at 1 mg / kg and 5 mg / kg showed normal histological appearance of the prostate gland. No increase in clearance or edema was observed, and the size and density of the prostate inside the epithelium were similar to normal prostate tissue.
[1395] conclusion
[1396] These results demonstrate that KGF-2 33 specifically induces prostate proliferation inside bladder epithelial cells and epithelial cells. This result also demonstrates that KGF-2 [Delta] 33 specifically reduces the ulcerated area in hemorrhagic cystitis induced by cyclophosphamide.
[1397] Example 31
[1398] Effect of KGF-2 on cell proliferation in normal rats
[1399] KGF-2, one of the FGF classes, induces the proliferation of normal human and rat keratinocytes. It is about 57% homologous to KGF-1 (one of the FGF classes). KGF-1 has been reported to induce epithelial proliferation in many organs. See Housley et al., Keratinocyte growth factor inducing proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract. J Clin INvest 94: 1764-1777 (1994); Ulich et al., Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo, J Clin Invest 93: 1298-1306 (1994); Ulich et al., Keratinocyte growth factor is a growth factor for mammary epithelium in vivo. The mammary epithelium of lactating rats is resistant to the proliferative action of keratinocyte growth factor. Am J Pathol 144: 862-868 (1994); Nguyen et al., Expression of keratinocyte growth factor in embryonic liver of transgenic mice causing changes in epithelial growth and differentiation result in polycystic kidneys and other organ malformations. Oncogene 12: 2109-2119 (1996); Yi et al., Keratinocyte growth factor induces pancreatic ductal epithelial proliferarion, AM J pathol 145: 80-85 (1994); and Yi et al., Keratinocyte growth factor proliferation of urothelium in vivo. J. Urology 154: 1455-1570 (1995)]. We conducted a similar experiment using KGF-2 to determine whether it induces normal epithelial proliferation in rats when administered systemically using subcutaneous and intraperitoneal routes.
[1400] Methods :
[1401] In this test, male Sprague-Dawley rats (160-220 g) were obtained from Harlan Sprague Dawley. KGF-2 33 (HG03411-E2) was administered at a dose of 5 mg / kg / day. KGF-2 33 or recombinant buffer (40 mM sodium acetate + 150 mM NaCl at pH 6.5) was administered daily for 1 to 7 days by intraperitoneal or subcutaneous injection and the next day the rats were sacrificed (see below). In order to investigate the reversibility of the effect induced by KGF-2 33, additional animals were dosed with KGF-2 33 or buffer daily for 7 days into the peritoneum and after 7 days without treatment, .
[1402] On the day of lethality, rats were intravenously administered BrdU 100 mg / kg. Two hours later, the rats were given an overdose of ether and the removed organs were selected. Tissue samples were fixed in 10% neutral buffered formalin for 24 h and paraffin-embedded. Immunohistochemical procedures were performed on 5 micron fragments using mouse anti-BrdU monoclonal antibody (Boehringer Mannheim) and ABC elite detection system (vector laura ratoliz) to detect the incorporation of BrdU into cloned cells. This fragment was slightly counter-stained with hematoxylin.
[1403] Fragments were read by a blind observer. The number of proliferating cells was counted in 10 random fields per animal at 10 times magnification in the following tissues: liver, pancreas, prostate and heart. For lung analysis, ten random fields were used, except that the proliferation was quantified at 20x magnification. Since kidneys have a number of functionally distinct regions, proliferation was assessed in control sections taken at the center of one kidney per animal. To evaluate the effect of KGF-2 33 on the esophagus and bladder, the sections of these tissues were prepared and the number of proliferating and non-proliferating cells were counted at 10 and 20 times magnification, respectively, in 10 random fields Respectively. Results are expressed as a percentage of labeled cells for unlabeled cells.
[1404] Data are presented as mean + SEM. Statistical analysis (unpaired t-test with two tails) was performed using the Star View software package (Abacus Concept Inc, Berkeley, Calif.) And statistical significance was defined as P <0.05.
[1405] Results :
[1406] Figure 56 shows an overview of the experimental protocol. Six animals were used in one group. However, at the time of the analysis by the blind observer, it was sometimes clear that the BrdU injection was unsuccessful. Before deciphering the results, data of 8 rats (or 7% of animals) out of 115 were excluded from the study and the size of the obtained population is shown in the table below.
[1407] The size of the population used in this study
[1408] n =
[1409] Processing time ipsc
[1410] KGF-2 △ 331 days 65
[1411] Buffer One day 66
[1412] KGF-2 △ 332 days 64
[1413] Buffer 2 days 66
[1414] KGF-2 △ 333 days 55
[1415] Buffer 3 days 55
[1416] KGF-2 △ 337 days 66
[1417] Buffer 7 days 65
[1418] KGF-2 △ 337 days + No processing for 7 days 6ND
[1419] 7 days for buffers + no treatment for 7 days 6ND
[1420] The liver . When administered intraperitoneally, KGF-2 33 after one injection induced rapid proliferation of hepatocytes (black squares) (Fig. 57), which increased the mitotic activity lasting for 3 days, Then came back to normal. In contrast to the rapid effect of intraperitoneal administration of KGF-2 performed in the liver when injected subcutaneously (black circle, FIG. 57), these growth factors showed a small effect. The proliferation increased after one day of treatment, but returned to normal levels after two days of daily injections.
[1421] Pancreas . In contrast to the rapid reversible effect of peritoneal administration of KGF-2 33 in the liver, this injection induced a prolonged pancreatic proliferation over a 14 day trial period (black squares, FIG. 58). Surprisingly, subcutaneous injection of KGF-2 33 (black circle) failed to induce proliferation at any time.
[1422] Kidney and bladder . KGF-2 △ 33 induced proliferation of the kidney epithelium when administered subcutaneously or intraperitoneally, but the former induced a greater effect. Subcutaneous administration resulted in a rapid increase in proliferation reaching the peak after 2 days and returning to normal after 7 days of treatment (black circle, FIG. 59). There was a significant increase in proliferation observed moderately but only on days 2 and 3 when KGF-2 33 was administered intraperitoneally (black squares). In addition, intraperitoneal injection of KGF-2 33 induced proliferation of bladder epithelial cells over a 7 day trial period (black square, FIG. 52). Subcutaneous administration induced a slight increase in proliferation, but failed to obtain statistical significance (black circle, FIG. 52).
[1423] Prostate . Both subcutaneous and intraperitoneal administration of KGF-2 33 induced significant proliferation of the prostate gland (Fig. 53), but this was normalized after two administrations.
[1424] Esophagus . KGF-2 33 administered subcutaneously or intraperitoneally induced proliferation of early and short-term (1 day and 2 days, respectively) returning rapidly to normal in the proliferation of esophageal cells (results not shown).
[1425] Other long - term . Systemic administration of KGF-2 △ 33 peritoneal and subcutaneous routes failed to induce pulmonary epithelial proliferation over a 7-day administration period.
[1426] Argument
[1427] When administered by the subcutaneous route, we observed normal epithelial proliferation stimulation in some organs (liver, kidney, esophagus and prostate), but most of these effects were short duration and reversible. Proliferation in these organs was reversed by daily subcutaneous administration of KGF-2 33.
[1428] The administration route had a remarkable effect on the observed proliferation. Daily peritoneal administration increased the rate of liver proliferation over a period of 3 days, whereas animals dosed daily subcutaneously with KGF-2 showed increased rates only after 1 day of treatment. Even more surprising was the response of the pancreas. The pancreas of animals receiving KGF-2 into the peritoneum exhibited a significantly increased level of proliferation over the 14 day test period. However, subcutaneous administration of KGF-2 did not induce an increase in mitotic activity in the pancreas. In addition, peritoneal administration, rather than subcutaneous administration of KGF-2, induced proliferation of bladder mucosa.
[1429] Peritoneal administration of KGF-2 induced short-term proliferation in the kidney centered on the area containing the collecting duct. Daily subcutaneous treatment resulted in prolonged proliferation in this area.
[1430] Example 32
[1431] Effect of KGF-2 △ 33 on lung cell proliferation by intratracheal administration
[1432] The purpose of this example is to show that KGF-2 33 can stimulate lung proliferation in normal rats following intratracheal administration (direct administration of KGF-2 33 in the lung).
[1433] Methods : Male Lewis rats (220-270 g), (n = 5 / treatment group) were used in this study. KGF-2 33 or placebo (40 mM sodium acetate + 150 mM NaCl at pH 6.5) was administered orally at a dose of 1 to 5 mg / kg of 0.6 ml volume followed by 3 ml of air. Treatment was administered on day 1 and day 2 of the experimental protocol.
[1434] On the third day of lethality, rats were intraperitoneally injected with BrdU 100 mg / kg. Two hours later, the rats were sacrificed by CO ₂ suffocation. The lungs were expanded with 10% buffered formalin through a bronchial catheter, and the area of the lungs was paraffin - embedded. To detect BrdU incorporation into cloned cells, immunohistochemical procedures were performed on the 5-micron region using mouse anti-BrdU monoclonal antibody and ABC elite detection system. This region was slightly counter-stained with hematoxylin.
[1435] This area was read by two blinded observers. The number of proliferating cells was counted in 10 random fields per fragment at 20x magnification. This result was expressed as the number of BrdU positive cells per field. Data are presented as means ± SEM. Statistical analysis (unpaired t-test) was performed using Instat v2.01 and statistical significance was defined as p < 0.05.
[1436] RESULTS : Intra-tracheal injection of KGF-2 △ 33 at 1 and 5 mg / kg resulted in an increase in lung epithelial cell proliferation as shown in Fig. The results of KGF-2 33 treatment showed that BrdU-positive cells / field (p = 0.0003) at 1 mg / kg 23.4 cells / field (p = 0.0002) and 5 mg / kg 10.3 cells / A statistically significant increase was noted in the number of fields.
[1437] Example 33
[1438] Topical KGF-2 in infected incision wound
[1439] Bacterial infections of the wound are consistently important in clinical practice. Under normal circumstances, the complex process of wound healing proceeds without a doubt. However, infection of the wound by the bacteria results in an imbalance of the cellular mediator in the inflammatory reaction and delays wound healing. Infection of open wounds interferes with wound healing processes characterized by reduced wound constriction and reduced tensile strength compared to normal wound collagen content. Male adult Sprague Dawley rats (n + 10 / group) were anesthetized with a combination of ketamine (5.3 mg / kg im) and xylazine (5.3 mg / kg im) on day 1. The backside was shaved and sterilized with 70% alcohol. Using a sterile, ten-point surgical scalpel, the patient underwent a surgical wound of 2.5 cm in total thickness (epithelium, dermis to subcutaneous layer) starting approximately 1 cm below the scapula. The wound was coated with three equidistant skin staples. The incision was then infected into the incision with Staphylococcus aureus (107 cfu / 50 [mu] l) in PBS. KGF-2 33 was topically applied at the time of wounding (0 day) at a volume of 0.1, 1 and 10 占 퐂 per wound in a volume of 50 占 퐇. The wound was then covered with a gas permeable occlusion dressing (Tegaderm). Animals were anesthetized with ketamine / xylazine and then sacrificed on day 5 by intratracheal administration of phenobarbital sodium salt (300 mg / kg). A 0.5 cm section of the wound was cut and frozen overnight to measure collagen. Two other wound pieces with a width of 0.5 cm were incised. Incision wound scrap was used to test the fracture strength using an Instron skin tensile tester. The fracture strength was defined as the maximum force that was blocked by each wound before rupture with 11 1 b load cells at a rate of 0 mm / sec. The two values for each animal were averaged to provide an average of the rupture strength values per wound. Statistical analysis was performed using an inexpensive t test (mean ± SE).
[1440] Application of staphylococcus aureus to the incision in the wound resulted in considerable obstacles to wound healing, as measured by breaking strength (136 ± 6 g uninfected wound treated with a bacterial vehicle in one experiment; Infected wound 87 ± 6 g; p <0.0001; uninfected wound treated with bacterial vehicle in other experiments 200 ± 14 g; infected wound 154 ± 10 gp = 0.1). The topical administration of KGF-2 was performed using KGF-2 buffer + S. aureus control (0.1 ug 152 ± 16 g (p = 0.002), 1 ug 135 ± 12 g (p = 0.003) of KGF-2 in one experiment; 10 190 190 ± 7 g (p = 0.009), 10 ㎍ 158 ± 10 g (p <0.0001), 0.1 ㎍ 185 ± 10 g ) Resulted in a statistically significant increase in fracture strength at doses of 0.1, 1 and 10 μg. Collagen analysis of the mid 0.5 cm wound fragment showed an increase in collagen content in wound treated with KGF-2. However, no statistically significant increase in collagen content was observed compared to the buffer control.
[1441] Example 33
[1442] Proliferative effect of two-day intravenous administration of 1 mg / kg of KGF-2 △ 33
[1443] Male Sprague Dawley rats were intravenously injected with 1 mg / kg of KGF-2 33 or one of the buffers. Animals were injected daily or every other day. Each treatment group was injected for one week and killed on the last day of the week. On the day of death, BrdU 100 mg / kg was injected into the peritoneum of the animals. Two hours later, the animals were sacrificed and serum was collected. Various tissues were collected and fixed in 10% neutral buffered formalin. Tissue was processed for histological evaluation. Tissues were stained with hematoxylin and eosin, periodic-acid-Schiff or alcian blue. Other fragments were immunohistochemically stained with anti-BrdU antibodies. Proliferation was quantified using image analysis spectra. Serum chemistry analysis was performed using an automated chemical analyzer. The following parameters were quantified: thyroid weight; Goblet cell proliferation of the small intestine (duodenum, plant and venous); Proliferation in parotid and mandibular lines; Serum chemical analyzes (glucose, BUN, calcium, total protein, albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, cholesterol and triglyceride).
[1444] In the small intestine and large intestine, KGF-2 daily treatment resulted in a significant increase in the number of goblet cells. Treatment once every two days caused a slight increase in the number of goblet cells, but this did not reach a statistically significant level. In salivary glands, an increase in cells was observed only in the parotid gland. There was no difference between treatment groups. There was an enlargement of the thyroid gland according to the dosage regimens of both sides. This increase was more pronounced in the daily treatment group. Daily treatment with KGF-2 resulted in a statistically significant increase in the following assays: triglyceride, alkaline phosphatase, calcium, albumin, total protein. Treatment every two days had no effect on these analytes. Cholesterol levels increased in both treatment groups. However, this increase was more pronounced in the daily treatment group. Markers of cell damage, such as ALT and AST, were similarly reduced in both treatment groups.
[1445] Example 34
[1446] Preparation of Polypeptides
[1447] The KGF-2 composition meets excellent medical technology, taking into account the clinical status of the individual patient (especially the side effects of treatment of the KGF-2 polypeptide alone), side effects of delivery, administration method, dosage regimen and other factors known to the skilled artisan &Lt; / RTI &gt; An &quot; effective amount &quot; for purposes herein is determined by this consideration.
[1448] As a general suggestion, the total pharmacologically effective amount of KGF-2 administered parenterally at the time of a single administration is from about 1 μg / kg / day to 10 mg / kg / day, based on the patient's body weight, This will depend on the judgment of the treatment. More preferably, the dose is 0.01 mg / kg / day or more, and most preferably 0.01 to 1 mg / kg / day in human. When administered consecutively, KGF-2 is usually administered at a dose rate of 1 μg / kg / day to 50 μg / kg / day using 1-4 mini-pumps or continuous subcutaneous infusion, do. Vein pocket solutions can also be used. The length of treatment needed to observe the change and the interval of treatment in which the response appears will vary depending on the effect of the treatment.
[1449] The pharmaceutical compositions comprising KGF-2 can be administered orally, rectally, parenterally, systemically, intravaginally, intraperitoneally, topically (by powder, ointment, gel, drip or transdermal patch) Administered as an oral or nasal spray. The term " pharmaceutically acceptable carrier " means a non-toxic reconstitutable, semi-solid or liquid filler, diluent, encapsulating material or any form of adjuvant composition. As used herein, the term " parenteral " refers to a mode of administration encompassing intravenous, intramuscular, intraperitoneal, intrasternal, and intraarterial injection or infusion.
[1450] KGF-2 is also suitable for administration as a sustained-release system. Suitable examples of sustained release compositions include semi-permeable polymeric matrices in the form of shaped articles such as films or microcapsules. Sustained-release matrices include polylactides (US Pat. No. 3,773,319, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl L-glutamate (Sidman, U. et al., Biopolymers 22; 547-556 (1983)), poly (2-hydroxyethyl methacrylate (R. langer et al., J. Biomed. Mater. Res. 15: 167-277 (1981) and R. Langer Chem. Tech. -105 (1982)), ethylene vinyl acetate (R. Langer et al.) Or poly-D- (-) = 3-hydroxybutyric acid (EP 133,988) -2 polypeptides. Liposomes containing KGF-2 are prepared by techniques known in the art: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Dci. USA 82: 3688-3692 1985); Hwang et al., Proc. Natl. Acad. Scid. USA 77: 4030-4034 (1980); Ep 52,322; EP 36,676; EP 88,406; EP 143,949; EP 142; 641; Japanese Patent Application No. 83-118008; U.S. Patent Nos. 4,485,045 and 4,544,545; And EP 102,324. Typically, the liposomes are in the form of a small (about 200-800 Angstroms) single layer with a lipid content of at least about 30 mole% cholesterol (selected ratios adjusted for optimal secretion polypeptide treatment).
[1451] In one embodiment of parenteral administration, KGF-2 is generally administered in a unit dose injection form (solution, suspension or emulsion) in the form of a pharmaceutically acceptable carrier (i. E., Compatible with the other ingredients in the composition and administered And non-toxic to the subject to which it is administered at a given dose and concentration). For example, it is preferred that the agent does not comprise an oxidizing agent and other compounds known to be detrimental to the polypeptide.
[1452] In general, the compositions are prepared by directly and uniformly bringing KGF-2 into contact with a liquid carrier or a uniformly distributed solid carrier, or both. Then, if necessary, the product is molded into a predetermined preparation. It is preferable that the carrier is a parenteral carrier, and a solution which is a solution of the subject to be administered and an isotonic solution is more preferable. Examples of such carrier vehicles include water, saline, Ringer's solution and dextrose solution. Non-aqueous vehicles and liposomes such as non-volatile oils and ethyl oleate are also useful herein.
[1453] The carrier is suitably a small amount of an additive, such as a material enhancing isotonicity and chemical stability. Such materials are non-toxic to the eye under test at the dosages and concentrations employed, and include phosphate, citrate, succinate, acetic acid, other organic acids or salts thereof; Antioxidants such as ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides, such as polyarginine or tripeptides; Proteins such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamic acid, aspartic acid or arginine; Cellulose or its derivatives, monosaccharides including glucose, mannose or dextrin, disaccharides and other carbohydrates; Sugar alcohols such as mannitol or sorbitol; Counter ions such as sodium; And / or anionic surfactants such as polysorbate, poloxamer, or PEG.
[1454] KGF-2 is typically formulated in such vehicles at a concentration of about 0.1 mg / ml to 100 mg / ml, preferably 1-10 mg / ml, at a pH of about 3-8. Use of the particular excipients, carriers or stabilizers described above will result in the formation of polypeptide salts.
[1455] KGF-2 used for therapeutic administration can be sterilized. Sterilization can be easily accomplished by filtration through sterile filtration membranes (e. G., 0.2 micron membranes). Therapeutic polypeptide compositions are generally placed into a vial having a sterile access port, for example, an intravenous solution bag, or a cap with a pierceable puncture needle.
[1456] KGF-2 polypeptides will typically be stored in unit or multi-dose containers, such as sealed ampoules or vials, as aqueous solutions or hydrating preparations. As an example of a thixotropic agent, a 10 ml vial is filled with 5 ml of sterile-filtered 1% (w / v) aqueous KGF-2 polypeptide solution and the resulting mixture is frozen. The infusion solution is prepared by hydrating KGF-2 polypeptide frozen using bacteriostatic water for injection.
[1457] The present invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the pharmaceutical composition of the present invention. In connection with such container (s), notices stipulated by a government agency regulating the manufacture, use or sale of drugs or biological agents shall include the permission of the government agency for manufacture, use or sale for human consumption. Reflect. In addition, KGF-2 may be used in combination with other therapeutic compounds.
[1458] The composition of the present invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that can be administered with the compositions of the present invention include, but are not limited to, other TNFs, chemotherapeutic agents, antibiotics, steroids and nonsteroidal anti-inflammatory agents, conventional immunotherapeutic agents, cytokines, and / But is not limited to. The combination is concurrently with the mixture; Each but simultaneously or incidentally; Or sequentially. This includes a presentation in which the combined medicament is administered with the therapeutic mixture, as well as how the combined medicament is administered separately but administered simultaneously (e. G., Administered to the same individual in a separate intravenous line). &Quot; Combination " administration also encompasses administering the second compound or medicament separately after first administering one of the given compound or medicament.
[1459] In one embodiment, the compositions of the invention are administered with other TNF classes. TNF, TNF-related or TNF-like molecules can be produced in soluble forms of TNF-alpha, lymphotoxin-alpha (LT-alpha or TNF-beta), LT- (International Patent Publication No. WO 96/14328), AIM-I (International Patent Publication No. WO 97/33899), endokin-1, (International Patent Publication No. WO 98/30694), OPG and Neutrokine-alpha (WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40, and 4 -IBB, TR2 (International Patent Publication WO 96/34096), DR3 (International Patent Publication WO 97/33904), DR4 (International Patent Publication WO 98/32856), TR5 (International Patent Publication WO 98/30693) TRANK, TR9 (International Patent Publication WO 98/56892), TR10 (International Patent Publication WO 98/54202), 312C2 (WO 98/30894), TR7 (International Patent Publication WO 98/41629) / 06842) and TR12 and soluble CD154, CD70 and CD153, The.
[1460] Typical non-specific immunosuppressive agents to which the present invention may be administered with the composition include steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methyl prednisone, prednisone, azathioprine, FK-506, 15-deoxy sper But are not limited to, other immunosuppressive agents that act by inhibiting the action of T cells,
[1461] In another embodiment, a composition of the invention is administered with an antibiotic. Antibiotics to be administered with the compositions of the present invention include but are not limited to tetracycline, metronidazole, amoxicillin, beta-lhamtamase, aminoglycoside, macrolide, quinazolone, fluoroquinolone, cephalosporin, erythromycin, ciprofloxacin and streptomycin Including, but not limited to.
[1462] In another embodiment, the compositions of the present invention are administered alone or in combination with an anti-inflammatory agent. Anti-inflammatory agents that may be administered with the compositions of the present invention include, but are not limited to, glucocorticoids and nonsteroidal anti-inflammatory agents, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acid derivatives, arylpropionic acid derivatives, pyrazole, pyrazolone , Salicylic acid derivatives, thiazinecarboxamide, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyrate, amicetin, vendas, benzydamine, But are not limited to, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, It is not limited thereto.
[1463] In another embodiment, a composition of the invention is administered with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the compositions of the present invention include antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); Antiestrogens (e. G. Tamoxifen); Antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, phloxuridine, interferon alpha-2b, glutamate, flicamycin, mercaptopurine and 6-thioguanine); Cytotoxic agents such as carmustine, BCNU, robustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, proccarbazine, mitomycin, Latin and vincristine sulfate); Hormones (for example, medroxyprogesterone, estramustine phosphate sodium salt, ethinylestradiol, estradiol, megestrol acetate, methyl testosterone, diethylstilbestrol diphosphate, chlorotrienicone, and testolactone) ; Nitrogen mustard derivatives (for example, mephalan, chlorambucil, mechlorethamin (nitrogen mustard) and thiotepa); But are not limited to, steroids and combinations (e. G., Betamethasone sodium phosphate) and other such as dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide, It is not limited thereto.
[1464] In another embodiment, the composition of the invention is administered with a cytokine. The cytokines that may be administered with the compositions of the present invention include IL2, IL3, IL4, IL5, IL6, IL7, ILlO, IL12, IL13, IL15, anti-CD40, CD40L, IFN-gamma and TNF- But is not limited to.
[1465] In another embodiment, a composition of the invention is administered with an angiogenic protein. The angiogenic proteins that may be administered with the compositions of the present invention include growth factors derived from glycoma (GDGF) as disclosed in EP-339816; Platelet derived growth factor-A (PDGF-A) as disclosed in EP-682110; Platelet derived growth factor-B (PDGF-B) as disclosed in EP-282317; Placental growth factor (PIGF) as disclosed in International Patent Publication WO 92/06194; Placental growth factor-2 (PIGF-2) as disclosed in Hauser et al., Growth facors, 4: 259-268 (1993); Vascular endothelial growth factor (VEGF) as disclosed in International Patent Publication No. WO 90/13649; Vascular endothelial growth factor-A (VEGF-A) as disclosed in EP-506477; Vascular endothelial growth factor-2 (VEGF-2) as disclosed in International Patent Publication WO 96/39515; Vascular endothelial growth factor B-186 (VEGF-B186) as disclosed in International Patent Publication WO 96/26736; Vascular endothelial growth factor-D (VEGF-D) as disclosed in International Patent Publication WO 98/02543; Vascular endothelial growth factor-D (VEGF-D) as disclosed in International Patent Publication WO 98/07832; And vascular endothelial growth factor-E (VEGF-E) as disclosed in DE19639601. The above-mentioned documents are incorporated herein by reference.
[1466] In another embodiment, the composition of the present invention is administered with a fibroblast growth factor. FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9 , FGF-10, FGF-11, FGF-12, FGF-13, FGF-14 and FGF-15.
[1467] In another embodiment, the compositions of the invention are administered in conjunction with other therapeutic or prophylactic therapies (e. G., Radiation therapy).
[1468] Example 35
[1469] How to Treat Reduction of KGF-2
[1470] The present invention also relates to a method of treating such an individual comprising administering to a subject in need thereof an increase in KGF-2 activity level in a body comprising a therapeutically effective amount of KGF-2 or an agonist thereof .
[1471] It is also understood that this can be treated by administration of KGF-2, preferably secretory form of KGF-2, to a condition caused by a decrease in the normal or normal expression level of KGF-2 in the subject. Thus, the present invention also encompasses the step of administering to such individuals a pharmaceutical composition comprising an amount of KGF-2 that increases the activity level of KGF-2 in an individual in need of an increase in KGF-2 polyprolidade levels, Provides a method of treating an individual.
[1472] For example, patients with reduced levels of KGF-2 polypeptide continue to receive 0.1 to 100 [mu] g / kg of polypeptide per day for 6 days. It is preferred that the polypeptide is secreted. More precise details of the dosage regimen based on administration and formulation are provided in Example 24.
[1473] Example 36
[1474] How to treat increased levels of KGF-2
[1475] The present invention also relates to a method of treating such an individual comprising administering a composition comprising a therapeutically effective amount of a KGF-2 antagonist to a subject in need of a reduction in the level of KGF-2 activity in the body. A preferred antagonist for use in the present invention is a KGF-2 specific antibody.
[1476] Antisense techniques are used to inhibit the production of KGF-2. This technique is one example of a method of reducing the level of KGF-2 polypeptide, preferably secretory KGF-2 polypeptide, due to various pathologies such as cancer.
[1477] For example, patients diagnosed with abnormally elevated levels of KGF-2 are given antisense polynucleotides at 0.5, 1.0, 1.5, 2.0, and 3.0 mg / kg / day for 21 days. If these treatments are well tolerated, repeat after a 7-day rest period. The antisense polynucleotide preparation is presented in Example 24.
[1478] Example 37
[1479] Therapeutic Methods Using Gene Therapy - In Vitro
[1480] One method of gene therapy is to transplant a fibroblast capable of expressing a KGF-2 polypeptide into a patient. In general, fibroblasts are obtained from a subject by skin biopsy. The tissue thus obtained is placed in a tissue culture medium and separated into small pieces. A small chunk of tissue is placed on the wet surface of the tissue culture flask and about ten pieces are placed in each flask. Turn the flask upside down, secure it tightly and place it at room temperature overnight. After 24 hours at room temperature, the flask was turned upside down and the tissue mass was fixed to the bottom of the flask and a fresh medium (containing, for example, Hams F12 medium, 10% FES, penicillin and streptomycin) do. The flask is then incubated at 37 [deg.] C for about one week.
[1481] At this point, a new medium is added and then changed every 7 days. After an additional two weeks in the medium, a monolayer of fibroblasts emerges. The monolayer is digested with trypsin and transferred to a larger flask.
[1482] PMV-7 (Kirschmeier, PT et al., DNA, 7: 219-25 (1988)) located on the side of the long-chain terminal repeat region of Molonimylin sarcoma virus was digested with EcoRI and HindIII, Treat with azeotrope. This linear vector is fractionated on an agarose gel using glass beads and purified.
[1483] PCR primers corresponding to the 5 ' and 3 ' end sequences, respectively, as described in Example 1 can be used to amplify cDNA encoding KGF-2. It is preferred that the 5 'primer comprises the EcoRI site and the 3' primer comprises the HindIII site. Equal amount of Molonimylin sarcoma virus linear main chain and amplified EcoRI and HindIII fragments are added together in the presence of T4 DNA ligase. The resulting mixture is maintained under conditions suitable for linking the two fragments. This is then inoculated onto an agar containing kanamycin to confirm that the vector is transformed with the germ-line GB101 using a ligation mixture, and then the vector contains KGF-2 intimately inserted.
[1484] Both amphotropic pA317 or GP + am12 packaging cells are grown at population density in modified eagles' medium (DMEM) of Dulbecco's containing 10% calf serum (CS), penicillin and streptomycin in tissue culture medium. Then, an MSV vector containing the KGF-2 gene is added to the medium, and the packaging cell is transduced with the vector. The packaging cells now produce infectious viral particles containing the KGF-2 gene (the packaging cells are now called production cells).
[1485] The new medium is added to the transfected producer cells, and then the medium is collected from the 10 cm plate of dense producer cells. After the spent medium containing the infectious viral particles is filtered through a millipore filter to remove adherent production cells, the medium is used to infect fibroblasts. The medium is removed from the small cluster plate of fibroblasts and the medium is quickly replaced from the producing cells. Remove this medium and replace with fresh medium. When the titer of the virus is high, virtually all fibroblasts will be infected, and no selection is required. If the titer is very low, it is necessary to use a retroviral vector with an optional marker, e. G. Neo or his. After fibroblasts are effectively infected, fibroblasts are analyzed to determine whether KGF-2 protein is produced.
[1486] The engineered fibroblasts are then implanted either alone on the dentition or after cloning on the Cydex 3 microcarrier beads to clusters.
[1487] Example 38
[1488] Gene therapy using endogenous KGF-2 gene
[1489] Another method of gene therapy in accordance with the present invention is described, for example, in U.S. Patent No. 5,641,670, issued June 24, 1997; International Patent Publication Wo 96/29411 (published September 26, 1996); International Patent Publication WO 94/12650 (published Aug. 4, 1994); See Koller et al., Proc. Natl. Acad. Sci. USA 86: 8923-8935 (1989); And operably combining the promoter and the endogenous KGF-2 sequence through homologous recombination as disclosed in Zijlstra et al., Nature 342: 435-438 (1989). This method involves activation of the gene present in the target cell, but it is not expressed in the cell or is expressed at a lower level than desired.
[1490] On the side of the promoter, a polynucleotide construct comprising a target sequence and a promoter having homology to the 5 ' unencrypted sequence of endogenous KGF-2 is prepared. The target sequence will be sufficiently contiguous to the 5 ' end of KGF-2 so that the promoter can be operatively associated with the endogenous sequence at homologous recombination. Promoters and target sequences can be amplified using PCR. It is preferred that the amplified promoter contains restriction enzyme sites that are distinguished on the 5 ' and 3 ' The 3'end of the first target sequence contains the same restriction enzyme site as the 5'end of the amplified promoter and the 5'end of the second target sequence contains the same restriction site as the 3'end of the amplified promoter Do. The amplified promoter and amplified target sequence are digested with appropriate restriction enzymes and then treated with calf intestinal phosphatase. In the presence of T4 DNA ligase, the degraded promoter and degraded target sequence are added together. The resulting mixture is maintained under conditions suitable for linking the two fragments. The structure is fractionated on an agarose gel by size and then purified by phenol extraction and ethanol precipitation.
[1491] In this example, the polynucleotide construct is administered as a naked polynucleotide by electrophoresis. However, the polynucleotide construct may also be administered with a transfection promoter (e. G., A liposome, viral sequence, viral particle, precipitant, etc.). Such delivery methods are well known in the art.
[1492] Once the cells are transfected, homologous recombination will occur in which the promoter is operably linked to the endogenous KGF-2 sequence. As a result, KGF-2 is expressed in the cells. Expression can be detected by immunological staining or by any method known in the art.
[1493] Fibroblasts are obtained from a subject by skin biopsy. The obtained tissues are placed in DMEM containing 10% fetal bovine serum. The initial stationary fibroblasts that grow at an exponential rate are digested with trypsin and washed using a nutrient medium from a flexible surface. The aliquot of the cell suspension is removed for counting and the remaining cells are centrifuged. The supernatant is aspirated and the pellet resuspended in 5 ml of electrophoresis buffer (20 mM hepes pH 7.3, 137 mM NaCl, 0.7 mM Na2HPO4, 6 mM Dextrose). The cells are re-centrifuged, the supernatant is aspirated, and the cells are resuspended in electrophoresis buffer containing 1 mg / ml acetylated serum albumin. Electrophoresis should be performed immediately after resuspension.
[1494] Plasmid DNA is prepared according to standard techniques. For example, plasmid pUC18 (MBI fermenter, Amherst, New York) is digested with HindIII to construct a plasmid that targets KGF-2 position. The CMV promoter is amplified by PCR using the XbaI site on the 5 'end and the BamHI site on the 3' end. Two KGF-2 noncoding sequences are amplified by PCR: One KGF-2 noncoding sequence (KGF-2 fragment 1) is amplified by the HindIII site at the 5 'end and the Xba site at the 3' The KGF-2 noncoding sequence (KGF-2 fragment 2) is amplified by the BamHI site at the 5 'end and the HindIII site at the 3' end. The CMV promoter and the KGF-2 fragment are digested with appropriate enzymes (CMV promoter-XbaI and BamHI; KGF-2 fragment 1-XbaI; KGF-2 fragment 2-BamHI) and ligated together. The resulting bound product is digested with HindIII and ligated into pUC18 plasmid digested with HindIII.
[1495] Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally above 120 [mu] g / ml. Then, add 0.5 ml of the cell suspension (containing about 1.5 x 10 ⁶ cells) to the cuvette, and gently mix the cell suspension and the DNA solution. Electrophoresis is performed using a Gene-pulser device (Bio-Rad). Capacitance and voltage are set to 960 μF and 250-300 V, respectively. As the voltage increases, the cell viability is reduced, but the percentage of viable cells stably incorporating the introduced DNA into their genome increases sharply. A vibration time of about 13-20 mSec is observed as a predetermined parameter.
[1496] The electrophoretic cells are maintained at room temperature for about 5 minutes and the contents of the cuvette are gently removed using a sterile transfer pipette. The cells are directly added to 10 ml of pre-warmed nutrient medium (DMEM containing 15% calf serum) in a 10 cm dish and cultured at 37 ° C. The next day, the medium is aspirated, replaced with 10 ml of fresh medium and incubated further for 16-24 hours.
[1497] The engineered fibroblasts are then infected to the host, either alone or in clusters on the cytosine 3 microcarrier beads, and then infected. This fibroblast produces a protein product. This fibroblast can be introduced into a patient as described above.
[1498] Example 39
[1499] Therapeutic methods using gene therapy - In vivo
[1500] The development of genetic research has led to the development of techniques for transferring and expressing genes in human cells. The ideal goal of gene therapy is to transduce normal genes to produce active proteins and to supplement deficiencies in human production (Gorecki, DC et al., Arch. Immunol. Ther. Exp. 45 (5-6): 375- 381 (1997)).
[1501] The transfer of genes encoding cytokines and growth factors associated with wound healing and other phases of tissue repair has the potential to alter the outcome of wound healing (Taub, TJ et al., J. Reconst. Microsur. 14 (6) : 387-390 (1998)). The use of growth factors and other cytokine cDNAs for wound healing and tissue repair is widely known (Tchorzewski, MT et al., J. Surg. Res. 77: 99-103 (1998)). The gene delivered by the vector can be used to produce a new cell line, to identify the transplanted cells, and to express growth factors or enzymes. One advantage of gene therapy is that it obtains a therapeutic concentration of the protein locally derived from the gene at the lesion site. Human recombinant KGF-2 protein has been shown to promote wound healing of other organs containing cells of the skin, gastrointestinal tract, and epithelial origin. The use of KGF-2 is expected to have a pharmacological profile similar to the recombinant protein. The KGF-2 gene may be involved in processes involved in tissue repair such as cell proliferation, migration, and the formation of an external matrix.
[1502] Transcripts and translated cDNA have been used to deliver the gene to the desired site. Some examples of genes used in this approach include FGF, BMP-7 (Britbart, AS et al., Ann. Plast. Surg. 24 (5): 488-495 (1999)). These cells were also inoculated into cell carriers containing biodegradable matrices (e. G., Polyglycolonic acid), tissue substitutes or equivalents (e. G., Artificial skin), artificial organs, collagen-derived matrices, Liposomes were used to carry cDNA. The PDGF-BB cDNA in Haemagglutinating virus-liposomal suspension in Japan (HVJ) has been studied in the treatment of patellar ligaments (Nakamura et al., Gene Ther. 5 (9): 1165-1170 (1998) ). In addition, the gene may be delivered directly to the site of action by direct injection (e. G., The heart).
[1503] Thus, another aspect of the invention is the use of gene therapy methods to treat disorders, diseases and conditions. This gene therapy method involves the introduction of a naked nucleic acid (DNA, RNA and antisense DNA or RNA) KGF-2 sequence into an animal to increase or decrease the expression of a KGF-2 polypeptide. The KGF-2 polynucleotide may be operatively linked to any other genetic element or promoter necessary for the expression of the KGF-2 polypeptide by the target tissue. Such gene therapy and delivery techniques and methods are described, for example, in WO 90/11092; WO 98/11779; U.S. Patent No. 5,693,622; 5,705, 151; 5,580, 859; Tabara, H., et al., Cardiovasc. Res. 35 (3): 470-479 (1997); Chao, J., et al., Pharmacol. Res. 25 (6): 517-522 (1997); Wolff, J. A., Neuromuscul. Disord. 7 (5): 314-318 (1997); Schwartz B., et al., Gene ther. 3 (5): 405-411 (1996); (Tsurumi, Y., et al., Circulation 94 (12): 3281-3290 (1996)), all of which are incorporated herein by reference.
[1504] The KGF-2 polynucleotide construct can be delivered by any method of delivering the injectable material to the cells of an animal, for example by injecting into the gap of the tissue (heart, muscle, skin, lung, liver, small intestine etc.) have. The KGF-2 polypeptide construct can be delivered in a pharmaceutically acceptable liquid or aqueous carrier.
[1505] A " naked " polypeptide, DNA, or RNA can be any delivery vehicle, including viral sequences, viral particles, liposomal preparations, lipofectin, or precipitating agents, that serve to aid, enhance, Quot; refers to a sequence having no nucleotide sequence. However, KGF-2 polypeptides can be delivered into liposomal preparations which can be prepared by any technique well known in the art. See, for example, Felgner P.L. et al., Ann. NY Acad. Sci. 772: 126-139 (1995) and Abdallab B. et al., Biol. Cell 85 (1): 1-7 (1995).
[1506] It is preferred that the KGF-2 polynucleotide vector construct used in the gene therapy method is a structure that is not inserted into the host genome and does not contain a sequence allowing replication. Any strong promoter known in the art can be used to regulate DNA expression. Unlike other gene therapy techniques, the great advantage of introducing naked nucleic acid sequences into target cells is the transient nature of polynucleotide synthesis in cells. Studies have shown that non-replicative DNA sequences can be introduced into cells to produce a given polypeptide for up to six months.
[1507] KGF-2 polynucleotide constructs can be used for the treatment of a wide variety of conditions including muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone, cartilage, pancreas, kidney, gallbladder, stomach, , The nervous system, the eye, the line and related tissues. Tissue gaps can be found in intracellular fluids, mucopolysaccharide matrices between reticulated fibers of organ tissues, flexible fibers in the walls of vessels or chambers, collagen fibers in fibrous tissues, or associated pores in bone or in muscle cells And a matrix within the tissue. These are similar to the space filled by the lymphatic fluid of the lymphatic channels and the circulating plasma. Delivery of the muscle cells to the gap is preferred for reasons described below. They may be conveniently delivered by injection into a tissue containing these cells. Although delivery and expression are carried out in undifferentiated or not completely differentiated cells such as, for example, blood stem cells or fibroblasts of the skin, they are preferably delivered and expressed in permanent, non-differentiated, non-dividing cells Do. In vivo muscle cells are particularly suited for the ability to capture and express polynucleotides.
[1508] For Naked KGF-2 polynucleotide injections, an effective amount of DNA or RNA will be in the range of about 0.05 g / kg (body weight) to 50 mg / kg (body weight). The dose is preferably about 0.005 to about 20 mg / kg, more preferably about 0.05 to about 5 mg / kg. Of course, one skilled in the art will appreciate that such capacity will vary depending upon the tissue site being injected. Such an appropriate and effective amount of the nucleic acid sequence can be readily determined by those skilled in the art, and may vary depending on the route of administration and the condition to be treated. The preferred route of administration is by a parenteral route of injection into the tissue gap. However, inhalation of aerosol preparations for delivery to other parenteral routes such as the lung or bronchial tissue, mucous membranes of the vagus or nasal cavity may be used. In addition, the naked KGF-2 polynucleotide construct can be delivered to the artery during angioplasty by a catheter used in this method.
[1509] The dose-response effect of injected KGF-2 polynucleotide in vivo can be determined as follows. KGF-2 template DNA suitable for the production of mRNA coding for KGF-2 polynucleotide is prepared according to the standard recombinant DNA method. Template DNA, either circular or linear, is used as naked DNA or in combination with liposomes. Subsequently, various amounts of template DNA are injected into the quadriceps muscle of the mouse.
[1510] Five to six week old female and male Balb / C mice are anesthetized by intraperitoneal injection of 0.3 ml of 2.5% avetin. Make a 1.5 cm incision on the anterior thigh and visualize the femoral head directly. Over a period of 1 minute, KGF-2 template DNA is injected through a 27 gauge needle at a depth of about 0.5 cm and a depth of about 0.2 cm from the distal insertion of the muscle to the knee in a 0.1 cc syringe in a 1 cc syringe. The injection site for future localization is sutured and the skin is closed with a stainless steel clip.
[1511] After a satisfactory incubation time (for example, 7 days) has elapsed, the muscle extract is prepared by incising the entire thigh. The 5th 15 μm cross section of each thigh is histologically stained for KGF-2 protein expression. The time course for KGF-2 protein expression can be performed in a similar fashion except that the thigh from other mice is collected at different times. The presence of KGF-2 DNA in muscle after injection can be measured by Southern blot analysis after preparation of HIRT supernatant and total cell DNA from injected and control mice. The results obtained in these mouse experiments can be used to extrapolate appropriate dosages and other therapeutic parameters in other animals and humans using KGF-2 naked DNA.
[1512] Example 40
[1513] KGF-2 Treatment in Inflammatory Bowel Disease
[1514] This example measures the inhibition of the pathological changes of the mouse colon caused by exposure to dextran sodium sulfate (DSS) in drinking water by systemic (intranasal) and intraperitoneal administration of KGF-2 polynucleotide.
[1515] Intranasal administration . Polynucleotides coding for KGF-2 33 are introduced into the nasal passages of female Swiss Webster mice (n = 10 / population) anesthetized via blunt 26-gauge needles in a volume of 1, 10 or 100 μg. Control polynucleotides are administered to each group of mice. When 5 days have passed since nasal administration of the polynucleotide, 5% DSS is added to the drinking water. Monitor mouse weight, hematocrit and stool scores. Mice are killed when 7 days have passed since exposure to DSS in drinking water. Perform a histological evaluation of the colon and small intestine. RT-PCR analysis is performed to measure the expression of KGF-2 in the liver, spleen, and large intestine.
[1516] Intraperitoneal administration . Polynucleotides encoding KGF-2 33 are injected into the peritoneum of a female Swiss Webster mouse (n = 10 / population) anesthetized on days 0 and 3 via blunt 26-gauge needles in a volume of 1, 10 or 100 μg. The control polynucleotides are administered to each group of mice using the same method. When 7 days have passed since nasal administration of the polynucleotide, 5% DSS is added to the drinking water. Monitor mouse weight, hematocrit and stool scores. On day 14, mice are sacrificed. Perform a histological evaluation of the colon and small intestine. RT-PCR analysis is performed to measure the expression of KGF-2 in liver, diaphragm, and large intestine.
[1517] The study described in this example is to test the activity in the KGF-2 33 polynucleotide. However, those skilled in the art will readily understand that the polynucleotides encoding the full length and mature KGF-2, KGF-2Δ28, and amino acids 77-208, 80-208, and 93-208 of KGF-2, KGF-2 polynucleotides, variants, fragments, agonists and / or antagonists; And other KGF-2 polynucleotides, including any of the KGF-2 mutations described above.
[1518] Example 41
[1519] KGF-2 treatment in surface disease of the eye
[1520] In this example, the effect of subconjugival administration of 33 KGF-2 polynucleotide on conjunctiva, cornea or lacrimal gland is measured.
[1521] The polynucleotide encoding 33 KGF-2 is injected into the subcoronal space of female Sprague Dawley rats (150-200 g body weight, 6 / treatment group) anesthetized with a dose of 1, 10 or 100 占 퐂. The control polynucleotides are administered in a similar manner to each group of control rats. When 3 days, 7 days and 14 days have passed since the injection, each rat group is sacrificed. Some rats are administered BrdU intraperitoneally 30 minutes before anesthesia. Remove the eye and surrounding tissue for histological analysis. Measure the degree of BrdU incorporation in the cornea, conjunctiva and lacrimal epithelium. The thickness of the epithelial layer is evaluated in the cornea and conjunctiva. Measure the number of goblet cells in the conjunctiva.
[1522] The study described above in this example tests activity in KGF-2 33 polynucleotides. However, those skilled in the art will readily understand that the polynucleotides encoding the full length and mature KGF-2, KGF-2Δ28, and amino acids 77-208, 80-208, and 93-208 of KGF-2, KGF-2 polynucleotides, variants, fragments, agonists and / or antagonists; And other KGF-2 polynucleotides, including any of the KGF-2 mutations described above.
[1523] Example 42
[1524] KGF-2 Treatment for Salivary Dysfunction
[1525] In this example, the effect of administration of KGF-2 polynucleotide on the epithelial cells of the duct and on the line of the line is measured into the papillary protrusions of the parotid salivary gland of normal rats.
[1526] Female Sprague Dawley rats (15-250 grams, 6 per group) are paralyzed by intramuscular injection of ketamine and xylazine. The polynucleotide encoding 33 KGF-2 is introduced into the papillary protuberance of the parotid salivary gland at a dose of 1, 10, or 100 占 using a 30 gauge steel gavage injection needle. This polynucleotide is injected at a rate of 1 쨉 l per minute over 10 minutes. A separate group of rats is given a control polynucleotide. Each rat group is killed at 3, 7, and 14 days after injection. BrdU is administered intraperitoneally 30 minutes before euthanasia. The salivary glands are weighed and the number of BrdU stained cells is counted in the tissue section. In a separate experiment, the saliva secretion stimulated with pyrocarnine was measured in the rat 7 days after injection.
[1527] The study described in this example tests the activity of the KGF-2 33 polynucleotide. However, those skilled in the art will appreciate that other KGF-2 polynucleotides, including polynucleotides encoding full length and mature KGF-2, KGF-2Δ28, and amino acids 77-208, 80-208, and 93-208 of KGF- ; And KGF-2 polypeptides, variants, fragments, agonists, and / or antagonists; And the studies exemplified to test the activity of any of the KGF-2 mutants disclosed herein can be readily modified.
[1528] Example 43
[1529] KGF-2 treatment for skin wound healing
[1530] In this example, the ability of KGF-2 polynucleotides to stimulate wound healing in normal rats and diabetic mice is measured.
[1531] Normal rats.
[1532] Anesthetized female Sprague Dawley rats (175-250 grams, 6 per treatment group) are wounded with an 8 mm biopsy punch. Transfer 33 KGF-2 polynucleotides (1, 10, or 30 μg) to the endothelium at four different sites along the wound. Control polynucleotides are administered in a similar manner to separate groups of rats. The wound is covered with a sterile ventilated fiber pad. After positioning the pad, a waterproof adhesive tape is wound around the center cut of the rat. Each rat group is killed on days 2 and 5 after injury. The wound tissue is fixed in 10% formalin fixed to paraffin. BrdU integration into epithelial cells proliferating in the epidermis and neonatal epidermis, and the length and thickness of the new epithelial tongue are measured.
[1533] Diabetic mouse.
[1534] They are wounded with 6 mm punches in diabetic mice (db + / db +, 10 per treatment group) and non-diabetic mice (db + / m +, 10 per treatment group). Transfer 33 KGF-2 polynucleotides (1, 10, or 30 μg) to the endothelium at four different sites along the wound. The control polynucleotide is administered to a separate group of mice in a similar manner. Cover the wound with a tergum (diabetic mouse) or a tegaderm plus adhesive tape (non diabetic mouse). Photographs of the wound at 0, 3, 7, 10 and 14 days after injury are taken. The surface area of the wound is measured by image analysis.
[1535] The study described in this example tests the activity of the KGF-2 33 polynucleotide. However, those skilled in the art will appreciate that other KGF-2 polynucleotides, including polynucleotides encoding full length and mature KGF-2, KGF-2 28, and amino acids 77-208, 80-208, and 93-208 of KGF-2; And KGF-2 polypeptides, variants, fragments, agonists, and / or antagonists; And the studies exemplified to test the activity of any of the KGF-2 mutants disclosed herein can be readily modified.
[1536] Example 44
[1537] Structure for KGF-2 Delivery
[1538] A suitable construct for KGF-2 gene therapy delivery is pVG I.0-KGF-2. This construct contains the entire KGF-2 native open reading frame cloned into the expression vector pVGI.0. pVGI.0 contains the kanamycin resistance gene, the CMV enhancer, and the RSV promoter. pVGI.0-KGF-2 was deposited with the ATCC Deposit No. PTA290 at the ATCC Patent Depository, University Boulevard 10801, Manassas, Va., 20110-2209, June 30, 1999. This construct was prepared by subcloning the KGF-2 ORF from the already proven KGF-2 construct into the expression vector pVGI-0 using known techniques.
[1539] Another construct suitable for KGF-2 delivery is pVGI-0-MPIFspKGF2 33. This construct contains the natural sequence of KGF-2 33 fused to the MPIF (CK 8) heterologous signal peptide cloned into the expression vector pVGI.0. pVGI.0-MPIFspKGF2 33 was deposited with ATCC Deposit No. PTA289 at the ATCC Patent Depository of University Boulevard 10801, Manassas, Va. 20110-2209, June 30, 1999. This structure was constructed using known primers and the following primers.
[1540] 5 'primer:
[1541] GAGCGCGGATCCGCCACCATGAAGGTCTCCGTGGCTGCCCTCTCCTGCCTCATGCTTGTTACTGCCCTTGGATCTCAGGCCAGCTACAATCACCTTCAAGGAGATG (SEQ ID NO: 149)
[1542] 3 'primer:
[1543] GAGCGCGGATCCCTATGAGTGTACCACCATTGGAAG (SEQ ID NO: 150)
[1544] Example 45
[1545] Angiogenesis during KGF-2 gene therapy
[1546] Characterization of various aspects of microvascular physiology in the transparent window system in mice has provided important data on angiogenesis, inflammation, microvascular transport, tissue rejection and tumor physiology. In this example, the occurrence of the vents during the wound healing reaction in the implanted collagen gel is assessed through direct observation of the tissue through the implanted skin window and the associated microvascular bed. This model is used to determine if KGF-2 gene therapy can simultaneously induce accelerated tissue regrowth and revascularization.
[1547] Skin biopsies of nude mice are digested with collagenase, and the resulting cell suspension is washed and cultured in DMEM with 10% FBS to obtain dermal fibroblasts. The confluent fibroblast cultures are transfected with KGF-2 or control polynucleotides, collected and washed in PBS. 106 cells are suspended in 20 占 퐇 of collagen matrix. The cell suspension sample is removed for western blot for confirmation of KGF-2 production. A 2 mm punch biopsy is made in the skin of the existing back skin and the skin is placed between two glass cover slips. The cell collagen mixture is placed on a circular wound and the chamber is sealed. The transplanted gels for angiogenesis are observed at regular intervals. Tissue re-growth within the wound is monitored by optical density changes of the collagen gel over three weeks. The tissue from the back chamber is removed after the conclusion of the study for histological evaluation. Control experiments add buffers or KGF-2 polypeptides into collagen gels instead of fibroblasts.
[1548] Mouse ready.
[1549] Surgical surgery is performed on Swiss nude mice. For surgical procedures, animals are paralyzed by subcutaneous injection of a mixture of 90 mg of ketamine and 9 mg of xylazine per kilogram body weight. All surgical procedures are performed under sterile conditions in a horizontal laminar flow hood, Steam, gas or chemical sterilization.
[1550] During surgery, the body temperature of the animal is held constant by the heated work surface. All mice are individually housed in a micro-separator and all manipulations are performed in a lamina flow hood. Buprenorphine (0.1 mg / kg q12h) is administered as an analgesic agent for 3 days after transplantation.
[1551] Place the mouse so that the chamber is positioned between the double layers of skin extending over the back surface. One layer of skin is removed with a circular area of about 15 mm in diameter. A second layer (composed of epithelium, fascia, and striated muscle) is placed on the chamber frame and covered with a sterile glass cover slip. Holds the chamber along with the hole along the top of the chamber and the nylon post through the extended skin. After 3 days, carefully remove the cover slip and insert the gel. A new, sterile cover slip is then placed on the observation surface. The measurement is performed by morphometric analysis using an enhanced CCD camera, S-VHS video cassette recorder and direct digital image acquisition. Mice with transplanted transformants were observed for 28 days.
[1552] Measure.
[1553] Mice are paralyzed by subcutaneous injection of a mixture of 90 mg of ketamine and 9 mg of xylazine per kg of body weight and placed in a sterile plastic sheeting. Using a permeation method or by injection of 100 μl of BSA-FITC (1 mg / ml, i.v.) and epithelial illumination, map the blood vessels of the window. Video recording of the vascular bed is made in a fixed magnification range (1x to 40x) as well as digital frames for off-line analysis. Angiogenesis measurements of grafted gels are made from offline analysis of videotapes.
[1554] The study described in this example tests the activity of the KGF-2 33 polynucleotide. However, those skilled in the art will appreciate that other KGF-2 polynucleotides, including polynucleotides encoding full length and mature KGF-2, KGF-2 28, and amino acids 77-208, 80-208, and 93-208 of KGF-2; And KGF-2 polypeptides, variants, fragments, agonists, and / or antagonists; And the studies exemplified to test the activity of any of the KGF-2 mutants disclosed herein can be readily modified.
[1555] Example 46
[1556] KGF-2 transgenic animal
[1557] KGF-2 polypeptides can be expressed in transgenic animals. Animal of any species including but not limited to mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cow and non-human primates (eg, baboons, monkeys, and chimpanzees) Can be used to create animals. In certain embodiments, polypeptides of the invention are expressed in humans as part of a gene therapy protocol, as disclosed herein or using techniques known in the art.
[1558] Any technique known in the art for introducing a transgene (e.g., a polynucleotide of the invention) into an animal to produce an ancestral line of transgenic animals may be used. Such techniques are described in detail in Paterson et al., Appl. Microbiol. Biotechnol. 40: 691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263-1270 Biotechnology (NY) 9: 830-834 (1991) and Hoppe et al., U.S. Patent No. 4,873,191 (1991)); Gene transfer into retroviral-mediated early embryogenesis, blastocyst or intrapusception (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82: 6148-6152 (1985)); Gene targeting in embryonic cells (Thompson et al., Cell 56: 313-321 (1989)); Electroporation of cells or embryos (Lo, Mol. Cell. Biol. 3: 1803-1814 (1983)); Introduction of polynucleotides of the invention using gene guns (Ulmer et al., Science 259: 1745 (1993)); Introducing a nucleic acid construct into multipotent hepatocytes of the embryo and transferring the hepatocytes into the blastocyst; And sperm-mediated gene transfer (Lavitrano et al., Cell 57: 717-723 (1989)); And the like. For a review of these techniques, see Gordon, " Transgenic Animals ", Intl. Rev. Cytol.115: 171-229 (1989), incorporated herein by reference.
[1559] In order to produce a transgenic clone containing the polynucleotides of the present invention, any of the methods known in the art, such as nuclear transfer from a cultured embryo, fetus, or dormantly derived adult cells into enucleated oocytes, (Campell et al., Nature 380: 64-66 (1996); Wilmut et al., Nature 385: 810-813 (1997)).
[1560] The present invention provides an animal (e.g., a mosaic animal or a chimera) carrying a transgene in a transgenic animal having a transgene in all of its cells and in some cells other than all of the cells. The transgene may be integrated as a single transgene, or it may be integrated into a concatamer such as a head-to-head tandem or a head-to-tail tandem. The transgene may be selectively introduced into specific cell types and activated according to the teachings of, for example, Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89: 6232-6236 . The regulatory sequences required for such cell type specific activation will depend on the particular cell type of interest and will be apparent to those skilled in the art. Where it is desired that the polynucleotide transgene is integrated into the chromosomal region of the endogenous gene, gene targeting is preferred.
[1561] In summary, when such techniques are used, vectors containing some nucleotide sequences that are homologous to the endogenous gene are designed to be integrated into the function of the nucleotide sequence of the endogenous gene through homologous recombination with the chromosomal sequence and destroyed. The transgene may also be selectively introduced into a particular cell type according to the teachings of Gu et al. (Gu et al., Science 265: 103-106 (1994)) to inactivate the endogenous gene only in that cell type It is possible. The regulatory sequence required for such cell type specific activation will depend on the particular cell type of interest and will be apparent to those skilled in the art. The contents of each of the documents cited in this paragraph are incorporated herein by reference in their entirety.
[1562] Once a transgenic animal is generated, the expression of the recombinant gene can be analyzed using standard techniques. Initial screening can be performed by analyzing animal tissues by Southern blot analysis or PCR techniques to demonstrate transgene integration. The degree of mRNA expression of the transgene in the tissues of the transgenic animals may also be determined by techniques that include, but are not limited to, Northern blot analysis of tissue samples obtained from animals, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR) . &Lt; / RTI &gt; Samples of transgenic gene expression tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
[1563] Once ancestral animals are produced, they can be breed, subjugated, crossbred, or crossed to produce colonies of specific animals. Examples of such mating strategies include crossing ancestral animals with one or more integration sites to establish separate lines; Producing a complex transgenic which expresses a higher level of transgene by the effect of the additional expression of each transgene, Crossing heterologous transgenic animals to produce homozygous animals for a given integration site to increase expression and eliminate the need to screen animals by DNA analysis; Crossing separate homogeneous lines to produce composite heterogeneous or homogeneous lines; And crossing to place the transgene in a separate background suitable for an experimental model of interest.
[1564] The transgenic animals of the present invention can be used to refine the biological function of a KGF-2 polypeptide, to study conditions and / or diseases associated with abnormal KGF-2 expression, and to screen for compounds effective to ameliorate such conditions and / Including but not limited to animal model systems.
[1565] Example 47
[1566] KGF-2 knock-out animals
[1567] Endogenous KGF-2 gene expression has also been shown to be associated with targeted homologous recombination (e.g., Smithies et al., Nature 317: 230-234 (1985); Thomas & Capecchi, Cell 51: 503-512 (1987); Thompson et al. Cells 5: 313-321 (1989), each of which is incorporated herein by reference in its entirety) to knock out or "knock-out" the KGF-2 gene and / or its promoter. For example, a mutant non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to an endogenous polynucleotide sequence may be used in vivo with or without an optional marker and / Cells expressing the polypeptide of the present invention can be transfected. In other embodiments, techniques known in the art can be used to generate knockouts in cells that contain or do not express the gene of interest. Insertion of the DNA construct through targeted homologous recombination results in inactivation of the targeted gene. Such an approach is particularly suitable for research and agriculture fields that can produce animal offspring with inactive targeting genes using modifications to germline cells (e. G., Thomas & Capecchi 1987 and Thompson 1989, supra). However, this approach can be readily adapted to use in humans if the recombinant DNA is directly administered or targeted to the desired site in vivo using appropriate viral vectors to be readily apparent to those skilled in the art.
[1568] In a further embodiment of the invention, a cell genetically engineered to express a polypeptide of the invention, or a cell genetically engineered to not express a polypeptide of the invention (e.g., knockout) is administered to a patient in vivo. Such a cell may be obtained from a patient (e.g., an animal including a human) or an MHC-compatible donor and may include fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, epithelial cells, But is not limited thereto. The cell may be transfected or transfected (including, but not limited to, plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.) Using a recombinant DNA technique to introduce the coding sequence of the polypeptide of the invention into the cell by means of recombinant DNA techniques, or to destroy the coding sequence and / or the relevant endogenous control sequences of the polypeptide of the invention. It is totally manipulated. The coding sequence of the polypeptide of the present invention can be placed under the control of a strong constitutive or inducible promoter or promoter / enhancer to achieve expression and preferably secretion of the KGF-2 polypeptide. Engineered cells expressing and preferably secreted the polypeptides of the present invention may be introduced into a patient systemically, for example, in a circulatory system or intraperitoneally.
[1569] On the other hand, the cells can be integrated into the matrix and transplanted into the body. That is, genetically engineered fibroblasts can be implanted as part of a skin graft, and genetically engineered endothelial cells can be implanted as part of lymphocytes or blood vessel grafts (e.g., Anderson et al., U.S. Pat. No. 5,399,349; and Mulligan &Amp; Wilson, U. S. Patent No. 5,460, 959, each incorporated herein by reference).
[1570] If the cells to be administered are non-magnetic or non-MHC compatible cells, they may be administered using known techniques to prevent the development of a host immune response to the introduced cells. For example, the cells may be introduced in an encapsulated form, which allows for the exchange of components with the immediate neighboring extracellular environment, while not allowing the introduced cells to be recognized by the host immune system.
[1571] The knockout animal of the present invention is useful for use as an animal model system useful for refining the biological function of KGF-2 polypeptides, for studying conditions and / or diseases associated with abnormal KGF-2 expression, and for effective treatment of conditions and / The use of the compound in the search, and the like.
[1572] Example 48
[1573] Composition of the KGF-2 mutation
[1574] To generate point mutations, the following primers were used in a PCR reaction using standard conditions known in the art. The resulting product was digested with Nde and Asp718, cloned into pHE4, or cut with BamHI and Xba and then cloned into pcDNA3. Any of the described KGF-2 variants can be produced in other vectors or produced by themselves using known methods.
[1575] Primer sequence
[1576] pHE4: KGF2: R80-S208 was constructed using the following primers:
[1577] 5 'primer: CCGGC CATATG CGTAAACTGTTCTCTTTCACC (SEQ ID NO: 151)
[1578] 3 'primer: CCGGCGGTACCTTATTATGAGTGTACCACCATTGG (SEQ ID NO: 152)
[1579] pHE4: KGF2: A63-S208 (R68G) was constructed using the following primers:
[1580] 5 'primer: GATCGC CATATG GCTGGTCGTCACGTTCGTTC (SEQ ID NO: 153)
[1581] 3 'primer: GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO: 154)
[1582] pHE4: KGF2: A63-S208 (R68S) was constructed using the following primers:
[1583] 5 'primer: GATCGC CATATG GCTGGTCGTCACGTTCGTTC (SEQ ID NO: 155)
[1584] 3 'primer: GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO: 156)
[1585] pHE4: KGF2: A63-S208 (R68A) was constructed using the following primers:
[1586] 5 'primer: GATCGC CATATG GCTGGTCGTCACGTTCGTTC (SEQ ID NO: 157)
[1587] 3 'primer: GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO: 158)
[1588] pHE4: KGF2: A63-S208 (R78R80K81A) was constructed using the following primers:
[1589] 5 'primer: GATCGC CATATG GCTGGTCGTCACGTTCGTTC (SEQ ID NO: 159)
[1590] 3 'primer: GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO: 160)
[1591] pcDNA3: KGF2 (K136137139144A) was constructed using the following primers:
[1592] 5 'primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ ID NO: 161)
[1593] 3 'primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ ID NO: 162)
[1594] pcDNA3: KGF2 (K151153R155A) was constructed using the following primers:
[1595] 5 'primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ ID NO: 163)
[1596] 3 'primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ ID NO: 164)
[1597] pcDNA3: KGF2 (R174K183A) was constructed using the following primers:
[1598] 5 'primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ ID NO: 165)
[1599] 3 'primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ ID NO: 166)
[1600] pcDNA3: KGF2 (R187R188A) was constructed using the following primers:
[1601] 5 'primer: GATCGCGGATCCGCCACCATGTGGAAATGGATACTGACACATTGTGC (SEQ ID NO: 167)
[1602] 3 'primer: GATCGCTCTAGATTATGAGTGTACCACCATTGGAAGAAAG (SEQ ID NO: 168)
[1603] pHE4: KGF2.A63 (K136137139144A) was constructed using the following primers:
[1604] 5 'primer: GATCGCCATATGGCTGGTCGTCACGTTCGTTC (SEQ ID NO: 169)
[1605] 3 'primer: GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO: 170)
[1606] pHE4: KGF2.A63 (K151153R155A) was constructed using the following primers:
[1607] 5 'primer: GATCGCCATATGCGTGGTCGTCACGTTCGTTC (SEQ ID NO: 171)
[1608] 3 'primer: GATCGCGGTACCTTATTATGAGTGTACCACCATTGGAAG (SEQ ID NO: 172)
[1609] Example 49
[1610] Use of KGF-2 to treat and / or prevent infertility
[1611] Transplantation is the single most important factor in successful pregnancy and is clinically and economically important. In humans, the largest fraction of 70% loss in life occurs in transplants. Mice are a selective model for mammalian transplantation studies. Three essential cell lines differentiate and divide into peri-transplanted mouse embryos. Abdomen, placenta and yolk sac precursor. Fibroblast growth factor (FGF) -4 is essential for the development of all three cell lines.
[1612] Using the 'transient transgenic' approach to transfer of the FGF receptor gene, the endogenous FGF signaling can be used for both mouse embryo transfer and placental lineage starting at the fifth cell division 2 days before transplantation Which is essential for cell division of hepatocytes.
[1613] Interestingly, a null mutation of fgfr-2 and fgf4 was found to die in the uterus within one day after transplantation and ICM also died. Embryo transfer into uterine cells in the pancreas and placental line requires FGF to continue to proliferate.
[1614] It is possible that one or several of the other 19 FGF ligands are transiently expressed in the pre-mouse embryo and that the ligand delays the effect of the co-mutation of fgfr-2 and fgf4 after transplantation. Six FGF ligands were tested using RT-PCR. To date, besides FGF-4, KGF-2 and FGF-8 are the only FGF ligands that are detected in pre-transplant embryos. KGF-2 mRNA is detected in the embryo post-transplantation after two cell steps.
[1615] KGF-2 co-mutation suggests that KGF-2 is not essential for survival during the expression of KGF-2 in perry-transplanted mouse embryos (Min et al., 1998; Sekine et al., 1999). However, other FGF-based components may replace KGF-2 or become redundant during peri-embryo development. A number of extra genetic effects have been observed during the analysis of co-mutations in mice and compensation in the gene system has also been observed (Thomas et al., 1995: Stein et al., 1994). KGF-2 may be more important in early development than suggested by KGF-2 co-mutation.
[1616] The best way to detect whether KGF-2 has a role in early development at a time when a ball mutation suggests no essential function is to perform a function acquisition experiment. These experiments have shown that KGF-2 inhibits the growth of endoderm cells (Rappolee et al., 1994), blastocysts and growth (Chai et al., 1998) and internal callus cells et al., 1994) have an effect on placental / tropoblast cells. The loss-of-function test can be done in a limited manner by using antisense oligonucleotides (Rappolee et al., 1992) or by blocking antibodies (LaFleur et al., 1996). It is known that the size of the embryos soon after transplantation, and the large positive and negative changes in cell number are always regulated in vivo (Rappolee, 1998). Suggesting that the small and less deadly KGF-2-dependent effect may be completely lost in the KGF-2 co-mutation. Functional loss and acquisition experiments are used to test periplanted mouse embryos for the effects of KGF-2.
[1617] To date, the detection of mRNA for growth factors in peritriated mouse embryos has led to the detection of corresponding proteins (Rappolee et al., 1998, 1992, 1994, organized in Rappolee 1998, 1999). Use a KGF-2 antibody that is suitable for immunocytochemistry to determine if KGF-2 protein is present (and where) in the vessel where KGF-2 mRNA is detected.
[1618] Example 50
[1619] Detection of KGF-2 in clinical samples
[1620] Purified chlorine PAb is diluted to 2 μg / ml in coating buffer (0.05 M NaHCO ₃ , Ph 9.5). 100 희 of diluted antibody is added per well of Immuno 4 microplate wells. Store the microplate at 4 ° C overnight. Discard the antibody solution from the plate. 200 [mu] l of blocking buffer (1% dry milk (BioRad) in coating buffer) is added to each well. Plates are incubated at room temperature for 2 hours. Discard the blocking buffer from the plate. The plate is vacuumed and completely dried at 32 [deg.] C for 1.5 hours in a vacuum chamber. The plate is removed from the vacuum chamber and sealed in a mylar pouch with three desiccant packs. Store plate at 4 ° C until use.
[1621] KGF-2 was diluted to 16 ng / ml with diluent 1 (0.1% Tween 20, 1XPBS, 1% BSA, and 0.001% Thimerosal) and subsequently diluted 2.5x consecutively for the next 7 dilutions. A concentration range of 16 ng / ml to 0.026 ng / ml is used as a standard. The background wells are made only of diluent without protein.
[1622] Unknown samples are diluted 10x, 50x, and 250x with diluent 1. The serial dilution standard solution and the unknown sample are added to the wells in an ELISA plate coated with 100 占 퐇 each. Plates are stored overnight at 4 ° C. Discard the solution from the plate. The plates are washed five times with wash buffer (0.1% Tween 20 and 1XPBS) using a Wheaton Instrument fixed at 1.6 ml. Between each wash, incubate for 15 seconds with wash buffer.
[1623] Dilute the biotinylated chicken anti-KGF-2 as diluent 1 to 0.5 μg / ml. 100 [mu] l of the diluted detector is added to each well. Plates are incubated at room temperature for 2 hours. Discard the solution and wash the plate 5 times with wash buffer as before. Between each wash, incubate for 15 seconds with wash buffer.
[1624] Peroxidase streptavidin is diluted 1: 2000 in diluent 1. Diluted peroxidase streptavidin is added to the plate in an amount of 100 μl per well and incubated at room temperature for 1 hour. Discard along the plate and wash 5 times with wash buffer. Between each wash, incubate for 15 seconds with wash buffer. Do not dry the plate.
[1625] An equivalent amount of a room temperature TMB peroxidase substrate and peroxidase solution B (TMB peroxidase microwell substrate system, KPL) are mixed. 100 혼합 of the mixed solution was added to each well and developed at room temperature for 10 minutes. 50 μl of 1 M H ₂ SO ₄ was added to each well to stop color development. Plates are read at 450 nm.
[1626] Example 51
[1627] Composition of E. coli optimized cut KGF-2
[1628] In order to increase the degree of expression of truncated KGF-2 in this E. coli expression system, the gene codon was optimized for the highly-used E. coli codon.
[1629] For example, the following construct called pHE4: KGF-2.A63-S608 was made.
[1630] 5 'CATATGGCTGGTCGTCACGTTCGTTCTTACAACCACCTGCAGGGT
[1631] GACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAA
[1632] AATCGAAAAAAACGGTAAAGTTTCTGGGACCAAGAAGGAGAACTG
[1633] CCCGTACAGCATCCTGGAGATAACATCAGTAGAAATCGGAGTTGTT
[1634] GCCGTCAAAGCCATTAACAGCAACTATTACTTAGCCATGAACAAGA
[1635] AGGGGAAACTCTATGGCTCAAAAGAATTTAACAATGACTGTAAGCT
[1636] GAAGGAGAGGATAGAGGAAAATGGATACAATACCTATGCATCATT
[1637] TAACTGGCAGCATAATGGGAGGCAAATGTATGTGGCATTGAATGG
[1638] AAAAGGAGCTCCAAGGAGAGGACAGAAAACACGAAGGAAAAACA
[1639] CCTCTGCTCACTTTCTTCCAATGGTGGTACACTCATAATAAGGTACC
[1640] 3 '(SEQ ID NO: 173)
[1641] A plasmid containing the cDNA having the nucleotide sequence of SEQ ID NO: 173 was deposited on July 3, 2000 with ATCC accession number Virginia, 20110-2209 Manassas University Boulevard 10801 ATCC Patent Depository.
[1642] Another construct called pHE4: KGF-2.A63-S208 cod.opt was constructed using the following primers:
[1643] Sense 5 'GACTACATATGGCTGGTCGTCACGTTCGTTCTTACAACCACCTGCA GG 3' (SEQ ID NO: 174)
[1644] Antisense 5 'CTAGTCTCTAGATTATGAGTGTACAACCATCGGCAGGAAGTGAG 3' (SEQ ID NO: 175)
[1645] The nucleotide sequence of pHE4: KGF-2.A63-208 cod.opt is as follows:
[1646] 5 'ATGGCTGGTCGTCACGTTCGTTCTTACAACCACCTGCAGGGTGACGTTCGTTGGCGTAAACTGTTCTCTTTCACCAAATACTTCCTGAAAATCGAAAAGAACGGTAAAGTTTCTGGTACCAAGAAAGAAAACTGCCCGTACTCTATCCTGGAAATCACCTCCGTTGAAATCGGTGTTGTAGCCGTTAAAGCCATCAACTCCAACTATTACCTGGCCATGAACAAAAAGGGTAAACTGTACGGCTCTAAAGAATTCAACAACGACTGCAAACTGAAAGAACGTATCGAAGAGAACGGTTACAACACCTACGCATCCTTCAACTGGCAGCACAACGGTCGTCAGATGTACGTTGCACTGAACGGTAAAGGCGCTCCGCGTCGCGGTCAGAAAACCCGTCGCAAAAACACCTCTGCTCACTTCCTGCCGATGGTTGTACACTCATAATAA 3' (SEQ ID NO: 176)
[1647] A plasmid containing the cDNA having the nucleotide sequence of SEQ ID NO: 176 was deposited on July 3, 2000 with the ATCC Accession No., Virginia, 20110-2209 Manasas University Boulevard 10801 ATCC Patent Depository.
[1648] Both structures disclosed in this example are useful for producing KGF-2 polypeptides, for example as described in Example 13. Nucleotides 4 to 444 of SEQ ID NO: 173 and Nucleotides 1 to 441 of SEQ ID NO: 176 encode amino acids 63 to 208 of SEQ ID NO: 2 and N-terminal methionine.
[1649] It will be apparent that the invention may be practiced otherwise than as specifically described in the foregoing description and examples.
[1650] Many modifications and variations of the present invention will be apparent to those skilled in the art from the foregoing description, and the invention may be practiced otherwise than as specifically described within the scope of the appended claims.
[1651] The entire disclosure of all documents (patents, patent applications, journal articles, experimental methods, books, or other documents) cited herein are hereby incorporated by reference.
[1652] Certificate of Microorganism Deposit (PCT Rule 13bis)
[1653] A. The following certificate relates to the microorganism mentioned on line 6 on page 5 of the specificationB. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: December 16, 1994Accession number: ATCC 75977 C. Additional proof (blank if not necessary) This information will continue on the attached sheet. DNA plasmid 366885A D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates listed below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For officeInternational Office This paper has been repaired with the international applicationThis paper has been repaired by the international collection office. AuthorizedAuthorized
[1654] Certificate of Microorganism Deposit (PCT Rule 13bis)
[1655] A. The following certificate relates to the microorganism mentioned on line 6 on page 26 of the specificationB. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: September 29, 1994Accession number: ATCC 75901 C. Additional proof (blank if not necessary) This information will continue on the attached sheet. DNA plasmids 366,885 D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For officeInternational Office This paper has been repaired with the international applicationThis paper has been repaired by the international collection office. AuthorizedAuthorized
[1656] Certificate of Microorganism Deposit (PCT Rule 13bis)
[1657] A. The following certificate relates to the microorganisms mentioned on the fifth line of the description on page 489B. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: June 30, 1999Accession number: ATCC PTA-290 C. Additional proof (blank if not necessary) This information will continue on the attached sheet. DNA plasmid pVGI-0: KGF2 (F.L.) (Ref.PF155) D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For officeInternational Office This paper has been repaired with the international applicationThis paper has been repaired by the international collection office. AuthorizedAuthorized
[1658] Certificate of Microorganism Deposit (PCT Rule 13bis)
[1659] A. The following certificate relates to the microorganism mentioned on line 12 on page 489 of the specificationB. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: June 30, 1999Accession number: ATCC PTA-289 C. Additional proof (blank if not necessary) This information will continue on the attached sheet. DNA plasmid pVGI-0: 33 KGF2 (Ref.PF155) D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For officeInternational Office This paper has been repaired with the international applicationThis paper has been repaired by the international collection office. AuthorizedAuthorized
[1660] Certificate of Microorganism Deposit (PCT Rule 13bis)
[1661] A. The following certificate relates to the microorganism mentioned on line 20 of the specification on page 187B. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: 1998. 1. 9Accession number: ATCC 209575 C. Additional proof (blank if not necessary) This information will continue on the attached sheet. DNA plasmid pHEKGF-2delta33 D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For officeInternational Office This paper has been repaired with the international applicationThis paper has been repaired by the international collection office. AuthorizedAuthorized
[1662] Certificate of Microorganism Deposit (PCT Rule 13bis)
[1663] A. The following certificate relates to the microorganism mentioned on line 19 on page 504 of the specificationB. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: July 3, 2000Accession number: Will be notified C. Additional proof (blank if not necessary) This information will continue on the attached sheet. (Will be notified) D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For officeInternational Office This paper has been repaired with the international applicationThis paper has been repaired by the international collection office. AuthorizedAuthorized
[1664] Certificate of Microorganism Deposit (PCT Rule 13bis)
[1665] A. The following certificate relates to the microorganism mentioned on line 17 on page 505 of the specificationB. Proof of Deposits Additional deposits are identified on the attached sheet. Name of the Depositary: American Bacterium Culture Collection Address (including postal code and country) of depositary institution: Virginia, USA 20110-2209 Manassas University Seat Bollevard 10801 Date of deposit: July 3, 2000Accession number: Will be notified C. Additional proof (blank if not necessary) This information will continue on the attached sheet. (Will be notified) D. Designation of the country requiring proof (if this proof is not for all designated States) E. Provide a separate certificate (blank if not necessary) The certificates presented below will then be submitted to the International Bureau (specify the general type of certificate, eg "Trust Number of Deposit") For officeInternational Office This paper has been repaired with the international applicationThis paper has been repaired by the international collection office. AuthorizedAuthorized

权利要求:
Claims (12)
[1" claim-type="Currently amended] A polynucleotide comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide is selected from the group consisting of R68G, R68S, R68A, R78A, R80A, K81A, K87A, K91A, K136A, K137A, K139A, K144A, K148E, K149E, K151A, K153A, K155A, Positive charge residues substituted with alanine therebetween including R174A, K183A, K183Q, K183E, R187A, R188A, R188E, K191E, R68 to K91, positively charged residues substituted with intervening neutral residues including R68 to K91, 2. A polynucleotide identical to the reference polypeptide of SEQ ID NO: 2 except for one or more mutations selected from the group consisting of positively charged residues substituted with negative charge residues therebetween including K91.
[2" claim-type="Currently amended] The polynucleotide of claim 1, wherein said reference polypeptide comprises amino acids 63 to 208, 69 to 208, 77 to 208, 80 to 208, or 93 to 208 of SEQ ID NO: 2.
[3" claim-type="Currently amended] A vector comprising the polynucleotide of claim 1.
[4" claim-type="Currently amended] A host cell comprising the polynucleotide of claim 1.
[5" claim-type="Currently amended] Culturing the host cell of claim 4 under conditions that allow the polypeptide to be expressed and recovering the polypeptide.
[6" claim-type="Currently amended] A polypeptide encoded by the polynucleotide of claim 1.
[7" claim-type="Currently amended] A method of stimulating epithelial cell proliferation in a patient comprising administering to the patient a polypeptide of claim 6.
[8" claim-type="Currently amended] The method of claim 7, wherein the patient is selected from the group consisting of a wound, mucositis, ulcer, inflammatory bowel disease, liver disease, lung injury, diabetes, oral injury, gastrointestinal injury, intestinal toxicity, vesicular epidermolysis, skin graft, Wherein the disease is selected from the group consisting of inflammation, injury, breast tissue damage, uterine damage, female reproductive tract disease, intestinal fibrosis, rectalitis, pulmonary fibrosis, pneumonia, pleural contraction, hemophilia, and bone marrow toxicity.
[9" claim-type="Currently amended] A method for treating or preventing ovarian damage, infertility, or hepatic fibrosis in a patient comprising administering to the patient an effective amount of a polypeptide of SEQ ID NO: 2, or an active fragment or variant thereof.
[10" claim-type="Currently amended] A method of promoting healing of an incision wound created during internal healing, donor site healing, internal surgical wound healing, or cosmetic surgery in a patient comprising administering to the patient an effective amount of the polypeptide of SEQ ID NO: 2 or an active fragment or variant thereof.
[11" claim-type="Currently amended] (a) inserting in the vector a polynucleotide encoding amino acids 63 to 208;
(b) transfecting said vector into a host cell;
(c) culturing the host cell under conditions in which the polypeptide is expressed;
(d) degrading said cells in the presence of guanidine hydrochloride; And
(e) recovering the polypeptide.
[12" claim-type="Currently amended] A polynucleotide comprising nucleotides 4 to 444 of SEQ ID NO: 173 or 1 to 441 of SEQ ID NO: 176.

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---

公开号 | 公开日

---

CA2399045A1|2001-01-11|

---

WO2001002433A1|2001-01-11|

---

AU5911700A|2001-01-22|

---

JP2003520572A|2003-07-08|

---

NZ516897A|2004-01-30|

---

EP1196441A1|2002-04-17|

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MXPA02000152A|2002-07-30|

---

CN1372569A|2002-10-02|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
1999-07-02|Priority to US14234399P

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1999-07-02|Priority to US60/142,343

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1999-07-14|Priority to US14364899P

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1999-07-14|Priority to US60/143,648

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1999-07-15|Priority to US60/144,024

---

1999-07-15|Priority to US14402499P

---

1999-08-12|Priority to US14862899P

---

1999-08-12|Priority to US60/148,628

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1999-08-19|Priority to US14993599P

---

1999-08-19|Priority to US60/149,935

---

1999-09-09|Priority to US????

---

1999-11-03|Priority to US60/163,375

---

1999-11-03|Priority to US16337599P

---

1999-12-22|Priority to US60/171,677

---

1999-12-22|Priority to US17167799P

---

2000-04-19|Priority to US60/198,322

---

2000-04-19|Priority to US19832200P

---

2000-05-19|Priority to US60/205,417

---

2000-05-19|Priority to US20541700P

---

2000-06-30|Priority to US09/610,651

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2000-07-03|Application filed by 추후제출, 휴먼 게놈 사이언시즈, 인크.

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2000-07-03|Priority to PCT/US2000/018328

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2002-04-10|Publication of KR20020026517A

优先权:

---

申请号 | 申请日 | 专利标题

---

US14234399P| true| 1999-07-02|1999-07-02|

---

US60/142,343|1999-07-02|

---

US14364899P| true| 1999-07-14|1999-07-14|

---

US60/143,648|1999-07-14|

---

US14402499P| true| 1999-07-15|1999-07-15|

---

US60/144,024|1999-07-15|

---

US14862899P| true| 1999-08-12|1999-08-12|

---

US60/148,628|1999-08-12|

---

US14993599P| true| 1999-08-19|1999-08-19|

---

US60/149,935|1999-08-19|

---

US????|1999-09-09|

---

US16337599P| true| 1999-11-03|1999-11-03|

---

US60/163,375|1999-11-03|

---

US17167799P| true| 1999-12-22|1999-12-22|

---

US60/171,677|1999-12-22|

---

US19832200P| true| 2000-04-19|2000-04-19|

---

US60/198,322|2000-04-19|

---

US20541700P| true| 2000-05-19|2000-05-19|

---

US60/205,417|2000-05-19|

---

US09/610,651|US6693077B1|1995-02-14|2000-06-30|Keratinocyte growth factor-2|

---

PCT/US2000/018328|WO2001002433A1|1999-07-02|2000-07-03|Keratinocyte growth factor-2|