Secreted and Transmembrane Polypeptide and Nucleic Acids Encoding the Same

专利摘要:
The present invention relates to novel polypeptides and nucleic acid molecules that encode the polypeptides. Also included herein are vectors and host cells comprising the nucleic acid sequences, chimeric polypeptide molecules comprising a polypeptide of the invention fused to a heterologous polypeptide sequence, antibodies that bind to the polypeptides of the invention, and methods of producing the polypeptides of the invention / RTI >
公开号:KR20010087359A
申请号:KR1020017003385
申请日:1999-09-15
公开日:2001-09-15
发明作者:지안 첸；오드리 고다드；오스틴 엘. 거니；켄네쓰 힐란；다이안 페니카；윌리엄 아이. 우드；진 유안
申请人:제넨테크, 인크.；
IPC主号:

专利说明:

Secreted and Transmembrane Polypeptides and Nucleic Acids Encoding the Same < RTI ID = 0.0 >
[2] Extracellular proteins play an important role in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, such as proliferation, migration, differentiation, or interaction with other cells, is typically dictated by information received from other cells and / or adjacent environments. Often, this information is transmitted by secreted polypeptides (e. G., Mitogen-promoting factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides and hormones) and is accommodated by various cell receptors or membrane- do. These secreted polypeptides or signaling molecules typically reach their action site in the extracellular environment through the secretory pathway of the cell.
[3] Secretory proteins have a variety of industrial applications, including drugs, diagnostics, biosensors and bioreactors. Most protein drugs currently available, such as thrombolytic agents, interferons, interleukins, erythropoietins, colony stimulating factors and various other cytokines, are secreted proteins. In addition, membrane proteins that are their receptors also have potential as therapeutic or diagnostic agents. Efforts to find new natural secreted proteins are being made by both industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to find the coding sequence of the novel secreted protein. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93: 7108-7113 (1996) and U.S. Patent No. 5,536,637).
[4] Membrane binding proteins and receptors may play an important role, among other things, in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, such as proliferation, migration, differentiation, or interaction with other cells, is typically dictated by information received from other cells and / or adjacent environments. Often, this information is transmitted by secreted polypeptides (e. G., Mitogen-promoting factors, survival factors, cytotoxic factors, differentiation factors, neuropeptides and hormones) and is accommodated by various cell receptors or membrane- do. Such membrane binding proteins and cell receptors include, but are not limited to, cytokine receptors, receptor kinases, receptor phosphatases, receptors involved in cell-cell interactions, and cell adhesin molecules such as selectins and integrins. For example, the introduction of signals that regulate cell growth and differentiation is partially regulated by phosphorylation of several cellular proteins. Protein tyrosine kinase, an enzyme that promotes this process, may act as a growth factor receptor. Examples include fibroblast growth factor receptor and nerve growth factor receptor.
[5] Membrane binding proteins and receptor molecules have a variety of industrial uses including drugs and diagnostic agents. For example, receptor mumonoadhesins can be used as therapeutic agents to block receptor-ligand interactions. In addition, membrane bound proteins may be used to screen potential peptides or small molecule inhibitors for the relevant receptor / ligand interactions.
[6] Efforts to identify new natural receptors or membrane binding proteins have been made by both industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to find coding sequences for novel receptors or membrane binding proteins.
[7] 1. PRO211 and PRO217
[8] Epidermal growth factor (EGF) is a common mitogen-promoting factor that promotes the proliferation of various types of cells, including epithelial cells and fibroblasts. EGF binds to and activates the EGF receptor (EGFR), which initiates intracellular signaling and results accordingly. EGFR is expressed not only in neurons in the cerebral cortex, cerebellum and hippocampus, but also in other areas of the central nervous system (CNS). EGF is also expressed in many other areas of the CNS. Thus, EGF acts not only on mitotic cells but also on neurons after mitosis. Indeed, many studies have shown that EGF plays a neurotrophic or neuronal regulatory role in several types of neurons in the CNS. For example, EGF acts directly on cultured cortical neurons and cerebellar neurons, thereby enhancing neurite outgrowth and survival. EGF, on the other hand, acts indirectly through neurons in other types of cells, including cholinergic neurons in the septum and dopaminergic neurons in the midbrain. Evidence for the action of EGF on neurons in the CNS has been accumulated, but the mechanism of action is inherently unknown. EGF-induced signal transduction in mitotic cells is more known than neurons after mitosis. Studies on cloned chromaffinocytoma PC12 cells and cultured cerebral cortex neurons suggest that EGF-induced neuronal nutrition is mediated by continued activation of EGFR and mitogen-activated protein kinase (MAPK) in response to EGF do. Continuous intracellular signaling is correlated with decreased EGFR down-regulation rate, which determines the response of neurons to EGF. EGF is considered to be a potent multiple growth factor acting on many types of cells, including mitotic cells and mitotic post-neurons.
[9] EGF is produced by salivary glands and gastrointestinal Brunner's glands, kidneys, pancreas, thyroid gland, pituitary gland and nervous system, and is used for body fluid such as saliva, blood, CSF, urine, amniotic fluid, prostate fluid, (Plata-Salaman, Peptide 12 : 653-663 (1991)).
[10] EGF is delivered by membrane-specific receptors including native tyrosine kinases (Stoscheck et al., J. Cell Biochem. 31: 135-152 (1986)]. EGF is believed to function to induce transmembrane signals and to activate intrinsic tyrosine kinases by binding to the extracellular portion of the receptor.
[11] Purification and sequencing of the EGF-like domain revealed the presence of six conserved cysteine residues that cross-linked to form three peptide loops (Savage et al., J. Biol. Chem. 248: 7669-7672 (1979)]. At present, it is common that the different generalized motifs X _n CX ₇ CX _4/5 CX ₁₀ CXCX ₅ GX ₂ CX _n (where X is any amino acid other than cysteine and n is a variable number of repeats) It is known to be able to react with a shared EGF receptor. Unsolicited peptides with such motifs include TGF- alpha, ampirirgulin, schwanoma-derived growth factor (SDGF), heparin-binding EGF-like growth factor and peptides that are coded for by certain viruses , Vaccinia virus (Reisner, Nature 313 : 801-803 (1985)), Shope fibrosis virus (Chang et al., Mol Cell Biol. 7: 535-540 (1987)], Molluscum contagiosum (Porter and Archard [J. Gen. Virol. 68 : 673-682 (1987)] and myxoma virus (Upton et al., J. Virol. 61 : 1271-1275 (1987) and Prigent and Lemoine, Prog. Growth Factor Res. 4 : 1-24 (1992)].
[12] EGF-like domains are not limited to growth factors but are also observed in a variety of cell-surface proteins and extracellular proteins with interesting characteristics in cell attachment, protein-protein interaction and growth (Laurence and Gustuson Gusterson, Tumor Biol. 11 : 229-261 (1990)]. These proteins include blood coagulation factors (Factors VI, IX, X, XII, Protein C, Protein S, Protein Z, Tissue plasminogen activator and urokinase), extracellular components (laminin, cytotaxin and enstatin) (LDL receptor and thrombomodulin receptor) and immuno-related proteins (complement C1r and euromodulin).
[13] More interestingly, the general structural pattern of EGF-like precursors is conserved throughout lower organisms and mammalian cells. A number of genes important for growth in invertebrates have been identified through EGF-like repeating structures. For example, the notch gene of Drosophila encodes 36 repeats in which 40 amino acids homologous to EGF are arranged in a row (Wharton et al., Cell 43 : 557-581 1985). The hydrocapsplot represents an estimated membrane-bound domain, in which the EGF-related sequence is located on the extracellular side of the membrane. Other homeotropic genes with EGF-like repeats include Delta, 95F and 5ZD identified with probes constructed based on the Notch gene, and receptors coding for receptors for growth signals transferred between two specific cells Lin-12, a nematode gene, is included.
[14] In particular, EGF has been implicated in the pathogenesis of necrotizing enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulcers and congenital microvilli atrophy (Guglietta and Sullivan, Eur. J. Gastroenterol Hepatol, 7 (10), 945-50 (1995)], as well as for the preservation and maintenance of gastrointestinal mucosa and for the repair of acute and chronic mucosal damage (Konturek et al. Eur.J.Gastroenterol Hepatol. 7 (10), 933-37 (1995)]. In addition, EGF is expressed in hair follicle differentiation [du Cros, J. Invest. Dermatol. 101 (1 Suppl.), 106S-113S (1993)], Hillier, Clin. Endocrinol. 33 (4), 427-28 (1990)], kidney function (Hamm et al., Semin. Nephrol. 13 (1): 109-15 (1993)], Harris, Am. J. Keyney Dis. 17 (6): 627-30 (1991)]], leakage [Bain Setten et al. Int. Ophthalmol 15 (6) 359-62 (1991)]], blood coagulation [Stenflo et al., Blood 78 (7): 1637-51 (1991)]. In addition, EGF has been implicated in a variety of skin diseases characterized by abnormal keratinocyte differentiation, such as squamous cell carcinoma of the psoriasis and lungs, and epithelial cancers such as epidermal carcinoma of the vulva and glioma (King et al. Am. J. Med. Sci. 296 : 154-158 (1998)].
[15] It is very important to find evidence that genetic changes in the signaling pathways of growth factors are closely related to developmental disorders and chronic diseases, including cancer (Aaronson, Science 254 : 1146-1153 (1991) ]]. For example, c-erb-2 (also known as HER-2), a proto-oncogene with close structural similarity to the EGF receptor protein, is overexpressed in human breast cancer (King et al. [Science 229 : 974-976 (1985)], Gullick's Hormones and their actions, and Cooke et al., Eds, Amsterdam, Elsvier, pp 349-360 (1986)].
[16] Herein we describe the identification and characterization of novel polypeptides (referred to herein as PRO211 and PRO217) that are homologous to EGF.
[17] 2. PRO230
[18] Nephritis is a symptom characterized by kidney inflammation that affects the structure and normal function of the kidney. These symptoms can be chronic or acute and are usually caused by infection, degenerative processes or vascular disease. In all cases, it is desirable that early detection and treatment of patients with nephritis can begin.
[19] One way to detect nephritis is to determine antigens and their antibodies that are associated with nephritis. (TIN-ag) of rabbit is recorded in Nelson, TR et al., J. Biol. Chem., 270 (27): 16265-70 (July 1995) (GENBANK / U24270) . In this study, we found that rabbit TIN-ag is a basement membrane glycoprotein with a putative amino acid sequence with a carboxy-terminal region that is 30% homologous to human preprokeaptinsin B, a member of the cysteine protease protein family. It is also possible that the rabbit TIN-ag comprises a domain comprising epidermal growth factor-like motifs that share homologies with laminin A and S chains, alpha 1 chain of type I collagen, von Willebrand's factor and mucin, End region, indicating structure and functional similarity. Studies were also performed in mice. However, it is desirable to find a human tubulointerstitial nephritis antigen to aid early detection of nephritis and improvement in treatment.
[20] Proteins that are homologous to tubular interstitial nephritis antigens are of particular interest in the medical and industrial sectors. Often, homologous proteins have similar functions. It is also important that homologous proteins do not have similar functions, which means that certain structural motifs represent information other than function, such as direction of function. Herein, the inventors describe the identification and characterization of novel polypeptides (referred to herein as PRO230) that are homologous to the tubular interstitial nephritis antigen.
[21] 3. PRO232
[22] Hepatocytes can be divided into three groups: (a) proliferation, (b) self-maintenance, (c) production of offspring of multiple differentiated functional tissues, (d) regenerative capacity of tissue after injury, and / Is an undifferentiated cell. Often, hepatocytes provide a method of identifying and isolating specific hepatocyte populations by expressing cell surface antigens that can serve as cell specific markers for identifying hepatocytes.
[23] If you have several hepatocyte populations, there will be many important uses. For example, identifying specific hepatocyte populations may identify growth factors and other proteins involved in their proliferation and differentiation. In addition, there are still unknown proteins involved in (1) the initial stage of hepatocyte to a specific lineage, (2) prevention of this process, and (3) reverse regulation of hepatocyte proliferation. . In addition, hepatocytes are an important and ideal target for gene therapy, and promote the health of individuals transplanted with hepatocytes through the inserted gene. Finally, hepatocytes can play an important role in transplantation of tissues or organs, such as liver regeneration and skin transplantation.
[24] Given the importance of hepatocytes in many different applications, both the industry and academia are now looking for new ways to provide specific cell surface markers that identify hepatocyte populations, as well as awareness of the function of hepatocyte antigens in cell proliferation and differentiation. Efforts are being made to find natural hepatocyte antigen proteins. Herein, the inventors describe the identification and characterization of novel polypeptides (referred to herein as PRO232 polypeptides) that are homologous to hepatocyte antigens.
[25] 4. PRO187
[26] Growth factors are molecular signals or mediators that bind to specific cell surface receptors alone or in combination to augment cell growth or proliferation. However, it is involved in not only growth but also other cell reactions after being expressed as a growth factor. As a result, growth factors are more likely to be multifunctional and potent cell regulators. His biological effects include the promotion of proliferation, chemotaxis and extracellular matrix production. Growth factors can both stimulate and inhibit. For example, modification of the growth factor (TGF-beta) results in highly multifunctional expression, which promotes proliferation in certain cells, particularly connective tissue, but strongly inhibits proliferation in other cells such as lymphocytes and epithelial cells.
[27] The physiological effects of growth promotion or inhibition by growth factors depend on the development and differentiation state of the target tissue. Local mechanisms of cellular regulation by typical endocrine molecules are involved in understanding autocrine, juxtacrine, neuronal, and paracrine (near-cell) pathways. Peptide growth factors are elements made up of complex biological languages and provide the basis for liaison between cells. This factor allows information to be transmitted between cells, mediates interactions between cells, and changes gene expression. The effect of these multifunctional and versatile factors will depend on the presence or absence of other peptides.
[28] FGF-8 is a member of fibroblast growth factor (FGF), a group of heparin-binding strong mitogens for both normal diploid fibroblasts and established cell lines (Gospodarowicz et al., 1984 ), Proc. Natl. Acad. Sci. USA 81 : 6963]. The FGF family includes, among others, acidic FGF (FGF-1), basic FGF (FGF-2), INT-2 (FGF-3), K-FGF / HST (FGF-4), FGF-5, FGF- FGF-7) and AIGF (FGF-8). All FGFs have two conserved cysteine residues and share 30-50% sequence homology at the amino acid level. These factors include granulosa cells, adrenocortical cells, chondrocytes, myoblasts, corneas and vascular endothelial cells (bovine or human), vascular smooth muscle cells, lens, retina and prostate epithelial cells, rare proximal glial cells, Is a cleavage promoter for a wide variety of normal diploid mesenchymal-derived and neural crest-derived cells, including myoblasts and osteoblasts.
[29] Fibroblast growth factors may also promote many types of cells in a different way than promoting mitosis. These activities include the promotion of cell migration (chemotaxis) to the wound site, the initiation of angiogenesis (angiogenesis), the regulation of neurotrophism (neuronal tendency), the regulation of endocrine function, Promoting or inhibiting protein expression, extracellular matrix production and cell survival (Baird and Bohlen, Handbook of Exp. Pharmacol. 95 (1): 369-418, Springer, (1990)]. These properties provide the basis for the use of fibroblast growth factors in therapeutic studies to promote wound healing, nerve regeneration, and collateral vessel formation. For example, fibroblast growth factor is recommended for minimizing myocardial damage in heart disease and surgery [US Pat. No. 4,378,347].
[30] FGF-8 is also known as the androgen-inducible growth factor (AIGF) and is an amino acid 215 protein that shares 30-40% sequence homology with other members of the FGF family. FGF-8 has been proposed to be under the control and induction of androgen in the mouse breast cancer cell line SC3 (Tanaka et al., Proc. Natl. Acad. Sci. USA 89 : 8928-8932 (1992) Sato et al., J. Steroid Biochem. Mol. Biol. 47 : 91-98 (1993)]. As a result, FGF-8 can act locally on a prostate known to be an androgen-reactive organ. In addition, FGF-8 may be tumorigenic as it exhibits active modification when transfected into NIH-3T3 fibroblasts (Kouhara et al., Oncogene 9 455-462 (1994)]. FGF-8 is detected in the heart, brain, lung, kidney, testes, prostate, and ovaries, and expression has been confirmed in the absence of exogenous androgens (Schmitt et al., J. Steroid Biochem. Mol. Biol. 57 (3-4): 173-78 (1996)].
[31] FGF-8 shares characteristics with several other FGFs expressed at different stages of development of ruminant embryos, supporting the theory that various FGFs play various, perhaps equally, roles in differentiation and embryogenesis. In addition, FGF-8 was confirmed to be a primary tumor gene cooperating with Wnt-1 in breast tumorigenesis (Shackleford et al., Proc. Natl. Acad. Sci. USA 90 , 740-744 ) And Heikinheimo et al., Mech. 48 : 129-138 (1994)).
[32] Unlike other FGFs, FGF-8 is present as three protein isoforms as a result of selective splicing of primary transcripts (Tanaka et al., Supra). Although normal FGF-8 expression is weak and confined to gonadal tissues, northern blot analysis revealed that FGF-8 mRNA was present for 10 to 12 days after gestation in rats and mice, (Heikinheimo et al., Mech. Dev. 48 (2): 129-138 (1994)). In situ hybridization between 8th and 16th postpartum was also observed in the first bronchial arch, frontal boss, frontal and midbrain-hindbrain junctions. On days 10-12, FGF-8 is expressed in the auricular processes of the forelegs and hind legs, the bovine and nasal pharynx, and the surface ectoderm of the dorsal, and capillaries, ganglia and hindbrain. Expression continues in the hind limb that develops for 13 days after pregnancy, but can not be detected thereafter. These results suggest that FGF-8 has a unique time and spatial pattern in embryogenesis, and suggests the role of the growth factor in various regions of exocrine differentiation of proliferating-posterior embryos.
[33] Herein, the inventors describe the identification of novel polypeptides (referred to herein as PRO187 polypeptides) that are homologous to FGF-8.
[34] 5. PRO265
[35] Protein-protein interactions include receptor-antigen complexes and signal transduction mechanisms. As is well known for the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be readily manipulated to control the specific consequences of their protein-protein interactions. Thus, the fundamental mechanisms of protein-protein interactions are of interest to the scientific and medical community.
[36] All proteins, including the lozengine repeat, are thought to be involved in protein-protein interactions. Lutein-rich repeats are short sequence motifs present in many proteins that differ in function and intracellular location. The crystal structure of the ribonuclease inhibitor protein reveals that the lozenge-rich repeats correspond to the beta-alpha structural units. These units form a parallel beta sheet with one side exposed to the solvent, resulting in a unique non-spherical shape. These two features have been found to be responsible for the protein-binding function of the protein, including the lozengine repeat. In this regard, Kobe and Deisenhofer, Trends Biochem. Sci. , 19 (10): 415-421 (Oct. 1994).
[37] According to one study report, the rich abundance of lutein proteoglycans functions as a tissue-forming body that directs and aligns collagen fibers during the development of an individual, and is involved in pathological processes such as wound healing, tissue repair, and tumor lipid formation [ Iozzo, RV, Crit. Rev. Biochem. Mol. Biol ., 32 (2): 141-174 (1997)). Other studies showing that a rich protein of lewis is involved in wound healing and tissue repair include De La Salle, who reported that a mutant lutein-rich motif in a complex related to the bleeding disorder Bernard-Soulier syndrome was mutated, C., et al., Vouv. Rev. Fr. Hematol . (Germany), 37 (4): 215-222 (1995)] and Chlemetson (KJ) Thromb. Haemost . (Germany), 74 (1): 111-116 (July 1995). Another particularly interesting protein reported to have a leucine rich repeats is the SLIT protein which has been reported to be useful in the treatment of neurodegenerative diseases such as Alzheimer's disease and neuronal damage such as in Parkinson's disease and in the diagnosis of cancer [ Artavanistsakonas, S. and Rothberg, JM, WO9210518-A1, Yale University]. Other reports on the biological function of proteins with lozengesic repeats include Tayar, N. et al., Mol. Cell Endocrinol. (Ireland), 125 (1-2): 65-70 (Dec. 1996)] (related to the gonadotropin receptor), Miura, Y. et al., Nippon Rinsho ): 1784-1789 (July 1996)] (related to apoptosis), Harris (PC) et al ., J. Am. Soc. Nephrol ., 6 (4): 1125-1133 (Oct. 1995)] (related to nephropathy) and degenerative growth factor β binding to the Lazolas Cancer Research Foundation reported to be involved in cancer treatment, wound healing and scar formation And WO 9110727A from Ruoslahti (EI) of La Jolla Cancer Research Foundation. It is also particularly interesting to use fibromodulin and its use to prevent or reduce skin scar formation. A study of pibromodulin is described in U.S. Patent No. 5,654,270 to Ruoslahti et al.
[38] To better understand protein-protein interactions, efforts to identify novel proteins with lozyne-rich repeats have been made by both industry and academia. Of particular interest is a protein having a lutein-rich repetition and being homologous to a known protein having a lysine-rich repeat, such as pibromodulin, SLIT protein and platelet glycoprotein V. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secreted and membrane bound proteins with lozyne-rich repeats. The present inventors herein describe the identification and characterization of novel polypeptides (referred to herein as PRO265 polypeptides) that are homologous to pibromodulin.
[39] 6. PRO219
[40] The human matrilin-2 polypeptide is a member of the von Willebrand factor type A-like module grandeur. Von Willebrand factor is a protein that plays an important role in maintaining hemostasis. More specifically, von Willebrand factor is a protein known to participate in platelet-vascular wall interactions at the site of vascular injury through its ability to interact with and form complexes with Factor VIII. Without the von Willebrand factor in the blood, platelets can not attach to the blood vessel walls at the site of vascular injury, resulting in platelet abnormalities. The result is a disease that includes bruises, nasal bleeding, intestinal bleeding, and von Willebrand disease.
[41] Considering the physiological importance of blood clotting factors, efforts to identify new natural proteins that may be involved in the coagulation process have been made by both industry and academia. The present inventors have described herein the identification of novel full-length polypeptides that are homologous to the human matrilin-2 precursor polypeptide.
[42] 7. PRO246
[43] The cell surface protein HCAR is a membrane-binding protein that acts as a receptor for subgroup C of the adenovirus and subclass B of the coxsackievirus. Thus, an HCAR can provide a method of modulating the viral infection of a cell in that the presence of the HCAR receptor on the cell surface facilitates viral infection by providing a binding site for the viral particle.
[44] In view of the physiological importance of membrane binding proteins, in particular proteins acting as cell surface receptors of viruses, efforts to identify new natural membrane binding receptor proteins are now being made by both industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel receptor proteins. Herein we describe a novel membrane binding polypeptide (referred to herein as PRO246) that is homologous to a variety of tumor antigens, including cell surface protein HCAR, and A33 and fetal carcinoma antigen, wherein the polypeptide is a novel cell surface Viral receptor or tumor antigen.
[45] 8. PRO228
[46] There are seven known transmembrane proteins, a family that includes CD97 and EMR1. CD97 is a transmembrane receptor that has cell ligands CD55, DAF and traverses the membrane seven times (Hamann et al., J. Exp. Med. (U.S.), 184 (3): 1189 (1996)]. In addition, CD97 has been shown to be indicative of dedifferentiation in human thyroid cancer and is associated with inflammation (Aust et al., Cancer Res. (U.S.), 57 (9): 1798 (1997) and Gray et al., J. Immunol. (US), 157 (12: 5438 (1996)].) In addition, CD97 is associated with the macrocystin receptor giant family, but unlike CD97 and EMR1, the known members of this family, several EGF domains at the N- (Hamam et al., Genomics, 32 (1): 144 (1996)), Hamann et al., J. Immunol., 155 EMR1 is also known from Lin et al. [Genomics, 41 (3): 301 (1997)] and Baud et al., Genomics, 26 (2): 334 (1995)]. Although CD97 and EMR1 appear to be associated with the secretin receptor, the known elements of the secretin family of G protein-coupled receptors include the alpha-ratoxin receptor and ratfoline, which are calcium-independent Have been described as being abundant in neural tissue (Lelianova et al., J. Biol. Chem., 272 (34), 21504 (1997)) and Davletov et al. .Chem. (US), 27 1 (38) 23239 (1996)]. [0003] Both the elements of the secretin receptor giants and the other elements associated with the secretin receptor macrogenius, or CRF and calcitonin receptors, are of interest, in particular by their homology with known proteins The new elements of this group are interesting.
[47] Efforts to identify transmembrane proteins with EGF repeats and large N-termini, which can belong to families of novel membrane-associated receptor proteins, specifically CD97 and EMR1, a member of the seven transmembrane proteins, . Herein we describe the identification and characterization of novel polypeptides (referred to herein as PRO228 polypeptides) that are homologous to CD97 and EMR1.
[48] 9. PRO533
[49] Growth factors are molecular signals or mediators that bind to specific cell surface receptors alone or in combination to augment cell growth or proliferation. However, after the expression of the growth factor, there is also a cell reaction other than growth. As a result, growth factors are more likely to be multifunctional and potent cell regulators. His biological effects include promotion of proliferation, chemotaxis, and extracellular matrix production. Growth factors can have both promoting and inhibitory effects. For example, modification of the growth factor (TGF-b) results in highly multipotent expression, which promotes proliferation in some cells, particularly connective tissue, but strongly inhibits proliferation in other cells such as lymphocytes and epithelial cells.
[50] The physiological effects of growth-promoting or inhibiting growth factors depend on the growth and differentiation state of the target tissue. Local mechanisms of cellular regulation by typical endocrine molecules are involved in understanding the pathways of self-secretion (same cells), adjacent secretions (neighboring cells), and side secretions (neighboring cells). Peptide growth factors are elements of complex biological languages and provide the basis for intracellular communication. This factor allows cells to transmit information to one another, mediate cell-cell interactions, and alter gene expression. The effect of these multifunctional and versatile factors will depend on the presence or absence of other peptides.
[51] Fibroblast growth factor (FGF) is a group of potent hepatonin-binding mitogens for both normal diploid fibroblasts and established cell lines (Gospodarowicz et al. (1984), Proc. Natl. Acad. Sci. USA 81 : 6963]. Among the FGF family members, acid FGF (FGF-1), basic FGF (FGF-2), INT-2 (FGF-3), K-FGF / HST (FGF- KGF (FGF-7) and AIGF (FGF-8). All FGFs have two conserved cysteine residues and share 30-50% sequence homology at the amino acid level. This factor is a cleavage promoting substance for a wide variety of normal diploid mesodermal-derived and neural-derived cells, including granulosa cells, adrenocortical cells, chondrocytes, myoblasts, corneas and vascular epithelial cells (bovine or human) , Vascular smooth muscle cells, lens, retina and prostate epithelial cells, glial cells, astrocytes, chondrocytes, myoblasts and osteoblasts.
[52] Fibroblast growth factors may also promote many types of cells in a different way than promoting mitosis. These activities include the promotion of cell migration (chemotaxis), the initiation of neovascularization (angiogenesis), the regulation of neuronal recovery and survival (neuronal tendency), the regulation of endocrine function, and the expression of specific cellular proteins, Promoting or inhibiting extracellular matrix production and cell survival (Baird and Bohlen, Handbook of Exp. Pharmacol. 95 (1): 369-418, Springer, (1990)]. These properties provide the basis for the use of fibroblast growth factors in therapeutic studies to promote wound healing, neural recovery, and collateral vessel formation. For example, fibroblast growth factor is recommended for minimizing myocardial damage in heart disease and surgery [US Pat. No. 4,378,437].
[53] Herein we describe the identification and characterization of novel polypeptides (referred to herein as PRO533 polypeptides) that are homologous to FGF.
[54] 10. PRO245
[55] Several of the most important proteins involved in the regulation and modulation of cellular responses described above are enzymes that regulate the level of protein phosphorylation in cells. For example, signal transduction regulating cell growth or differentiation is known to be regulated, at least in part, by phosphorylation and dephosphorylation of various cellular proteins. Enzymes that facilitate this process include protein kinases that function to phosphorylate various cellular proteins and protein phosphatases that function to remove phosphate residues from various cellular proteins. Thus, the balance of protein phosphorylation levels in cells is regulated by the relative activity of these two types of enzymes.
[56] Although many protein kinase enzymes have been identified, the physiological role played by many of these catalytic proteins is now being addressed. However, it is well known that many known protein kinases function to exhibit various other effects by phosphorylating the tyrosine residue of a protein. Perhaps most importantly, protein tyrosine kinases have been of great interest since the discovery that many tumor gene products and growth factors have unique protein tyrosine kinase activity. Thus, it is required to identify new elements of the protein tyrosine kinase family.
[57] Given the physiological importance of protein kinases, efforts to identify novel natural kinase proteins have been made both by industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel kinase proteins. The present inventors herein describe the identification and characterization of novel polypeptides (designated PRO245 herein) that are homologous to the tyrosine kinase protein.
[58] 11. PRO220, PRO221 and PRO227
[59] Protein-protein interactions include complexes of receptors and antigens and signaling mechanisms. As is well known for the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be readily manipulated to control the specific consequences of their protein-protein interactions. Thus, the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community.
[60] All proteins, including the lozengine repeat, are thought to be involved in protein-protein interactions. Lutein-rich repeats are short sequence motifs present in many proteins that differ in function and intracellular location. The crystal structure of the ribonuclease inhibitor protein reveals that the lozengine repeat is consistent with the beta-alpha structural unit. These units form a parallel beta sheet with one side exposed to the solvent, resulting in a unique non-spherical shape. These two features have been found to be responsible for the protein-binding function of the protein, including the lozengine repeat. In this regard, Kobe and Deisenhofer, Trends Biochem. Sci. , 19 (10): 415-421 (Oct. 1994).
[61] According to one study, a leucine-rich proteoglycan functions as a tissue-forming organ that directs and aligns the collagen fibrils during the development of an individual, and is involved in pathological processes such as wound healing, tissue repair, and tumor lipid formation [Iozzo, RV, Crit. Rev. Biochem. Mol. Biol ., 32 (2): 141-174 (1997)). Other studies that have shown that lutein-rich proteins are involved in wound healing and tissue repair include De La Salle, C., et al., Who reported that mutations in the lysine-rich motif in the complex associated with the bleeding disorder Bernard-Soullier syndrome Vouv. Rev. Fr. Hematol . (Germany), 37 (4): 215-222 (1995)] and Chlemetson (KJ) thrombocytopenia reported to have a leucine rich repeat . Haemost . (Germany), 74 (1): 111-116 (July 1995). Another protein reported to have a leucine rich repeats is the SLIT protein, which has been reported to be useful in the treatment of neurodegenerative diseases such as Alzheimer's disease, nerve damage such as in Parkinson's disease, and in the diagnosis of cancer Artavanistsakonas, S. and Rothberg, JM, WO9210518-A1, Yale University]. Other reports on the biological function of proteins with lozengesic repeats include Tayar, N. et al ., Mol. Cell Endocrinol. (Ireland), 125 (1-2): 65-70 (Dec. 1996)] (related to the gonadotropin receptor), Miura, Y. et al., Nippon Rinsho ): 1784-1789 (July 1996)] (related to apoptosis), Harris (PC) et al ., J. Am. Soc. Nephrol ., 6 (4): 1125-1133 (Oct. 1995)] (related to nephropathy) and degenerative growth factor β binding to the Lazolas Cancer Research Foundation reported to be involved in cancer treatment, wound healing and scar formation And WO 9110727A such as Ruoslahti (EI) of La Jolla Cancer Research Foundation.
[62] Thus, efforts to identify novel proteins with lozengine repeat moieties have been made by both industry and academia to better understand protein-protein interactions. Of particular interest is a protein homologous to a known protein having a lutein-rich repeating portion, such as the SLIT protein and the platelet glycoprotein V,
[63] 12. PRO258
[64] Immunoglobulins are antibody molecule proteins that act as receptors for antigens and secretory substances for plasma cells in the B-cell membrane. Like all antibody molecules, immunoglobulins perform two main functions: Immunoglobulins bind specifically to an antigen and participate in a limited number of biological effector functions. Thus, the new elements of the Ig group are always of interest. Molecules acting as receptors by various viruses and molecules functioning to regulate immune function are of particular interest. In particular, molecules that are homologous to known Ig elements that act as viral receptors or modulate immune function are of interest. Thus, CRTAM and CD166 (ligands for lymphocyte antigen CD6), which are molecules homologous to the poliovirus receptor, are of particular interest.
[65] Extracellular proteins and membrane-bound proteins play an important role in the formation, differentiation and maintenance of multicellular organisms. The fate of many individual cells, such as proliferation, migration, differentiation, or interaction with other cells, is typically governed by information received from other cells and / or adjacent environments. This information is often transmitted by secreted polypeptides (e. G., Mitogenic, survival, cytotoxic, differentiating, neuropeptides and hormones) and is ultimately accommodated by various cell receptors or membrane binding proteins It is deciphered. These secreted polypeptides or signaling molecules generally reach the action site in the extracellular environment, usually a membrane-bound receptor protein, via a cell secretory pathway.
[66] The present inventors herein describe the identification and characterization of novel polypeptides (designated herein as PRO258 polypeptides) that are homologous to CRTAM.
[67] 13. PRO266
[68] Protein-protein interactions include complexes of receptors and antigens and signaling mechanisms. As is well known for the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be readily manipulated to control the specific consequences of their protein-protein interactions. Thus, the underlying mechanism of protein-protein interactions is of interest to the scientific and medical community.
[69] All proteins, including the lozengine repeat, are thought to be involved in protein-protein interactions. Lutein-rich repeats are short sequence motifs present in many proteins that differ in function and intracellular location. The crystal structure of the ribonuclease inhibitor protein reveals that the lozengine repeat is consistent with the beta-alpha structural unit. These units have a unique non-spherical shape because they form a parallel beta sheet with one side exposed to the solvent. These two features have been found to be responsible for the protein-binding function of the protein, including the lozengine repeat. In this regard, Kobe and Deisenhofer, Trends Biochem. Sci. , 19 (10): 415-421 (Oct. 1994).
[70] According to one study, a leucine-rich proteoglycan functions as a tissue-forming organ that directs and aligns the collagen fibrils during the development of an individual, and is involved in pathological processes such as wound healing, tissue repair, and tumor lipid formation [Iozzo, RV, Crit. Rev. Biochem. Mol. Biol ., 32 (2): 141-174 (1997)). Other studies that have shown that lutein-rich proteins are involved in wound healing and tissue repair include De La Salle, C., et al., Who reported that mutations in the lysine-rich motif in the complex associated with the bleeding disorder Bernard-Soullier syndrome Vouv. Rev. Fr. Hematol . (Germany), 37 (4): 215-222 (1995)] and Chlemetson (KJ) thrombocytopenia reported to have a leucine rich repeat . Haemost . (Germany), 74 (1): 111-116 (July 1995)]. Another protein reported to have a lutein-rich repeats is the SLIT protein, which has been reported to be useful in the treatment of neurodegenerative diseases such as Alzheimer's disease and nerve damage such as in Parkinson's disease, and in the diagnosis of cancer Artavanistsakonas, S. and Rothberg, JM, WO9210518-A1, Yale University]. Other reports on the biological function of proteins with lozengesic repeats include Tayar, N. et al ., Mol. Cell Endocrinol. (Ireland), 125 (1-2): 65-70 (Dec. 1996)] (related to the gonadotropin receptor), Miura, Y. et al., Nippon Rinsho ): 1784-1789 (July 1996)] (related to apoptosis), Harris (PC) et al ., J. Am. Soc. Nephrol ., 6 (4): 1125-1133 (Oct. 1995)] (related to nephropathy) and degenerative growth factor β binding to the Lazolas Cancer Research Foundation reported to be involved in cancer treatment, wound healing and scar formation And WO 9110727A such as Ruoslahti (EI) of La Jolla Cancer Research Foundation.
[71] Thus, efforts to identify novel proteins with lozengine repeat moieties have been made by both industry and academia to better understand protein-protein interactions. Of particular interest is a protein homologous to a known protein having a lutein-rich repeating portion and a lutein-like repeating portion such as SLIT. Herein we describe a novel polypeptide (referred to herein as a PRO266 polypeptide) that is homologous to SLIT.
[72] 14. PRO269
[73] Trombomodulin combines with thrombin to regulate its activity. This is important in the control of blood clotting. Trombomodulin functions as a natural anticoagulant by promoting the activation of protein C by thrombin. Soluble trombomodulin may have therapeutic use as an antithrombotic agent and is less prone to bleeding than heparin. Trombomodulin is a transmembrane glycoprotein on the surface of cells present in endothelial cells and platelets. A smaller, functionally active form of thrombomodulin is circulating in plasma and also found in urine (Haeberli, Human Protein Data, VCH Oub., N.Y., 1992). Particularly preferred are peptides that are homologous to thrombomodulin.
[74] Herein we describe the identification and characterization of novel polypeptides (referred to herein as PRO269 polypeptides) that are homologous to trombomodulin.
[75] 15. PRO287
[76] The procollagen C-proteinase enhancer protein binds to the bone formation protein " BMP1 " / procollagen C-proteinase (PCP) to increase its activity. This protein is involved in extracellular matrix deposition. The BMP1 protein can be used to induce bone and / or cartilage formation, and for wound healing and tissue repair. Thus, the procollagen C-proteinase enhancer protein BMP1 and its homologous proteins are of interest in the scientific and medical field.
[77] Herein we describe the identification and characterization of novel polypeptides (herein referred to as PRO287 polypeptides) that are homologous to the procollagen C-proteinase protein precursor and the procollagen C-proteinase enhancer protein .
[78] 16. PRO214
[79] Growth factors are molecular signals or mediators that bind to specific cell surface receptors alone or in combination to augment cell growth or proliferation. However, after the expression of the growth factor, there is also a cell reaction other than growth. As a result, growth factors are more likely to be multifunctional and potent cell regulators. His biological effects include promotion of proliferation, chemotaxis, and extracellular matrix production. Growth factors can have both promoting and inhibitory effects. For example, modification of the growth factor (TGF-b) results in highly multipotent expression, which promotes proliferation in some cells, particularly connective tissue, but strongly inhibits proliferation in other cells, such as lymphocytes and epithelial cells.
[80] The physiological effects of growth-promoting or inhibiting growth factors depend on the growth and differentiation state of the target tissue. Local mechanisms of cellular regulation by typical endocrine molecules are involved in understanding the pathways of self-secretion (same cells), adjacent secretions (neighboring cells), and side secretions (neighboring cells). Peptide growth factors are elements of complex biological languages and provide the basis for intracellular communication. This factor allows cells to transmit information to one another, mediate cell-cell interactions, and alter gene expression. The effect of these multifunctional and versatile factors will depend on the presence or absence of other peptides.
[81] Epidermal growth factor (EGF) is a common cleavage promoting factor that promotes the proliferation of various types of cells including epithelial cells and fibroblasts. EGF binds to and activates the EGF receptor (EGFR), which initiates intracellular signaling and results accordingly. EGFR is expressed not only in neurons in the cerebral cortex, cerebellum and hippocampus, but also in other areas of the central nervous system (CNS). In addition, EGF is also expressed in various regions of the CNS. Thus, EGF acts not only on mitotic cells but also on neurons after mitosis. Indeed, many studies have shown that EGF exerts neurotrophic or neurotoxic effects on various types of neurons in the CNS. For example, EGF acts directly on the cultured cerebral cortex and cerebellar neurons, thereby enhancing neurite outgrowth and survival. EGF, on the other hand, acts indirectly through neurons in other types of cells, including cholinergic neurons in the septum and dopaminergic neurons in the midbrain. Evidence for the effect of EGF on CNS neurons has been accumulated, but the mechanism of action is inherently unknown. EGF-induced signaling is better known in mitotic cells than in neurons after mitosis. Studies on cloned chromaffinocytoma PC12 cells and cultured cerebral cortical neurons suggest that EGF-induced neurotrophic effects are modulated by the continuous activation of EGFR and mitogen-activated protein kinase (MAPK) on EGF do. Continuous intracellular signaling is correlated with decreased EGFR down-regulation rate, which determines the response of neurons to EGF. EGF is considered to be a potent multiple growth factor acting on many types of cells, including mitotic cells and mitotic post-neurons.
[82] EGF is produced by salivary glands and gastrointestinal Brunner's glands, kidneys, pancreas, thyroid gland, pituitary gland and nervous system and is used for body fluids such as saliva, blood, CSF, urine, amniotic fluid, prostate fluid, (Plata-Salaman, Peptide 12 : 653-663 (1991)).
[83] EGF is delivered by membrane-specific receptors including native tyrosine kinases (Stoscheck et al., J. Cell Biochem. 31: 135-152 (1986)]. EGF is believed to function to induce transmembrane signals and to activate intrinsic tyrosine kinases by binding to the extracellular portion of the receptor.
[84] Purification and sequencing of the EGF-like domain revealed the presence of six conserved cysteine residues that cross-linked to form three peptide loops (Savage et al., J. Biol. Chem. 248: 7669-7672 (1979)]. At present, it is now generally believed that EGF, which shares several different peptides with the same generalized motif X _n CX ₇ CX _4/5 CX ₁₀ CXCX ₅ GX ₂ CX _n , where X is an amino acid other than cysteine and n is a repeat variable It is known to be able to react with receptors. Unsolicited peptides with such motifs include TGF-a, ampirirgulin, schwanoma-derived growth factor (SDGF), heparin-binding EGF-like growth factor and proteins encoded by a particular virus For example, Vaccinia virus (Reisner, Nature 313: 801-803 (1985)), Shope fibrosis virus (Chang et al., Mol Cell Biol. 7: 535-540 (1987)], Molluscum contagiosum (Porter and Archard [J. Gen. Virol. 68 : 673-682 (1987)] and myxoma virus (Upton et al., J. Virol. 61 : 1271-1275 (1987) and Prigent and Lemoine, Prog. Growth Factor Res. 4 : 1-24 (1992)].
[85] EGF-like domains are not limited to growth factors and are observed in a variety of cell-surface proteins and extracellular proteins with interesting characteristics in cell attachment, protein-protein interaction and growth (Laurence and Gustuson Gusterson, Tumor Biol. 11 : 229-261 (1990)]. These proteins include coagulation factors (Factors VI, IX, X, XII, Protein C, Protein S, Protein Z, Tissue plasminogen activator and urokinase), extracellular matrix components (laminin, cytotaxin and enstatin) (LDL receptor and thrombomodulin receptor) and immuno-related proteins (complement C1r and euromodulin).
[86] Even more interestingly, the general structural pattern of EGF-like precursors is conserved not only in lower organisms but also in mammalian cells. Many genes important for growth have been identified in invertebrates with EGF-like repetition. For example, the notch gene of Drosophila codes for 36 repeats in which 40 amino acids are arranged in a row, which shows homology with EGF (Wharton et al., Cell 43 : 557-581 (1985)]. The hydro-patch plot represents an estimated membrane-bound domain, with EGF-related sequences located on the extracellular side of the membrane. Other homotypic genes with EGF-like repeats include Delta, 95F and 5ZD and include Notch genes and nematode genes encoding presumptive receptors for growth signals transmitted between two specific cells Using Lin-12-based probes.
[87] In particular, EGF has been implicated in the pathogenesis of necrotizing enterocolitis, Zollinger-Ellison syndrome, gastrointestinal ulcers and congenital microvilli atrophy (Guglietta and Sullivan, Eur. J. Gastroenterol Hepatol, 7 (10), 945-50 (1995)], as well as the preservation and maintenance of gastrointestinal mucosa and the repair of acute and chronic mucosal damage (Konturek et al., Eur.J. Gastroenterol Hepatol. 7 (10), 933-37 (1995)]. In addition, EGF has been shown to be effective against hair follicle differentiation [du Cru et al., J. Invest. Dermatol. 101 (1 Suppl.), 106S-113S (1993)], Hillier, Clin. Endocrinol. 33 (4), 427-28 (1990)], kidney function (Hamm et al., Semin. Nephrol. 13 (1): 109-15 (1993)], Harris, Am. J. Keyney Dis. 17 (6): 627-30 (1991)]], leakage [Bain Setten et al. Int. Ophthalmol 15 (6) 359-62 (1991)]], blood coagulation [Stenflo et al. Blood 78 (7): 1637-51 (1991)]. In addition, EGF has been implicated in a variety of skin diseases characterized by abnormal keratinocyte differentiation, such as squamous cell carcinomas such as squamous cell carcinoma of the psoriasis and lung, epidermal carcinoma of the vulva and glioma (King et al. [Am.J.Med.Sci. 296 : 154-158 (1998)].
[88] It is interesting to prove that genetic changes in the signaling pathways of growth factors are closely related to developmental disorders and chronic diseases, including cancer (Aaronson, Science 254 : 1146-1153 (1991) ]. For example, c-erb-2 (also known as HER-2), a proto-oncogene having a close structural similarity to the EGF receptor protein, is overexpressed in human breast cancer [King et al. Science 229: 974 -976 (1985), Gullick's Hormones and their actions, and Cooke BA et al., Eds, Amsterdam, Elsvier, pp 349-360 (1986)].
[89] 17. PRO317
[90] One family of secreted proteins, the TGF-beta giant family, or simply the TGF-beta giant family, contains a large number of related growth and differentiation factors expressed in virtually all phylum. Macrophages induce their multifunctional cytokine effect by binding to specific cell surface receptors and activating signal transduction mechanisms (Kolodziejczyk and Hall, Biochem. Cell. Biol. , 74 : 299-314 (1996)], Attisano and Wrana, Cytokine Growth Factor Rev. , 7 : 327-339 (1996) and Hill, Cellular Signaling , 8 : 533-544 (1996)]. Elements of this group include five different forms of TGF-beta (Sporn and Roberts, Peptide Growth Factors and Their Receptors , Sporn and Roberts, eds. (Springer-Verlag: Berlin, 1990) pp. 419-472)] as well as the differentiation factor vg1 (Weeks and Melton, Cell , 51 : 861-867 (1987)] and DPP-C polypeptides [Padgett et al. ( Nature , 325 : 81-84 (1987)], hormone actibin and inhivin [Mason et al. , Nature , 318 : 659-663 (1985), Mason, Growth Factors , 1 : 77-88 (1987)], Mullerian Inhibitor (MIS) [Cate et al., Cell , 45 : 685-698 (1986)], bone marrow protein (BMP) [Wozney et al., Science , 242 : 1528-1534 (1988) and PCT WO 88/00205 published January 14, 1988; US 4,877,864 issued October 31, 1989], growth regulator Vgr-1 [Lyons et al ., Proc. Natl. Acad. Sci. USA . 86 : 4554-4558 (1989)] and Vgr-2 [Jones et al ., Molec. Endocrinol. , 6 : 1961-1968 (1992)], mouse growth differentiation factor (GDF) such as GDF-3 and GDF-9 [Kingsley, Genes Dev. , 8 : 133-146 (1994) and McPherron and Lee, J. Biol. Chem. , 268 : 3444-3449 (1993)], mouse lefty / Stra1 [Meno et al. , Nature , 381 : 151-155 (1996)], Bouillet et al . Biol. , 170 : 420-433 (1995)], glial cell line-induced neurotrophic factor (GDNF) [Lin et al., Science , 260 : 1130-1132 (1993)], neurturin (Kotzbauer et al., Nature, 384: 467-470 (1990)), and endometrial bleeding-related factors (EBAF) [Kothapalli et al., J. Clin. Invest. , ≪ / RTI > 99 : 2342-2350 (1997)]. The subset BMP-2A and BMP-2B are approximately 75% sequence homologous to DPP-C and may represent mammals equivalent to this protein.
[91] The TGF- [beta] macromolecule protein has a large hydrophobic signal sequence, several hundred amino acids of a relatively long conserved long N-terminal pro region, a cleavage site (usually several bases) and a more conserved C- A disulfide-linked homodimer or heterodimer that is encoded by a precursor polypeptide chain. This C-terminal region corresponds to the processed mature protein and contains about 100 amino acids with characteristic cysteine residues, i.e., seven of the nine cysteine residues of TGF- [beta] are conserved among all known family elements. The position of the cleavage site between the mature and pro regions is different between these elements, and all C-terminal ends of these proteins terminate at the same position with the sequence Cys-X-Cys-X, but in all cases the TGF-beta consensus C -Terminal Cys-Lys-Cys-Ser [Sporn and Roberts, 1990, supra .].
[92] At present, there are five or more types of TGF-β identified, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 and TGF-β5. The active form of TGF- [beta] l is a homodimer formed by dimerization of the carboxyl-terminal 112 amino acids of a precursor of 390 amino acids. Recombinant TGF- 1 was cloned (Derynck et al. , Nature , 316 : 701-705 (1985)] and expressed in Chinese hamster ovary cells [Gentry et al ., Mol. Cell. Biol. , 7 : 3418-3427 (1987)). In addition, [Martin et al., Having (deMartin) [EMBO J., 6 : 3673 (1987)]] recombinant human TGF-β2 et al., As well as human and porcine TGF-β3 [de rinkeu (Derynck) [EMBO J. , 7 : 3737-3743 (1988)], ten Dijke et al ., Proc. Natl. Acad. Sci. USA , 85 : 4715 (1988)]. TGF- [beta] 2 is a precursor form of 414 amino acids and is also processed from 112 carboxy-terminal amino acids that share about 70% homology with the active form of TGF- [beta] to form homodimers (Marquardt Et al ., J. Biol. Chem. , 262 : 12127 (1987)]. Cell , 48 : 409-415 (1987)], European Patent Nos. 200,341, 169,016, 268,561 and 267,463, US Patent 4,774,322, Cheifetz et al. Jakowlew et al ., Molecular Endocrin. , 2 : 747-755 (1988)], Derynck et al ., J. Biol. Chem. , 261 : 4377-4379 (1986)], Sharples et al. , DNA , 6 : 239-244 (1987), Derynck et al., Nucl. Acids. Res. , 15 : 3188-3189 (1987)], Derynck et al., Nucl. Acids. Res. , 15 : 3187 (1987)], Seyedin et al ., J. Biol. Chem. , 261 : 5693-5695 (1986), Madisen et al., DNA , 7 : 1-8 (1988), and Hanks et al ., Proc. Natl. Acad. Sci. (USA) , 85 : 79-82 (1988).
[93] TGF-β4 and TGF-β5 were isolated from chick chondrocytic cDNA library (Jakowlew et al ., Molec. Endocrinol. , 2 : 1186-1195 (1988)] and frog oocyte cDNA libraries, respectively.
[94] The pro region of TGF-ss is non-covalently associated with mature TGF-beta dimers (Wakefield et al ., J. Biol. Chem. , 263: 7646-7654 (1988) and Wakefield et al., (Wakefield) [Growth Factors, 1: 203-218 (1989)], a pro region of both active mature TGF-β and activin solution dimer [Gray and Mason, Science , 247 : 1328-1330 (1990)]]. ≪ / RTI > The link between the mature and pro-regions of TGF- [beta] blocks the biological activity of mature dimers and forms an inactive potential form. The potential is not constant in the TGF-β giant group because the presence of the pro region does not affect the biological activity of actin or inhivin.
[95] A constant characteristic of the biology of proteins from the TGF-β family is their ability to regulate the growth process. TGF-beta has been shown to exert a number of modulatory effects on a wide variety of normal or tumor cells. TGF-beta is multifunctional and can promote or inhibit cellular proliferation and differentiation and other important processes in cell function (Sporn and Roberts, supra ).
[96] EBAF, a member of the TGF-beta giant family, is expressed exclusively during endometriosis and abnormal endometrial bleeding at the delayed release phase (Kothapalli et al., J. Clin. Invest. , ≪ / RTI > 99 : 2342-2350 (1997)]. The human endometrium is unique in that it is the only tissue that causes bleeding at regular intervals in the human body. In addition, abnormal endometrial bleeding is one of the most common signs of gynecological disease and is a major indication for hysterectomy. As a result of in situ hybridization, EBAF mRNA was expressed in stroma and many mRNAs were not expressed in endometrium or endothelial cells.
[97] The predicted amino acid sequence of EBAF was found to be highly homologous with the protein encoded by mouse lefty / stra3 of the TGF-β giant group. A study of the motif revealed that the expected EBAF protein is conserved among TGF-β-related proteins and contains most of the cysteine residues essential for the formation of cysteine knot structures. The EBAF sequence contains 12 amino acids upstream from the conserved cysteine residue at the front which is another cysteine residue. The only other group of elements known to contain other cysteine residues are TGF-beta, Inhibin and GDF-3. EBAF is similar to LEFTY, GDF-3 / Vgr2 and GDF-9, and has no cysteine residues known to form intramolecular disulfide bonds. Thus, EBAF appears to be another element of the TGF-β giant family with an intact cysteine residue that can not exist as a dimer. However, hydrophobic contacts between the two monomeric subunits can promote dimer formation. As a result of fluorescent in situ hybridization, the ebaf gene was found to be located in the band q42.1 of human chromosome 1.
[98] Industry and academia are investigating other factors associated with the TGF-β giant family, such as EBAF. Herein we describe the identification and characterization of novel polypeptides (referred to herein as PRO317 polypeptides) that are homologous to EBAF.
[99] 18. PRO301
[100] Expansion of cancer incidence is accelerating considerable resource consumption and discovery of new therapeutic drugs. One particular method involves the generation of tumor or cancer-specific monoclonal antibodies (mAbs) specific for tumor antigens. These mAbs, which can differentiate normal and cancer cells, are useful in the diagnosis, prognosis and treatment of disease. Certain antigens are known to be associated with a teratogenic disorder such as colorectal cancer.
[101] The specific antigen, A33 antigen, is expressed in over 90% of normal colon or metastatic colon cancer as well as normal colon epithelium. Since colon cancer is a widespread disease, early diagnosis and treatment are important medical goals.
[102] Diagnosis and treatment of colon cancer can be performed using a specific monoclonal antibody (mAb) having an antigenic, nuclear magnetic or radioactive tag. A radioactive gene, toxin and / or drug-tagged mAb can be used to treat with a minimum patient rating. It may also be diagnosed using mAbs during the diagnosis and treatment of colon cancer. For example, if the serum level of the A33 antigen in a patient is elevated, a postoperative reduction in the concentration indicates successful resection of the tumor. On the other hand, the elevation of postoperative serum A33 antigen concentration suggests that the original cancer may be metastasized or a new primary tumor may appear. Such monoclonal antibodies may be used in lieu of, or in conjunction with, surgery and / or other chemotherapy. For example, U.S. Patent No. 4,579,827 and Soviet Patent No. 424,991 (European Patent No. 199,141) are directed to the therapeutic administration of monoclonal antibodies and the latter relates to the use of anti-A33 mAbs.
[103] Many cancers of the epithelium have adenovirus receptors. Indeed, adenovirus-derived vectors have been proposed as a means of inserting antisense nucleic acids into tumors [US Pat. No. 5,518,885]. Thus, the binding of a viral receptor to a neoplastic tumor is not expected.
[104] Herein we describe the identification and characterization of novel polypeptides (designated herein as PRO301 polypeptides) that are homologous to several cancer-associated antigens.
[105] 19. PRO224
[106] Absorption of cholesterol can be significantly related to the health of an individual. Cholesterol absorption provides most of the cholesterol needed for cell membrane synthesis to cells. When this absorption is blocked, cholesterol can accumulate in the blood and form an atherosclerotic plaque in the blood vessel wall. Most cholesterol is carried in the blood and binds to a complex form of protein known as low density lipoprotein (LDL). LDL is introduced into the cell via the LDL receptor protein. Therefore, LDL receptor proteins and proteins that are homologous thereto are of interest in the scientific and medical fields.
[107] Membrane binding proteins and receptors may play an important role in the formation, differentiation and maintenance of multicellular organisms. LDL receptors are an example of membrane binding proteins involved in the synthesis and formation of cell membranes where the health status of an individual is directly or indirectly influenced by the function of the LDL receptor. Many membrane binding proteins act as receptors such as LDL receptors. These receptors can function as intracellular transfer of substrates or as receptors in channels. Other membrane binding proteins function as signals or antigens.
[108] Membrane binding proteins and receptor molecules have a variety of industrial uses including drugs and diagnostic agents. In addition, membrane bound proteins may be used to screen for potential peptides or small molecular modulators in the relevant receptor / ligand interaction. In the case of LDL receptors, it is desirable to find molecules that reduce blood colosterol concentration and flake formation by increasing intracellular entry. It is also desirable to find a molecule that inhibits intracellular entry so that an individual with a high blood cholesterol concentration can avoid or control the molecules. Also, polypeptides that are homologous to lipoprotein receptors but that do not function as lipoprotein receptors are of interest in determining the function of the fragments that display homology.
[109] Reports on such known low density lipoprotein receptors and related proteins, including apolipoproteins, are described in Sawamura et al., Nippon Chemiphar Co, Japan patent application J09098787, Novak, S., et al. J. Biol. Chem. , 69 (11) 7244-7 (Nov. 1995), Scott, J., et al ., Blaas, J. Inherit. Metab. Dis . (UK), 9 / Supp. 1 (3-16) (1986), Yamamoto et al., Cell , 39: 27-38 (1984), Rebece et al., Neurobiol. Aging , 15: 5117 (1994)], Novak, S. et al ., J. Biol. Chemistry , 271: 11732-11736 (1996), and Sestavel and Fruchart, Cell Mol. Biol ., 40 (4): 461-81 (June 1994). These publications and other documents published prior to this application provide additional background to the peptides already known in the art.
[110] Efforts to identify novel natural membrane-bound receptor proteins, particularly those that are homologous to lipoprotein receptors, are being made by both industry and academia. The present inventors herein describe the identification and characterization of novel polypeptides (referred to herein as PRO244 polypeptides) that are homologous to lipoprotein receptors.
[111] 20. PRO222
[112] Complement is found in the blood and is a group of proteins that are important in humoral immune and inflammatory responses. The complement protein is sequentially activated by an antigen-antibody complex or proteolytic enzyme. When activated, the complement protein kills bacteria and other microorganisms and influences vascular permeability to release histamines, thereby attracting leukocyte cells. In addition, when the complement binds to the target cell, the phagocytosis is increased. To prevent damage to autologous tissue cells, the complement activity pathway is precisely regulated.
[113] Control of complement activation or defects in the complement protein itself can cause immune-complex disorders such as systemic lupus erythematosus and may increase susceptibility to viral infection. In all cases, initial discovery of complement defects is desirable to enable the patient to begin treatment. Therefore, research efforts are now being made to identify soluble membrane proteins that regulate complement activation.
[114] Proteins known to be important in regulating complement activity in humans include Factor H and complement receptor type 1 (CR1). Factor H is a 150 kD soluble serum protein that acts as a cofactor in the factor I-mediated cleavage of the complement protein C4b, interacting with the complement protein C3b promoting the degradation of the C3 convertase. The complement receptor type I is a membrane binding protein of 190-280 kD found in mast cells and most blood cells. CR1 interacts with the complement proteins C3b, C4b and iC3b to accelerate the degradation of the C3 convertase and acts as a cofactor in the factor I-mediated cleavage of C3b and C4b, and in combination with the immune complex promotes its degradation and phagocytosis .
[115] Proteins that are homologous to complement proteins are of particular interest in the medical and industrial fields. Often, homologous proteins have similar functions. It may also be interesting if homologous proteins do not have similar functions, suggesting that certain structural motifs can provide information such as functional sites in addition to functions.
[116] Efforts to identify novel natural secretion and membrane binding proteins, particularly those that are homologous to known proteins involved in complement activity, are being made by both the industry and academia. Proteins involved in the complement pathway are described in Birmingham DJ, (1995), Critical Reviews in Immunology , 15 (2): 133-154) and Abbas AK et al. (1994) Cellular and Molecular Immunology , 2nd Ed. WB Saunders Company, Philadelphia, pp 295-315.
[117] The present inventors herein describe the identification and characterization of novel polypeptides (referred to herein as PRO222 polypeptides) that are homologous to the complement receptor.
[118] 21. PRO234
[119] The successful function of many systems in multicellular organisms depends on cell-cell interactions. This interaction is influenced by the arrangement of specific ligands and specific receptors in a manner that enables cell-cell adhesion by ligand-receptor binding. Protein-protein interactions in cell recognition have often been recognized, but the role of carbohydrates in physiologically relevant recognition reactions has only recently been widely considered (BK Brandley et al ., J. Leuk. Biol. 40 : 97 (1986) and N. Sharon et al. Science 246 : 227 (1989)). Oligosaccharides, when properly positioned, act to recognize new lectins due to their cell surface location and structural diversity. Many oligosaccharide structures can be generated through the different activities of a few glycosyltransferases. Oligosaccharides of various structures can be generated by the transfer of relatively few gene products, suggesting that oligosaccharides are an excellent mechanism involved in a wide range of cell-cell interactions. Examples of different expression of cell surface carbohydrates and putative carbohydrate binding proteins (lectins) for interacting cells are described in J. Dodd and TM Jessel, J. Neurosci. 5 : 3278 (1985)], LJ Regan et al ., Proc. Natl. Acad. Sci. USA 83 : 2248 (1986), M. Constantine-Paton et al., Nature 324 : 459 (1986), and M. Tiemeyer et al ., J. Biol. Chem. 263 : 1671 (1989). One interesting element in the Lectin family is selectin.
[120] Migration of leukocytes to acute or chronic inflammatory sites involves an adhesion interaction between these cells and the endothelium. This specific attachment is very important in the controlled defense of the organism because it is the first phenomenon in the cascade that is initiated by inflammatory damage.
[121] The types of cell adhesion molecules that are involved in the interaction between leukocytes and endothelium during inflammatory reactions are currently (1) selectin, (2) ligand (carbohydrate and glycoprotein) ligands for selectin, (3) integrin and (4) integrin There are four kinds of ligands, these are elements of the immunoglobulin gene large group.
[122] Selectin is a structurally and functionally unified cell adhesion molecule. Structurally, selectins are characterized by including calcium-dependent lectins (C-lectins), epidermal growth factor (egf) -like domains and some complement-binding-like domains (Bevilacqua, Science 243: 1160-1165 (1989)] , Johnston (Johnston) et al., [Cell 56: 1033-1044 et al., (1989), referred to the ski (Lasky) [Cell 56: 1045-1055 (1989), Siegalman, M. et al . , Science 243 : 1165-1172 (1989); Stoolman, LM. Cell 56 : 907-910 (1989)]. Functionally, selectin shares a common feature in its ability to mediate cell binding through its interaction between its lectin domain and cell surface carbohydrate ligands (Brandley, B. et al., Cell 63 , 861 ( Nature 349 , 19-197 (1991)], Bevilacqua (MP) and Nelson (RM), Springer, T. and Lasky J. Clin. Invest. 91 379-387 (1993) and Tedder et al ., J. Exp. Med. 170 : 123-133 (1989)].
[123] (PnHR), LEC-CAM-1, LAM-1, gp90 ^MEL , gp100 ^MEL , gp110 ^MEL , and MEL-14 antigens in the selectin family of cell adhesion molecules. (LEC-CAM-2, LECAM-2, ELAM-1) and P-selectin (LEC-CAM-3, LECAM-3, GMP-140, PADGEM).
[124] With the identification of the C-lectin domain, much effort has been devoted to finding carbohydrate binding ligands for proteins containing this domain. E-selectin is thought to recognize the carbohydrate sequence NeuNAcα2-3Galβ1-4 (Fucα1-3) GlcNAc (sialyl-Lewis x, or sLe ^x ) and related oligosaccharides (Berg et al. J. Biol. Chem. 265 : 14869-14872 (1991), Lowe et al. Cell 63 : 475-484 (1990), Phillips et al. Science 250 : 1130-1132 (1990) ( Tiemeyer , Proc. Natl. Acad. Sci. USA 88 : 1138-1142 (1991)].
[125] Selectin containing one lectin domain performs its attachment function by recognizing carbohydrate-containing ligands in endothelial cells. L-selectin is expressed on the surface of leukocytes such as lymphocytes, neutrophils, monocytes and eosinophils, and the migration of lymphocytes into peripheral lymphoid tissues ( Gallatin et al., Nature 303 : 30-34 (1983) Acute neutrophil-induced inflammatory response [Watson, SR, Nature 349 : 164-167 (1991)]. The amino acid sequence of L-selectin and the nucleic acid sequence encoding it are described, for example, in U.S. Patent No. 5,098,833, issued March 24, 1992.
[126] L-selectin (LECAM-1) is of particular interest due to its ability to block neutrophil influx [Watson et al., Nature 349 : 164-167 (1991)]. He is expressed in chronic lymphocytic leukemia cells that bind HEV (Spertini et al., Nature 349 : 691-694 (1991)]. It is also believed that the HEV structure at the site of chronic inflammation is also associated with the symptoms of diseases such as rheumatoid arthritis, ringworm and multiple sclerosis.
[127] E-selectin (ELAM-1) is particularly interesting because it reacts with IL-1 or TNF and is transiently expressed in endothelial cells (Bevilacqua et al., Science 243 : 1160 (1989)]. The time course of this induction (2 to 8 hours) suggests a role for the receptor in early neutrophil-derived extravasation in response to infection and wounding. In addition, anti-ELAM-1 antibodies have been reported to be beneficial in preventing airway obstruction resulting from inflammatory reactions because they block neutrophil entry into the primate asthma model [Gundel et al., J. Clin. Invest. 88 : 1407 (1991)).
[128] Adherence of circulating neutrophils to accelerated vascular endothelium is a major process of inflammatory response. P-selectin has been reported to recognize the Lewis x structure (Gal beta 1-4 (Fuc alpha 1-3) GlcNAc) (Larsen et al., Cell 63 : 467-474 (1990)). Other documents have reported that sialic acid to which the other terminal is connected is required for high affinity binding (Moore et al ., J. Cell. Biol. 112 : 491-499 (1991)]. P-selectin is known to be important in acute lung injury. Anti-P-selectin antibodies show potent protective effects in the rodent lung injury model [MS Mulligan et al., J. Clin. Invest. 90 : 1600 (1991)].
[129] The present inventors herein describe the identification and characterization of novel polypeptides (designated herein as PRO234 polypeptides) that are homologous to the lectin proteins.
[130] 22. PRO231
[131] Some of the most important proteins involved in the regulation and regulation described above for cellular processes are enzymes that regulate the level of protein phosphorylation in cells. For example, at least some of the signal transduction regulating cell growth and differentiation is known to be regulated by phosphorylation and dephosphorylation of various cellular proteins. Enzymes that facilitate this process include protein kinases that function to phosphorylate various cellular proteins and protein phosphatases that function to remove phosphate residues from various cellular proteins. Thus, the balance of protein phosphorylation levels in cells is regulated by the relative activity of these two types of enzymes.
[132] Protein phosphatase is a large family of enzymes found in many different forms including both membrane-bound and soluble forms. Although many protein phosphatases have been described, they have only begun to understand some functions (Tonks, Semin. Cell Biol. 4: 373-453 (1993) and Dixon, Recent Prog. Horm. Res. 51: 405-414 (1996)]. However, in general, many protein phosphatases appear to function to regulate positive or negative signals induced by various protein kinases. Thus, protein phosphatase is likely to play an important role in many different cell processes.
[133] Given the physiological importance of protein phosphatase, efforts to identify novel natural phosphatase proteins have been made by both industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel phosphatase proteins. Examples of screening methods and techniques are described in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996), and U.S. Patent No. 5,536,637).
[134] The present inventors herein describe the identification and characterization of novel polypeptides (designated herein as PRO231 polypeptides) that are homologous to acid phosphatases.
[135] 23. PRO229
[136] Scavenger receptors are known to protect IgG molecules from catabolic degradation [Riechmann and Hollinger, Nature Biotechnology , 15: 617 (1997)]. In particular, studies on the CH2 and CH3 domains show that certain sequences in these domains are important in determining the half-life of the antibody (Ellerson et al ., J. Immunol ., 116: 510 (1976) Yasmeen et al ., J. Immunol . 116: 518 (1976), Pollock et al ., Eur. J. Immunol ., 20: 2021 (1990)]. In addition, scavenger receptor proteins and antibodies thereto are reported in Krieger et al., U.S. Patent No. 5,510,466. Because of the ability of the scavenger receptor to increase the half-life of the polypeptide and its relevance in the immune response, molecules that are homologous to scavenger receptors are important in the scientific and medical arenas.
[137] Efforts to identify novel natural secretion and membrane-bound receptor proteins, particularly those that are homologous to scavenger receptors, are being made by both industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane bound receptor proteins. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996) and U.S. Patent No. 5,536,637).
[138] The present inventors herein describe the identification and characterization of novel polypeptides (referred to herein as PRO229 polypeptides) that are homologous to scavenger receptors.
[139] 24. PRO238
[140] Oxygen free radicals and antioxidants appear to play an important role in the central nervous system after cerebral ischemia and reperfusion. In addition, cardiac damage associated with ischemia and reperfusion has been reported to be caused by the action of free radicals. There is also a report that the oxidation-reduction state of the cells is the main determinant of the fate of cells. In addition, those containing reactive oxygen are cytotoxic and have been reported to cause inflammatory diseases including tissue necrosis, organ failure, atherosclerosis, infertility, birth defects, premature aging, mutations and malignant tumors. Thus, modulation of oxidation and reduction is important for many reasons including seizures, heart disease, oxidative stress and regulation and prevention of hypertension. In this respect, the reductase, especially the oxidoreductase, is interesting. Further publications describing these subject matter include Kelsey et al ., Br. J. Cancer , 76 (7): 852-4 (1997)], Friedrich and Weiss, J. Theor. Biol ., 187 (4): 529-40 (1997) and Pieulle et al ., J. Bacteriol. , 179 (18): 5684-92 (1997)].
[141] Efforts to identify secreted proteins that are homologous to new natural secretion and membrane-bound receptor proteins, particularly reductases, are being made by both the industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane bound receptor proteins. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996), and U.S. Patent No. 5,536,637).
[142] The present inventors herein describe the identification and characterization of novel polypeptides (referred to herein as PRO238 polypeptides) that are homologous to the reductase.
[143] 25. PRO233
[144] There is a report that the oxidation-reduction state of cells is an important determinant of cell fate. In addition, those containing reactive oxygen are cytotoxic and have been reported to cause inflammatory diseases including tissue necrosis, organ failure, atherosclerosis, infertility, birth defects, progeny, mutations and malignant tumors. Thus, modulation of oxidation and reduction is important for many reasons including seizures, heart disease, oxidative stress and the control and prevention of hypertension. Oxygen free radicals and antioxidants appear to play an important role in the central nervous system after cerebral ischemia and reperfusion. In addition, cardiac damage associated with ischemia and reperfusion has been reported to be caused by the action of free radicals. In this respect, the reductase, especially the oxidoreductase, is interesting. In addition, the transcription factors NF-kappa B and AP-1 are regulated by the oxidation-reduction state and are known to affect the expression of many various genes thought to be involved in the development of AIDS, cancer, atherosclerosis and diabetic complications have. Further publications describing these subject matter include Kelsey et al ., Br. J. Cancer , 76 (7): 852-4 (1997)], Friedrich and Weiss, J. Theor. Biol ., 187 (4): 529-40 (1997) and Pieulle et al ., J. Bacteriol. , 179 (18): 5684-92 (1997)]. Given the physiological importance of the oxidation-reduction reaction in vivo, efforts to identify new natural proteins involved in oxidation-reduction reactions have been made by both industry and academia. The present inventors herein describe the identification of a novel polypeptide (referred to herein as a PRO233 polypeptide) that is homologous to a reductase.
[145] 26. PRO223
[146] The carboxypeptidase family of exopeptidases consists of several classes of enzymes that hydrolyze carboxyl-terminal amide bonds in polypeptides, and many mammalian tissues produce this enzyme. Exhibit more potent cleavage properties for certain amino acids of many carboxy peptidase enzyme polypeptides identified to date. For example, carboxypeptidase enzymes have been found to prefer amino acids with lysine, arginine, serine, or aromatic or branched chain aliphatic side chains as substrates at the carboxyl end of the polypeptide.
[147] For serine carboxypeptidases, the amino acid specific enzymes have been identified from a variety of mammals and from other non-mammalian species. Mammalian serine carboxypeptidase enzymes play an important role in many other biological processes including, for example, proteolysis, activation, regulation of activity or regulation of peptide hormone activity, and physical properties of proteins and enzymes.
[148] In view of the physiological importance of serin carboxypeptidase, efforts to identify novel natural secretion and membrane-bound receptor proteins, particularly novel carboxypeptidases, have been made by both industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane bound receptor proteins. The present inventors herein describe a novel polypeptide (referred to herein as a PRO223 polypeptide) that is homologous to one or more serine carboxy peptidase polypeptides.
[149] 27. PRO235
[150] Flexcin was first identified as a membrane glycoprotein known to modulate cell attachment through the binding mechanism of specific antibodies in the presence of calcium ions in the Xenopus tadpole neuron. For plexins, evolutionary conservation was observed to be significant between genopus, mice, and homologues (Kaneyama et al., Biochem. And Biophys. Res. Comm. 226: 524-529 (1996)]. Considering the physiological importance of the cell attachment mechanism in vivo, efforts are currently being made to identify new natural proteins involved in cell adhesion. Herein we describe the identification of a novel polypeptide (referred to herein as PRO235) that is homologous to flexin.
[151] 28. PRO236 and PRO262
[152] β-Galactosidase is a well-known enzyme protein that functions to hydrolyze β-galactoside molecules. β-Galactosidase has been used in a variety of different applications both in vitro and in vivo and has proven to be a very useful research tool. As such, it is interesting to obtain novel polypeptides that are homologous to -Galactosidase polypeptides.
[153] Given the great interest in obtaining new polypeptides that are homologous to -galactosidase, efforts to identify novel novel -galactosidase homologous proteins have been made both by industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel -Galactosidase-like proteins. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996) and U.S. Patent No. 5,536,637). Herein we describe a novel polypeptide (termed herein PRO236 and PRO262 polypeptides) which has considerable homology with the -Galactosidase enzyme.
[154] 29. PRO239
[155] Densin is a glycoprotein isolated from the brain with all the features of the adhesion molecule. He is highly concentrated at the synaptic region of the brain and is significantly expressed in the dendrites of the growing neurons. Densin is a member of the O-linked sialoglycoprotein as a member. Densin is associated with medically important processes such as regeneration. Given the physiological importance of synaptic processes and cell attachment mechanisms in vivo, efforts are now being made to identify new natural proteins involved in synaptic sites and cell adhesion. The present inventors herein describe the identification of a novel polypeptide (referred to herein as a PRO239 polypeptide) that is homologous to densin.
[156] 30. PRO257
[157] Ebnerine is a cell surface protein associated with von Willebrand line in mammals. Efforts are being made by both industry and academia to identify proteins that have sequences that are homologous to cell surface receptor proteins, particularly cell surface proteins such as Ebnerin. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel receptor proteins. Herein we describe the identification of a novel polypeptide (termed PRO257 polypeptide herein) that is homologous to the parenaphysin-related protein Ebnerine.
[158] 31. PRO260
[159] Fucosidase is an enzyme that removes fucose residues from fucose-containing proteoglycans. In some pathological conditions such as cancer, rheumatoid arthritis and diabetes, serum proteins are abnormally fucosylated. Thus, proteins that are homologous to fucosidase, and fucosidase are important in the study and elimination of these symptoms. Fucosidase and fucosidase inhibitors are described further in U.S. Patent Nos. 5,637,490, 5,382,709, 5,240,707, 5,153,325, 5,100,797, 5,096,909 and 5,017,704. . Such studies are also described in Valk et al ., J. Virol ., 71 (9): 6796 (1997), Aktogu et al ., Monaldi. Arch. Chest Dis . (Italy), 52 (2): 118 (1997) and Focarelli et al., Biochem. Biophys. Res. Commun . (US), 234 (1): 54 (1997).
[160] Efforts to identify new natural secretion and membrane-bound receptor proteins have been made by both industry and academia. Proteins that are homologous to the alpha-1-fucosidase precursor are of particular interest. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane bound receptor proteins. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , ≪ / RTI > 93 : 7108-7113 (1996); U.S. Pat. 5,536, 637)).
[161] Herein we describe the identification and characterization of novel polypeptides (designated herein as PRO260) that are homologous to fucosidases.
[162] 32. PRO263
[163] CD44 is a cell surface adhesion molecule involved in cell-cell and cell-substrate interactions. Hyaluronic acid, a component of the extracellular matrix, is a major ligand. Other ligands include collagen, fibronectin, laminin, chondroitin sulfate, mucoadhesive, seriglycine and osteoponin. In addition, CD44 is important in regulating cell transport, lymph node engraftment, growth signaling, and the delivery of chemokines and growth factors to circulating cells. CD44 surface proteins bind to metastatic tumors, and CD44 is used as a marker for HIV infection. Some splice variants are associated with metastasis of cancer and poor prognosis in cancer patients. Thus, molecules that are homologous to CD44 are of particular interest because such homology indicates that he can perform functions related to the function of CD44. CD44 is also described in US 5,506,119, 5,504,194 and 5,108,904, Gerberick et al ., Toxicol. Appl. Pharmacol ., 146 (1): 1 (1997)], Wittig et al., Immunol. Letters (Netherlands), 57 (1-3): 217 (1997) and Oliveira and Odell, Oral Oncol . (England), 33 (4): 260 (1997).
[164] Efforts to find new natural secretion and membrane-bound receptor proteins, particularly transmembrane proteins that are homologous to the CD44 antigen, have been made by both industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to find the coding sequence for the novel secretion and membrane-bound receptor proteins. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93: 7108-7113 (1996) and U.S. Patent No. 5,536,637).
[165] Herein we describe the identification and characterization of novel polypeptides (designated herein as PRO263 polypeptides) that are homologous to the CD44 antigen.
[166] 33. PRO270
[167] Thioredoxin affects the oxidation-redox state. Many diseases are potentially associated with oxidation-reduction states, and those containing reactive oxygen can play an important role in many important biological processes. The transcription factors NF-kappa B and AP-1 are regulated by the oxidation-reduction state and are known to affect the expression of many different genes thought to be involved in the pathogenesis of AIDS, cancer, atherosclerosis and diabetic complications have. These proteins may play a role in apoptosis as well as pathological symptoms, including oxidative stress, such as defense against antioxidants in cells and seizures and inflammation. Thus, thioredoxin and its homologous proteins are of interest in the scientific and medical field.
[168] The present inventors herein describe the identification and characterization of novel polypeptides (referred to herein as PRO270 polypeptides) that are homologous to thioredoxin.
[169] 34. PRO271
[170] Proteoglycan-linked proteins are proteins that are tightly bound to a variety of extracellular matrix proteins, more specifically proteins such as collagen. For example, one major component of collagen is a large proteoglycan called agrecane. This molecule is retained by binding to the glycosaminoglycan hyaluronan through the G1 globular domain of the amino terminus of the core protein. This binding is stabilized by a proteoglycan-linked protein, a protein also associated with other tissues containing hyaluronan-binding proteoglycans such as < RTI ID = 0.0 >
[171] Linked proteins have been identified as potential targets for autoimmune antibodies in persons suffering from juvenile rheumatoid arthritis [Guerassimov et al., J. Rheumatology 24 (5): 959-964 (1997) ]). As such, it is of great interest to identify novel proteins that are homologous to the linking protein. Herein we describe the identification and characterization of novel polypeptides (termed herein as PRO271 polypeptides) that have this homology.
[172] 35. PRO272
[173] Reticulo calvin is an endoplasmic reticulum protein that can participate in protein transport and protein processing of the lumen. In the lumen of the endoplasmic reticulum, the reticulo calvin residue is known to bind calcium and may be involved in the lumen retention mechanism of the endoplasmic reticulum. He includes six domains of EF-hand motifs associated with high affinity calcium binding. The present inventors herein describe the identification and characterization of novel polypeptides (designated herein as PRO272) that are homologous to reticulo calvin proteins.
[174] 36. PRO294
[175] Collagen, a natural protein, has a wide range of industrial applications. Chemically hydrolyzed natural collagen can be denatured and re-denatured by heating and cooling to produce gelatin for other uses in photography and medicine. Collagen has important properties such as the ability to form aggregates in a chain having a form called a triple helix. Herein we describe the identification and characterization of novel polypeptides (designated herein as PRO294) that are homologous to a portion of the collagen molecule.
[176] 37. PRO295
[177] Integrins include a large gene family of cell-surface glycoprotein receptors that promote cell adhesion. Each cell has a number of receptors that determine its ability to attach cells. Integrins are involved in a wide variety of interactions between cells and other cells or substrate components. Integrin is particularly important in regulating the migration and function of immune system cells. Platelet IIb / IIIA integrin complexes are particularly important for controlling platelet aggregation. Among many Integrins, integrin β-6 is expressed in epithelial cells and regulates the epithelial inflammatory response. Other integrins, leukocyte-associated antigen-1 (LFA-1), are important for lymphocyte adhesion during the immune response. Integrins are expressed as heterodimers of non-covalently associated alpha and beta subunits. Considering the physiological importance of the cell attachment mechanism in vivo, efforts are now being made to identify new natural proteins involved in cell adhesion. The present inventors herein describe the identification and characterization of novel polypeptides (referred to herein as PRO295) that are homologous to integrins.
[178] 38. PRO293
[179] Protein-protein interactions include receptor-antigen complexes and signal transduction mechanisms. As is well known for the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be readily manipulated to control the specific consequences of their protein-protein interactions. Thus, the underlying mechanisms of protein-protein interactions are of interest to the scientific and medical community.
[180] All proteins, including the lozengine repeat, are thought to be involved in protein-protein interactions. Lutein-rich repeats are short sequence motifs present in many proteins that differ in function and intracellular location. The crystal structure of the ribonuclease inhibitor protein reveals that the lozengine repeat is equivalent to the beta-alpha structural unit. These units form a parallel beta sheet with one side exposed to the solvent, resulting in a unique non-spherical shape. These two features have been found to be responsible for the protein-binding function of the protein, including the lozengine repeat. In this regard, Kobe and Deisenhofer, Trends Biochem. Sci. , 19 (10): 415-421 (Oct. 1994).
[181] According to one study report, the rich abundance of lutein proteoglycans functions as a tissue-forming body that directs and aligns collagen fibers during the development of an individual, and is involved in pathological processes such as wound healing, tissue repair, and tumor lipid formation [ Iozzo, RV, Crit. Rev. Biochem. Mol. Biol ., 32 (2): 141-174 (1997)). Other studies that have implicated leucine rich proteins in wound healing and tissue repair include De La Salle, C., who reported that mutations in the Louis X-rich motif in complexes associated with bleeding disorders, Bernard-Soullier syndrome, Et al., Vouv. Rev. Fr. Hematol . (Germany), 37 (4): 215-222 (1995)] and Chlemetson (KJ) thrombocytes reported to have a lutein rich repeat . Haemost . (Germany), 74 (1): 111-116 (July 1995). Another particularly interesting protein reported to have a lucin-rich repeat is the SLIT protein, which has been reported to be useful in the treatment of neurodegenerative diseases such as Alzheimer's disease and neuronal damage such as in Parkinson's disease, and in the diagnosis of cancer See Artvanistsakonas, S. and Rothberg, JM, WO9210518-A1, Yale University]. Other reports on the biological function of proteins with lozengesic repeats include Tayar, N. et al ., Mol. Cell Endocrinol. (Ireland), 125 (1-2): 65-70 (Dec. 1996)] (related to the gonadotropin receptor), Miura, Y. et al., Nippon Rinsho ): 1784-1789 (July 1996)] (related to apoptosis), Harris (PC) et al ., J. Am. Soc. Nephrol ., 6 (4): 1125-1133 (Oct. 1995)] (related to nephropathy) and degenerative growth factor β binding to the Lazolas Cancer Research Foundation reported to be involved in cancer treatment, wound healing and scar formation And WO 9110727A such as Ruoslahti (EI) of La Jolla Cancer Research Foundation.
[182] Thus, efforts to identify novel proteins with lozengine repeat moieties have been made by both industry and academia to better understand protein-protein interactions. Of particular interest is a protein that has a lutein rich repeat and is homologous to a known repeat lutein-rich repeat protein. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane-bound proteins with lozine-rich repeats. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996), and U.S. Patent No. 5,536,637).
[183] Here we describe the identification and characterization of novel polypeptides (referred to herein as PRO293) that are homologous to the leucine rich repeat protein.
[184] 39. PRO247
[185] Protein-protein interactions include complexes of receptors and antigens and signaling mechanisms. As is well known for the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be readily manipulated to control the specific consequences of their protein-protein interactions. Thus, the underlying mechanism of protein-protein interactions is of interest to the scientific and medical community.
[186] All proteins, including the lozengine repeat, are thought to be involved in protein-protein interactions. Lutein-rich repeats are short sequence motifs present in many proteins that differ in function and intracellular location. The crystal structure of the ribonuclease inhibitor protein reveals that the lozengine repeat is equivalent to the beta-alpha structural unit. These units form a parallel beta sheet with one side exposed to the solvent, resulting in a unique non-spherical shape. These two features have been found to be responsible for the protein-binding function of the protein, including the lozengine repeat. In this regard, Kobe and Deisenhofer, Trends Biochem. Sci. , 19 (10): 415-421 (Oct. 1994).
[187] According to one study report, the rich abundance of lutein proteoglycans functions as a tissue-forming body that directs and aligns collagen fibers during the development of an individual, and is involved in pathological processes such as wound healing, tissue repair, and tumor lipid formation [ Iozzo, RV, Crit. Rev. Biochem. Mol. Biol ., 32 (2): 141-174 (1997)). Other studies that have implicated leucine rich proteins in wound healing and tissue repair include De La Salle, C., who reported that mutations in the Louis X-rich motif in complexes associated with bleeding disorders, Bernard-Soullier syndrome, Et al., Vouv. Rev. Fr. Hematol . (Germany), 37 (4): 215-222 (1995)] and Chlemetson (KJ) thrombocytes reported to have a lutein rich repeat . Haemost . (Germany), 74 (1): 111-116 (July 1995). Another protein reported to have a lucin-rich repeat is the SLIT protein, which has been reported to be useful in the treatment of neurodegenerative diseases such as Alzheimer's disease, nerve damage in Parkinson's disease, and the like, and in the diagnosis of cancer See Artvanistsakonas, S. and Rothberg, JM, WO9210518-A1, Yale University]. Other reports on the biological function of proteins with lozengesic repeats include Tayar, N. et al ., Mol. Cell Endocrinol. (Ireland), 125 (1-2): 65-70 (Dec. 1996)] (related to the gonadotropin receptor), Miura, Y. et al., Nippon Rinsho ): 1784-1789 (July 1996)] (related to apoptosis), Harris (PC) et al ., J. Am. Soc. Nephrol ., 6 (4): 1125-1133 (Oct. 1995)] (related to nephropathy) and degenerative growth factor β binding to the Lazolas Cancer Research Foundation reported to be involved in cancer treatment, wound healing and scar formation And WO 9110727A such as Ruoslahti (EI) of La Jolla Cancer Research Foundation.
[188] Densin is a glycoprotein isolated from the brain with all the features of the adhesion molecule. He is highly concentrated at the synaptic region of the brain and is significantly expressed in the dendrites of the growing neurons. Densin is a member of the O-linked sialoglycoprotein as a member. Densin is associated with medically important processes such as regeneration. Given the physiological importance of synaptic processes and cell attachment mechanisms in vivo, efforts are now being made to identify new natural proteins involved in synaptic sites and cell adhesion. Tenshin is described in Kennedy, MB, Trends Neurosci . (England), 20 (6): 264 (1997) and Apperson et al ., J. Neurosci. , ≪ / RTI > 16 (21): 6839 (1996).
[189] Thus, efforts to identify novel proteins with lozengine repeat moieties have been made by both industry and academia to better understand protein-protein interactions. Of particular interest is a protein having a lutein-rich repeat and a protein homologous to a known protein having a lutein-rich repeat, such as KIAA0231 and densin. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane-bound proteins with lozine-rich repeats. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996), and U.S. Patent No. 5,536,637).
[190] Herein we describe the identification and characterization of novel polypeptides (referred to herein as PRO247) that are homologous to the leucine rich repeat protein.
[191] 40. PRO302, PRO303, PRO304, PRO307 and PRO343
[192] Proteases are enzyme proteins that are involved in a number of very important biological processes in mammals and in organisms other than mammals. Many other protease enzymes have been identified and characterized from organisms other than a variety of other mammals and mammals. Mammalian protease enzymes play an important role in many other biological processes including, for example, proteolysis, activation, regulation of activity or modulation of peptide hormone activity, and changes in the physical properties of proteins and enzymes.
[193] Regarding the important physiological role played by protease enzymes, efforts to identify novel natural protease analogues are now being made both by industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane bound receptor proteins. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996), and U.S. Patent No. 5,536,637). Herein we describe the identification of novel polypeptides (herein termed PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides) that are homologous to various protease enzymes.
[194] 41. PRO328
[195] The GLIP protein family is characterized by containing a zinc-finger protein that plays an important role in embryogenesis. This protein can function as a transcriptional regulatory protein and is known to be amplified in a subset of human tumors. The glioma-producing protein is structurally related to a group of plant disease-associated proteins. He is highly expressed in glioblastomas [Ellington, AD, et al. , Nature , 346 (1986), U.S. Patent No. 5,582,981 issued December 10,1996 and 5,322,801 issued June 21, : 818 (1990), Grindley, JC et al ., Dev. Biol. , 188 (2) : 337 (1997)], Marine (JC) et al ., Mech. Dev. , 63 (2) : 211 (1997)). The CRISP or cystine-rich secreted protein family is also a family of proteins structurally related to a group of plant disease-causing proteins (Schwidetzky, U., Biochem. J. , 321 : 325 (1997)], Pfisterer, P. Mol. Cell Biol. , 16 (11) : 6160 (1996)], Kratzschmar, J. Eur. J. Biochem. , 236 (3) : 827 (1996)). The present inventors herein describe the identification of novel polypeptides (designated herein as PRO328 polypeptides) that are homologous to GLIP and CRISP.
[196] 42. PRO335, PRO331 and PRO326
[197] Protein-protein interactions include complexes of receptors and antigens and signaling mechanisms. As is well known for the structural and functional mechanisms underlying protein-protein interactions, protein-protein interactions can be readily manipulated to control the specific consequences of their protein-protein interactions. Thus, the underlying mechanism of protein-protein interactions is of interest to the scientific and medical community.
[198] All proteins, including the lozengine repeat, are thought to be involved in protein-protein interactions. Lutein-rich repeats are short sequence motifs present in many proteins that differ in function and intracellular location. The crystal structure of the ribonuclease inhibitor protein reveals that the lozengine repeat is consistent with the beta-alpha structural unit. These units form a parallel beta sheet with one side exposed to the solvent, resulting in a unique non-spherical shape. These two features have been found to be responsible for the protein-binding function of the protein, including the lozengine repeat. In this regard, Kobe and Deisenhofer, Trends Biochem. Sci. , 19 (10): 415-421 (Oct. 1994).
[199] According to one study report, the rich abundance of lutein proteoglycans functions as a tissue-forming body that directs and aligns collagen fibers during the development of an individual, and is involved in pathological processes such as wound healing, tissue repair, and tumor lipid formation [ Iozzo, RV, Crit. Rev. Biochem. Mol. Biol ., 32 (2): 141-174 (1997)). Other studies that have shown that lewyx-rich proteins are involved in wound healing and tissue repair include De La Salle, C., et al., Who reported that mutations in the lysine-rich motif in complexes associated with bleeding disorders Bernard-Soullier syndrome Gt; Vouv. ≪ / RTI > Rev. Fr. Hematol . (Germany), 37 (4): 215-222 (1995)] and Chlemetson (KJ) thrombocytopenia reported to have a leucine rich repeat . Haemost . (Germany), 74 (1): 111-116 (July 1995)] and decolin that binds to the growth factor beta are reported to be involved in the treatment of cancer, wound healing and scar formation, La Jolla Cancer Research Foundation WO 9110727A such as Ruoslahti (EI) of Research Foundation. Insulin-like growth factor (IGF) has been shown to be useful in wound healing and related therapies that are associated with regrowth of tissues such as connective tissue, skin and bone, in promoting body growth and other growth-related processes in humans and animals The function of the above group is related. In addition, the subunit (ALS) of the IGF that is unstable to the acid is interesting in that it increases the half-life of IGF and is part of the IGF complex in vivo.
[200] Another protein reported to have a lucin-rich repeats is the SLIT protein, which has been reported to be useful in the treatment of neurodegenerative diseases such as Alzheimer's disease and in nerve damage such as in Parkinson's disease, and in the diagnosis of cancer Artavanistsakonas, S. and Rothberg, JM, WO9210518-A1, Yale University]. Of particular interest is LIG-1, a membrane glycoprotein that is specifically expressed in glial glia of the mouse brain and has a leucine rich repeat and an immunoglobulin-like domain [Suzuki et al. Biol. Chem. (US), 271 (37): 22522 (1996)]. Other research reports on the biological function of proteins with lozengesic repeats include Tayar, N. et al ., Mol. Cell Endocrinol. (Ireland), 125 (1-2): 65-70 (Dec. 1996)] (related to the gonadotropin receptor), Miura, Y. et al., Nippon Rinsho ): 1784-1789 (July 1996)] (related to apoptosis), and Harris (PC) et al ., J. Am. Soc. Nephrol ., 6 (4): 1125-1133 (Oct. 1995)] (related to kidney disease).
[201] Thus, efforts to identify novel proteins with lozengine repeat moieties have been made by both industry and academia to better understand protein-protein interactions. Of particular interest is a protein that has a lutein-rich repeat and is homologous to a known protein having a lutein-rich repeat, such as LIG-1, ALS, and decolin. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane-bound proteins with lozine-rich repeats. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996), and U.S. Patent No. 5,536,637).
[202] Herein we describe the identification and characterization of novel polypeptides (referred to herein as PRO335, PRO331, and PRO326 polypeptides) that are homologous to a leucine rich repeat macrophage protein.
[203] 43. PRO332
[204] Secretory proteins comprising an abdomen characterized by an arrangement of conserved lysine residues (leucine-rich repeat motif) have diverse biological roles. Several proteoglycans, such as biglycans, pibromodulin and decolin, are characterized, for example, by the presence of a leucine-rich repeating moiety consisting of about 24 amino acids (Ruoslahti, Ann. Rev. Cell. Biol. 4 229-255 (1988), Oldberg et al. EMBO J. 8 , 2601-2604 (1989)]. In general, proteoglycans are thought to play a role in regulating extracellular matrix, cartilage and bone function. Proteoglycan decolin binds collagen types I and II and affects the formation rate of the fibrils. In addition, pibromodulin combines with collagen to delay fibril formation. Fibromodulin and decolin all inhibit the activity of transforming growth factor beta (TGF-β) [US Pat. No. 5,583,103, published December 10, 1996]. TGF-beta is known to play a central role in the induction of extracellular matrix and is involved in the expansion of fibrotic diseases such as cancer and glomerulonephritis. Therefore, proteoglycans are proposed for the treatment of fibrotic cancer, based on their ability to inhibit the growth-promoting activity of TGF-beta on cancer cells. The proteoglycans may also be used in the treatment of rheumatoid arthritis, atherosclerosis, adult respiratory distress syndrome, cirrhosis, pulmonary fibrosis, post-myocardial infarction, cardiac fibrosis, restenosis after angioplasty, renal fibrosis, Or other proliferative diseases including some dermal fibrosis symptoms such as keloid and scar formation which may result from restoration surgery [US Pat. No. 5,654,270, published on August 5, 1997 ].
[205] The present inventors herein describe the identification and characterization of novel polypeptides (designated herein as PRO332 polypeptides) that are homologous to a leucine rich repeat macrolide protein.
[206] 44. PRO334
[207] Microfibril bundles and proteins, especially adhesion molecules, found with this bundle are of interest in the field of dermatology, particularly in skin studies that have been damaged by aging, scarring or sunlight. Fibriline microfibrils form a resilient, continuous network of skin and are a measurable elastin-free microfibril bundle extending from the skin-epithelial junction, present in the dermis as a component of thick elastic fibers present in the deep mesangial dermis . In addition, Marfan syndrome is associated with mutations that interfere with the polymerization of fibrillar monomers or other connective tissue elements.
[208] Pibulin-1 is a modular glycoprotein having four possible carboxyl terminal ends with an amino-terminal terminal anapatoxin-like module followed by nine epidermal growth factor (EGF) -like modules and with selective splicing. Pibulin-2 is a novel extracellular matrix protein that appears to be tightly bound to microfibrils, often including fibronectin or fibrillar. Thus, it is of particular interest to use fibrillin, pibulin, and related molecules to prevent skin damage, particularly from aging, wounds or sunlight, or to restore damaged skin therefrom. In addition, such molecules are generally of interest in the study of connective tissue and adhesion molecules, and related mechanisms. Fibrillin, piburin and related molecules are described by Adams et al ., J. Mol. Biol ., 272 (2): 226-36 (1997)], Kielty and Shuttleworth, Microsc . Res. Tech ., 38 (4): 413-27 (1997)) and Child, J. Card. Surg,. 12 (2Supp.): 131-5 (1997).
[209] At present, efforts are being made by both industry and academia to identify new natural secretion and membrane-bound receptor proteins, particularly secretory proteins that are homologous to piburin and fibrin. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane bound receptor proteins. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996), and U.S. Patent No. 5,536,637).
[210] Herein we describe the identification and characterization of novel polypeptides (referred to herein as PRO334 polypeptides) that are homologous to pibuline and fibril.
[211] 45. PRO346
[212] The widespread cancer outbreak is fueling considerable resource consumption and the discovery of new therapeutic drugs. One particular method involves the production of tumor or cancer specific monoclonal antibodies (mAbs) specific for tumor antigens. These mAbs, which can distinguish normal cells from cancer cells, are useful in the diagnosis, prediction and treatment of disease. Certain antigens are known to be associated with tumor diseases, such as rectal colon cancer and breast cancer. Because colon cancer is a widespread disease, early diagnosis and treatment are important medical goals. Thus, the diagnosis and treatment of cancer can be performed using a specific monoclonal antibody (mAb) having a fluorescent, nuclear magnetic or radioactive tag. A radioactive gene, toxin and / or drug-tagged mAb can be used to treat with a minimum patient rating.
[213] Fetal cancer antigen (CEA) is a glycoprotein found in the digestive organs of human colon cancer and two to six month old embryos. CEA is a known human tumor marker and is widely used for the diagnosis of tumor diseases such as colon cancer. For example, when the patient's CEA serum level is elevated, a reduction in post-operative CEA levels indicates that tumor resection is successful. On the other hand, an increase in serum CEA concentration after surgery suggests that the original tumor may be metastasized or a new primary tumor may develop. CEA may also be the target of mAbs and antisense nucleotides.
[214] 46. PRO268
[215] Protein disulfide isomerase is an enzyme protein involved in promoting correct refolding of proteins through establishment of accurate disulfide bond formation. Protein disulfide isomerase was first identified based on its ability to promote rearrangement of the modified modified RNAse (Goldberger et al ., J. Biol. Chem. 239: 1406-1410 (1964) and Epstein et al ., Cold Spring Harbor Symp. Quant. Biol. 28: 439-449 (1963)]. Protein disulfide isomerase is an enzyme present in the endoplasmic reticulum retained in the endoplasmic reticulum via the -KDEL or -HDEL amino acid sequence at its C-terminal end.
[216] Considering the importance of the disulfide bond-forming enzyme and its potential use in increasing the yield of many different fields, for example, the correct refolding of recombinantly produced proteins, it has been found that homology with the protein disulfide isomerase Efforts to identify new natural proteins that are present are now being made by both industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel protein disulfide isomerase analogs. The present inventors herein describe a novel polypeptide (designated PRO268 herein) that is homologous to a protein disulfide isomerase.
[217] 47. PRO330
[218] Prolyl 4-hydroxylase is an enzyme that acts on post-translational hydroxylated proline residues at the Y position of the amino acid sequence Gly-XY, which is a sequence of three repeating amino acids found in both collagen and procollagen. Hydroxylation of the proline residue at the Y-position of the Gly-XY amino acid triplet should form a 4-hydroxylproline residue at this position before the newly synthesized collagen polypeptide chain folds into its appropriate three-dimensional triple-helix form . If hydroxylation does not occur, then the synthesized collagen polypeptide remains non-spiral, less secreted by the cell, and can not be assembled into stable functional collagen fibrils (Vuorio et al ., Proc. Natl. Acad. Sci. USA 89: 7467-7470 (1992)]. The prolyl 4-hydroxylase consists of at least two different polypeptide subunits, alpha and beta.
[219] Efforts to identify novel secreted and membrane-bound receptor proteins have been made by both industry and academia. Much of this effort has focused on screening mammalian recombinant DNA libraries to identify coding sequences for novel secretion and membrane bound receptor proteins. Examples of screening methods and techniques are described, for example, in Klein et al ., Proc. Natl. Acad. Sci. , 93 : 7108-7113 (1996), and U.S. Patent No. 5,536,637). Based on this effort, the present inventors have identified and described a novel polypeptide (designated herein as PRO330) that is homologous to the alpha subunit of prolyl 4-hydroxylase.
[220] 48. PRO339 and PRO310
[221] Fringe is a protein that specifically blocks the three-rate-mediated activation of Notch at the dorsal site of a virtual disk of a floss wing (Fleming et al., Development, 124 (15): 2973- 81 (1997)]. Thus, fringe is of interest both in its role in development and in its ability to regulate serum levels, particularly the signaling ability of the serum. Also of interest are new polypeptides that can play a role in development and / or regulation of serrate-like molecules. In particular, novel polypeptides (referred to herein as PRO339 and PRO310 polypeptides) that are homologous to the fringes identified and described herein are of interest.
[222] 49. PRO244
[223] Lectins are a class of proteins that contain regions that specifically and non-covalently bind carbohydrates. Many lectins with membrane binding and solubility have been identified in higher animals and have been associated with various cell-recognition phenomena and tumor metastasis.
[224] Most lectins can be classified as C-type (calcium-dependent) or S-type (thiol-dependent).
[225] Lectins are thought to play a role in regulating cellular phenomena that occur at the level of the plasma membrane. For example, molecules bound to the plasma membrane are involved in the activation of various subsets of lymphoid cells, such as T-lymphocytes, and cell surface molecules are known to activate these cells and react them during the immune response .
[226] Selectin, a specific group of cell adhesion molecules, belongs to a large family of C-type lectins. These groups include L-selectin (pnHR), LEC-CAM-1, LAM-1, gp90 ^MEL , gp100 ^MEL , gp110 ^MEL , MEL-14 antigen, Leu-8 antigen, TQ- (LEC-CAM-3), E-selectin (LEC-CAM-2, LECAM-2, ELAM-1) and P-selectin (LEC-CAM-3, LECAM-3, GMP-140 and PADGEM). The structure of selectin consists of the C-type lectin (carbohydrate binding) domain, epidermal growth factor-like (EGF-like) motifs, and variable complement control (CR) motifs. Selectin can be used to inhibit leukocyte adhesion, e. G., The attachment of neutrophils to venous epithelial cells near the site of inflammation (E-selectin) or the transport of lymphocytes from the blood into second lymphatic organs such as lymph nodes and Peyer's patches L-selectin).
[227] An example of another lectin is Mac-2, a cell-binding macrophage antigen thought to be involved in cell adhesion and immune responses. Macrophages also express lectins that recognize Tn Ag, a human cancer-associated epitope.
[228] Another C-type lectin is CD95 (Fas antigen / APO-1), an important modulator of immunologically modulated or programmed cell death (apoptosis). &Quot; Apoptosis " is a non-necrotic apoptosis that occurs after the activation of a unique apoptotic program in the remnant cells. Cloning of Fas antigens is described in PCT Publication No. WO 91/10448 and European Patent No. EP 510691. The mature Fas molecule is composed of 319 amino acids, 157 of which are extracellular amino acids, 17 are transmembrane domains, and 145 are intracellular amino acids. Increased levels of Fas expression on T-cell surfaces are associated with tumor cells and HIV-infected cells. Ligation of CD95 initiates apoptosis in the presence of interleukin-1 (IL-2). In addition, C-type lectins include receptors for oxidized low density lipoprotein (LDL). This suggests a possible role in the development of atherosclerosis.
[229] The present inventors herein describe the identification and characterization of novel polypeptides (referred to herein as PRO244 polypeptides) that are homologous to C-type lectins.
[230] SUMMARY OF THE INVENTION [
[231] 1. PRO211 and PRO217
[232] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as "PRO211" and "PRO217" polypeptides) that is homologous to EGF.
[233] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO211 or PRO217 polypeptide. In one aspect, the isolated nucleic acid comprises an EGF-like homologue of Figure 2 (SEQ ID NO: 2) and / or Figure 4 (SEQ ID NO: 4) shown in Figure 1 (SEQ ID NO: PRO211 and PRO217 polypeptides, or are complementary to such coding nucleic acid sequences and are stably associated therewith, at least under appropriate conditions, optionally under stringent conditions.
[234] In another embodiment, the invention provides isolated PRO211 and PRO217 EGF-like homologues PRO211 and PRO217 polypeptides. In particular, the invention provides isolated natural sequences PRO211 and PRO217 EGF-like (SEQ ID NO: 2) comprising an amino acid sequence comprising residues 1 to 353 of Figure 2 (SEQ ID NO: 2) or residues 1 to 379 of Figure 4 Thereby providing a homologue.
[235] 2. PRO230
[236] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO230 ".
[237] In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding a PRO230 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO230 polypeptide having amino acid residues 1 to 467 of Figure 6 (SEQ ID NO: 12) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[238] In another embodiment, the invention provides an isolated PRO230 polypeptide. In particular, the invention provides an isolated native sequence PRO230 polypeptide comprising an amino acid sequence comprising residues 1 through 467 of Figure 6 (SEQ ID NO: 12) in one embodiment.
[239] In another embodiment, the invention provides an expressed sequence tag (EST) comprising a nucleotide sequence of SEQ ID NO: 13 (Figure 7) and referred to herein as PRO20088.
[240] 3. PRO232
[241] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO232 ".
[242] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO232 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO232 polypeptide having amino acid residues 1-114 of Figure 9 (SEQ ID NO: 18) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[243] In another embodiment, the invention provides an isolated PRO232 polypeptide. In particular, the invention provides an isolated native sequence PRO232 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1-114 of Figure 9 (SEQ ID NO: 18).
[244] 4. PRO187
[245] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO187 ".
[246] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO187 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO187 polypeptide of Figure 11 (SEQ ID NO: 23) or is complementary to such a coding nucleic acid sequence and is at least in a stable condition, optionally under stringent conditions, have. In another aspect, the invention provides a nucleic acid comprising a coding sequence of Figure 10 (SEQ ID NO: 22), or a complement thereof. In another aspect, the invention provides a nucleic acid of the full-length protein of clone DNA27864-1155 deposited at ATCC Deposit No. 209375, or a coding sequence of clone DNA27864-1155 deposited at ATCC Deposit No. 209375.
[247] In another embodiment, the invention provides an isolated PRO187 polypeptide. In particular, the invention provides an isolated native sequence PRO187 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 205 of Figure 11 (SEQ ID NO: 23). Alternatively, the invention provides a polypeptide encoded by a nucleic acid deposited with ATCC Deposit No. 209375.
[248] 5. PRO265
[249] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO265 ".
[250] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO265 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO265 polypeptide having amino acid residues 1 to 660 of Figure 13 (SEQ ID NO: 28) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[251] In another embodiment, the invention provides an isolated PRO265 polypeptide. In particular, the invention provides an isolated native sequence PRO265 polypeptide comprising an amino acid sequence comprising residues 1 through 660 of Figure 13 (SEQ ID NO: 28) in one embodiment. A further embodiment of the invention relates to the isolated extracellular domain of a PRO265 polypeptide.
[252] 6. PRO219
[253] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO219 ".
[254] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO219 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO219 polypeptide having amino acid residues 1 to 915 of Figure 15 (SEQ ID NO: 34) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[255] In another embodiment, the invention provides an isolated PRO219 polypeptide. In particular, the invention provides an isolated native sequence PRO219 polypeptide comprising an amino acid sequence comprising residues 1 through 915 of Figure 15 (SEQ ID NO: 34) in one embodiment.
[256] 7. PRO246
[257] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO246 ".
[258] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO246 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO246 polypeptide having amino acid residues 1 to 390 of Figure 17 (SEQ ID NO: 39) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[259] In another embodiment, the invention provides an isolated PRO246 polypeptide. In particular, the invention provides an isolated native sequence PRO246 polypeptide comprising an amino acid sequence comprising residues 1 through 390 of Figure 17 (SEQ ID NO: 39) in one embodiment. A further embodiment of the invention relates to the isolated extracellular domain of a PRO246 polypeptide.
[260] 8. PRO228
[261] We have found a cDNA clone that encodes a novel polypeptide (designated herein as " PRO228 ") that is homologous to CD97, EMR1 and latrophylline.
[262] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO228 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO228 polypeptide having amino acid residues 1 to 690 of Figure 19 (SEQ ID NO: 49) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[263] In another embodiment, the invention provides isolated PRO228 polypeptides. In particular, the invention provides an isolated native sequence PRO228 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 690 of Figure 19 (SEQ ID NO: 49). A further embodiment of the invention relates to the isolated extracellular domain of a PRO228 polypeptide.
[264] In another embodiment, the invention provides an expressed sequence tag (EST) comprising the nucleotide sequence of SEQ ID NO: 50 and referred to herein as PRO21951.
[265] 9. PRO533
[266] We have found a cDNA clone (DNA49435-1219) which encodes a novel polypeptide referred to herein as PRO533.
[267] (A) a DNA molecule encoding a PRO533 polypeptide comprising a sequence of amino acids 23 to 216 of Figure 22; or (b) a complement of a DNA molecule of (a). In one embodiment, RTI ID = 0.0 > 80% < / RTI > sequence identity. Sequence identity is preferably about 85%, more preferably about 90%, and most preferably about 95%. In one aspect, the isolated nucleic acid is at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 80%, more preferably at least about 90% Has about 95% or more sequence identity. Preferably, the region of highest degree of sequence identity is the secreted portion (Figure 22, amino acids 23 to 216 of SEQ ID NO: 59). In another embodiment, the isolated nucleic acid molecule comprises DNA that encodes a PRO533 polypeptide having amino acid residues 1 to 216 of Figure 22 (SEQ ID NO: 59), or is complementary to such a coding nucleic acid sequence and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI > In another aspect, the invention provides a nucleic acid of the full-length protein of clone DNA 49435-1219 deposited with ATCC Accession No. 209480.
[268] In another embodiment, the invention provides an isolated PRO533 polypeptide. In particular, the invention provides an isolated native sequence PRO533 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 23 to 216 of Figure 22 (SEQ ID NO: 59). Specifically, natural PRO533 polypeptide with or without the native signal sequence (amino acids 1 to 22 of FIG. 22 (SEQ ID NO: 59)) and native methionine-containing or free native PRO533 polypeptide are included. Alternatively, the present invention provides a PRO533 polypeptide encoded by a nucleic acid deposited with ATCC Accession No. 209480.
[269] 10. PRO245
[270] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO245 ".
[271] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO245 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO245 polypeptide having amino acid residues 1 to 312 of Figure 24 (SEQ ID NO: 64) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[272] In another embodiment, the invention provides an isolated PRO245 polypeptide. In particular, the invention provides an isolated native sequence PRO245 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 312 of Figure 24 (SEQ ID NO: 64).
[273] 11. PRO220, PRO221 and PRO227
[274] We have found a cDNA clone that encodes a novel polypeptide, each having a lucine rich repeat. These polypeptides are referred to herein as PRO220, PRO221 and PRO227.
[275] In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding PRO220, PRO221 and PRO227, respectively. In one aspect, the present application is directed to a nucleic acid sequence encoding a PRO220 polypeptide having amino acid residues 1 to 708 of Figure 26 (SEQ ID NO: 69), or a sequence encoding a PRO220 polypeptide complementary to such a coding nucleic acid sequence, To provide an isolated nucleic acid that is stably bound. The present invention also encompasses a DNA encoding a PRO221 polypeptide having amino acid residues 1 to 259 of Figure 28 (SEQ ID NO: 71), or comprises or is complementary to such a coding nucleic acid sequence and is stable at least under appropriate, Lt; RTI ID = 0.0 > isolated < / RTI > nucleic acid. Further, the present application is intended to include a DNA encoding a PRO227 polypeptide having amino acid residues 1 to 620 of Figure 30 (SEQ ID NO: 73), or alternatively, complementary to such a coding nucleic acid sequence and being stable at least under appropriate, Lt; RTI ID = 0.0 > isolated < / RTI > nucleic acid.
[276] In another embodiment, the present invention provides isolated PRO220, PRO221 and PRO227 polypeptides. In particular, the disclosure provides isolated natural sequences for PRO220 polypeptides comprising an amino acid sequence comprising residues 1 through 708 of Figure 26 (SEQ ID NO: 69) in one embodiment. Also provided herein are isolated natural sequences for PRO221 polypeptides comprising an amino acid sequence comprising residues 1 through 259 of Figure 28 (SEQ ID NO: 71) in one embodiment. In addition, this disclosure provides an isolated natural sequence for a PRO227 polypeptide comprising an amino acid sequence comprising residues 1 through 620 of Figure 30 (SEQ ID NO: 73) in one embodiment.
[277] 12. PRO258
[278] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO258 ") that is homologous to CRTAM and a poliovirus receptor precursor.
[279] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO258 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO258 polypeptide having amino acid residues 1 to 398 of Figure 32 (SEQ ID NO: 84) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[280] In another embodiment, the invention provides an isolated PRO258 polypeptide. In particular, the invention provides an isolated native sequence PRO258 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 398 of Figure 32 (SEQ ID NO: 84). A further embodiment of the invention relates to the isolated extracellular domain of a PRO258 polypeptide.
[281] 13. PRO266
[282] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO266 ".
[283] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO266 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO266 polypeptide having amino acid residues 1 to 696 of Figure 34 (SEQ ID NO: 91) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[284] In another embodiment, the invention provides an isolated PRO266 polypeptide. In particular, the invention provides an isolated native sequence PRO266 polypeptide comprising an amino acid sequence comprising residues 1 through 696 of Figure 34 (SEQ ID NO: 91) in one embodiment.
[285] 14. PRO269
[286] We have found a cDNA clone that encodes a novel polypeptide referred to herein as PRO269.
[287] In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding a PRO269 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO269 polypeptide having amino acid residues 1 to 490 of Figure 36 (SEQ ID NO: 96) and is at least in a suitable condition, optionally in a highly stringent condition Lt; RTI ID = 0.0 > stable < / RTI >
[288] In another embodiment, the invention provides an isolated PRO269 polypeptide. In particular, the invention provides an isolated native sequence PRO269 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 490 of Figure 36 (SEQ ID NO: 96). A further embodiment of the invention is directed to the isolated extracellular domain of a PRO269 polypeptide.
[289] 15. PRO287
[290] We have found a cDNA clone that encodes a novel polypeptide referred to herein as PRO " 287 ".
[291] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO287 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding a PRO287 polypeptide having amino acid residues 1 to 415 of Figure 38 (SEQ ID NO: 104), or is complementary to such a coding nucleic acid sequence and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[292] In another embodiment, the invention provides an isolated PRO287 polypeptide. In particular, the invention provides an isolated native sequence PRO287 polypeptide comprising an amino acid sequence comprising residues 1 through 415 of Figure 38 (SEQ ID NO: 104) in one embodiment.
[293] 16. PRO214
[294] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO214 ".
[295] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO214 polypeptide. In one aspect, the isolated nucleic acid comprises DNA encoding the PRO214 polypeptide of FIG. 40 (SEQ ID NO: 109), or is complementary to such a coding nucleic acid sequence and is at least under stable conditions, optionally under stringent conditions, have. In another aspect, the invention provides a nucleic acid comprising a coding sequence of Figure 39 (SEQ ID NO: 108), or a complement thereof. In another aspect, the invention provides a nucleic acid of the full-length protein of clone DNA 32286-1191 deposited at ATCC Accession No. 209385.
[296] In another embodiment, the invention provides an isolated PRO214 polypeptide. In particular, the invention provides an isolated native sequence PRO214 polypeptide comprising, in one embodiment, an amino acid sequence comprising the residue of Figure 40 (SEQ ID NO: 109). Alternatively, the present invention provides a polypeptide encoded by the nucleic acid deposited at ATCC Deposit No. 209385.
[297] 17. PRO317
[298] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO317 ".
[299] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO317 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO317 polypeptide having the amino acid residues 1 to 366 of Figure 42 (SEQ ID NO: 113), or is complementary to such a coding nucleic acid sequence and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[300] In another embodiment, the invention provides an isolated PRO317 polypeptide. In particular, the invention provides isolated natural-sequence PRO317 polypeptides comprising an amino acid sequence comprising residues 1 through 366 of Figure 42 (SEQ ID NO: 114) in one embodiment.
[301] In another embodiment, the invention provides a method of detecting the presence of PRO317 in a sample comprising: a) contacting a detectable anti-PRO317 antibody with a sample suspected of comprising PRO317 and b) Detecting the binding of the antibody to a sample selected from the group consisting of blood, body fluids, tissue samples, cell extracts, and cell culture media.
[302] In another embodiment, there is provided a method of determining the presence of PRO317 mRNA in a sample comprising: a) contacting a sample suspected of containing PRO317 mRNA with a detectable nucleic acid that hybridizes with PRO317 mRNA under appropriate conditions or stringency conditions Contacting the probe with the probe and b) detecting hybridization of the probe to the sample.
[303] Preferably, in this method the sample is a tissue sample and the detection step is by in situ hybridization, or the sample is a cell extract and the detection is by Northern analysis.
[304] The present invention also relates to a method of treating a PRO317-related disorder, comprising administering to a mammal an effective amount of an antibody that specifically binds to a PRO317 polypeptide or composition thereof, or a PRO317 agonist or a PRO317 antagonist, such as PRO317 Provide a treatment method.
[305] 18. PRO301
[306] We have found a cDNA clone (DNA40628-1216) encoding a novel polypeptide herein referred to as " PRO301 ".
[307] (A) a DNA molecule encoding a PRO301 polypeptide comprising a sequence of amino acids 28 to 258 of Figure 44 (SEQ ID NO: 119), or (b) a complement of a DNA molecule of RTI ID = 0.0 > 80% < / RTI > sequence identity. Sequence identity is preferably about 85%, more preferably about 90%, and most preferably about 95%. In one aspect, the isolated nucleic acid is at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 80%, more preferably at least about 90% Has about 95% or more sequence identity. Preferably, the highest degree of sequence identity is the extracellular domain (Figure 44, amino acids 28 to 258 of SEQ ID NO: 119). In another embodiment, the isolated nucleic acid molecule comprises or is complementary to a DNA encoding a PRO301 polypeptide having amino acid residues 28 to 299 of Figure 44 (SEQ ID NO: 119) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI > In another aspect, the invention provides a nucleic acid of the full-length protein of clone DNA 40628-1216 deposited at ATCC Accession No. 209432, or a coding sequence of clone DNA 40628-1216 deposited at ATCC Accession No. 209432.
[308] In another embodiment, the invention provides an isolated PRO301 polypeptide. In particular, the present invention provides an isolated native sequence PRO301 polypeptide comprising an amino acid sequence comprising extracellular domain residues 28 to 258 of Figure 44 (SEQ ID NO: 119) in one embodiment. Specifically, natural PRO301 polypeptide with or without the native signal sequence (amino acids 1 to 27 of FIG. 44 (SEQ ID NO: 119)) and native PRO301 polypeptide with or without initiation methionine are included. 44, residues 236 to about 258 of SEQ ID NO: 119) and / or the intracellular domain (Figure 44, residues 259 to about 299 of SEQ ID NO: 119). Alternatively, the present invention provides a PRO301 polypeptide encoded by a nucleic acid deposited with ATCC Accession No. 209432.
[309] 19. PRO224
[310] We have found a cDNA clone that encodes a novel polypeptide herein referred to as " PRO224 ".
[311] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO224 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO224 polypeptide having amino acid residues 1 to 282 of Figure 46 (SEQ ID NO: 127) and is at least in a suitable condition, optionally in a high stringency condition Lt; RTI ID = 0.0 > stable < / RTI >
[312] In another embodiment, the invention provides an isolated PRO224 polypeptide. In particular, the invention provides an isolated native sequence PRO224 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 282 of Figure 46 (SEQ ID NO: 127).
[313] 20. PRO222
[314] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO222 ".
[315] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO222 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO222 polypeptide having amino acid residues 1 to 490 of Figure 48 (SEQ ID NO: 132) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[316] In another embodiment, the invention provides an isolated PRO222 polypeptide. In particular, the invention provides an isolated native sequence PRO222 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 490 of Figure 48 (SEQ ID NO: 132).
[317] 21. PRO234
[318] We have found a cDNA clone that encodes a novel lectin polypeptide molecule referred to herein as " PRO234 ".
[319] In one embodiment, the invention provides an isolated nucleic acid encoding a novel lectin comprising DNA encoding a PRO234 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO234 polypeptide having amino acid residues 1 to 382 of Figure 50 (SEQ ID NO: 137) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI > In another aspect, the invention provides an isolated nucleic acid molecule comprising the nucleotide sequence of Figure 49 (SEQ ID NO: 136).
[320] In another embodiment, the invention provides isolated novel PRO234 polypeptides. In particular, the invention provides an isolated native sequence PRO234 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 382 of Figure 50 (SEQ ID NO: 137).
[321] In another embodiment, the invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences.
[322] 22. PRO231
[323] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO231 ") that is homologous to the putative acid phosphatase.
[324] In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding a PRO231 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO231 polypeptide having amino acid residues 1 to 428 of Figure 52 (SEQ ID NO: 142) and is at least in a suitable condition, optionally in a high stringency condition Lt; RTI ID = 0.0 > stable < / RTI >
[325] In another embodiment, the invention provides an isolated PRO231 polypeptide. In particular, the invention provides an isolated native sequence PRO231 polypeptide comprising an amino acid sequence comprising residues 1 through 428 of Figure 52 (SEQ ID NO: 142) in one embodiment.
[326] 23. PRO229
[327] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO229 ") that is homologous to a scavenger receptor.
[328] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO229 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO229 polypeptide having amino acid residues 1 to 347 of Figure 54 (SEQ ID NO: 148) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[329] In another embodiment, the invention provides an isolated PRO229 polypeptide. In particular, the invention provides an isolated native sequence PRO229 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 347 of Figure 54 (SEQ ID NO: 148).
[330] 24. PRO238
[331] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO238 ") that is homologous to a reductase.
[332] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO238 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO238 polypeptide having amino acid residues 1 to 310 of Figure 56 (SEQ ID NO: 153) and is at least in a suitable condition, optionally in a high stringency condition Lt; RTI ID = 0.0 > stable < / RTI >
[333] In another embodiment, the invention provides an isolated PRO238 polypeptide. In particular, the present invention provides an isolated native sequence PRO238 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 310 of Figure 56 (SEQ ID NO: 153).
[334] 25. PRO233
[335] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO233 ".
[336] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO233 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO233 polypeptide having amino acid residues 1 to 300 of Figure 58 (SEQ ID NO: 159) and is at least in a suitable, Lt; RTI ID = 0.0 > stable < / RTI >
[337] In another embodiment, the present invention provides an isolated PRO233 polypeptide. In particular, the invention provides an isolated native sequence PRO233 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 300 of Figure 58 (SEQ ID NO: 159).
[338] 26. PRO223
[339] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO223 ") that is homologous to a serine carboxypeptidase polypeptide.
[340] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO223 polypeptide. In one aspect, the isolated nucleic acid comprises DNA that encodes a PRO223 polypeptide having residues 1 through 476 of Figure 60 (SEQ ID NO: 164), or is complementary to such a coding nucleic acid sequence and is capable of hybridizing under stringent conditions, And is stably coupled thereto.
[341] In another embodiment, the invention provides an isolated PRO223 polypeptide. In particular, the invention provides an isolated native sequence PRO223 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 476 of Figure 60 (SEQ ID NO: 164).
[342] 27. PRO235
[343] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO235 ".
[344] In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding a PRO235 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO235 polypeptide having amino acid residues 1 to 552 of Figure 62 (SEQ ID NO: 170) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[345] In another embodiment, the invention provides an isolated PRO235 polypeptide. In particular, the invention provides an isolated native sequence PRO235 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 552 of Figure 62 (SEQ ID NO: 170).
[346] 28. PRO236 and PRO262
[347] The present inventors have found a cDNA clone that encodes a novel polypeptide (referred to herein as "PRO236" and "PRO262") that is homologous to β-galactosidase.
[348] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO236 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO236 polypeptide having amino acid residues 1 to 636 of Figure 64 (SEQ ID NO: 175) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[349] In another embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO262 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO262 polypeptide having amino acid residues 1 to 654 of Figure 66 (SEQ ID NO: 177) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[350] In another embodiment, the invention provides an isolated PRO236 polypeptide. In particular, the invention provides, in one embodiment, an isolated native sequence PRO236 polypeptide comprising an amino acid sequence comprising residues 1 to 636 of Figure 64 (SEQ ID NO: 175).
[351] In another embodiment, the invention provides an isolated PRO262 polypeptide. In particular, the invention provides an isolated native sequence PRO262 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1-654 of Figure 66 (SEQ ID NO: 177).
[352] 29. PRO239
[353] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO239 ".
[354] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO239 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO239 polypeptide having amino acid residues 1 to 501 of Figure 68 (SEQ ID NO: 185) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[355] In another embodiment, the invention provides an isolated PRO239 polypeptide. In particular, the invention provides an isolated native sequence PRO239 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 501 of Figure 68 (SEQ ID NO: 185).
[356] 30. PRO257
[357] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO257 ".
[358] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO257 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO257 polypeptide having amino acid residues 1 to 607 of Figure 70 (SEQ ID NO: 190) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[359] In another embodiment, the invention provides an isolated PRO257 polypeptide. In particular, the present invention provides an isolated native sequence PRO257 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 607 of Figure 70 (SEQ ID NO: 190). A further embodiment of the invention relates to the isolated extracellular domain of a PRO257 polypeptide.
[360] 31. PRO260
[361] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO260 ".
[362] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO260 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO260 polypeptide having amino acid residues 1 to 467 of Figure 72 (SEQ ID NO: 195) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[363] In another embodiment, the invention provides an isolated PRO260 polypeptide. In particular, the invention provides an isolated native sequence PRO260 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 467 of Figure 72 (SEQ ID NO: 195).
[364] 32. PRO263
[365] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO263 ") that is homologous to the CD44 antigen.
[366] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO263 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO263 polypeptide having amino acid residues 1 to 322 of Figure 74 (SEQ ID NO: 201) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[367] In another embodiment, the invention provides an isolated PRO263 polypeptide. In particular, the invention provides an isolated native sequence PRO263 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 322 of Figure 74 (SEQ ID NO: 201). One further embodiment of the invention is directed to the isolated extracellular domain of a PRO263 polypeptide.
[368] 33. PRO270
[369] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO270 ".
[370] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO270 polypeptide. In one aspect, the isolated nucleic acid comprises or comprises a DNA comprising a sequence encoding a PRO270 polypeptide having amino acid residues 1 to 296 of Figure 76 (SEQ ID NO: 207), or is complementary to such a coding nucleic acid sequence, Optionally stably bonded thereto under high stringency conditions.
[371] In another embodiment, the invention provides an isolated PRO270 polypeptide. In particular, the invention provides an isolated native sequence PRO270 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 296 of Figure 76 (SEQ ID NO: 207).
[372] 34. PRO271
[373] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO271 ") that is homologous to the proteoglycan-linked protein.
[374] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO271 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO271 polypeptide having amino acid residues 1 to 360 of Figure 78 (SEQ ID NO: 213) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[375] In another embodiment, the invention provides an isolated PRO271 polypeptide. In particular, the invention provides an isolated native sequence PRO271 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 360 of Figure 78 (SEQ ID NO: 213).
[376] 35. PRO272
[377] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO272 ".
[378] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO272 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO272 polypeptide having amino acid residues 1 to 328 of Figure 80 (SEQ ID NO: 221) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[379] In another embodiment, the invention provides an isolated PRO272 polypeptide. In particular, the invention provides an isolated native sequence PRO272 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 328 of Figure 80 (SEQ ID NO: 211).
[380] 36. PRO294
[381] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO294 ".
[382] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO294 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO294 polypeptide having amino acid residues 1-550 of Figure 82 (SEQ ID NO: 227) and is at least in a suitable, Lt; RTI ID = 0.0 > stable < / RTI >
[383] In another embodiment, the invention provides an isolated PRO294 polypeptide. In particular, the invention provides an isolated native sequence PRO294 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1-550 of Figure 82 (SEQ ID NO: 227).
[384] 37. PRO295
[385] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO295 ".
[386] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO295 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO295 polypeptide having amino acid residues 1 to 350 of Figure 84 (SEQ ID NO: 236) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[387] In another embodiment, the invention provides an isolated PRO295 polypeptide. In particular, the invention provides an isolated native sequence PRO295 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 350 of Figure 84 (SEQ ID NO: 236).
[388] 38. PRO293
[389] We have found a cDNA clone that encodes a leucine rich repeat polypeptide of new human neurons, referred to herein as " PRO293 ".
[390] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO293 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO293 polypeptide having amino acid residues 1 to 713 of Figure 86 (SEQ ID NO: 245) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[391] In another embodiment, the invention provides an isolated PRO293 polypeptide. In particular, the invention provides an isolated native sequence PRO293 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 713 of Figure 86 (SEQ ID NO: 245). One further embodiment of the invention relates to the isolated extracellular domain of the PRO293 polypeptide.
[392] 39. PRO247
[393] We have found a cDNA clone that encodes a novel polypeptide having a lucine rich repeat and referred to herein as " PRO247 ".
[394] In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding a PRO247 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO247 polypeptide having amino acid residues 1 to 546 of Figure 88 (SEQ ID NO: 250) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[395] In another embodiment, the invention provides an isolated PRO247 polypeptide. In particular, the invention provides an isolated native sequence PRO247 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 546 of Figure 88 (SEQ ID NO: 250). One further embodiment of the invention relates to the isolated extracellular domain of the PRO247 polypeptide.
[396] 40. PRO302, PRO303, PRO304, PRO307 and PRO343
[397] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as "PRO302", "PRO303", "PRO304", "PRO307", and "PRO343" polypeptides) that is homologous to several proteases.
[398] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO302 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO302 polypeptide having amino acid residues 1 to 452 of Figure 90 (SEQ ID NO: 255) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[399] In another embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO303 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO303 polypeptide having amino acid residues 1 to 314 of Figure 92 (SEQ ID NO: 257) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[400] In another embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO304 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO304 polypeptide having amino acid residues 1 to 556 of Figure 94 (SEQ ID NO: 259) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[401] In another embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO307 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO307 polypeptide having amino acid residues 1 to 383 of Figure 96 (SEQ ID NO: 261) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[402] In another embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO343 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO343 polypeptide having amino acid residues 1 to 317 of Figure 98 (SEQ ID NO: 263) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[403] In another embodiment, the invention provides an isolated PRO302 polypeptide. In particular, the invention provides an isolated native sequence PRO302 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 452 of Figure 90 (SEQ ID NO: 255).
[404] In another embodiment, the invention provides an isolated PRO303 polypeptide. In particular, the invention provides an isolated native sequence PRO303 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 314 of Figure 92 (SEQ ID NO: 257).
[405] In another embodiment, the invention provides an isolated PRO304 polypeptide. In particular, the invention provides an isolated native sequence PRO304 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 556 of Figure 94 (SEQ ID NO: 259).
[406] In another embodiment, the invention provides an isolated PRO307 polypeptide. In particular, the invention provides an isolated native sequence PRO307 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 383 of Figure 96 (SEQ ID NO: 261).
[407] In another embodiment, the invention provides an isolated PRO343 polypeptide. In particular, the invention provides an isolated native sequence PRO343 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 317 of Figure 98 (SEQ ID NO: 263).
[408] 41. PRO328
[409] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO328 ".
[410] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO328 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO328 polypeptide having amino acid residues 1 to 463 of Figure 100 (SEQ ID NO: 285) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[411] In another embodiment, the invention provides isolated PRO328 polypeptides. In particular, the invention provides an isolated native sequence PRO328 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 through 463 of Figure 100 (SEQ ID NO: 285). One further embodiment of the invention is directed to the isolated extracellular domain of a PRO306 polypeptide.
[412] 42. PRO335, PRO331 and PRO326
[413] We have found three cDNA clones, each encoding a novel polypeptide having a lucine rich repeat and homologous to LIG-1 and ALS, respectively. These polypeptides are referred to herein as PRO335, PRO331 and PRO326, respectively.
[414] In one embodiment, the invention provides three isolated nucleic acid molecules each comprising DNA encoding PRO335, PRO331 and PRO326, respectively. In one aspect, the present application is directed to a nucleic acid sequence encoding a PRO335 polypeptide having amino acid residues 1 to 1059 of Figure 102 (SEQ ID NO: 290), comprising or encoding a PRO335 polypeptide complementary to such a coding nucleic acid sequence, To provide an isolated nucleic acid that is stably bound. Further, the present application is intended to include a DNA encoding a PRO331 polypeptide having amino acid residues 1 to 640 of Figure 104 (SEQ ID NO: 292), or a complementary to such a coding nucleic acid sequence and which is stable at least under appropriate conditions, Lt; RTI ID = 0.0 > isolated < / RTI > nucleic acid. The present application also includes DNA encoding PRO326 polypeptide having amino acid residues 1-1119 of Figure 106 (SEQ ID NO: 294), or is complementary to such a coding nucleic acid sequence and is stable at least under appropriate, Lt; RTI ID = 0.0 > isolated < / RTI > nucleic acid.
[415] In another embodiment, the invention provides isolated PRO335, PRO331 and PRO326 polypeptides, or an extracellular domain thereof. In particular, the invention provides an isolated natural sequence for a PRO335 polypeptide comprising an amino acid sequence comprising residues 1 to 1059 of Figure 102 (SEQ ID NO: 290) in one embodiment. In addition, the disclosure provides isolated natural sequences for a PRO331 polypeptide comprising an amino acid sequence comprising residues 1 through 640 of Figure 104 (SEQ ID NO: 292) in one embodiment. In addition, the disclosure provides isolated natural sequences for PRO326 polypeptides comprising an amino acid sequence comprising residues 1-1119 of Figure 106 (SEQ ID NO: 294) in one embodiment.
[416] 43. PRO332
[417] We have found a cDNA clone (DNA 40982-1235) encoding a novel polypeptide herein referred to as " PRO332 ".
[418] (A) a DNA molecule encoding a PRO358 polypeptide comprising the sequence of amino acids 49 to 642 of Figure 108 (SEQ ID NO: 310), or (b) a complement of a DNA molecule of RTI ID = 0.0 >% < / RTI > sequence identity. Sequence identity is preferably about 85%, more preferably about 90%, and most preferably about 95%. In one aspect, the isolated nucleic acid comprises at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 80%, more preferably at least about 90% Has about 95% or more sequence identity. Preferably, the highest sequence identity is the leucine-rich repeat domain (Figure 108, amino acids 116 to 624 of SEQ ID NO: 310). In another embodiment, the isolated nucleic acid molecule comprises or is complementary to DNA encoding a PRO332 polypeptide having amino acid residues 49 to 642 of Figure 108 (SEQ ID NO: 310) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[419] In another embodiment, the invention provides an isolated PRO332 polypeptide. In particular, the invention provides an isolated native sequence PRO332 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 49 to 624 of Figure 108 (SEQ ID NO: 310). Specifically, a native PRO332 polypeptide with or without a native signal sequence (Figure 108, amino acids 1 to 48 of SEQ ID NO: 310) and a native PRO332 polypeptide with or without an initiation methionine are included.
[420] 44. PRO334
[421] The present inventors have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO334 ") that is homologous to pibuline and fibrillin.
[422] In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding a PRO334 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO334 polypeptide having amino acid residues 1 to 509 of Figure 110 (SEQ ID NO: 315) and is at least in a suitable, Lt; RTI ID = 0.0 > stable < / RTI >
[423] In another embodiment, the invention provides an isolated PRO334 polypeptide. In particular, the invention provides an isolated native sequence PRO334 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 509 of Figure 110 (SEQ ID NO: 315).
[424] 45. PRO346
[425] We have found a cDNA clone (DNA44167-1243) which encodes a novel polypeptide herein referred to as " PRO346 ".
[426] (A) a DNA molecule encoding a PRO346 polypeptide comprising a sequence of amino acids 19 to 339 of Figure 112 (SEQ ID NO: 320), or (b) a complement of a DNA molecule of RTI ID = 0.0 >% < / RTI > sequence identity. Sequence identity is preferably about 85%, more preferably about 90%, and most preferably about 95%. In one aspect, the isolated nucleic acid is at least about 80%, preferably at least about 85%, more preferably at least about 90%, most preferably at least about 80%, more preferably at least about 90% Has about 95% or more sequence identity. Preferably, the highest sequence identity is the extracellular domain (Figure 112, amino acids 19 to 339 of SEQ ID NO: 320). In another embodiment, the polypeptide for which the homology is measured comprises residues 1 to 339, 19 to 360, or 19 to 450 of Figure 112 (SEQ ID NO: 320). In a further embodiment, the isolated nucleic acid molecule is a PRO346 polypeptide having amino acid residues 19 to 339 of Figure 112 (SEQ ID NO: 320), or residues 1 to 339, 19 to 360, or 19 to 450 of Figure 112 (SEQ ID NO: 320) Or is complementary to such a coding nucleic acid sequence and is stably associated with it under at least the appropriate conditions and optionally the high stringency conditions. In another aspect, the invention provides a nucleic acid of the full-length protein of clone DNA44167-1243 deposited at ATCC Accession No. 209434 or the coding sequence of clone DNA44167-1243 deposited at ATCC Accession No. 209434.
[427] In another embodiment, the invention provides an isolated PRO346 polypeptide. In particular, the invention provides an isolated native sequence PRO346 polypeptide comprising an amino acid sequence comprising residues 19 to 339 of Figure 112 (SEQ ID NO: 320) in one embodiment. Specifically, a native PRO346 polypeptide with or without an intrinsic signal sequence (residues 1 to 18 of Figure 112 (SEQ ID NO: 320)), a native PRO346 polypeptide with or without an initiation methionine, a transmembrane domain (residues 340 to 360) Native PRO346 polypeptide, and native PRO346 polypeptide with or without the intracellular domain (residues 361-450). Alternatively, the present invention provides a PRO346 polypeptide encoded by a nucleic acid deposited with ATCC Accession No. 209434.
[428] 46. PRO268
[429] The present inventors have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO268 ") that is homologous to a protein disulfide isomerase.
[430] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO268 polypeptide. In one aspect, the isolated nucleic acid comprises, or is complementary to, a DNA encoding a PRO268 polypeptide having amino acid residues 1-280 of Figure 114 (SEQ ID NO: 325) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[431] In another embodiment, the invention provides an isolated PRO268 polypeptide. In particular, the invention provides an isolated native sequence PRO268 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1-280 of Figure 114 (SEQ ID NO: 325). One further embodiment of the invention is directed to the isolated extracellular domain of the PRO268 polypeptide.
[432] 47. PRO330
[433] We have found a cDNA clone that encodes a novel polypeptide (referred to herein as " PRO330 ") that is homologous to the alpha subunit of prolyl 4-hydroxylase.
[434] In one embodiment, the present invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO330 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO330 polypeptide having amino acid residues 1 to 533 of Figure 116 (SEQ ID NO: 332) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[435] In another embodiment, the invention provides an isolated PRO330 polypeptide. In particular, the invention provides an isolated native sequence PRO330 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 533 of Figure 116 (SEQ ID NO: 332).
[436] 48. PRO339 and PRO310
[437] The present inventors have found two cDNA clones each encoding a novel polypeptide (referred to herein as "PRO339" and "PRO310") that is homologous to fringe.
[438] In one embodiment, the invention provides isolated nucleic acid molecules comprising DNA encoding PRO339 and / or PRO310 polypeptides. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO339 polypeptide having amino acid residues 1 to 772 of Figure 118 (SEQ ID NO: 339) and is at least in a suitable condition, optionally in a high stringency condition Lt; RTI ID = 0.0 > stable < / RTI > In another aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO310 polypeptide having amino acid residues 1 to 318 of Figure 120 (SEQ ID NO: 341) and is at least in a suitable condition, Lt; RTI ID = 0.0 > stable < / RTI >
[439] In another embodiment, the invention provides isolated PRO339 and isolated PRO310 polypeptides. In particular, the invention provides an isolated native sequence PRO339 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 772 of Figure 118 (SEQ ID NO: 339). The invention also provides an isolated native sequence PRO310 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 318 of Figure 120 (SEQ ID NO: 341).
[440] 49. PRO244
[441] We have found a cDNA clone that encodes a novel polypeptide referred to herein as " PRO244 ".
[442] In one embodiment, the invention provides an isolated nucleic acid molecule comprising DNA encoding a PRO244 polypeptide. In one aspect, the isolated nucleic acid comprises or is complementary to a DNA encoding a PRO244 polypeptide having amino acid residues 1 to 219 of Figure 122 (SEQ ID NO: 377) and is at least in a suitable condition, optionally in a high stringency condition Lt; RTI ID = 0.0 > stable < / RTI >
[443] In another embodiment, the invention provides an isolated PRO244 polypeptide. In particular, the invention provides an isolated native sequence PRO244 polypeptide comprising, in one embodiment, an amino acid sequence comprising residues 1 to 219 of Figure 122 (SEQ ID NO: 377).
[444] 50. Additional embodiments
[445] In another embodiment of the present invention, the present invention provides a vector comprising DNA encoding any of the above polypeptides. Also provided is a host cell comprising such an arbitrary vector. By way of example, the host cell may be a CHO cell, E. coli or yeast. Also provided is a method of producing any of the polypeptides described herein, comprising culturing the host cell under conditions suitable for expression of the polypeptide of interest and recovering the polypeptide of interest from the cell culture.
[446] In another embodiment, the invention provides chimeric molecules comprising any polypeptide described herein fused to a heterologous polypeptide or amino acid sequence. Examples of such chimeric molecules include epitope tag sequences or any of the polypeptides described herein fused to the Fc region of an immunoglobulin.
[447] In another embodiment, the invention provides an antibody that specifically binds to any of the above or below polypeptides. Optionally, the antibody is a monoclonal antibody, humanized antibody, antibody fragment or single chain antibody.
[448] In another embodiment, the present invention provides oligonucleotide probes useful for isolating genomic and cDNA nucleotide sequences, which probes can be derived from any of the above or below nucleotide sequences.
[449] In another embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a PRO polypeptide.
[450] In one aspect, the isolated nucleic acid molecule comprises (a) a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking a signal peptide as disclosed herein, or a signal peptide containing or no signaling peptide transporter protein as disclosed herein (B) a DNA molecule encoding a PRO polypeptide having an extracellular domain, or (b) a sequence identity of at least about 80%, preferably at least about 81%, more preferably at least about 82% , More preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86%, even more preferably at least about 87% Is at least about 88%, more preferably at least about 89%, more preferably at least about 90%, more preferably at least about 91%, even more preferably at least about 92%, even more preferably at least about 93% , , More preferably at least about 94%, more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97%, even more preferably at least about 98% 99% or more nucleotide sequence.
[451] In another aspect, the isolated nucleic acid molecule comprises (a) a coding sequence of a full-length PRO polypeptide cDNA as disclosed herein, a coding sequence of a PRO polypeptide lacking a signal peptide as disclosed herein, or a signal peptide Or (b) the complement of the DNA molecule of (a) is at least about 80%, preferably at least about 81% , More preferably at least about 82%, even more preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 85%, even more preferably at least about 86% , More preferably at least about 87%, more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91%, even more preferably at least about 92%, Preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97% 98% or more, and more preferably about 99% or more of the nucleotide sequence.
[452] (A) a DNA molecule encoding the same mature polypeptide as that encoded by any of the ATCC deposited human protein cDNAs as disclosed herein, or (b) a DNA molecule encoding a DNA molecule of More preferably at least about 82%, more preferably at least about 83%, even more preferably at least about 84%, even more preferably at least about 80%, more preferably at least about 80%, even more preferably at least about 82% More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90% , More preferably at least about 91%, more preferably at least about 92%, even more preferably at least about 93%, even more preferably at least about 94%, even more preferably at least about 95% Is at least about 96%, more preferably About 97%, more preferably at least about 98% to, more preferably, relates to an isolated nucleic acid molecule comprising a nucleotide sequence at least about 99%.
[453] In another aspect, the invention provides an isolated nucleic acid molecule comprising, or complementary to, a nucleotide sequence that encodes a PRO polypeptide having a transmembrane domain deleted or fused, wherein the transmembrane domain of the polypeptide comprises Lt; / RTI > Thus, the soluble extracellular domains of the PRO polypeptides described herein are contemplated.
[454] Yet another embodiment is a fragment of a PRO polypeptide coding sequence that can be used as, for example, a hybridization probe, or that encodes a fragment of a PRO polypeptide capable of optionally coding a polypeptide comprising a binding site for an anti-PRO antibody, . The length of such nucleic acid fragments will generally be at least about 20 nucleotides, preferably at least about 30 nucleotides, more preferably at least about 40 nucleotides, more preferably at least about 50 nucleotides, more preferably at least about 60 nucleotides Preferably at least about 70, more preferably at least about 80, more preferably at least about 90, more preferably at least about 100, even more preferably at least about 110, even more preferably at least about 120 More preferably at least about 130, more preferably at least about 140, more preferably at least about 150, even more preferably at least about 160, even more preferably at least about 170, More preferably at least about 190, more preferably at least about 200, more preferably at least about 250, even more preferably at least about 300, even more preferably at least about 350 More preferably about 400, , More preferably at least about 450, more preferably at least about 500, even more preferably at least about 600, even more preferably at least about 700, even more preferably at least about 800, About 900 or more, more preferably about 1000 or more, and the term " about " means a length obtained by adding or subtracting 10% of this length to the above-mentioned nucleotide sequence length. A new fragment of the PRO polypeptide-coding nucleotide sequence can be obtained by aligning the PRO polypeptide-coding nucleotide sequence and other known nucleotide sequences using any of a number of well-known sequence alignment programs to determine whether the PRO polypeptide- coding nucleotide sequence fragment is novel Or < / RTI > All such PRO polypeptide-coding nucleotide sequences are contemplated herein. Also contemplated are PRO polypeptide fragments encoded by this nucleotide molecule fragment, preferably PRO polypeptide fragments comprising a binding site for an anti-PRO antibody.
[455] In another embodiment, the invention provides isolated PRO polypeptides that are encoded by any of the isolated nucleic acid sequences identified above.
[456] In one particular aspect, the invention provides a method for detecting the presence or absence of a signal peptide-containing or non-containing transmembrane protein, such as a full-length amino acid sequence as disclosed herein, an amino acid sequence lacking a signal peptide as disclosed herein, Domain identity with a PRO polypeptide having a domain of about 80% or more, preferably about 81% or more, more preferably about 82% or more, still more preferably about 83% or more, still more preferably about 84% or more, , More preferably at least about 85%, even more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% More preferably at least about 90%, more preferably at least about 91%, even more preferably at least 92%, even more preferably at least about 93%, even more preferably at least about 94%, even more preferably at least about 95% Phase, and more preferably relates to a PRO polypeptide is isolated comprising the amino acid sequence at least about 96%, more preferably at least about 97%, more preferably at least about 98%, more preferably about 99%.
[457] In a further aspect, the invention provides an isolated nucleic acid molecule having at least about 80% sequence identity, preferably at least about 81% sequence identity, more preferably at least about 81% sequence identity with an amino acid sequence encoded by any of the ATCC deposited human protein cDNAs as disclosed herein , Preferably at least about 82%, more preferably at least about 83%, more preferably at least about 84%, more preferably at least about 85%, even more preferably at least about 86%, even more preferably at least about 87% , More preferably at least about 88%, even more preferably at least about 89%, even more preferably at least about 90%, even more preferably at least about 91%, even more preferably at least 92% Is at least about 93%, more preferably at least about 94%, more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97%, even more preferably at least about 98% , More preferably about 99% RTI ID = 0.0 > PRO < / RTI >
[458] In a further aspect, the invention provides a method for detecting a signal peptide comprising the amino acid sequence of the full-length amino acid sequence as disclosed herein, a signal peptide lacking the signal peptide as disclosed herein, or an extracellular domain of a transmembrane protein with or without a signal peptide as disclosed herein Preferably about 81% or more, more preferably about 82% or more, more preferably about 83% or more, and more preferably about 80% or more, more preferably about 80% or more, More preferably at least about 84% positive, more preferably at least about 85% positive, more preferably at least about 86% positive, even more preferably at least about 87% positive, even more preferably at least about 88% positive, About 89% or more positive, more preferably about 90% or more positive, more preferably about 91% or more positive, The crab is preferably at least 92% positive, more preferably at least about 93% positive, even more preferably at least about 94% positive, more preferably at least about 95% positive, even more preferably at least about 96% positive, Quot; refers to an isolated PRO polypeptide comprising an amino acid sequence that is at least about 97% positive, more preferably at least about 98% positive, more preferably at least about 99% positive.
[459] In one particular aspect, the invention provides isolated PRO polypeptides that are encoded by a nucleotide sequence encoding an amino acid sequence as described above and which do not have an N-terminal signal sequence and / or an initiation methionine. Also described herein are methods of making the isolated PRO polypeptides, wherein the host cells comprising a vector comprising a suitable encoding nucleic acid molecule are cultured under conditions suitable for expression of the PRO polypeptide and cultured from the cell culture And recovering the PRO polypeptide.
[460] In another aspect, the invention provides an isolated PRO polypeptide wherein the transmembrane domain is deleted or deleted. Also described herein are methods of making the isolated PRO polypeptide, wherein the host cells comprising a vector comprising a suitable encoding nucleic acid molecule are cultured under conditions suitable for expression of the PRO polypeptide, and the cell culture RTI ID = 0.0 > PRO < / RTI >
[461] In another embodiment, the invention relates to agonists and antagonists of native PRO polypeptides as defined above. In one particular embodiment, the agonist or antagonist is an anti-PRO antibody or small molecule.
[462] In a further embodiment, the invention relates to a method for identifying an agonist or antagonist for a PRO polypeptide, comprising contacting the PRO polypeptide with a candidate molecule and monitoring the biological activity modulated by the PRO polypeptide. Preferably, the PRO polypeptide is a native PRO polypeptide.
[463] In a further embodiment, the invention relates to a composition comprising a PRO polypeptide, or an agonist or antagonist of a PRO polypeptide as defined herein, together with a carrier. Optionally, the carrier is a pharmaceutically acceptable carrier.
[464] Another embodiment of the present invention is the use of a PRO polypeptide, or an agonist or antagonist thereof as described above, or an anti-PRO antibody useful for treating a disease responsive to a PRO polypeptide, an agonist or antagonist or an anti-PRO antibody To the use in the manufacture of a medicament.
[1] The present invention relates to identification and isolation of novel DNA, and to recombinant production methods of novel polypeptides encoded by the DNA.
[465] Figure 1 shows the nucleotide sequence (SEQ ID NO: 1) of the native sequence PRO211 cDNA, wherein SEQ ID NO: 1 is a clone referred to herein as " DNA32292-1131 ".
[466] Fig. 2 shows the amino acid sequence (SEQ ID NO: 2) derived from the coding sequence of SEQ ID NO: 1 shown in Fig.
[467] Figure 3 shows the nucleotide sequence (SEQ ID NO: 3) of the native sequence PRO217 cDNA, wherein SEQ ID NO: 3 is a clone referred to herein as " DNA33094-1131 ".
[468] Fig. 4 shows the amino acid sequence (SEQ ID NO: 4) derived from the coding sequence of SEQ ID NO: 3 shown in Fig.
[469] Figure 5 shows the nucleotide sequence (SEQ ID NO: 11) of the native sequence PRO230 cDNA, wherein SEQ ID NO: 11 is a clone referred to herein as " DNA33223-1136 ".
[470] Fig. 6 shows the amino acid sequence (SEQ ID NO: 12) derived from the coding sequence of SEQ ID NO: 11 shown in Fig.
[471] Figure 7 shows the nucleotide sequence referred to herein as DNA20088 (SEQ ID NO: 13).
[472] Figure 8 shows the nucleotide sequence (SEQ ID NO: 17) of the native sequence PRO232 cDNA, wherein SEQ ID NO: 17 is a clone referred to herein as " DNA34435-1140 ".
[473] Figure 9 shows the amino acid sequence (SEQ ID NO: 18) derived from the coding sequence of SEQ ID NO: 17 shown in Figure 8.
[474] Figure 10 shows the nucleotide sequence (SEQ ID NO: 22) of the native sequence PRO187 cDNA, wherein SEQ ID NO: 22 is a clone referred to herein as " DNA27864-1155 ".
[475] Fig. 11 shows the amino acid sequence (SEQ ID NO: 23) derived from the coding sequence of SEQ ID NO: 22 shown in Fig.
[476] Figure 12 shows the nucleotide sequence (SEQ ID NO: 27) of the native sequence PRO265 cDNA, wherein SEQ ID NO: 27 is a clone referred to herein as " DNA36350-1158 ".
[477] Fig. 13 shows the amino acid sequence (SEQ ID NO: 28) derived from the coding sequence of SEQ ID NO: 27 shown in Fig.
[478] Figure 14 shows the nucleotide sequence (SEQ ID NO: 33) of the native sequence PRO219 cDNA, wherein SEQ ID NO: 33 is a clone referred to herein as " DNA32290-1164 ".
[479] Fig. 15 shows the amino acid sequence (SEQ ID NO: 34) derived from the coding sequence of SEQ ID NO: 33 shown in Fig.
[480] Figure 16 shows the nucleotide sequence (SEQ ID NO: 38) of the native sequence PRO246 cDNA, wherein SEQ ID NO: 38 is a clone referred to herein as " DNA35639-1172 ".
[481] 17 shows the amino acid sequence (SEQ ID NO: 39) derived from the coding sequence of SEQ ID NO: 38 shown in Fig.
[482] Figure 18 shows the nucleotide sequence (SEQ ID NO: 48) of the native sequence PRO228 cDNA, wherein SEQ ID NO: 48 is a clone referred to herein as " DNA33092-1202 ".
[483] 19 shows the amino acid sequence (SEQ ID NO: 49) derived from the coding sequence of SEQ ID NO: 48 shown in FIG.
[484] Figure 20 shows the nucleotide sequence referred to herein as DNA21951 (SEQ ID NO: 50).
[485] Figure 21 shows the nucleotide sequence (SEQ ID NO: 58) of the native sequence PRO533 cDNA, wherein SEQ ID NO: 58 is a clone referred to herein as " DNA49435-1219 ".
[486] Fig. 22 shows the amino acid sequence (SEQ ID NO: 59) derived from the coding sequence of SEQ ID NO: 58 shown in Fig.
[487] Figure 23 shows the nucleotide sequence (SEQ ID NO: 63) of the native sequence PRO245 cDNA, wherein SEQ ID NO: 63 is a clone referred to herein as " DNA35638-1141 ".
[488] Fig. 24 shows the amino acid sequence (SEQ ID NO: 64) derived from the coding sequence of SEQ ID NO: 63 shown in Fig.
[489] Figure 25 shows the nucleotide sequence (SEQ ID NO: 68) of the native sequence PRO220 cDNA, wherein SEQ ID NO: 68 is a clone referred to herein as " DNA32298-1132 ".
[490] 26 shows the amino acid sequence (SEQ ID NO: 69) derived from the coding sequence of SEQ ID NO: 68 shown in FIG.
[491] Figure 27 shows the nucleotide sequence (SEQ ID NO: 70) of the native sequence PRO221 cDNA, wherein SEQ ID NO: 70 is a clone referred to herein as " DNA33089-1132 ".
[492] 28 shows the amino acid sequence (SEQ ID NO: 71) derived from the coding sequence of SEQ ID NO: 70 shown in FIG.
[493] Figure 29 shows the nucleotide sequence (SEQ ID NO: 72) of the native sequence PRO227 cDNA, wherein SEQ ID NO: 72 is a clone referred to herein as " DNA33786-1132 ".
[494] Fig. 30 shows the amino acid sequence (SEQ ID NO: 73) derived from the coding sequence of SEQ ID NO: 72 shown in Fig.
[495] Figure 31 shows the nucleotide sequence of the native sequence PRO258 cDNA (SEQ ID NO: 83), wherein SEQ ID NO: 83 is a clone referred to herein as " DNA35918-1174 ".
[496] 32 shows the amino acid sequence (SEQ ID NO: 84) derived from the coding sequence of SEQ ID NO: 83 shown in Fig.
[497] Figure 33 shows the nucleotide sequence (SEQ ID NO: 90) of the native sequence PRO266 cDNA, wherein SEQ ID NO: 90 is a clone referred to herein as " DNA37150-1178 ".
[498] 34 shows the amino acid sequence (SEQ ID NO: 91) derived from the coding sequence of SEQ ID NO: 90 shown in Fig.
[499] Figure 35 shows the nucleotide sequence (SEQ ID NO: 95) of the native sequence PRO269 cDNA, wherein SEQ ID NO: 95 is a clone referred to herein as " DNA38260-1180 ".
[500] Fig. 36 shows the amino acid sequence (SEQ ID NO: 96) derived from the coding sequence of SEQ ID NO: 95 shown in Fig.
[501] Figure 37 shows the nucleotide sequence (SEQ ID NO: 103) of the native sequence PRO287 cDNA, wherein SEQ ID NO: 103 is a clone referred to herein as " DNA39969-1185 ".
[502] Fig. 38 shows the amino acid sequence (SEQ ID NO: 104) derived from the coding sequence of SEQ ID NO: 103 shown in Fig.
[503] Figure 39 shows the nucleotide sequence (SEQ ID NO: 108) of the native sequence PRO214 cDNA, wherein SEQ ID NO: 108 is a clone referred to herein as " DNA32286-1191 ".
[504] 40 shows the amino acid sequence (SEQ ID NO: 109) derived from the coding sequence of SEQ ID NO: 108 shown in Fig.
[505] 41 shows the nucleotide sequence (SEQ ID NO: 113) of the native sequence PRO317 cDNA, wherein SEQ ID NO: 113 is a clone referred to herein as " DNA33461-1199 ".
[506] Figure 42 shows the amino acid sequence (SEQ ID NO: 114) derived from the coding sequence of SEQ ID NO: 113 shown in Figure 41.
[507] Figure 43 shows the nucleotide sequence (SEQ ID NO: 118) of the native sequence PRO301 cDNA, wherein SEQ ID NO: 118 is a clone referred to herein as " DNA40628-1216 ".
[508] Figure 44 shows the amino acid sequence (SEQ ID NO: 119) derived from the coding sequence of SEQ ID NO: 118 shown in Figure 43.
[509] Figure 45 shows the nucleotide sequence (SEQ ID NO: 126) of the native sequence PRO224 cDNA, wherein SEQ ID NO: 126 is a clone referred to herein as " DNA33221-1133 ".
[510] Figure 46 shows the amino acid sequence (SEQ ID NO: 127) derived from the coding sequence of SEQ ID NO: 126 shown in Figure 45.
[511] Figure 47 shows the nucleotide sequence (SEQ ID NO: 131) of the native sequence PRO222 cDNA, wherein SEQ ID NO: 131 is a clone referred to herein as " DNA33107-1135 ".
[512] Figure 48 shows the amino acid sequence (SEQ ID NO: 132) derived from the coding sequence of SEQ ID NO: 131 shown in Figure 47.
[513] 49 shows the nucleotide sequence (SEQ ID NO: 136) of the native sequence PRO234 cDNA, wherein SEQ ID NO: 136 is a clone referred to herein as " DNA35557-1137 ".
[514] Figure 50 shows the amino acid sequence (SEQ ID NO: 137) derived from the coding sequence of SEQ ID NO: 136 shown in Figure 49.
[515] Figure 51 shows the nucleotide sequence (SEQ ID NO: 141) of the native sequence PRO231 cDNA, wherein SEQ ID NO: 141 is a clone referred to herein as " DNA34434-1139 ".
[516] 52 shows the amino acid sequence (SEQ ID NO: 142) derived from the coding sequence of SEQ ID NO: 141 shown in FIG.
[517] Figure 53 shows the nucleotide sequence (SEQ ID NO: 147) of the native sequence PRO229 cDNA, wherein SEQ ID NO: 147 is a clone referred to herein as " DNA33100-1159 ".
[518] 54 shows the amino acid sequence (SEQ ID NO: 148) derived from the coding sequence of SEQ ID NO: 147 shown in FIG.
[519] Figure 55 shows the nucleotide sequence (SEQ ID NO: 152) of the native sequence PRO238 cDNA, wherein SEQ ID NO: 152 is a clone referred to herein as " DNA35600-1162 ".
[520] 56 shows the amino acid sequence (SEQ ID NO: 153) derived from the coding sequence of SEQ ID NO: 152 shown in FIG.
[521] Figure 57 shows the nucleotide sequence (SEQ ID NO: 158) of the native sequence PRO233 cDNA, wherein SEQ ID NO: 158 is a clone referred to herein as " DNA34436-1238 ".
[522] Figure 58 shows the amino acid sequence (SEQ ID NO: 159) derived from the coding sequence of SEQ ID NO: 158 shown in Figure 57.
[523] Figure 59 shows the nucleotide sequence (SEQ ID NO: 163) of the native sequence PRO223 cDNA, wherein SEQ ID NO: 163 is a clone referred to herein as " DNA33206-1165 ".
[524] Figure 60 shows the amino acid sequence (SEQ ID NO: 164) derived from the coding sequence of SEQ ID NO: 163 shown in Figure 59.
[525] Figure 61 shows the nucleotide sequence (SEQ ID NO: 169) of the native sequence PRO235 cDNA, wherein SEQ ID NO: 169 is a clone referred to herein as " DNA35558-1167 ".
[526] Figure 62 shows the amino acid sequence (SEQ ID NO: 170) derived from the coding sequence of SEQ ID NO: 169 shown in Figure 61.
[527] Figure 63 shows the nucleotide sequence (SEQ ID NO: 174) of the native sequence PRO236 cDNA, wherein SEQ ID NO: 174 is a clone referred to herein as " DNA35599-1168 ".
[528] Figure 64 shows the amino acid sequence (SEQ ID NO: 175) derived from the coding sequence of SEQ ID NO: 174 shown in Figure 63.
[529] Figure 65 shows the nucleotide sequence (SEQ ID NO: 176) of the native sequence PRO262 cDNA, wherein SEQ ID NO: 176 is a clone referred to herein as " DNA36992-1168 ".
[530] Figure 66 shows the amino acid sequence (SEQ ID NO: 177) derived from the coding sequence of SEQ ID NO: 176 shown in Figure 65.
[531] 67 shows the nucleotide sequence (SEQ ID NO: 184) of the native sequence PRO239 cDNA, wherein SEQ ID NO: 184 is a clone referred to herein as " DNA34407-1169 ".
[532] Figure 68 shows the amino acid sequence (SEQ ID NO: 185) derived from the coding sequence of SEQ ID NO: 184 shown in Figure 67.
[533] Figure 69 shows the nucleotide sequence (SEQ ID NO: 189) of the native sequence PRO257 cDNA, wherein SEQ ID NO: 189 is a clone referred to herein as " DNA35841-1173 ".
[534] Figure 70 shows the amino acid sequence (SEQ ID NO: 190) derived from the coding sequence of SEQ ID NO: 189 shown in Figure 69.
[535] Figure 71 shows the nucleotide sequence (SEQ ID NO: 194) of the native sequence PRO260 cDNA, wherein SEQ ID NO: 194 is a clone referred to herein as " DNA33470-1175 ".
[536] 72 shows the amino acid sequence (SEQ ID NO: 195) derived from the coding sequence of SEQ ID NO: 194 shown in FIG. 71;
[537] Figure 73 shows the nucleotide sequence of the native sequence PRO263 cDNA (SEQ ID NO: 200), wherein SEQ ID NO: 200 is a clone referred to herein as " DNA34431-1177 ".
[538] 74 shows the amino acid sequence (SEQ ID NO: 201) derived from the coding sequence of SEQ ID NO: 200 shown in FIG.
[539] Figure 75 shows the nucleotide sequence (SEQ ID NO: 206) of the native sequence PRO270 cDNA, wherein SEQ ID NO: 206 is a clone referred to herein as " DNA39510-1181 ".
[540] 76 shows the amino acid sequence (SEQ ID NO: 207) derived from the coding sequence of SEQ ID NO: 206 shown in FIG.
[541] Figure 77 shows the nucleotide sequence (SEQ ID NO: 212) of the native sequence PRO271 cDNA, wherein SEQ ID NO: 212 is a clone referred to herein as " DNA39423-1182 ".
[542] 78 shows the amino acid sequence (SEQ ID NO: 213) derived from the coding sequence of SEQ ID NO: 212 shown in FIG. 77;
[543] 79 shows the nucleotide sequence (SEQ ID NO: 220) of the native sequence PRO272 cDNA, wherein SEQ ID NO: 220 is a clone referred to herein as " DNA40620-1183 ".
[544] 80 shows the amino acid sequence (SEQ ID NO: 221) derived from the coding sequence of SEQ ID NO: 220 shown in FIG.
[545] Figure 81 shows the nucleotide sequence (SEQ ID NO: 226) of the native sequence PRO294 cDNA, wherein SEQ ID NO: 226 is a clone referred to herein as " DNA40604-1187 ".
[546] 82 shows the amino acid sequence (SEQ ID NO: 227) derived from the coding sequence of SEQ ID NO: 226 shown in FIG.
[547] Figure 83 shows the nucleotide sequence (SEQ ID NO: 235) of the native sequence PRO295 cDNA, wherein SEQ ID NO: 235 is a clone referred to herein as " DNA38268-1188 ".
[548] 84 shows the amino acid sequence (SEQ ID NO: 236) derived from the coding sequence of SEQ ID NO: 235 shown in Fig.
[549] 85 shows the nucleotide sequence (SEQ ID NO: 244) of the native sequence PRO293 cDNA, wherein SEQ ID NO: 244 is a clone referred to herein as " DNA37151-1193 ".
[550] 86 shows the amino acid sequence (SEQ ID NO: 245) derived from the coding sequence of SEQ ID NO: 244 shown in Fig.
[551] Figure 87 shows the nucleotide sequence (SEQ ID NO: 249) of the native sequence PRO247 cDNA, wherein SEQ ID NO: 249 is a clone referred to herein as " DNA35673-1201 ".
[552] 88 shows the amino acid sequence (SEQ ID NO: 250) derived from the coding sequence of SEQ ID NO: 249 shown in Fig.
[553] Figure 89 shows the nucleotide sequence (SEQ ID NO: 254) of the native sequence PRO302 cDNA, wherein SEQ ID NO: 254 is a clone referred to herein as " DNA40370-1217 ".
[554] 90 shows the amino acid sequence (SEQ ID NO: 255) derived from the coding sequence of SEQ ID NO: 254 shown in FIG.
[555] 91 shows the nucleotide sequence (SEQ ID NO: 256) of the native sequence PRO303 cDNA, wherein SEQ ID NO: 256 is a clone referred to herein as " DNA42551-1217 ".
[556] 92 shows the amino acid sequence (SEQ ID NO: 257) derived from the coding sequence of SEQ ID NO: 256 shown in FIG.
[557] 93 shows the nucleotide sequence (SEQ ID NO: 258) of the native sequence PRO304 cDNA, wherein SEQ ID NO: 258 is a clone referred to herein as " DNA39520-1217 ".
[558] 94 shows the amino acid sequence (SEQ ID NO: 259) derived from the coding sequence of SEQ ID NO: 258 shown in Fig.
[559] 95 shows the nucleotide sequence of the native sequence PRO307 cDNA (SEQ ID NO: 260), wherein SEQ ID NO: 260 is a clone referred to herein as " DNA41225-1217 ".
[560] 96 shows the amino acid sequence (SEQ ID NO: 261) derived from the coding sequence of SEQ ID NO: 260 shown in FIG.
[561] 97 shows the nucleotide sequence (SEQ ID NO: 262) of the native sequence PRO343 cDNA, wherein SEQ ID NO: 262 is a clone referred to herein as " DNA43318-1217 ".
[562] 98 shows the amino acid sequence (SEQ ID NO: 263) derived from the coding sequence of SEQ ID NO: 262 shown in Fig.
[563] 99 shows the nucleotide sequence (SEQ ID NO: 284) of the native sequence PRO328 cDNA, wherein SEQ ID NO: 284 is a clone referred to herein as " DNA40587-1231 ".
[564] Figure 100 shows the amino acid sequence (SEQ ID NO: 285) derived from the coding sequence of SEQ ID NO: 284 shown in Figure 99.
[565] Figure 101 shows the nucleotide sequence (SEQ ID NO: 289) of the native sequence PRO335 cDNA, wherein SEQ ID NO: 289 is a clone referred to herein as " DNA41388-1234 ".
[566] Figure 102 shows the amino acid sequence (SEQ ID NO: 290) derived from the coding sequence of SEQ ID NO: 289 shown in Figure 101.
[567] Figure 103 shows the nucleotide sequence (SEQ ID NO: 291) of the native sequence PRO331 cDNA, wherein SEQ ID NO: 291 is a clone referred to herein as " DNA40981-1234 ".
[568] 104 shows the amino acid sequence (SEQ ID NO: 292) derived from the coding sequence of SEQ ID NO: 291 shown in Fig.
[569] Figure 105 shows the nucleotide sequence (SEQ ID NO: 293) of the native sequence PRO326 cDNA, wherein SEQ ID NO: 293 is a clone referred to herein as " DNA37140-1234 ".
[570] 106 shows the amino acid sequence (SEQ ID NO: 294) derived from the coding sequence of SEQ ID NO: 293 shown in Fig.
[571] 107 shows the nucleotide sequence of the native sequence PRO332 cDNA (SEQ ID NO: 309), wherein SEQ ID NO: 309 is a clone referred to herein as " DNA40982-1235 ".
[572] Figure 108 shows the amino acid sequence (SEQ ID NO: 310) derived from the coding sequence of SEQ ID NO: 309 shown in Figure 107.
[573] 109 shows the nucleotide sequence (SEQ ID NO: 314) of the native sequence PRO334 cDNA, wherein SEQ ID NO: 314 is a clone referred to herein as " DNA41379-1236 ".
[574] Figure 110 shows the amino acid sequence (SEQ ID NO: 315) derived from the coding sequence of SEQ ID NO: 314 shown in Figure 109.
[575] Figure 111 shows the nucleotide sequence (SEQ ID NO: 319) of the native sequence PRO346 cDNA, wherein SEQ ID NO: 319 is a clone referred to herein as " DNA44167-1243 ".
[576] 112 shows the amino acid sequence (SEQ ID NO: 320) derived from the coding sequence of SEQ ID NO: 319 shown in FIG.
[577] Figure 113 shows the nucleotide sequence (SEQ ID NO: 324) of the native sequence PRO268 cDNA, wherein SEQ ID NO: 324 is a clone referred to herein as " DNA39427-1179 ".
[578] Figure 114 shows the amino acid sequence (SEQ ID NO: 325) derived from the coding sequence of SEQ ID NO: 324 shown in Figure 113.
[579] Figure 115 shows the nucleotide sequence (SEQ ID NO: 331) of the native sequence PRO330 cDNA, wherein SEQ ID NO: 331 is a clone referred to herein as " DNA40603-1232 ".
[580] Figure 116 shows the amino acid sequence (SEQ ID NO: 332) derived from the coding sequence of SEQ ID NO: 331 shown in Figure 115.
[581] 117 shows the nucleotide sequence (SEQ ID NO: 338) of the native sequence PRO339 cDNA, wherein SEQ ID NO: 338 is a clone referred to herein as " DNA43466-1225 ".
[582] Figure 118 shows the amino acid sequence (SEQ ID NO: 339) derived from the coding sequence of SEQ ID NO: 338 shown in Figure 117.
[583] Figure 119 shows the nucleotide sequence of the native sequence PRO310 cDNA (SEQ ID NO: 340), wherein SEQ ID NO: 340 is a clone referred to herein as " DNA43046-1225 ".
[584] 120 shows the amino acid sequence (SEQ ID NO: 341) derived from the coding sequence of SEQ ID NO: 340 shown in FIG.
[585] Figure 121 shows the nucleotide sequence of the native sequence PRO244 cDNA (SEQ ID NO: 376), wherein SEQ ID NO: 376 is a clone referred to herein as " DNA35668-1171 ".
[586] Figure 122 shows the amino acid sequence (SEQ ID NO: 377) derived from the coding sequence of SEQ ID NO: 376 shown in Figure 121.
[587] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [
[588] Ⅰ. Justice
[589] &Quot; PRO polypeptide " and " PRO ", as used herein, are intended to refer to various polypeptides immediately followed by a number, wherein the full designation (i. E., PRO / number) means the particular polypeptide sequence described herein. As used herein, the terms " PRO / numeric polypeptide " and " PRO / number " (where the numerical portion is represented by the actual number) are terms including native sequence polypeptides and polypeptide variants (further defined herein). The PRO polypeptides described herein can be isolated from a variety of sources, such as human tissue types or other sources, or can be prepared by recombinant or synthetic methods.
[590] A " native sequence PRO polypeptide " includes a polypeptide having the same amino acid sequence as the corresponding PRO polypeptide derived from nature. Such native sequence PRO polypeptides can be isolated from nature or can be prepared by recombinant or synthetic methods. The term " native sequence PRO polypeptide " specifically refers to a naturally occurring truncated or secreted form of a particular PRO polypeptide (e. G., Extracellular domain sequence), a naturally occurring variant form of such a polypeptide , Differently spliced forms) and naturally occurring allelic variants. In various embodiments of the invention, the native sequence PRO polypeptides described herein are mature or full length native sequence polypeptides comprising the full length amino acid sequence shown in the accompanying figures. Start and stop codons are shown in bold and underlined in the figure. However, while the PRO polypeptide disclosed in the figures accompanying the present document is shown beginning with a methionine residue referred to as amino acid position 1 in the figure, other methionine residues located in the upper or lower portion from the amino acid position 1 in the figure represent a departure from the PRO polypeptide It is conceivable and possible that it can be used as an amino acid residue.
[591] PRO polypeptide " extracellular domain " or " ECD " refers to a form of a PRO polypeptide that is essentially free of transmembrane or cytoplasmic domains. Typically, the PRO polypeptide ECD comprises less than 1% of the transmembrane and / or cytoplasmic domains, preferably less than 0.5% of said domains. It will be appreciated that all transmembrane domains identified in the PRO polypeptides of the present invention have been identified in accordance with criteria routinely used in the art to identify hydrophobic domain types. The exact borderline of the transmembrane domain may be different, but most of it is only present at about 5 amino acids at the end of the domain first identified herein. Thus, the extracellular domain of the PRO polypeptide may optionally contain up to about 5 amino acids on either side of the transmembrane / extracellular domain boundaries identified in the Examples and in the description, Polypeptides, and nucleic acids encoding them are encompassed by the present invention.
[592] The approximate positions of the " signal peptides " of the various PRO polypeptides disclosed herein are shown in the accompanying figures. However, although the C-terminal boundary of the signal peptide may be different, it should be noted that most are only about five amino acids on either side of the signal peptide C-terminal boundary first identified herein, wherein the C-terminal boundary of the signal peptide Can be identified according to criteria commonly used in the art for identifying the type of amino acid sequence element (see, for example, Nielsen et al ., Prot. Eng. 10: 1-6 (1997) and Heinje et al., Nucl. Acids. Res. 14: 4683-4690 (1986)]. In addition, it should be appreciated that in some cases the cleavage of the signal sequence of the secreted polypeptide is not entirely identical, resulting in one or more secreted polypeptides. Mature polypeptides in which signal peptides have been cleaved in only about five amino acids on either side of the C-terminal boundary of the signal peptide identified herein, and polynucleotides encoding them, are encompassed by the present invention.
[593] &Quot; PRO polypeptide variant " refers to a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide without a signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with or without a signal peptide as disclosed herein, As used herein means an active PRO polypeptide as defined above or below, having an amino acid sequence identity of at least about 80% with any other fragment of the full-length PRO polypeptide sequence set forth in SEQ ID NO. Such PRO polypeptide variants include, for example, PRO polypeptides in which one or more amino acid residues are added or deleted at the N-terminus or C-terminus of the full-length native amino acid sequence, and the like. Typically, a PRO polypeptide variant is a full-length native sequence PRO polypeptide sequence as disclosed herein, a PRO polypeptide sequence without a signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with or without a signal peptide as disclosed herein, Amino acid sequence identity with any other fragment of the disclosed full-length PRO polypeptide sequence of at least about 80%, preferably at least about 81%, more preferably at least about 82%, more preferably at least about 83% Is at least about 84%, more preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 89% , More preferably at least about 90%, more preferably at least about 91%, even more preferably at least 92%, even more preferably at least about 93% , More preferably at least about 94%, more preferably at least about 95%, more preferably at least about 96%, more preferably at least about 97%, even more preferably at least about 98%, most preferably at least about 99% . Typically, a PRO variant polypeptide is one whose length is at least about 10 amino acids, often at least about 20 amino acids, more often at least about 30 amino acids, more often at least about 40 amino acids, more often at least about 50 amino acids, More usually at least about 70 amino acids, more often at least about 80 amino acids, more often at least about 90 amino acids, more often at least about 100 amino acids, more often at least about 150 amino acids, More usually more than about 200 amino acids, more often more than about 300 amino acids or more.
[594] &Quot; Amino acid sequence identity (%) " for a PRO polypeptide sequence disclosed herein refers to the alignment of a candidate sequence with the same PRO polypeptide sequence and its amino acid residues, introducing a gap to obtain a maximum of the percent sequence identity, Is defined as the percentage of amino acid residues of the candidate sequence that are identical to the amino acid residues of a particular PRO polypeptide sequence without any conservative substitutions considered as part of the same sequence. Alignment to determine amino acid sequence identity can be accomplished using a variety of methods in the art, such as publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art will be able to determine parameters that are suitable for measuring alignment, including any algorithms needed to maximally align over the entire length of the compared sequences. However, for purposes of this disclosure, the amino acid sequence identity (%) is determined using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Table 10 below. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. The source code shown in Table 10 is kept in the user documentation of the US Copyright Office (located in Washington, DC 20559) It is registered under the US Copyright Registration No. TXU510087. The ALIGN-2 program may be publicly available through Genentech, Inc. (South San Francisco, Calif.), Or may be edited from the source code described in Table 10. The ALIGN-2 program can be edited and used on a UNIX operating system, preferably Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.
[595] If ALIGN-2 is used for amino acid sequence comparison, the amino acid sequence identity (%) of a given amino acid sequence A to a given amino acid sequence B versus a given amino acid sequence B, or a given amino acid sequence B (given amino acid sequence B May be expressed as a given amino acid sequence A, with or with a given amino acid sequence B, or with a constant amino acid sequence identity (%) relative to a given amino acid sequence B) is calculated as follows:
[596] X / Y x 100
[597] Where X is the number of amino acid residues recorded as being equally matched by the sequence alignment program ALIGN-2 in the program alignment of A and B, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, it will be understood that the amino acid sequence identity (%) of A to B is not the same as the amino acid sequence identity (%) of B to A. As an example of the calculation of amino acid sequence identity (%) using this method, the following Tables 11 and 12 list the amino acid sequence identity (%) of the amino acid sequence referred to as " comparison protein "Lt; / RTI >
[598] Unless specifically stated otherwise, all amino acid sequence identity values (%) used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph. However, the amino acid sequence identity (%) can also be determined using the WU-BLAST-2 computer program [Altschul et al., Methods in Enzymology 266: 460-480 (1996) have. Most of the WU-BLAST-2 search parameters are set to default values. Those that are not set to default values, that is, adjustable parameters, are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is used, the amino acid sequence identity value (%) is determined by (a) comparing the amino acid sequence of the PRO polypeptide having the sequence derived from the native PRO polypeptide with the amino acid sequence of the comparative amino acid sequence Is determined by WU-BLAST-2, and (b) divided by the total number of amino acid residues of the PRO polypeptide in question. For example, a polypeptide comprising the amino acid sequence A having an amino acid sequence identity of 80% or more with the amino acid sequence B ", the amino acid sequence A is the comparative amino acid sequence and the amino acid sequence B is the amino acid sequence of the PRO polypeptide.
[599] Amino acid sequence identity (%) can also be calculated using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)]. This NCBI-BLAST2 sequence comparison program can be downloaded from the website (http://www.ncbi.nlm.nih.gov.). NCBI-BLAST2 uses several search parameters, such as all unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01, constant for multi-pass = 25, dropoff for final gapped alignment = 25, and scoring matrix = BLOSUM62.
[600] When NCBI-BLAST2 is used for amino acid sequence comparison, the amino acid sequence identity (%) of a given amino acid sequence A with respect to a given amino acid sequence B versus a given amino acid sequence B, or against a given amino acid sequence B (or a given amino acid sequence Which may be expressed as a given amino acid sequence A with or with a given amino acid sequence B, or with a given amino acid sequence identity (%) relative to a given amino acid sequence B, is calculated as follows:
[601] X / Y x 100
[602] Where X is the number of amino acid residues recorded as being equally matched by the sequence alignment program NCBI-BLAST2 in the NCBI-BLAST2 program alignment of A and B, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, it will be understood that the amino acid sequence identity (%) of A to B is not the same as the amino acid sequence identity (%) of B to A.
[603] A " PRO variant polynucleotide " or " PRO variant nucleic acid sequence " is a nucleic acid molecule that encodes an active PRO polypeptide as defined below, comprising a full length native sequence PRO polypeptide sequence as disclosed herein, a full length native A nucleic acid sequence identity between a sequence PRO polypeptide sequence, an extracellular domain of a signal sequence-containing or free PRO polypeptide as disclosed herein, or any other fragment of the full-length PRO polypeptide sequence disclosed herein, is about 80 Or more. Typically, a PRO variant polynucleotide is a full-length native sequence PRO polypeptide sequence as disclosed herein, a full-length native sequence PRO polypeptide sequence without a signal peptide as disclosed herein, an extracellular domain of a PRO polypeptide with or without a signal sequence as disclosed herein Domain, or a nucleic acid sequence encoding any other fragment of the full-length PRO polypeptide sequence disclosed herein, is at least about 80%, more preferably at least about 81%, more preferably at least about 82% More preferably at least about 85%, more preferably at least about 86%, even more preferably at least about 87%, even more preferably at least about 88%, even more preferably at least about 85% , More preferably at least about 89%, more preferably at least about 90%, even more preferably at least about 91%, even more preferably at least about 92% , Preferably at least about 93%, more preferably at least about 94%, more preferably at least about 95%, even more preferably at least about 96%, even more preferably at least about 97%, even more preferably at least about 98% %, And most preferably at least about 99%. Variants do not contain natural nucleotide sequences.
[604] Typically, a PRO variant polynucleotide is about 30 nucleotides or more in length, more often about 60 nucleotides or more, more often about 90 nucleotides or more, more often about 120 nucleotides or more, more often about 150 or more nucleotides Nucleotides, more usually at least about 180 nucleotides, more often at least about 210 nucleotides, more often at least about 240 nucleotides, more often at least about 270 nucleotides, more often at least about 300 nucleotides, More than about 450 nucleotides, more often more than about 600 nucleotides, more often more than about 900 nucleotides, or more.
[605] The "nucleic acid sequence identity (%)" for the PRO-coding nucleic acid sequence identified herein can be determined by aligning the PRO nucleic acid sequence with the candidate sequence and, if necessary, introducing a gap to maximize sequence identity, Is defined as the ratio of the nucleotides of the same candidate sequence as the nucleotides. Sorting to determine nucleic acid sequence identity (%) can be accomplished using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software in a variety of ways known in the art . However, for purposes of this disclosure, nucleic acid sequence identity values (%) can be obtained using the sequence comparison computer program ALIGN-2, wherein the complete source codes for the ALIGN-2 program are listed in Table 10 below. The ALIGN-2 sequence comparison computer program was developed by Genentech, Inc. The source code shown in Table 10 below is stored as a user document of the US Copyright Office (located in Washington, DC 20559), which is a registered trademark of US Copyright Registration No. TXU510087 . The ALIGN-2 program is publicly available through Genentech, Inc. (South San Francisco, Calif.) Or can be edited from the source code described in Table 10 below. The ALIGN-2 program can be edited and used on a UNIX operating system, preferably Digital UNIX V4.0D. All sequence comparison parameters are set with the ALIGN-2 program and do not change.
[606] When ALIGN-2 is used for nucleic acid sequence comparison, the nucleic acid sequence identity (%) of a given nucleic acid sequence C to a given nucleic acid sequence D relative to a given nucleic acid sequence D, or against a given nucleic acid sequence D May be represented by the given nucleic acid sequence D in SEQ ID NO: D, or a given nucleic acid sequence C, which has or comprises a constant nucleic acid sequence identity to a given nucleic acid sequence D) is calculated as follows:
[607] W / Z x 100
[608] Where W is the number of nucleotides that are equally recorded by the sequence alignment program ALIGN-2 in the program alignment of C and D, and Z is the total number of nucleotides in D; If the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, it will be understood that the nucleic acid sequence identity (%) of C to D is not the same as the nucleic acid sequence identity (%) of D to C. As examples of nucleic acid sequence identity (%) calculations, Tables 13 and 14 below show a method for calculating the nucleic acid sequence identity (%) of a nucleic acid sequence referred to as " comparison DNA " .
[609] Unless specifically stated otherwise, all nucleic acid sequence identity values (%) used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph. However, the nucleic acid sequence identity (%) can also be determined using the Wu-BLAST-2 computer program [Altschul et al., Methods in Enzymology 266: 460-480 (1996) . Most of the WU-BLAST-2 search parameters are set to default values. Those that are not set to default values, that is, adjustable parameters, are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = 11, and scoring matrix = BLOSUM62. When WU-BLAST-2 is used, the nucleic acid sequence identity value (%) is determined by (a) the identity of the PRO polypeptide-encoding nucleic acid molecule having a sequence derived from the native sequence PRO polypeptide-encoding nucleic acid by WU-BLAST- Determining the number of identical nucleotides matched between the nucleic acid sequence and the comparative nucleic acid molecule (i. E., The sequence in which the PRO polypeptide-coding nucleic acid molecule is compared is a variant PRO nucleotide), and (b) Divided by the total number of nucleotides of the nucleic acid molecule. For example, in the case of an isolated nucleic acid molecule comprising a nucleic acid sequence A having a nucleic acid sequence identity of 80% or more with the nucleic acid sequence B, the nucleic acid sequence A is the comparative nucleic acid sequence and the nucleic acid sequence B is the corresponding PRO polypeptide- ≪ / RTI >
[610] Nucleic acid sequence identity (%) can also be calculated using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997)]. The NCBI-BLAST2 sequence comparison program can be downloaded from the website (http://www.ncbi.nlm.nih.gov.). NCBI-BLAST2 uses several search parameters, such as unmask = yes, strand = all, expected occurrences = 10, minimum low complexity length = 15/5, multi-pass e-value = 0.01 , constant for multi-pass = 25, dropoff for final gapped alignment = 25, and scoring matrix = BLOSUM62.
[611] If NCBI-BLAST2 is used for nucleic acid sequence comparison, the nucleic acid sequence identity (%) of a given nucleic acid sequence C to a given nucleic acid sequence D relative to a given nucleic acid sequence D, or against a given nucleic acid sequence D (or, D, with a given nucleic acid sequence D, or with a given nucleic acid sequence C that has or comprises a constant nucleic acid sequence identity to a given nucleic acid sequence D) is calculated as follows:
[612] W / Z x 100
[613] Where W is the number of nucleotides recorded as being equally matched by the sequence alignment program NCBI-BLAST2 in the program alignment, and Z is the total number of nucleotides of D. If the length of the nucleic acid sequence C is not equal to the length of the nucleic acid sequence D, then the nucleic acid sequence identity (%) of C to D will not be equal to the nucleic acid sequence identity (%) of D to C.
[614] In another embodiment, a PRO variant polynucleotide is a nucleic acid molecule that encodes an active PRO polypeptide capable of hybridizing (preferably in stringent hybridization and washing conditions) with a nucleotide sequence encoding the full length PRO polypeptide disclosed herein. The PRO mutant polypeptide may be one which is encoded by a PRO mutant polynucleotide.
[615] The term " positive " with respect to sequence comparisons performed as described above means that the residues of the compared sequences have similar but similar characteristics (for example, see Table 1 below as a result of conservative substitutions) ). To achieve the object, the positive value (%) is a positive value between the PRO polypeptide amino acid sequence having the sequence derived from the native PRO polypeptide sequence and the comparative amino acid sequence (i. E., The amino acid sequence to which the PRO polypeptide sequence is compared) Is determined in the BLOSUM 62 matrix of WU-BLAST-2 and then divided by the total number of amino acid residues of the PRO polypeptide in question.
[616] Unless specifically stated otherwise, positive values (%) are calculated as described in the immediately preceding paragraph. However, amino acid sequence identity assays performed as described for ALIGN-2 and NCBI-BLAST2 include amino acid residues of comparable sequences that are not only identical, but have similar characteristics. Amino acid residues in which a positive value for the amino acid residue is recorded are the same as the amino acid residue or the preferred substituents of the amino acid residue (described in Table 1 below).
[617] For amino acid sequence comparisons using ALIGN-2 or NCBI-BLAST2, the positive values (%) of a given amino acid sequence A relative to a given amino acid sequence B against a given amino acid sequence B, or against a given amino acid sequence B Which may be expressed as a given amino acid sequence A, which has or contains a constant positive value (%) for B, B and / or B) is calculated as follows:
[618] X / Y x 100
[619] Where X is the number of amino acid residues that record the positive value defined above by the sequence alignment program ALIGN-2 or NCBI-BLAST2 in the program alignment of A and B, and Y is the total number of amino acid residues in B. If the length of the amino acid sequence A is not equal to the length of the amino acid sequence B, it will be understood that the positive value (%) of A with respect to B is not equal to the positive value (%) with respect to A.
[620] &Quot; Isolated " as used to describe the various polypeptides disclosed herein means that the polypeptide has been identified and has been isolated and / or recovered from its natural environment elements. Pollution factors in the natural environment of the polypeptide are typically substances that interfere with the diagnostic or therapeutic use of the polypeptide, including enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide is purified (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a spinning cup sequencer, or (2) Coomassie blue or preferably Using a silver stain, to a single band on SDS-PAGE under non-reducing or reducing conditions. Because the PRO polypeptide will not have more than one element of its natural environment, the isolated polypeptide comprises a polypeptide present in the recombinant cell. However, isolated polypeptides will typically be obtained by one or more purification steps.
[621] An " isolated " PRO polypeptide-encoding nucleic acid is a nucleic acid molecule identified and isolated from one or more contaminating nucleic acid molecules that are typically associated with a PRO polypeptide nucleic acid of natural origin. The isolated PRO polypeptide nucleic acid molecule differs from the form or state found in nature. Thus, isolated PRO polypeptide nucleic acid molecules are distinguished from certain PRO polypeptide nucleic acid molecules present in native cells. However, isolated PRO polypeptide nucleic acid molecules include, for example, PRO polypeptide nucleic acid molecules that are present at chromosomal locations that differ from native cell chromosomes and are typically contained in cells expressing PRO polypeptides.
[622] The term " regulatory sequence " is a DNA sequence necessary for the expression of a coding sequence operably linked to a particular host organism. Regulatory sequences suitable for prokaryotes include, for example, promoters, operator sequences as the case may be, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
[623] A nucleic acid is " operably linked " when placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a whole protein that participates in the secretion of the polypeptide, and the promoter or enhancer is one in which the coding sequence The ribosome binding site is operably linked to a coding sequence if it is placed to facilitate translation. Generally, " operably linked " means that the DNA sequences to be ligated are contiguous and, in the case of a secretory leader, is located adjacent and within the same reading region. However, enhancers need not be contiguous. The connection is accomplished by ligation at a convenient restriction site. If such sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.
[624] The term " antibody " is used in its broadest sense and specifically includes, for example, one anti-PRO monoclonal antibody (including agonists, antagonists and neutralizing antibodies), an anti-PRO antibody composition with polyepitope specificity, Anti-PRO antibodies, and fragments of anti-PRO antibodies (see below). The term " monoclonal antibody " as used herein refers to an individual antibody that is a group of antibodies, except for antibodies derived from a population of substantially allogeneic antibodies, i. E., Possible spontaneous mutations that may be present in minor amounts.
[625] The " stringency " of the hybridization reaction is readily determinable by those skilled in the art and is generally an empirical calculation that depends on probe length, wash temperature, and salt concentration. Generally longer probes require higher temperatures for proper annealing and shorter probes require lower temperatures. Generally, hybridization will depend on the ability of the denatured DNA to be annealed again when the complementary strand is in an environment below its melting temperature. The higher the degree of desired homology between the probe and the hybridizable sequence, the higher the relative temperature available. As a result, reaction conditions become more stringent at higher relative temperatures, while reaction conditions become less stringent at lower relative temperatures. For further information and description of the severity of the hybridization reaction, see Ausubel et al. Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995).
[626] "Strict conditions" or "high stringency conditions" as defined herein means (1) conditions of low ionic concentration and high temperature conditions for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate (2) Formamide during hybridization, for example, 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone containing 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C, 50 mM (3) a condition using 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M citric acid) at 42 < 0 > C, (50 μg / ml), 0.1% SDS, and 10% dextran sulphate (50 μg / ml), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, The pate was used at 42 [deg.] C and 0.2 x SSC (sodium chloride / citric acid , And 50% formamide at 55 占 폚, followed by a high-stringency wash with EDTA-containing 0.1 x SSC at 55 占 폚.
[627] The term " intermediate stringent conditions " is the same as described in Sambrook et al., Molecular Cloning: A Laboratory Manual , New York: Cold Spring Harbor Press, 1989, For example, temperature, ion concentration and% SDS). Examples of medium stringent conditions include 20% formamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt solution, 10% dextran sulphate Followed by incubation at 37 캜 overnight in a solution containing 20 mg / ml of fetal and denatured truncated salmon semen DNA, followed by washing the filter with 1 x SSC at about 37 to 50 캜. One skilled in the art will appreciate how to adjust the temperature, ion concentration, and the like necessary to meet factors such as probe length and the like.
[628] As used herein, the term " epitope tagged " means a chimeric polypeptide comprising a PRO polypeptide fused with a " tag polypeptide ". A " tag polypeptide " has sufficient residues to provide an epitope that allows the antibody to be made, but is sufficiently short so as not to interfere with the activity of the polypeptide to which it is fused. It is also preferred that the tag polypeptide is highly specific so that the antibody against it does not actually cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues, and typically have about 8 to 50 amino acid residues (preferably about 10 to 20 amino acid residues).
[629] As used herein, the term " immunoadhesin " refers to an antibody-like molecule that combines the effector function of an immunoglobulin constant domain with the binding specificity of a heterologous protein (adhesin). Structurally, the immunoadhesin comprises a fusion of an immunoglobulin constant domain sequence with an amino acid sequence having a predetermined binding specificity that is not (i. E., Heterologous) an antigen recognition and binding site of the antibody. The adhered portion of the immunoadhesin molecule is typically a contiguous amino acid sequence comprising at least the binding site of the receptor or ligand. Immunoglobulin constant domain sequences of immunoadhesins include IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, such as IgA (including IgA-1 and IgA-2), IgE, IgD or IgM Can be obtained from any immunoglobulin.
[630] The term " active " or " activity ", as used herein, means a form of a PRO polypeptide having biological and / or immunological activity of a natural or naturally occurring PRO, Refers to a biological function (inhibitory or promoting function) triggered by a natural or naturally-occurring PRO, not an ability to induce antibody production against an antigenic epitope possessed by a PRO. &Quot; Immunological "Quot; refers to the ability of the PRO to induce antibody production against the antigenic epitope it possesses.
[631] The term " antagonist " is used in its broadest sense and includes any molecule that partially or completely blocks, inhibits, or neutralizes the biological activity of the native PRO polypeptide disclosed herein. Likewise, the term " agonist " is used in its broadest sense and includes all molecules which mimic the biological activity of the native PRO polypeptides disclosed herein. Suitable agonist or antagonist molecules include, in particular, agonist or antagonist antibodies, or antibody fragments, fragments or amino acid sequence variants of native PRO polypeptides, peptides, antisense oligonucleotides, small organic molecules and the like. Methods of identifying an agonist or antagonist of a PRO polypeptide can include contacting the PRO polypeptide with a candidate agonist molecule or a candidate antagonist molecule and measuring a detectable change in one or more biological activities typically associated with the PRO polypeptide.
[632] &Quot; Treatment " means treatment and prevention or prevention measures performed for the purpose of preventing or slowing (alleviating) pathological symptoms or diseases of the subject. Subjects requiring treatment include those who are already susceptible to disease, as well as those who are already suffering from the disease.
[633] &Quot; Chronic " administration refers to the administration of a substance in a continuous mode as opposed to an acute mode, maintaining the initial therapeutic effect (activity) for a prolonged period of time. &Quot; Intermittent " administration refers to procedures that are performed in a substantially periodic manner, rather than continuing without interruption.
[634] A " mammal " for treatment refers to a mammal including, but not limited to, humans, livestock and rabbits, and mammals including zoo, competition or pets such as dogs, cats, cows, horses, sheep, pigs, goats, And the like. A preferred mammal is a human.
[635] The " combined " administration with one or more other therapeutic agents includes simultaneous (co-administration) or sequential administration in any order.
[636] As used herein, " carrier " includes pharmaceutically acceptable carriers, excipients or stabilizers which are non-toxic to the cells or mammals exposed to the dosages and concentrations employed. Physiologically acceptable carriers are often pH buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids; Antioxidants including ascorbic acid; Low molecular weight (less than about 10 residues) polypeptides; Proteins such as serum albumin, gelatin or immunoglobulin; Hydrophilic polymers such as polyvinylpyrrolidone; Amino acids such as glycine, glutamine, asparagine, arginine or lysine; Monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrin; Chelating agents such as EDTA; Sugar alcohols such as mannitol or sorbitol; Salt-forming counter ions such as sodium; And / or non-ionic surfactants such as TWEEN, polyethylene glycol (PEG) and PLURONICS.
[637] An " antibody fragment " includes a portion of an intact antibody, preferably an antigen binding region or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') ₂ and Fv fragments, diabodies, linear antibodies (Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)], multispecific antibodies formed from single chain antibody molecules and antibody fragments.
[638] Papain degradation of the antibody produces two identical antigen binding fragments, a "Fab" fragment with a single antigen-binding site, and a residual "Fc" fragment named reflecting its ability to crystallize readily. Treatment with pepsin produces an F (ab ') ₂ fragment that has two antigen-binding sites and is still capable of cross-linking with the antigen.
[639] &Quot; Fv " is a minimal antibody fragment comprising a complete antigen-recognition and antigen-binding site. This region consists of one heavy chain non-covalently bound heavy chain variable domain and one light chain variable domain dimer. In this form, three CDRs of each variable domain interact to form an antigen-binding site on the surface of the V _H -V _L dimer. In conclusion, six CDRs confer antigen-binding specificity to the antibody. However, a single variable domain (or half of the Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen even though it is less affinity than the entire binding site.
[640] In addition, the Fab fragments comprise the constant domain of the light chain and the first constant domain (CHl) of the heavy chain. Fab fragments differ from Fab 'fragments in that several residues are added to the carboxy terminus of the heavy chain CH1 domain, which contains at least one cysteine from the antibody hinge region. Fab'-SH herein refers to Fab 'in which the cysteine residue of the constant domain retains a free thiol group. The F (ab ') ₂ antibody fragment was originally made up of several pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
[641] The "light chain" of an antibody (immunoglobulin) from any vertebrate species can belong to one of two distinct types called kappa (κ) and lambda (λ) based on the amino acid sequence of its constant domain .
[642] Immunoglobulin can belong to another class depending on the amino acid sequence of the constant domain of its heavy chain. There are five major classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, some of which are subclassed (isotype), for example IgG1, IgG2, IgG3, IgG4, IgA and IgA2 .
[643] A "single chain Fv" or "sFv" antibody fragment comprises the V _H and V _L domains of an antibody present in a single polypeptide chain. Preferably, the Fv polypeptide additionally comprises a polypeptide linker between the V _H and V _L domains that allows the sFv to form the required structure for antigen binding (see Pluckthan, The Pharmacology for sFv, of Monoclonal Antibodies , vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)].
[644] The term "dia body (diabody)" in the same polypeptide chain (V _H -V _L) in the light chain variable domain (V _L) small antibody fragments with two antigen-binding site comprising a heavy chain variable domain (V _H) connected to a . Using two linkers that are too short to pair two domains in the same chain, the domains are paired with the complementary domains of the other chain to generate two antigen-binding sites. Diabodies are described, for example, in EP 404,097, WO 93/11611 and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993).
[645] An " isolated " antibody is an antibody that has been identified, isolated and / or recovered from elements of its natural environment. Contaminants in his natural environment are substances that interfere with the diagnostic or therapeutic use of antibodies, and may include enzymes, hormones and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the antibody is (1) directly more than 95% by weight, most preferably more than 99% by weight as measured by the Lowry method, (2) using a spinning cup sequencer To a sufficient degree to obtain at least 15 residues of the N-terminal or internal amino acid, or (3) by means of SDS-PAGE under reduced or nonreducing conditions using coomassie blue, or preferably silver staining, It will be refined. An isolated antibody includes an antibody in a recombinant cell since there will be no more than one component present in the natural environment of the antibody. However, the isolated antibody will typically be produced by one or more purification steps.
[646] As used herein, the term " label " refers to a detectable compound or composition that is directly or indirectly linked to an antibody to produce a " labeled " The label may be detected by itself (e. G., A radioactive isotope label or fluorescent label) or, if it is an enzyme label, a chemical modification of the detectable substrate compound or composition.
[647] &Quot; Solid phase " means a non-aqueous matrix to which an antibody of the invention may be attached. Examples of solid phases included herein include those formed partially or wholly of glass (e.g., controlled void glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol, and silicon . In certain embodiments, the solid phase may comprise a well of the assay plate, depending on the situation, and in other embodiments, the solid phase is a purification column (e. G., An affinity chromatography column). The term also encompasses discrete, non-continuous, solid particles such as those described in U.S. Patent No. 4,275,149.
[648] &Quot; Liposomes " are small vesicles composed of lipids, phospholipids and / or surfactants useful for delivering drugs (e. G., PRO polypeptides or antibodies thereto) to mammals. The components of the liposome are typically arranged in a bilayer form similar to the lipid arrangement of the biological membrane.
[649] As used herein, " small molecule " is defined as having a molecular weight of less than about 500 daltons.
[650] &Quot; PRO317-related disorder " refers to a pathological condition or disease in which PRO317 is over-expressed or under-expressed. Such diseases include the genital or endometrial pathologies of mammalian females including hyperplasia, endometritis and endometriosis wherein the patient is suffering from abnormal bleeding such as endometrial factors, endometrial and endometrial cancers, especially gynecological diseases There is a risk of infertility due to illness including illness. In addition, the disease includes diseases including angiogenesis, wherein angiogenesis causes pathological symptoms such as cancer including solid tumors (the treatment of this disease reduces the angiogenesis and the proliferation of various cancers And transitions are reduced). Alternatively, angiogenesis can be beneficial, for example, in ischemia, especially coronary artery ischemia. Therefore, the above diseases include patients suffering from coronary artery disease, which is not an optimal candidate for angioplasty and coronary artery bypass surgery, and patients whose blood supply is blocked due to atherosclerotic coronary artery disease, Include those that occur in patients who are functioning normally but who have low blood perfusion. In addition, the disease includes diseases associated with or originating from the kidney, such as polycystic kidney disease, and chronic and acute renal failure.
[651] II. Compositions and Methods of the Invention
[652] A. Whole length PRO polypeptide
[653] The present invention relates to newly identified and isolated nucleotide sequences encoding polypeptides referred to herein as PRO polypeptides. In particular, cDNAs encoding various PRO polypeptides were identified and isolated, as described in more detail in the Examples below. Although the PRO number of proteins produced in separate expression rounds may vary, the UNQ number is unique to any given DNA and coded protein and will not change. And other natural homologues and variants of PRO included in the above definition will be referred to as " PRO / number " regardless of their source and method of manufacture.
[654] Various cDNA clones were deposited with the ATCC, as described in the Examples below. One of ordinary skill in the art can readily determine the actual nucleotide sequence of the clone by analyzing the sequence of the deposited clone using common methods in the art. General techniques can be used to determine the expected amino acid sequence from the nucleotide sequence. In the case of the PRO polypeptides described herein and the nucleic acids encoding the same, the present inventors confirmed that they are regarded as the best readable frames using sequence information available at the time.
[655] 1. Full length PRO211 and PRO217 polypeptides
[656] The present invention provides newly identified and isolated nucleotide sequences that encode polypeptides referred to herein as PRO211 and PRO217. Specifically, the inventors identified and isolated cDNAs encoding PRO211 and PRO217 polypeptides, as described in more detail in the Examples below. Using the BLAST (Fast A format) sequence alignment computer program, the present inventors have found that the cDNA sequence encoding full length native sequences PRO211 and PRO217 is homologous to known proteins with EGF-like domains. Specifically, the cDNA sequence DNA32292-1131 (FIG. 1, SEQ ID NO: 1) has a specific identity with PAC6_RAT and a Blast value of 209 and has a specific identity and a Blast value of 206 with the pibulin-1 isoform c precursor. The cDNA sequence DNA33094-1131 (FIG. 3, SEQ ID NO: 3) has 36% identity and 346 Blast value with Eastern Newttenacin, 37% identity with the human tenascin-X precursor, and Blast value of 331. Thus, as currently contemplated, the PRO211 and PRO217 polypeptides disclosed herein are newly identified members of the EGF-like family and have the typical characteristics of EGF-like protein families.
[657] 2. Whole-length PRO230 polypeptide
[658] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO230. We have identified and isolated cDNA encoding the PRO230 polypeptide, particularly as described in more detail in the Examples below. Using a known program such as the BLAST and FastA sequence alignment computer programs, the present inventors have found that the cDNA encoding the full length native sequence PRO230 has 48% amino acid identity with the rabbit tubular interstitial nephritis antigen precursor. Thus, as currently contemplated, the PRO230 polypeptides disclosed herein are newly identified members of the interstitial nephritis antigen family and can be recognized by human autoantibodies in certain forms of tubular interstitial inflammation.
[659] 3. Whole-length PRO232 polypeptide
[660] The present invention provides newly identified and isolated nucleotide sequences that encode polypeptides referred to herein as PRO232. We have identified and isolated cDNA encoding the PRO232 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that a portion of the full-length native sequence PRO232 (Figure 9, SEQ ID NO: 18) has 35% sequence identity with the hepatocellular surface antigen of Gallusgallus. Thus, as currently contemplated, the PRO232 polypeptides disclosed herein may be newly identified hepatocyte antigens.
[661] 4. Whole-length PRO187 polypeptide
[662] The present invention provides newly identified and isolated nucleotide sequences that encode polypeptides referred to herein as PRO187. We have identified and isolated cDNA encoding the PRO187 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that the full length native sequence PRO187 (Figure 15) is 74% amino acid sequence identity with several antigen-inducing growth factors and FGF-8 and has a BLAST value of 310. Thus, as currently contemplated, the PRO187 polypeptides disclosed herein are a newly identified member of the family of FGF-8 proteins and may have the typical activities and characteristics of FGF-8-like protein families.
[663] 5. Whole-length PRO265 polypeptide
[664] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO265. We have identified and isolated cDNA encoding the PRO265 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO265 polypeptide are highly homologous to the fibromodulin and pibromodulin precursor proteins. We have also found that the PRO265 polypeptide is highly homologous to platelet glycoprotein V, one of the leucine rich protein families involved in skin and wound repair. Thus, the PRO265 polypeptide disclosed herein is a newly identified member of the leucine rich repeat subpopulation family and is currently believed to be capable of protein-protein binding as well as skin and wound repair as well as protein families.
[665] 6. Whole-length PRO219 polypeptide
[666] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO219. We have identified and isolated cDNA encoding the PRO219 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO219 polypeptide are highly homologous to mouse and human matrilin-2 precursor polypeptides. Thus, the PRO219 polypeptides disclosed herein are presently thought to be related to matrilin-2 precursor polypeptides.
[667] 7. Whole-length PRO246 polypeptide
[668] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO246. We have identified and isolated cDNA encoding the PRO246 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that some of the PRO246 polypeptides are highly homologous to human cell surface protein HCAR. It is therefore presently contemplated that the PRO246 polypeptides disclosed herein may be newly identified membrane bound virus receptors or tumor cell specific antigens.
[669] 8. Whole-length PRO228 polypeptide
[670] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO228. We have identified and isolated cDNA encoding the PRO228 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several parts of the PRO228 polypeptide are highly homologous to the EMR1 protein. The inventors have also found that the DNA encoding the PRO228 polypeptide is highly homologous with the rat filopin, the macrophage-restricted cellular glycoprotein, the B0457.1 and the leukocyte antigen CD97 precursor. Thus, the PRO228 polypeptides disclosed herein are now a newly discovered member of the seven membrane transmembrane family and are currently thought to possess the typical properties and functionality of this family of proteins. In particular, PRO228 is thought to be a new member of a subgroup within this protein family to which CD97 and EMR1 belong.
[671] 9. Whole-length PRO533 polypeptide
[672] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO533. We have identified and isolated cDNA encoding the PRO533 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, we found that the full length native sequence PRO533 (Figure 22, SEQ ID NO: 59) has a BLAST value of 509 and a sequence identity of 539 with Fibroblast Growth Factor (FGF). It is therefore presently contemplated that PRO533 disclosed herein is a newly discovered member of the fibroblast growth factor family and may have typical activity in such polypeptides.
[673] 10. Whole-length PRO245 polypeptide
[674] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO245. We have identified and isolated cDNA encoding the PRO245 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that a portion of the amino acid sequence of the PRO245 polypeptide has 60% amino acid identity with human c-myb protein. It is presently contemplated that the PRO245 polypeptide disclosed herein may be a newly discovered member of the transmembrane protein tyrosine kinase family.
[675] 11. Full-length PRO220, PRO221 and PRO227 polypeptides
[676] The present invention provides newly identified and isolated nucleotide sequences that encode polypeptides referred to herein as PRO220, PRO221 and PRO227. We have identified and isolated cDNAs encoding PRO220, PRO221 and PRO227 polypeptides, respectively, as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that PRO220 has 87% sequence identity with the amino acid sequence of the leucine rich protein. PRO220 also has a sequence identity of 55% with the neuronal lucin-rich protein. This neuron-lucine rich protein is further described in the article (Taguchi, et al., Mol. Brain Res., 35: 31-40 (1996)).
[677] PRO221 has amino acid identity with the SLIT protein precursor, which is 39%, 38%, 34%, 31%, and 30%, respectively, of the two proteins.
[678] PRO227 has amino acid identity with the amino acid sequence of the platelet glycoprotein V precursor. The same results were obtained for human glycoprotein V. [ The identity of these two proteins is 30%, 28%, 28%, 31%, 35%, 39% and 27%, respectively.
[679] Thus, the PRO220, PRO221 and PRO227 polypeptides disclosed herein are a newly discovered member of the leucine rich repeat subpopular macrolide population, each of which is currently believed to have the typical protein-protein binding capacity of the leucine rich repeat subpopulation macrolide. It is also believed that they have similar abilities to the leucine rich repeat protein SLIT and human glycoprotein V. [
[680] 12. Whole-length PRO258 polypeptide
[681] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO258. We have identified and isolated cDNA encoding the PRO258 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the present inventors have found that several parts of the PRO258 polypeptide are highly homologous to CRTAM and poliovirus receptors. Thus, the PRO258 polypeptides disclosed herein are now a newly discovered member of the Ig giant family and are now thought to have viral receptor capabilities or modulate immune function as they are.
[682] 13. Whole-length PRO266 polypeptide
[683] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO266. We have identified and isolated cDNA encoding the PRO266 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several parts of the PRO266 polypeptide are highly homologous to the SLIT protein of Drosophila. Thus, the PRO266 polypeptide disclosed herein is a newly discovered member of the leucine rich repeat subpopulation family, which has the typical ligand-ligand binding activity of this protein family and is currently thought to be involved in neuronal development. Since SLIT has been shown to be useful in the study and treatment of Alzheimer's disease, as mentioned above, PRO266 may be involved in the study and treatment of this disease.
[684] 14. Whole-length PRO269 polypeptide
[685] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO269. We have identified and isolated cDNA encoding the PRO269 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, we found that the amino acid sequence encoded by nucleotides 314 to 1783 of the full-length native sequence PRO269 (Figure 35 and SEQ ID NO: 95) was identical to the human urine thrombomodulin and several thrombomodulin analogs ≪ RTI ID = 0.0 > homologous < / RTI > Thus, the PRO269 polypeptides disclosed herein are now considered to be a newly discovered member of the trombomodulin family.
[686] 15. Full-length PRO287 polypeptide
[687] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO287. We have identified and isolated cDNA encoding the PRO287 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the present inventors have found that several parts of the PRO287 polypeptide have substantial homology with the type 1 procollagen C-proteinase enhancer protein precursor and the type 1 procollagen C-proteinase enhancer protein . Thus, the PRO287 polypeptides disclosed herein are currently believed to be a newly identified member of the C-proteinase enhancer family.
[688] 16. Full-length PRO214 polypeptide
[689] The present invention provides newly identified and isolated nucleotide sequences that encode polypeptides referred to herein as PRO214. We have identified and isolated cDNA encoding the PRO214 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the present inventors have found that the full length native sequence PRO214 polypeptide (Figure 40 and Figure 109) has a sequence identity of 49% with HT, a known protein of the EGF family. As a result, the BLAST value was 920 and 150 nucleotides were identical. Thus, it is presently believed that the PRO214 polypeptide disclosed herein is a newly discovered member of a family of proteins comprising the EGF domain and can have the typical activity or properties of an EGF domain containing protein family.
[690] 17. Full-length PRO317 polypeptide
[691] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO317. We have identified and isolated a cDNA encoding a PRO317 polypeptide, particularly as described in more detail in the Examples below. We have used the BLAST (trademark) and FastA (trademark) sequence alignment computer program to find that the full length native sequence PRO317 (Figure 42 and Figure 114) has an amino acid sequence identity of 92% with EBAF-1. It is also closely aligned with the TGF giant and many other members.
[692] Thus, it is presently contemplated that the PRO317 disclosed herein is a newly discovered member of the TGF-giant family and may have therapeutically useful properties in conditions such as uterine bleeding. Thus, PRO317 can be used to diagnose and treat abnormal bleeding associated with gynecological diseases, for example, to avoid or reduce the need for hysterectomy. Since PRO317 may also be useful as an agonist generally affecting angiogenesis, it may be useful for treating antitumor indications, or inverted coronary ischemic conditions.
[693] The ESTs and probes used to obtain consensus DNA to generate the PRO317 primers were obtained from normal tissues (uterus, prostate, colon and pancreas), multiple tumors (colon, brain (2X), pancreatic and somatic cells) It was found in a library of heart. PRO317 was also found in many tissues but was found to have a higher concentration in the uterus. Thus, PRO317 may be more widely used in the body than EBAF-1. PRO317 is expected to have the opposite effect of EBAF-1 for at least some symptoms.
[694] 18. Whole-length PRO301 polypeptide
[695] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO301. We have identified and isolated cDNA encoding the PRO301 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that the full length native sequence PRO301 (Figures 44 and 119) has a Blast value of 246 corresponding to 30% amino acid identity with the human A33 antigen precursor. Thus, it is presently believed that the PRO301 polypeptide disclosed herein is a newly discovered member of the A33 antigenic protein family and can be expressed in human neoplastic disease, such as colorectal cancer.
[696] 19. Full-length PRO224 polypeptide
[697] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO224. We have identified and isolated cDNA encoding the PRO224 polypeptide, particularly as described in more detail in the Examples below. The present inventors have found amino acid identity with the human apoji protein E receptor 2906 using known programs such as BLAST and FastA sequence alignment computer programs. Alignment of the different parts of these two polypeptides resulted in amino acid identity of 37%, 36%, 30%, 44%, 44% and 28%, respectively. Whole-length native PRO224 (Figure 46, SEQ ID NO: 127) is also amino acid identical to the very low-density lipoprotein receptor precursor of bile. Alignment of the other parts of these two polypeptides resulted in amino acid identity of 38%, 37%, 42%, 33% and 37%, respectively. In addition, the full-length native PRO224 (FIG. 46, SEQ ID NO: 127) has amino acid identity with the chicken oocyte receptor P95 of Gallus gallus. The alignment of the other parts of these two polypeptides resulted in amino acid identity of 38%, 37%, 42%, 33% and 37%, respectively. Furthermore, full-length native PRO224 (Figure 46, SEQ ID NO: 127) has sequence identity to the short form of the human very low density lipoprotein receptor. Alignment of the other parts of these two polypeptides results in amino acid identity of 32%, 38%, 34%, 45% and 31%, respectively. Thus, the PRO224 polypeptides disclosed herein are now a newly discovered member of the low density lipoprotein receptor family and are currently thought to have the structural properties necessary to recognize the typical low density lipoproteins of the low density lipoprotein receptor family and to have endocytosis function. (Scoring parameters T = 7, S + 65, S2 = 36, Matrix: BLOSUM62 were used for the above mentioned alignment.)
[698] 20. Whole-length PRO222 polypeptide
[699] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO222. We have identified and isolated cDNA encoding the PRO222 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have determined that the sequence coding for the full length native sequence PRO222 (Figure 48 and Figure 132) is 25-26% amino acid identity with the mouse complement factor h precursor and the amino acid sequence with the complement receptor Found that the amino acid identity with the long form precursor of the mouse complement C3b receptor type 2 is 25-47% and the amino acid identity with the human virtual protein kiaa0247 is 40%. Thus, the PRO222 polypeptide disclosed herein is a newly discovered member of the complement receptor family and is currently thought to have the typical activity of a complement receptor family.
[700] 21. Full-length PRO234 polypeptide
[701] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO234. We have identified and isolated cDNA encoding the PRO234 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST (FastA format) sequence alignment computer program, the present inventors have found that the cDNA encoding the full length native sequence PRO234 is 31% identical to the E-selectin precursor and has a Blast value of 134. Thus, the PRO234 polypeptide disclosed herein is a newly discovered member of the lectin / selectin family and is currently thought to have the typical activity of the lectin / selectin family.
[702] 22. Whole-length PRO231 polypeptide
[703] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO231. We have identified and isolated cDNA encoding the PRO231 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, we found that the full length native sequence PRO231 polypeptide (Figures 52 and 142) had amino acid identity of 30% and 31% with human and rat prostate acid phosphatase precursor protein, respectively did. Thus, it is presently believed that the PRO231 polypeptide disclosed herein can be a newly discovered member of the acid phosphatase protein family.
[704] 23. Whole-length PRO229 polypeptide
[705] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO229. We have identified and isolated cDNAs encoding PRO229 polypeptides, as described in more detail in the Examples below, in more detail. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO229 polypeptide are highly homologous to the antigen wc1.1, M130 antigen, T cell surface glycoproteins CD6 and CD6. It is also related to SP alpha. Thus, the PRO229 polypeptide disclosed herein is a newly discovered member of a family of scavenger receptor homologous sequence motifs found in a number of proteins involved in immune function, so that the degradation-suppressing segments and / or immune functions . ≪ / RTI >
[706] 24. The full-length PRO238 polypeptide
[707] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO238. We have identified and isolated cDNA encoding the PRO238 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO238 polypeptide are highly homologous with the reductase, including the oxidoreductase and the fatty acid acyl-CoA reductase. Thus, the PRO238 polypeptide disclosed herein is a newly discovered member of the reductase family and is currently thought to have the typical reducing activity of the reductase family.
[708] 25. The full-length PRO233 polypeptide
[709] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO233. We have identified and isolated cDNA encoding the PRO233 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO233 polypeptide are highly homologous to the reductase protein. We have also found that the DNA encoding the PRO233 polypeptide is highly homologous to the protein of Caenorhabditis elegans . Thus, the PRO233 polypeptide disclosed herein is a newly discovered member of the reductase family and is currently thought to have the typical ability of the reductase family to affect the redox state of the cells.
[710] 26. The full-length PRO223 polypeptide
[711] The present invention provides newly identified and isolated nucleotide sequences that encode polypeptides referred to herein as PRO223. We have identified and isolated cDNA encoding the PRO223 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that the full length native sequence PRO223 polypeptide is highly homologous to several serine carboxy peptidase polypeptides. Thus, the PRO223 polypeptide disclosed herein is presently considered to be a newly identified serine carboxypeptidase.
[712] 27. The full-length PRO235 polypeptide
[713] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO235. We have identified and isolated cDNA encoding the PRO235 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO235 polypeptide are highly homologous to several flexin proteins. Thus, the PRO235 polypeptides disclosed herein are now a newly identified member of the fringe family and are currently thought to have the typical cell adhesion of the felcines.
[714] 28. The full-length PRO236 and PRO262 polypeptides
[715] The present invention provides newly identified and isolated nucleotide sequences that encode polypeptides referred to herein as PRO236 and PRO262. We have identified and isolated cDNAs encoding PRO236 and PRO262 polypeptides, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO236 and PRO262 polypeptides are highly homologous to several -Galactosidase and -Galactosidase precursor polypeptides. Thus, the PRO236 and PRO262 polypeptides disclosed herein are now contemplated as newly identified beta -galactosidase analogs.
[716] 29. The full-length PRO239 polypeptide
[717] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO239. We have identified and isolated cDNA encoding the PRO239 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several parts of the PRO239 polypeptide are highly homologous to the tansine proteins. Thus, the PRO239 polypeptide disclosed herein is a newly identified tenninic member and is currently believed to have the ability to perform the snapping process and cell adhesion, as does the tennin family.
[718] 30. Whole-length PRO257 polypeptide
[719] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO257. We have identified and isolated cDNA encoding the PRO257 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO257 polypeptide are highly homologous to the Ebnerine precursor. Thus, the PRO257 polypeptide disclosed herein is currently considered to be a newly identified protein associated with the < RTI ID = 0.0 > Ebnerin < / RTI >
[720] 31. The full-length PRO260 polypeptide
[721] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO260. We have identified and isolated cDNA encoding the PRO260 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the present inventors have found that several parts of the PRO260 polypeptide are highly homologous to alpha-1-fucosidase precursors. Thus, the PRO260 polypeptides disclosed herein are newly identified proteins of the fucosidase family and are currently thought to have enzyme activities related to the typical fucosyl residues of the fucosidase family.
[722] 32. The full-length PRO263 polypeptide
[723] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO263. We have identified and isolated cDNA encoding the PRO263 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the present inventors have found that several parts of the PRO263 polypeptide are highly homologous to the CD44 antigen and related proteins. Thus, the PRO263 polypeptides disclosed herein are newly identified proteins of the CD44 antigen class and are useful for the characterization of these antigens, such as cancer and HIV markers, cell-cell interactions, cell-substrate interactions, cellular traffic regulation, lymph node homing, Delivery of a growth signal, presentation of a growth factor to a cell under migration, and growth factor.
[724] 33. The full-length PRO270 polypeptide
[725] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO270. We have identified and isolated cDNA encoding the PRO270 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several parts of the PRO270 polypeptide are highly homologous to several thioredoxin proteins. Thus, the PRO270 polypeptides disclosed herein are currently believed to have the typical ability of the thioredoxin family to be a newly identified protein of the thioredoxin family and affecting the reduction-oxidation (redox) state.
[726] 34. The full-length PRO271 polypeptide
[727] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO271. We have identified and isolated cDNA encoding the PRO271 polypeptide, as described in more detail in the Examples below, in more detail. Using the BLAST and FastA sequence alignment computer programs, the present inventors have found that several parts of the PRO271 polypeptide are highly homologous to multiple linking proteins and their precursors. Thus, the PRO271 polypeptide disclosed herein is currently considered as a newly identified link protein homolog.
[728] 35. The full-length PRO272 polypeptide
[729] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO272. We have identified and isolated cDNA encoding the PRO272 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO272 polypeptide are highly homologous to human reticulocalcalipin proteins and their precursors. We have also found that the DNA encoding the PRO272 polypeptide is highly homologous to the mouse reticulo-calvin precursor protein. Thus, the PRO272 polypeptide disclosed herein is a newly identified reticulocalcaline protein and is currently thought to have the typical calcium binding capacity of this family.
[730] 36. The full-length PRO294 polypeptide
[731] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO294. We have identified and isolated cDNA encoding the PRO294 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several parts of the PRO294 polypeptide are highly homologous to various parts of many collagen proteins. Thus, the PRO294 polypeptide disclosed herein is currently considered as a newly identified collagen family protein.
[732] 37. The full-length PRO295 polypeptide
[733] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO295. We have identified and isolated a cDNA encoding the PRO295 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several parts of the PRO295 polypeptide are highly homologous to the integrin protein. Thus, the PRO295 polypeptide disclosed herein is a newly identified integrin protein and is currently thought to have the typical cell adhesion potential of the Integrins.
[734] 38. The full-length PRO293 polypeptide
[735] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO293. We have identified and isolated cDNA encoding the PRO293 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that portions of the PRO293 polypeptide are highly homologous to the neuronin-rich repeat proteins 1 and 2 (NLRR1 and NLRR-2), particularly NLRR-2 . Thus, the PRO293 polypeptide disclosed herein is a newly identified protein of the neurolancein rich repeat subpopulation family and is currently thought to have the typical ligand-ligand binding activity of the NRLL protein family.
[736] 39. The full-length PRO247 polypeptide
[737] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO247. We have identified and isolated cDNA encoding the PRO247 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO247 polypeptide are highly homologous to densin. We have also found that the DNA encoding the PRO247 polypeptide is highly homologous to many other proteins, including KIAA0231. Thus, the PRO247 polypeptide disclosed herein is a newly identified protein of the leucine rich repeat subpopulation family and is currently thought to have the typical ligand binding capacity of this family.
[738] 40. The full-length PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides
[739] The present invention provides newly identified and isolated nucleotide sequences which encode polypeptides referred to herein as PRO302, PRO303, PRO304, PRO307 or PRO343. Specifically, the inventors identified and isolated cDNAs encoding PRO302, PRO303, PRO304, PRO307 or PRO343 polypeptides, as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides are highly homologous to several protease proteins. Thus, the PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides disclosed herein are currently believed to be newly identified protease proteins.
[740] 41. The full-length PRO328 polypeptide
[741] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO328. Specifically, the inventors identified and isolated cDNA encoding the PRO328 polypeptide, as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO328 polypeptide are highly homologous to human glial subclass protein (" GLIP "). We also found that several parts of the PRO328 polypeptide are highly homologous to the cysteine rich secreted protein (" CRISP ") through BLAST homology [ECCRISP3_1, S68683 and CRS3_HUMAN]. Thus, the PRO328 polypeptide disclosed herein is a newly identified protein of the GLIP or CRISP family and is currently thought to have a typical transcription regulatory activity of the GLIP or CRISP family.
[742] 42. The full-length PRO335, PRO331 and PRO326 polypeptides
[743] The present invention provides newly identified and isolated nucleotide sequences which encode polypeptides referred to herein as PRO335, PRO331 or PRO326. Specifically, the inventors identified and isolated cDNAs encoding PRO335, PRO331, or PRO326 polypeptides, as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO335, PRO331, or PRO326 polypeptides are highly homologous to LIG-1, ALS, and PRO331 is also highly homologous to decolin. Thus, the PRO335, PRO331, and PRO326 polypeptides disclosed herein are newly identified proteins of the leucine rich repeat subpopular macrolide family, particularly those associated with LIG-1 and have a biological function of this family as discussed and recited herein It is thought now.
[744] 43. The full-length PRO332 polypeptide
[745] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO332. Specifically, the inventors identified and isolated cDNA encoding the PRO332 polypeptide, as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, we found that the full length native sequence PRO332 (Figure 108 and SEQ ID NO: 310) contained several species of the fibridolandlin and pibromodulin precursor sequences (FMOD_BOVIN, FMOD_CHICK, FMOD_RAT, FMOD_MOUSE (PTU48360_1, AF022890_1), corneal proteoglycans (AF022256_1), FMO_HUMAN, P_R36773), osteo-modulin sequences (AB000114_1, AB007848_1), decolin sequences (CFU83141_1, OCU03394_1, P_R42266, P_R42267, P_R42260, P_R89439), keratan sulfate proteoglycans And a series of known proteoglycan sequences, including bone / cartilage proteoglycans and proteoglycan precursors (PGS1_BOVIN, PGS2_MOUSE, PGS2_HUMAN), which are about 30-40% amino acid identity. Thus, it is presently believed that the PRO332 disclosed herein is a novel proteoglycan-type molecule and may be involved in extracellular matrix, cartilage and / or bone function.
[746] 44. The full-length PRO334 polypeptide
[747] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO334. We have identified and isolated cDNA encoding the PRO334 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several parts of the PRO334 polypeptide are highly homologous to fibrin and fibril. Thus, the PRO334 polypeptide disclosed herein is a newly identified protein of the epithelial growth factor family and is currently thought to have the typical properties and activities of this family.
[748] 45. The full-length PRO346 polypeptide
[749] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO346. We have identified and isolated cDNA encoding the PRO346 polypeptide, particularly as described in more detail in the Examples below. We have used the BLAST and FastA sequence alignment computer programs to find that the full length native sequence PRO346 (Figure 112 and SEQ ID 320) has an amino acid sequence identity of 28% with the marine antigen. Thus, the PRO346 polypeptide disclosed herein is a newly identified protein of the marine protein family and is currently thought to be capable of being expressed in association with neoplastic tissue disorders.
[750] 46. The full-length PRO268 polypeptide
[751] The present invention provides a newly identified and isolated nucleotide sequence encoding a polypeptide referred to herein as PRO268. We have identified and isolated cDNA encoding the PRO268 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that portions of the PRO268 polypeptide are highly homologous to several protein disulfide isomerase proteins. Thus, the PRO268 polypeptide disclosed herein is currently considered to be a homolog of the protein disulfide isomerase p5 protein.
[752] 47. The full-length PRO330 polypeptide
[753] The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to herein as PRO330. We have identified and isolated a cDNA encoding a PRO330 polypeptide, particularly as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that several portions of the PRO330 polypeptide are highly homologous to the rat prolyl 4-hydroxylase alpha-II subunit protein. Thus, the PRO330 polypeptide disclosed herein is currently believed to be a novel prolyl 4-hydroxylase subunit polypeptide.
[754] 48. The full-length PRO339 and PRO310 polypeptides
[755] The present invention provides newly identified and isolated nucleotide sequences that encode polypeptides referred to herein as PRO339 and PRO310. We have identified and isolated cDNA encoding the PRO339 polypeptide, particularly as described in more detail in the Examples below. We also identified and isolated cDNA encoding the PRO310 polypeptide, as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, the inventors have found that various portions of the PRO339 and PRO310 polypeptides are highly homologous to the small secretory proteins of C. elegans and are remotely related to fringe. The sequences used to identify PRO310 are referred to herein as DNA40533 and DNA42267 and likewise exhibit homology to the C. elegans protein. Thus, the PRO339 and PRO310 polypeptides disclosed herein are now a newly identified member of the family of genes involved in development and are now thought to have regulatory abilities similar to their ability to regulate serrate.
[756] 49. The full length PRO0244 polypeptide
[757] The present invention provides a newly identified and isolated nucleotide sequence encoding a C-type lectin referred to herein as PRO0244. Specifically, the inventors identified and isolated cDNA encoding the PRO244 polypeptide, as described in more detail in the Examples below. Using the BLAST and FastA sequence alignment computer programs, we found that the full-length native sequence PRO244 (Figure 122 and SEQ ID NO: 377) has 43% amino acid sequence identity with Glenectin Gallus gallus (LECH- CHICK) and HIV gp120- ) Was found to be 42%. Thus, the PRO244 disclosed herein is a newly identified protein of the C-lectin macropeptide and is currently thought to be capable of engaging in the development of immune function, apoptosis or atherosclerosis. In addition, PRO244 may be useful in identifying tumor-associated epitopes.
[758] B. PRO polypeptide variants
[759] It is contemplated that, in addition to the full-length native sequence PRO polypeptides described herein, PRO variants can be produced. PRO mutants can be produced by introducing suitable nucleotide changes into PRO DNA and / or by synthesis of the desired PRO polypeptides. Those skilled in the art will appreciate that amino acid changes such as changes in the number or location of glycosylation sites or changes in membrane anchoring properties can alter the post-translational processing of RPO.
[760] Mutations in various domains of the full-length native sequence PRO or PRO described herein may be made using conservative and non-conservative mutagenesis techniques and instructions, for example, as described in U.S. Patent No. 5,364,934. A mutation can be a substitution, deletion or insertion of one or more codons that encode a PRO that changes the amino acid sequence of PRO relative to the native sequence PRO. In some cases, the mutation is generated by substituting one or more amino acids in one or more domains of PRO with any other amino acid. Amino acid residues that can be inserted, substituted or deleted without deleterious effects on the desired activity can be obtained by comparing the sequence of PRO with a sequence of known homologous protein molecules and minimizing the number of amino acid sequence changes produced in regions of high homology You can decide. Amino acid substitutions can be the substitution of one amino acid with another amino acid having similar structure and / or chemical properties, e. G., Replacement of lysine with serine, i. E. Conservative substitution of amino acids. Insertion or deletion may optionally occur at about 1 to 5 amino acids. Acceptable variations can be determined by systematically inserting, deleting, or substituting amino acids within the sequence and testing the activity of the resulting variant displayed by full-length or mature native sequences.
[761] The subject provides PRO polypeptide fragments. For example, when compared to full-length native proteins, these fragments may be truncated at the N-terminus or at the C-terminus and internal residues may be deleted. Some fragments lack amino acid residues that are not essential for the desired biological activity of the PRO polypeptides of the invention.
[762] PRO fragments can be prepared by any of many conventional techniques. The desired peptide fragments can be chemically synthesized. Another method is an enzymatic degradation method, for example, treating the protein with an enzyme known to cleave the protein at a site defined by a specific amino acid residue, or by cleaving the DNA with a suitable restriction enzyme to produce a PRO fragment, Lt; / RTI > However, another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment by polymerase chain reaction (PCR). Oligonucleotides that define the desired end of the DNA fragment are used as 5 ' and 3 ' primers in PCR. Preferably, the PRO polypeptide fragment shares one or more biological and / or immunological activities with the native PRO polypeptide disclosed herein.
[763] In a specific embodiment, conservative substitutions of the subject are shown in Table 1 with the heading of preferred substitutions. When the biological activity is changed by such substitution, a more substantial change, which is named as a substitution example in Table 1 below or is described in more detail below for the type of amino acid, is introduced and the product screened.
[764] Original residue Substitution example Preferred substitution examples Ala (A) val, leu, with val Arg (R) lys, gln, asn lys Asn (N) gln, his, lys, arg gln Asp (D) glu glu Cys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro, ala ala His (H) asn, gln, lys, arg arg Ile (I) leu, val, met, ala, phe, norleucine leu Leu (L) norleucine, with val, met, ala, phe with Lys (K) arg, gln, asn arg Met (M) leu, phe, with leu Phe (F) leu, val, with, ala, tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr, phe tyr Tyr (Y) trp, phe, thr, ser phe Val (V) with leu, met, phe, ala, norleucine leu
[765] Substantial modifications of the function or immunological identity of the PRO polypeptide can be accomplished by (a) maintaining the structure of the polypeptide backbone in the replacement domain, e.g., in the form of a sheet or spiral, or (b) maintaining the charge or hydrophobicity of the molecule at the target site Or (c) selecting a substitution that significantly changes its effect of retaining most of the side chains. Naturally occurring residues can be divided into the following groups according to their common branching characteristics:
[766] (1) hydrophobicity: norleucine, met, ala, val, leu, ile;
[767] (2) Neutral hydrophilic: cys, ser, thr;
[768] (3) Acid: asp, glu;
[769] (4) Basicity: asn, gln, his, lys, arg;
[770] (5) Residues that affect chain orientation: gly, pro; And
[771] (6) aromatic; trp, tyr, phe.
[772] Non-conservative substitutions will replace the same kind of component with another kind. In addition, the thus substituted moiety can be introduced into the conservative substitution moiety or, more preferably, can be introduced into the remaining (non-conserved) moiety.
[773] Variations can be made using methods known in the art such as oligonucleotide-mediated (locating) mutagenesis, alanine scanning and PCR mutagenesis. Positioning mutagenesis [Carter et al., Nucl. Acids Res. , 13 : 4331 (1986)], Zoller et al., Nucl. Acids Res. , 10: 6487 (1987)], cassette mutagenesis [Wells (Wells) including literature [Gene, 34 of 315 (1985)], restriction et selection mutagenesis [Wells (Wells) [Philos. Trans. R. Soc. London Ser A , 317 : 415 (1986)] or other known techniques to the cloned DNA to produce the PRO polypeptide mutant DNA of the present invention.
[774] In addition, one or more amino acids can be identified along the contiguous sequence using a scanning amino acid assay. Preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Typically, alanine is the preferred scanning amino acid because it is less likely to remove the side chains outside the beta-carbon and alter the backbone sequence of the variant (Cunningham and Wells, Science , 244 : 1081-1085 )]]. In addition, alanine is preferred because it is usually the most common amino acid. Alanine is also frequently found in both buried and exposed sites (Creighton, TheProteins , (WH Freeman & Co., NY); Chothia, J. Mol. Biol. , ≪ / RTI > 150 : 1 (1976)]. An isoteric amino acid can be used if the alanine substitution does not produce a suitable amount of the variant.
[775] C. Variation of PRO
[776] Covalent modifications of PRO are included within the scope of the present invention. One form of covalent modification includes reacting a target amino acid residue of the PRO polypeptide with an organic derivatizing agent capable of reacting with a selected side chain or N-terminal or C-terminal residue of PRO. Derivatization with bifunctional agents is useful, for example, for cross-linking PRO to the water-insoluble support matrix or surface for use in anti-PRO polypeptide antibody purification methods. Commonly used crosslinking agents are, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example esters with 4- Homobifunctional imidoesters including disuccinimidyl esters such as 3,3'-dithiobis (succinimidyl propionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and Methyl-3 [(p-azidophenyl) dithio] propioimidate.
[777] Other variations include deamidation of the glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, hydroxylation of the proline and lysine, phosphorylation of the hydroxyl group of the seryl or threonyl residue, lysine, Methylation of alpha-amino groups of arginine and histidine side chains [see TE Creighton, Proteins: Structure and Molecular Properties , WH Freeman & Co., San Francisco, 79-86 (1983)], acetylation of the N-terminal amine and amidation of the C-terminal carboxyl group.
[778] Other types of covalent modifications of the PRO polypeptides included within the scope of the present invention include altering the native glycosylation pattern of the polypeptide. By " altering the native glycosylation pattern " is meant a deletion (deletion of a potential glycosylation site or deletion of the glycosylation by chemical and / or enzymatic methods) of one or more carbohydrate moieties found in the native sequence PRO and / Quot; means the addition of one or more glycosylation sites that are not present in the native sequence PRO. The term also includes qualitative changes in the glycosylation of natural proteins, including changes in the nature and proportions of the various carbohydrate residues present.
[779] Addition of a glycosylation site to a PRO polypeptide can be achieved by altering the amino acid sequence. The change can be made, for example, by the addition or substitution of one or more serine or threonine residues in the native sequence PRO (for O-linked glycosylation sites). The PRO amino acid sequence can be arbitrarily altered at varying levels of DNA by mutating the DNA encoding the PRO polypeptide in a preselected base to produce a codon specifically translated into the desired amino acid.
[780] Another means of increasing the number of carbohydrate moieties on the PRO polypeptide is by chemically or enzymatically coupling the glycoside to the polypeptide. Such methods are described, for example, in WO 87/05330, published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem. , pp. 259-306 (1981).
[781] Removal of the carbohydrate moieties present in the PRO polypeptide can be accomplished either chemically or by enzymatic or substitution by mutation of codons encoding amino acid residues that function as glycosylation targets. Chemical deglycosylation techniques are well known in the art and are described, for example, in Hakimuddin et al . Arch. Biochem. Biophys. , 259 : 52 (1987) and Edge et al ., Anal. Biochem. , 118 : 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved using a variety of endoglycosidases and exoglycosidases (Thotakura et al ., Meth. Enzymol. , ≪ / RTI > 138 : 350 (1987)].
[782] Other classes of covalent modifications of PRO include various non-proteinaceous polymers in the manner described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192 or 4,179,337, examples For example, linking the PRO polypeptide to one of polyethylene glycol (PEG), polypropylene glycol or polyoxyalkylene.
[783] In addition, the PRO of the present invention may be modified in such a way as to form a chimeric molecule comprising a PRO fused to another heterologous polypeptide or amino acid sequence.
[784] In another embodiment, such a chimeric molecule comprises a fusion of a PRO with a tag polypeptide that provides an epitope to which the anti-tag antibody can selectively bind. The epitope tag is generally located at the amino or carboxyl terminus of PRO. The presence of the PRO in the form with the epitope tag can be detected using an antibody against the tag polypeptide. In addition, when an epitope tag is introduced, PRO can be easily purified by affinity purification using an anti-tag antibody or other affinity matrix that binds to an epitope tag. A variety of tag polypeptides and their respective antibodies are known in the art. Examples include poly-histidine (poly-his) or poly-his-gly tag, flu HA tag polypeptide and its antibody 12CA5 [Field et al . Cell. Biol. , 8: 2159-2165 (1988)] ], c-myc tag and hence of 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies [Evan (Evan) documents [Molecular and Cellular Biology, 5, such as: 3610-3636 (1985)], and the herpes simplex virus glycoprotein D (gD) tag and its antibody (Paborsky et al., Protein Engineering , 3 (6): 547-553 (1990) have. Other tag polypeptides include Flag-peptides (Hopp et al., BioTechnology , 6 : 1204-1210 (1988)], KT3 epitope peptides [Martin et al. Science 255 : 192-194 )]], Alpha-tubulin epitope peptides [Skinner et al ., J. Biol. Chem. , 266 : 15163-15166 (1991)] and the T7 gene 10 protein tag [Lutz-Freyermuth et al ., Proc. Natl. Acad. Sci. USA , 87 : 6393-6397 (1990)].
[785] In another embodiment, the chimeric molecule comprises a fusion of a specific region of an immunoglobulin or immunoglobulin with PRO. In the case of a bimodal form of chimeric molecule (also referred to as " immunoadhesin "), the fusant may be the Fc region of an IgG molecule. The Ig fusions preferably include substitution of at least one region of the variable region in the Ig molecule with the soluble (deleted or inactivated transmembrane domain) form of the PRO polypeptide. In a particularly preferred embodiment, the immunoglobulin fusions comprise the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of the IgG1 molecule. For methods of producing immunoglobulin fusions, see U.S. Patent No. 5,428,130, issued June 27, 1995.
[786] D. Manufacture of PRO
[787] The following description relates to a method for producing PRO by culturing cells transfected or transfected with a vector containing mainly PRO nucleic acid. Of course, PRO can be prepared using other methods known in the art. For example, PRO sequences or fragments thereof can be prepared by direct peptide synthesis using solid phase techniques (Stewart et al., Solid-Phase Peptide Synthesis , WH Freeman Co., San Francisco, Calif. 1969) and Merrifield, J. Am. Chem. Soc. , 85 : 2149-2154 (1963)]. In vitro protein synthesis can be performed by manual or automated methods. Automated synthesis can be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) according to the manufacturer's instructions. The different fragments of PRO can be chemically synthesized separately and combined with chemical or enzymatic methods to produce full-length PRO.
[788] 1. Isolation of DNA encoding PRO
[789] DNA encoding PRO can be obtained from a cDNA library prepared from tissues that possess PRO mRNA and are expected to express it at a detectable level. Thus, human PRO DNA can conveniently be obtained from cDNA libraries prepared from human tissue as described in the Examples. PRO coding genes can also be obtained from genomic libraries or obtained by known synthetic methods (e. G., Automated nucleic acid synthesis methods).
[790] The library may be screened using a probe designed to identify the gene of interest or a protein encoded by the gene (e.g., an antibody to the desired PRO or an oligonucleotide consisting of about 20-80 bases). Screening of the cDNA or genomic library using the selected probe can be carried out using standard methods as described for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989) . Other means for isolating the gene encoding PRO is to use PCR method [the literature, such as fountain Brook (Sambrook); Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Haror Laboratory Press, 1995)].
[791] The following examples illustrate techniques for screening cDNA libraries. The oligonucleotide sequence selected as the probe should be of sufficient length and sufficiently distinct sequence to minimize false results. The oligonucleotides are preferably labeled so that they can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art and include the use of radiolabeled, biotinylated or enzymatic labels such as ³² P-labeled ATP. Hybridization conditions, including moderate stringency and high stringency is provided in the described supra, such as literature Sam Brook, (Sambrook).
[792] The sequence identified in the library screening method can be aligned with a public database such as GenBank or other known sequences that are deposited and available in other proprietary databases. Sequence identity (at the amino acid or nucleotide level) within a defined region of a molecule or across a full-length sequence can be determined using methods known in the art and described herein.
[793] Nucleic acid having protein coding sequence is a cDNA or genomic library selected using conventional methods of primer extension as described in described supra, such as fountain Brook (Sambrook) for first detecting the estimated amino acid sequence and the precursor, if necessary, as disclosed herein, Screening and processing an intermediate of the mRNA that has not been reverse transcribed with the cDNA.
[794] 2. Selection and Transformation of Host Cells
[795] The host cell is transfected or transformed with the expression or cloning vector described herein for PRO production and cultured in a conventional nutrient medium modified to suit the promoter induction, transformant selection or gene amplification encoding the desired sequence. Those skilled in the art can select culture conditions such as medium, temperature, pH, etc., without performing unnecessary experiments. In general, principles, protocols, and techniques for maximizing the productivity of cell cultures are described in Mammalian Cell Biotechnology: a Practical Approach , M. Butler, ed. Can be found in (IRL Press, 1991)] and Sam Brook et al., (Sambrook) [supra].
[796] Eukaryotic transfection methods and prokaryotic transformation methods, such as CaCl ₂ , CaPO ₄ , liposome-mediated methods and electroporation, are known to those skilled in the art. Depending on the host cell used, transformation is carried out using standard techniques suitable for the cell. Sam Brook (Sambrook) described supra, or electroporation, calcium treatment using calcium chloride method described in, such as is generally used for prokaryotes. Transfection using Agrobacterium tumefaciens can be carried out as described in Shaw et al. Gene , 23 : 315 (1983) and in WO 89/05859 published June 29, It is used to transform certain plant cells. For the mammalian cell without such a cell wall, the calcium phosphate precipitation method of Graham and van der Eb, Virology , 52 : 456-457 (1978) can be used. Generic characteristics of mammalian cell host system transformation are described in U.S. Patent No. 4,399,216. Transformation into yeast is generally performed according to the method of Van Solingen et al., J. Bact. , 130 : 949 (1977) and Hsiao et al ., Proc. Natl. Acad. Sci. (USA) , 76 : 3829 (1979). However, other methods of introducing DNA into cells, such as nuclear microinjection, electroporation, protoplast fusion with bacterial protoplasts, or polycations, such as polybrene and polyornithine, may also be used. Several techniques for transforming mammalian cells are described in Keown et al., Methods in Enzymology , 185: 527-537 (1990), and Mansour et al. , Nature , 336: 348-352 (1988).
[797] Suitable host cells for cloning or expressing DNA in the vector herein are prokaryotic, yeast or higher eukaryotic cells. Suitable prokaryotic cells include true bacteria, such as gram-negative or gram-positive organisms, such as Enterobacteriaceae, e. Cola, but are not limited thereto. A variety of this. Coli strains, e. E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31, 537); Coli strains W3110 (ATCC 27,325) and K5 772 (ATCC 53,635) are readily available. Other suitable prokaryotic host cells include Escherichia, e. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium, Serratia, For example, marcescans and Shigella, and Bacillus, e. G. Subtilis and non-subtilis. Licheniformis (e.g., B. riciniformis 41P described in DD 266,710, published April 12, 1989), Pseudomonas such as p. Aeruginosa and Streptomyces. ≪ / RTI > These examples are merely illustrative and not restrictive. The strain W3110 is a particularly preferred host or parental host since it is a host strain common to recombinant DNA product fermentation. Preferably, the host cell secretes a minimal amount of proteolytic enzyme. For example, strain W3110 can be modified to result in a mutation of the gene encoding the endogenous protein of the host, and an example of such a host is E. coli having the complete genotype tonA. E. coli W3110 strain 1A2, with complete genotype tonA ptr3. E. coli W3110 strain 9E4, complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan ^r . E. coli W3110 strain 27C7 (ATCC 55,244), complete genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT rbs7 ilvG kan ^r . E. coli W3110 strain 37D6, strain 37D6 with a non-kanamycin resistant degP deletion mutant. E. coli W3110 strain 40B4, and periplasmic protease variants disclosed in U.S. Patent No. 4,946,783, issued Aug. 7, Coli strains. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are suitable.
[798] In addition to prokaryotes, eukaryotic microbes such as fibrous fungi or yeast are suitable as cloning or expression hosts for PRO coding vectors. Saccharomyces cerevisiae is a lower eukaryotic host microorganism commonly used. Other microorganisms include Schizosaccharomyces pombe (Beach and Nurse, Nature , 290: 140 [1989]); European Patent No. 139,383, published May 2, 1985]; Kluyveromyces host [U.S. Patent No. 4,943,529; Fleer et al., Bio / Technology , 9: 968-975 (1991); Lactis [MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Baceriol. , ≪ / RTI > 737 [1983]), Kay. Fragilis (ATCC 12,424), Kay. Bulgaricus (ATCC 16,045), k. Wickeramii (ATCC 24,178), k. Waltii (ATCC 56,500), Kay. Drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology , 8: 135 (1990), K. Thermotolerans and K. Marxianus; Yarrowia (European Patent No. 402,226); Pichia pastoris [European Patent No. 183,070; Sreekrishna et al ., J. Basic Microbiol. , 28: 265-278 [1988]; Candida; Trichoderma marocia [European Patent No. 244,234]; Neurospora crassa (Case et al ., Proc. Natl. Acad. Sci. USA , 76: 5259-5263 [1979]); Schwanniomyces, e.g., Siebenia mume occidentalis (European Patent 394,538, published October 31, 1990); And filamentous fungi such as Neurospora, Penicillium, Tolypocladium (WO 91/00357 published Jan. 10, 1991) and Aspergillus host, e.g., Listen to this. Nidulans (Ballance et al., Biochem. Biophys. Res. Commun. , ≪ / RTI > 112: 284-289 [1983]; Tilburn et al., Gene , 26: 205-221 [1983]; Yelton et al ., Proc. Natl. Acad. Sci. USA , 81: 1470-1474 [1984]) and A. < RTI ID = 0.0 > Niger (Kelly and Hynes, EMBO J. , 4: 475-479 [1985]). Methyl auxotrophic yeast is suitable and has a genus consisting of Hansenula, Candida, Kloeckera, Pichia, Carolomyces, Torulopsis and Rhodotorula But not limited to, yeast that can grow on methanol. A list of specific species that are examples of these types of yeast is given in C. Anthony, The Biochemistry of Methylotrophs , 269 (1982).
[799] Suitable host cells for expression of glycosylated PRO are derived from multicellular organisms. Examples of invertebrate cells are insect cells and plant cells such as Drosophila S2 and Spodoptera Sf9. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) cells and COS cells. More specific examples include the monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed by SV40, the human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al. J. Gen Virol. , 36: 599 (1997)), Chinese hamster ovary cells / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA , 77: 4216 )), mouse Sertoli cells (TM4, mateo, Biol Reprod, 23:.. 243-251 (1980)), human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, HB 8065) and mouse mammary Tumor (MMT 060562, ATCC CCL51). One skilled in the art can readily select suitable host cells.
[800] 3. Choosing and Using Replicable Vectors
[801] A nucleic acid (e. G., CDNA or genomic DNA) encoding a PRO is inserted into a replicable vector for cloning (amplification of DNA) or expression. Various vectors can be easily obtained. For example, the vector may be in the form of a plasmid, a cosmid, a viral particle, or a phage. Suitable nucleic acid sequences may be inserted into the vector by a variety of methods. Generally, DNA is inserted into the appropriate restriction endonuclease site (s) using techniques known in the art. The vector component generally includes, but is not limited to, a signal sequence, a replication origin, one or more marker genes, an enhancer component, a promoter, and a transcription termination sequence. The preparation of suitable vectors comprising one or more of the above components employs standard ligation techniques known to those skilled in the art.
[802] PRO can be produced as a fusion polypeptide with a heterologous polypeptide that can be produced by direct recombination methods as well as other polypeptides or signal sequences that have a specific cleavage site at the N terminus of the mature protein or polypeptide. Generally, the signal sequence may be a component of a vector or it may be part of a PRO polypeptide DNA inserted into a vector. The signal sequence may be, for example, a prokaryotic signal sequence selected from the group of alkaline phosphatase, penicillinase, lpp or thermostable enterotoxin II leaders. For yeast secretion, the signal sequence may be, for example, a yeast invertase leader, an alpha factor leader (Saccharomyces and Kluyveromyces alpha-factor leader (US Patent 5,010,182)) or an acid phosphatase Reader, Mr.. It may be a signal sequence as described in C. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990) or WO 90/13646 published Nov. 15, 1990. In mammalian cell expression, signal sequences from secreted polypeptides of mammalian signal sequences, e. G. The same or related species, and viral secretory leader can be used to direct secretion of the protein.
[803] Both the expression and cloning vectors contain nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Such sequences are known for a variety of bacteria, yeast and viruses. The origin of replication from plasmid pBR322 fits most Gram negative bacteria, the 2μ plasmid origin fits yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) clone vectors in mammalian cells useful.
[804] Expression and cloning vectors will typically contain a selectable gene, also referred to as a selectable marker. A representative selection gene, for example, in the case of Bacillus, is a gene encoding D-alanine racemase that (a) is a protein that confers resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, b) proteins that complement nutritional deficiencies, or (c) proteins that supply important nutrients that are not available from complex media.
[805] Examples of suitable selectable markers for mammalian cells are those which enable the identification of cells capable of receiving PRO coding nucleic acid, e. G. DHFR or thymidine kinase. When wild-type DHFR is used, a suitable host cell is a CHO cell line lacking DHFR activity and is described by Urlaub et al ., Proc. Natl. Acad. Sci. USA , 77: 4216 (1980)]. A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al. , Nature , 282: 39 (1979)); Kingsman et al., Gene , 7: 141 (1979); Tschemper et al. Gene , 10: 157 (1980)]. The trp1 gene provides selection markers for yeast mutants (e.g., ATCC 44076 or PEP4-1) lacking the ability to grow into tryptophan (Jones, Genetics , 85: 12 (1977)).
[806] The expression and cloning vector contains a promoter operably linked to a PRO coding nucleic acid sequence, directing mRNA synthesis. Promoters recognized by a variety of potential host cells are known. Promoters suitable for use in prokaryotic hosts include the -Lactamase and lactose promoter systems (Chang et al. , Nature , 275: 615 (1978); Goeddel et al. , Nature , 281: 544 (1979)], alkaline phosphatase, tryptophan promoter system [Goeddel, Nucleic acid Res. , ≪ / RTI > 8: 4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al ., Proc. Natl. Acad. Sci. USA , 80: 21-25 (1983)]. In addition, the promoter used in the bacterial system will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the PRO.
[807] Examples of promoter sequences suitable for use in yeast hosts include 3-phosphoglycerate kinase [Hitzeman et al ., J. Biol. Chem. , 255: 2073 (1980)] or other sugar degrading enzymes [Hess et al ., J. Adv. Enzyme Reg. , ≪ / RTI > 7: 149 (1968); Holland, Biochemistry , 17: 4900 (1978)], for example, enolase, glyceraldehyde-3- phosphate dihydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose- 6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosulfate isomerase, phosphoglucose isomerase, and a promoter for glucokinase.
[808] Other yeast promoters that are inducible promoters with additional transcriptional advantages that are regulated by growth conditions include alcohol dehydrogenase 2, isocytocrome C, acid phosphatase, degrading enzymes related to nitrogen metabolism, metallothionein, glyceraldehyde- 3-phosphate dehydrogenase, and promoter regions for enzymes that act on maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.
[809] PRO transcription from vectors in mammalian host cells may be carried out using viruses, e. G., Poliomavirus, puetal virus (UK 2,211,504, issued July 5, 1989), adenovirus (e. G., Adenovirus 2) From a genome of papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and monkey virus 40 (SV40), from heterologous mammalian promoters such as actin promoters or immunoglobulin promoters, from heat-shock promoters And is regulated by the obtained promoter which is suitable for the host cell system.
[810] Transcription of the DNA encoding PRO by higher eukaryotic cells can be increased by inserting an enhancer sequence into the vector. Enhancers are generally cis-acting components of DNA of about 10 to 300 bp that act on the promoter to increase transcription. Many enhancer sequences are known to result from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, an enhancer will usually be used which is derived from eukaryotic viruses. Examples include the SV40 enhancer (bp 100-270) on the back of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the back of the origin of replication, and the adenovirus enhancer. The enhancer can be spliced to the vector at the 5 'or 3' position in the PRO coding sequence, but is preferably located at the 5 'site from the promoter.
[811] In addition, expression vectors used in eukaryotic host cells (polynuclear cells derived from yeast, fungi, insects, plants, animals, humans or other multicellular organisms) may contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are typically obtained from eukaryotic cells, or 5 'and sometimes 3' untranslated regions of viral DNA or cDNA. These regions contain a nucleotide fragment that is transcribed as a polyadenylation fragment in the untranslated portion of the mRNA encoding the PRO.
[812] Other methods, vectors, and host cells suitable for application in the synthesis of PRO in recombinant vertebrate cell culture are described in Gething et al. , Nature , 293: 620-625 (1981); Mantei et al. , Nature , 281: 40-46 (1979); European Patent No. 117,060 and European Patent No. 117,058.
[813] 4. Detection of gene amplification / expression
[814] Gene amplification and / or expression can be performed, for example, by conventional Southern blotting, northern blotting to quantitate transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA , 77: 5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization using appropriately labeled probes based on the sequences provided herein. Alternatively, antibodies capable of recognizing a particular double chain, including DNA duplexes, RNA duplexes and DNA-RNA hybrid duplexes or DNA-protein duplexes, can be used. In other words, an assay can be performed that can label the antibody, bind the duplex to the surface, and detect the presence of antibodies bound to the double strand when the duplex is formed on the surface.
[815] Alternatively, gene expression can be measured by analysis of cell culture fluids or body fluids to directly quantitate the expression of the gene product and immunological methods such as immunohistochemical staining of cells or tissue sections. Antibodies useful for immunohistochemical staining and / or analysis of sample fluids may be monoclonal or polyclonal antibodies, and may be prepared in any mammal. Conveniently, an antibody against a native PRO polypeptide or a synthetic peptide based on the DNA sequence provided herein or an exogenous sequence fused to a PRO DNA and encoding a specific antibody epitope can be produced.
[816] 5. Purification of the polypeptide
[817] The form of PRO may be recovered from the culture medium or the bacterial solution for host cells. When bound to the membrane, it can be released from the membrane by using a suitable detergent solution (e.g., Triton-X 100) or by cleavage of the enzyme. Cells used for the expression of PRO may be pulverized by various physical or chemical means such as freeze-thaw cycles, sonication, mechanical grinding or cell homogenization.
[818] It may be desirable to purify the PRO from a recombinant cell protein or polypeptide. Examples of suitable purification methods include fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins such as chromatography on DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, Gel filtration using G-75, a protein A Sepharose column for removing contaminants such as IgG, and a metal chelating column for binding an epitope tag form of PRO. A variety of protein purification methods can be used, such methods are well known in the art and are described, for example, in Deutscher, Methods in Enzymology , 182 (1990); Scopes, Protein Purification: Principles and Practice , Springer-Verlag, New York (1982). The purification step (s) selected will depend, for example, on the production method used and on the characteristics of the particular PRO produced.
[819] Uses of E. PRO
[820] The nucleotide sequence coding for PRO (or its complement) has a variety of uses in the field of molecular biology, including chromosome and gene mapping, and in the production of antisense RNA and DNA, including use as hybridization probes. In addition, the PRO coding nucleic acid will also be useful for the production of PRO polypeptides by recombinant techniques as described herein.
[821] The full-length native sequence PRO gene or fragment thereof may be isolated from a full-length PRO cDNA or other cDNA (e. G., A gene encoding a PRO from a naturally-occurring variant of PRO or another species) having the desired sequence identity with the native PRO sequence disclosed herein Can be used as a hybridization probe for the cDNA library to be used. Optionally, the length of the probe will be from about 20 to about 50 bases. The hybridization probe may be derived from a genomic sequence comprising at least a portion of a full-length natural nucleotide sequence, wherein the region may be determined without undue experimentation, or a promoter, enhancer component and intron of the native sequence PRO. For example, the screening method will include isolating the coding region of the PRO gene using a known DNA sequence to synthesize a selected probe of about 40 bases. Hybridization probes can be labeled with a variety of labels, including enzymatic labels such as alkaline phosphatase coupled to probes via radioactive nucleotides such as ³² P or ³⁵ S or avidin / biotin coupling systems. A library of human cDNA, genomic DNA or mRNA can be screened using a labeled probe having a sequence complementary to the PRO gene of the present invention to determine the member of the library in which the probe hybridizes. The hybridization technique is described in more detail in the following examples.
[822] Any of the EST sequences disclosed herein may similarly be used as probes using the methods described herein.
[823] Other useful fragments of a PRO nucleic acid include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (RNA or DNA) capable of binding to a target PRO mRNA (sense) or PRO DNA (antisense) sequence. Antisense or sense oligonucleotides according to the present invention comprise fragments of the coding region of PRO DNA. This fragment generally comprises at least about 14 nucleotides, preferably about 14 to 30 nucleotides. The ability to derive antisense or sense oligonucleotides based on cDNA sequences encoding a given protein is described, for example, in Stein and Cohen ( Cancer Res. 48: 2659, 1988) and van der Krol et al. ( BioTechniques 6: 958, 1988).
[824] The binding of the antisense or sense oligonucleotide to the target nucleic acid sequence forms a double strand that blocks transcription or translation of the target sequence by one of several methods or other methods including enhanced resolution of the double strand, early termination of transcription or translation . Thus, antisense oligonucleotides can be used to block the expression of the PRO protein. The antisense or sense oligonucleotides also include oligonucleotides having a modified sugar-phosphodiester backbone (or other sugar chains, for example, the sugar chains disclosed in WO 91/06629), wherein such sugar chains are internal nucleases There is resistance to the subject. Such oligonucleotides with a resistant sugar chain retain sequence homology that is stable in vivo (i.e., resistant to enzymatic degradation) but capable of binding to a target nucleotide sequence.
[825] Other examples of sense or antisense oligonucleotides include, but are not limited to, residues disclosed in WO 90/10048 and other residues that increase the affinity of the oligonucleotide for the target nucleic acid sequence, such as poly- (L-lysine) Covalently linked oligonucleotides are included. In addition, an intercalator, such as elliticin, and an alkylating agent or metal complex can be linked to a sense or antisense oligonucleotide to alter the binding specificity of the antisense or sense oligonucleotide to the target nucleotide sequence.
[826] For example, CaPO _4-mediated DNA transfection, in any gene transfer method, including Electra troponin illustration or Epstein-Bar cell containing the target nucleic acid sequence by using a gene transfer vector, such as a (Epstein-Barr) virus RTI ID = 0.0 > oligonucleotides < / RTI > In a preferred method, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. Cells containing the target nucleic acid sequence are contacted with the recombinant retroviral vector in vivo or ex vivo. Suitable retroviral vectors include vectors derived from murine and retroviral M-MuLV, N2 (retroviruses derived from M-MuLV), or double copy vectors named DCT5A, DCT5B and DCT5C (see WO 90/13641) But are not limited to.
[827] In addition, as disclosed in WO 91/04753, a sense or antisense oligonucleotide can be introduced into a cell comprising a target nucleotide sequence by complexing with a ligand binding molecule. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, proliferation factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the linkage of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to the corresponding molecule or receptor, nor does it block the intracellular entry of the sense or antisense oligonucleotide or its complex form.
[828] Alternatively, a sense or antisense oligonucleotide can be introduced into a cell containing a target nucleic acid sequence by forming an oligonucleotide-lipid complex, as disclosed in WO 90/10448. Such sense or antisense oligonucleotide-lipid complexes are preferably separated by an internal lipase in the cells.
[829] The antisense RNA or DNA molecule generally has a length of about 5 bases, about 10 bases, about 15 bases, about 20 bases, about 25 bases, about 30 bases, about 35 bases, about 40 bases, About 45 bases, about 80 bases, about 85 bases, about 90 bases, about 65 bases, about 70 bases, about 75 bases, about 80 bases, about 85 bases, about 90 bases, About 95 bases, about 100 bases or more.
[830] Probes can also be used in PCR technology to generate a set of sequences for the identification of closely related PRO coding sequences.
[831] In addition, the nucleotide sequence coding for PRO can be used to prepare a hybridization probe for gene mapping of a gene encoding a PRO and genetic analysis of an individual having a genetic disease. The nucleotide sequences provided herein can be mapped to specific regions of chromosomes and chromosomes using known techniques such as in situ hybridization, linkage analysis to known chromosome markers, and hybridization screening using libraries.
[832] When the coding sequence of PRO encodes a protein that binds to another protein (e.g., where PRO is a receptor), PRO may be used in assays to identify other proteins or molecules involved in such binding interactions. By this method, inhibitors of receptor / ligand binding interactions can also be identified. In addition, proteins involved in the binding interactions can be used to screen for small molecule inhibitors or agonists of peptides or binding interactions. In addition, similar ligands can be isolated using the receptor PRO. Screening assays can be designed to find lead compounds that mimic the biological activity of a receptor for native PRO or PRO. Such screening assays will include high throughput screening assays for chemical libraries and will be particularly suitable for identifying small molecule drug candidates. Small molecules include synthetic organic or inorganic compounds. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell-based assays, which are characterized in the art.
[833] In addition, nucleic acids encoding PRO or variants thereof can be used to generate transgenic animals or " knock out " animals useful in the development and screening of useful therapeutic agents. A transgenic animal (e. G., A mouse or rat) is an animal having cells that contain a transgene, the transgene being introduced into the animal or an ancestor of the animal in the fetal phase, e.g., . The transgene is DNA that is integrated into the genome of the cell, from which transgenic animals develop. In one embodiment, genomic DNA encoding PRO can be cloned according to established techniques using cDNA encoding PRO, and transgenic cells containing cells expressing the PRO encoding DNA using this genomic sequence Animals can be created. In particular, methods for generating transgenic animals such as mice or rats are conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, specific cells are targeted for the introduction of a PRO transgene with a tissue specific enhancer. Transgenic animals containing transcripts of one copy encoding a PRO introduced into the reproductive line of an animal at the embryonic stage can be used to investigate the increased expression of PRO encoding DNA. Such animals can be used, for example, as test animals for reagents that are thought to protect against pathological conditions associated with overexpression of PRO polypeptides. According to this aspect of the invention, treating the animal with the reagent results in a reduction in the incidence of pathological symptoms compared to untreated animals carrying the transgene, which indicates potential therapeutic intervention for the pathological symptoms.
[834] Alternatively, the non-human homolog of PRO may be used to have a defect or modified gene encoding PRO as a result of homologous recombination between the endogenous gene encoding PRO and the modified genomic DNA encoding PRO introduced into the embryonic stem cell of this animal PRO Can be used to make "knock-out" animals. For example, genomic DNA encoding PRO can be cloned according to established techniques using cDNA encoding PRO. Some of the genomic DNA encoding PRO may be deleted or replaced with other genes, such as genes encoding an optional marker that can be used to monitor integration. Generally, unmodified flanking DNA (at both the 5 'and 3' ends) of thousands of bases is included in the vector (see, for example, Thomas and Capecchi, Cell , 51: 503 (1987)). This vector is introduced into the embryonic stem cell line (for example, by electroporation), and cells in which the introduced DNA and the endogenous DNA are homologously recombined are selected (see, for example, Li et al., Cell , 69: 915 (1992)). Selected cells are then injected into blastocysts of animals (e. G. Mice or rats) to form aggregation chimeras (see, for example, Bradley, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, EJ Robertson, Oxford, 1987), pp. 113-152). The chimeric embryos were then transplanted into suitable primate surrogate animal to produce " knock-out " animals. The progeny harboring homologous recombinant DNA in germ cells can be identified by standard techniques and used to breed animals in which all cells contain homologous recombinant DNA. Rust-out animals are characterized, for example, by the ability to defend against certain pathological conditions and the occurrence of pathological symptoms due to the absence of PRO polypeptides.
[835] The nucleic acid encoding the PRO polypeptide may also be used in gene therapy. When used in gene therapy, a gene is introduced into a cell to achieve, for example, a defective gene in order to achieve in vivo synthesis of a therapeutically effective gene product. &Quot; Gene therapy " encompasses both traditional gene therapy where a sustained effect is achieved by a single treatment, and administration of a gene therapy agent, including once or repeated administration of therapeutically effective DNA or mRNA. Antisense RNA and DNA can be used as therapeutic agents to block the expression of a specific gene in vivo. It has already been shown that the absorption of short antisense oligonucleotides through cell membranes is limited, so that despite their low intracellular concentrations of these oligonucleotides, they can act as inhibitors when introduced into cells (Zamecnik et al., Proc. Natl. Acad Sci. USA 83, 4143-4146 (1986)). Such oligonucleotides can increase the uptake of oligonucleotides by modification, for example, by replacing their negatively charged phosphodiester groups with non-charged groups.
[836] There are a variety of techniques that can be used to introduce nucleic acids into living cells. This technique depends on whether the nucleic acid is delivered to the cultured cells in vitro or to the cells of the desired host in vivo. Suitable techniques for delivering nucleic acids into mammalian cells in vitro include liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, and the like. Current preferred in vivo gene transfer techniques include transfection using virus (usually retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 1993). In some cases, it is desirable to provide the nucleic acid source with an agent that targets the target cell, such as a cell surface membrane protein or an antibody specific for the target cell, a ligand for a receptor on the target cell, and the like. When liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis can be used for targeting and / or promoted uptake, examples of which include, but are not limited to, An antibody to a protein that undergoes internalization during circulation, a protein that targets the intracellular location and increases intracellular half-life, and the like. Receptor-mediated endocytosis techniques are described for example in Wu et al., J. Biol. Chem. 262, 4429-4432 (1987), and Wagner et al., Proc. Natl. Acad. -3414 (1990). For a review of gene marking and gene therapy protocols, see Anderson et al., Science 256, 808-813 (1992).
[837] The PRO polypeptides disclosed herein may also be used as molecular weight markers for protein electrophoresis, and the isolated nucleic acid sequences can be used to recombinantly express these markers.
[838] Nucleic acid molecules or fragments thereof encoding the PRO polypeptides disclosed herein are useful for identification of chromosomes. In this regard, there is a continuing need to identify new chromosomal markers because the available chromosomal marking reagents based on actual sequence data are currently relatively small. Each of the PRO nucleic acid molecules of the present invention can be used as a chromosome marker.
[839] The PRO polypeptides and nucleic acid molecules of the present invention may also be used for tissue typing, wherein the PRO polypeptides of the invention may be differentially expressed in one tissue relative to other tissues. The PRO nucleic acid molecule will be used to generate probes for PCR, Northern analysis, Southern analysis and Western analysis.
[840] The PRO polypeptides disclosed herein may also be used as therapeutic agents. The PRO polypeptides of the present invention may be formulated according to known methods to produce pharmaceutically useful compositions wherein the PRO product is combined with a pharmaceutically acceptable carrier vehicle. The therapeutic formulations are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the active ingredient with the desired purity with any physiologically acceptable carrier, excipient or stabilizer ( Remington ' s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients or stabilizers are non-toxic to the patient at the dosages and concentrations employed and include antioxidants such as phosphates, citrates and other organic acids, ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, serum albumin , Proteins such as gelatin or immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine, monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrin, Or a salt-forming counterion such as sodium hydroxide, and / or a nonionic surfactant such as Tween (trademark), Pluronics (trademark) or PEG, and the like .
[841] Preparations used for intravenous administration should be sterilized. This is readily accomplished by filtration through a sterile filtration membrane before or after lyophilization and reconstitution.
[842] The therapeutic compositions herein may be placed in a container with a sterile access port, for example a vial containing an intravenous solution bag or a stopper pierceable by a hypodermic needle.
[843] The route of administration may be a known method, for example, by injection or injection by intravenous, intraperitoneal, intracerebral, intramuscular, intravenous, intraarterial, or intralesional routes, topical administration, or sustained release.
[844] The dosage of the pharmaceutical composition of the present invention and the desired drug concentration may vary depending on the particular application intended. Determination of the appropriate dosage and route of administration will be well known to those skilled in the art. The results of animal experiments provide reliable guidance in determining effective doses for human treatment. An effective dose scaling of the species is described in Mordenti, J. and Chappell, W. " The use of interspecies scaling in toxicokinetics " in Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, , pp. 42-96).
[845] When a PRO polypeptide or agonist or antagonist thereof is used in vivo, the typical dosage will depend on the route of administration and will range from about 10 ng to 100 mg per kg of body weight of the mammal, or preferably about 1 Kg / day to 10 mg / kg / day. Instructions for specific dosages and delivery methods are provided in the literature, for example, in U.S. Pat. Nos. 4,657,760, 5,206,344 or 5,225,212. It is contemplated that other agents may be effective for other therapeutic compounds and other conditions, and administration targeting one organ or tissue may require delivery in a different manner than administration to, for example, another organ or tissue do.
[846] Microencapsulation of PRO polypeptides is contemplated where sustained release administration of a PRO polypeptide is required for an agent having release characteristics suitable for the treatment of a disease or disorder requiring administration of the PRO polypeptide. Microencapsulation of recombinant proteins for delayed release has been successfully performed using human growth hormone (rhGH), interferon (rhIFN), interleukin-2 and MN rgp120 (Johnson et al., Nat. Med. , 2: 795- Cleland, " Design and Production of Single Immunization Vaccines Using "(1996); Yasuda, Biomed. Ther. , 27: 1221-1223 (1993); Hora et al., Bio / Technology , 8: 755-758 Polyactide Polyglycolide Microsphere Systems, "in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds, (Plenum Press: New York, 1995), pp 439-462:. WO 97/03692, WO 96/40072, WO 96 / 07399 and U.S. Patent No. 5,654,010).
[847] The sustained-release preparation of the protein was developed using poly-lactic acid-co-glycolic acid (PLGA) polymer in consideration of biocompatibility and broad biodegradability. Lactic acid and glycolic acid, degradation products of PLGA, can be rapidly removed in the human body. The resolution of the polymer can also be adjusted to months to years depending on its molecular weight and composition (Lewis, " Controlled release of bioactive agents from lactide / glycolide polymer, " in M. Chasin and R. Langer (Eds. Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41).
[848] The present invention includes a method of screening for compounds that mimic (antagonist) mimics or inhibits the effects of PRO polypeptides (agonists). Screening assays for antagonist drug candidates were designed to identify compounds that bind or complex with the PRO polypeptides encoded by the genes identified herein or that interfere with the interaction of the encoded polypeptides with other intracellular proteins. Such screening assays include assays capable of high throughput screening for chemical libraries, which would be particularly suitable for identifying small molecule drug candidates.
[849] Such assays may be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and cell-based assays, which are characterized in the art.
[850] One common feature of all assays for antagonists is that the PRO polypeptide encoded by the nucleic acid identified herein should be contacted with the candidate drug under conditions and for a time sufficient for them to interact with each other.
[851] In the binding assay, the interaction is the binding, and the complex formed can be isolated or detected in the reaction mixture. In certain embodiments, the PRO polypeptides or drug candidates encoded by the genes identified herein are immobilized on a solid phase, e. G., A microtiter plate, by covalent attachment or noncovalent attachment. Non-covalent attachment is generally accomplished by coating the solid surface with a PRO polypeptide solution and drying. Alternatively, a monoclonal antibody specific for an immobilized antibody, e. G., A fixed PRO polypeptide, can be used to anchor it to a solid surface. This assay can be performed by adding a non-immobilizable component which can be labeled with a detectable label, to a coding surface comprising a fixed component, for example an anchored component. When the reaction is terminated, unreacted components are removed, for example by washing, and complexes anchored to the solid surface are detected. Detection of a label immobilized on a surface indicates that a complexing reaction has taken place if the original, non-immobilized component carries a detectable label. If the original non-immobilized component does not carry the label, for example, a labeled antibody that specifically binds to the immobilized complex can be used to detect the complexation reaction.
[852] If the candidate compound interacts with, but does not bind to, the specific PRO polypeptide encoded by the gene identified herein, the interaction of the candidate compound with the polypeptide may be analyzed by well known methods for detecting protein-protein interactions . Such assays include conventional methods, such as cross-linking, co-immunoprecipitation, and co-purification through gradient or chromatographic columns. Protein-protein interactions have also been reported by Fields and co-workers (Fields and Song, Nature (London) , 340: 245-246 (1989); Chien et al., Proc. Natl. Acad. : 9578-9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA , 89: 5789-5793 (1991)). Many transcription activators, such as yeast GAL4, consist of two physically distinct modular domains, one acting as a DNA-binding domain and the other acting as a transcription-active domain. A yeast expression system (commonly referred to as a "2-hybrid system") described in the above publication utilizes this property and uses two hybrid proteins, one of which is the target protein with the DNA-binding domain of GAL4 And the other is that the candidate active protein is fused with the active domain. Expression of the GAL1-lacZ reporter gene under the control of the GAL4-active promoter depends on the reconstitution of the GAL4 activity through protein-protein interactions. Colonies containing interacting polypeptides are detected using a chromogenic substrate for beta -galactosidase. A complete kit (MATCHMAKER TM) that verifies protein-protein interactions between two specific proteins using the 2-hybrid technique is available from Clontech. In addition, this system can be extended to map protein domains involved in specific protein interactions, as well as to pinpoint the position of amino acid residues critical to such interactions.
[853] In the following way, compounds that interfere with the interaction of other genes with the intracellular or extracellular components of the genes encoding the PRO polypeptides identified herein can be tested. A reaction mixture comprising the product of the gene encoding the PRO polypeptide and the intracellular or extracellular component was prepared under conditions and for a time allowing interaction and binding of the two products. To test the ability of the candidate compound to inhibit binding, the reaction was carried out in the presence and absence of the test compound. Placebo can also be added to a third reaction mixture that serves as a positive control. Binding (complex formation) between intracellular or extracellular components and test compounds present in the mixture was monitored as described above. A complex formed in the control reaction but no complex formed in the reaction mixture containing the test compound indicates that the test compound interferes with the interaction of the test compound with its reaction counterpart.
[854] To analyze the antagonist, the PRO polypeptide could be added to the cell with the compound to screen for specific activity, and the ability of the compound to inhibit the desired activity in the presence of the PRO polypeptide indicates that the compound is an antagonist to the PRO polypeptide. Alternatively, antagonists could be detected by binding PRO polypeptides and potential antagonists with membrane-bound PRO polypeptide receptors or recombinant receptors under appropriate conditions for competitive inhibition analysis. PRO polypeptides can be labeled, for example, with radioactive isotopes, to determine the effect of potential antagonists using the number of PRO polypeptide molecules that bind to the receptor. Genes encoding the receptor could be identified by several methods known to those skilled in the art, for example, the ligand panning method and the FACS taxonomy (Coligan et al., Current Protocols in Immun. , 1 (2): Chapter 5 (1991) . Preferably, expression cloning is used wherein the polyadenylated RNA is produced from a cell that is reactive to a PRO polypeptide, and the cDNA library generated from the RNA is classified as a PUO to a COS cell that is unreactive to a PRO polypeptide or It is used to transfect other cells. Transfected cells proliferating on a glass slide are exposed to the labeled PRO polypeptide. The PRO polypeptide could be labeled by several methods including iodination or encapsulation of the recognition site on the site-specific protein kinase. After immobilization and incubation, the slides were analyzed by autoradiography. Positive pools were identified to produce sub-pools and transfected again using interactive sub-fooing and re-screening methods, resulting in a single clone encoding the putative receptor.
[855] As another way of identifying the receptor, the labeled PRO polypeptide could be photo-chemically conjugated with an extractant sample expressing the cell membrane or receptor molecule. The crosslinked material was analyzed by PAGE and exposed to x-ray film. The labeled complexes containing the receptor could be cut and digested into peptide fragments for protein micro-sequencing. By designing a set of degenerate oligonucleotide probes using the amino acid sequence obtained from micro-sequencing, a cDNA library could be screened and genes encoding the putative receptor could be identified.
[856] As another antagonist assay method, mammalian cells or membrane samples expressing the receptor in the presence of the candidate compound were incubated with the labeled PRO polypeptide. Thereafter, the ability of the compound to enhance or block the interaction could be measured.
[857] More specific examples of potential antagonists include oligonucleotides that bind to fusions of immunoglobulins and PRO polypeptides, and in particular poly- and monoclonal antibodies, and antibody fragments, side chain antibodies, anti-genotype antibodies, Chimeric or humanized forms, and antibodies, including human antibodies and antibody fragments. Alternatively, a potential antagonist may be a mutated form of a PRO polypeptide that competitively inhibits the action of a PRO polypeptide, although it does not pertain to a closely related protein, e. G., A receptor.
[858] Another potential PRO polypeptide antagonist is an antisense RNA or DNA construct prepared using antisense technology wherein an antisense RNA or DNA molecule acts to directly block translation of mRNA by hybridizing with the target mRNA to prevent protein translation . Antisense technology can be used to regulate gene expression through triple helix formation or antisense DNA or RNA, all based on the binding of polynucleotides to DNA or RNA. For example, the 5 'coding portion of the polynucleotide sequence encoding mature PRO nucleotides herein may be used to design antisense RNA oligonucleotides of about 10 to 40 bases in length. 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)); (Dervan et al, Science, 251:. 1360 (1991).)) refer to) to prevent transcription and the production of the PRO polypeptide antisense RNA oligonucleotide his mRNA and hybridizes in vivo to a To block translation of mRNA molecules into PRO polypeptides (antisense - Okano, Neurochem. , 56: 560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton, FL, 1988) It is possible to inhibit the production of PRO polypeptide by expressing the antisense RNA or DNA in vivo by transferring the described oligonucleotide to the cells. When antisense DNA is used, the transcription-initiation site, e. G. 10 months +10 The oligonucleotide is preferably oxy-ribonucleotide was used derived from a location.
[859] Potential antagonists include small molecules that bind to the active site, a receptor binding site, or small molecules that bind to the proliferation factors or other related binding sites of the PRO polypeptide to block the normal biological activity of the PRO polypeptide. Examples of small molecules include, but are not limited to, small peptide or peptide-based molecules, preferably soluble peptides, and synthetic non-peptidyl organic or inorganic compounds.
[860] Ribozymes are enzyme RNA molecules capable of catalyzing specific cleavage of RNA. Ribozymes function by sequencing-specific hybridization with complementary target RNA followed by cleavage by endonuclease. A known technique could identify specific ribozyme cleavage sites within a potential RNA target. For further details, see, for example, Rossi, Current Biology , 4: 469-471 (1994) and PCT publication No. WO 97/33551 (published September 18, 1997).
[861] The triple helical nucleic acid molecule used to inhibit transcription must be single-stranded and must be composed of deoxynucleotides. The base composition of these oligonucleotides is designed to promote triple helix formation through the Hoogsteen base pairing rule. Triple helix formation generally requires a purine or pyrimidine stretch of considerable size on one of the double strands. For further details, see, for example, PCT publication No. WO 97/33551, supra.
[862] The small molecules may be identified by any one or more of the screening assays described above and / or by any other screening technique well known to those skilled in the art.
[863] With respect to PRO211 and PRO217 polypeptides, therapeutic indications may include disorders related to the preservation and maintenance of gastric mucosa, and disorders associated with acute, chronic mucosal lesions (e.g., small bowel colitis, Sollinger's Ellison syndrome, gastric ulcer and congenital microvilli atrophy), abnormal keratinocyte differentiation , Such as psoriasis, epithelial cancers such as lung cancer cell lines, vulvar epidermis, and glioma repair.
[864] Since the PRO232 polypeptide and the nucleic acid encoding it are homologous to the cell surface hepatocyte antigen and its coding nucleic acid, probes based on the PRO232 nucleotide sequence can be used to identify other novel hepatocyte surface antibody proteins. The soluble forms of PRO232 polypeptides can be used as antagonists of membrane bound PRO232 activity both in vitro and in vivo. PRO232 polypeptides can be used in screening assays designed to identify agonists or antagonists of native PRO232 polypeptides, and such assays can take the form of any conventional cell type or biochemical binding assay. Moreover, PRO232 polypeptides can function as molecular markers for tissues in which the polypeptide is specifically expressed.
[865] With respect to the PRO187 polypeptides disclosed herein, FGF-8 has been found to be involved in cell differentiation and embryogenesis, including pattern formation that occurs during limb formation. Therefore, the FGF-8 and PRO187 molecules of the present invention may have a strong influence on cell growth and development. Diseases related to cell growth and differentiation are therefore appropriate targets of therapy based on functionality similar to FGF-8. For example, there are diseases related to the growth or survival of nerve cells such as Parkinson's disease, Alzheimer's disease, ALS, neuropathy. It is also expected that diseases associated with uncontrolled cell growth such as cancer will also be targeted.
[866] With respect to the PRO265 polypeptides disclosed herein, other methods of using PRO265 are described in U.S. Patent No. 5,654,270 (Ruoslahti et al.). In particular, PRO265 can be used to compare the effect of the fibromodulin described herein on skin abatement and other properties (subject to presence and non-binding or non-binding) compared to the fibromodulin disclosed herein have.
[867] The PRO219 polypeptide of the present invention has a regulatory function in the blood coagulation process and can thus be used for therapeutic purposes in vitro and in vivo. Those skilled in the art will know how to use the PRO219 polypeptide for this purpose.
[868] The PRO246 polypeptides of the present invention may serve as cell surface receptors for one or more viruses and have other uses. For example, extracellular domains derived from these PRO246 polypeptides can be used therapeutically in vivo to mitigate the effects of cell infections. Since the PRO246 polypeptide functions as a tumor-specific antibody, it can be used as a therapeutic target for an antitumor drug. Those skilled in the art will know how to use the PRO246 polypeptide for this purpose.
[869] Assays in which connective growth factors and other growth factors are commonly used may be performed using PRO261. The assay to determine whether TGF beta induces PRO261 (indicating its role in cancer) is carried out as is known in the art. Wound restoration and tissue growth analysis can also be performed using PRO261. The result is applied accordingly.
[870] PRO228 polypeptides may be used in assays to determine their relative activity using EMR1, CD97, and lactlofilin. The results can be applied accordingly. For example, competitive binding assays for PRO228 and CD97 can be performed using ligands for CD97, CD55.
[871] Native PRO533 is a 216 amino acid polypeptide, of which residues 1-22 are signal sequences. Residues 3 to 216 have a Blast value of 509 corresponding to 53% homology to the fibroblast growth factor. At the nucleotide level, the EST, DNA47412, was observed to map 11p15, and a PCR oligomer was generated from the EST to isolate the full-length DNA49435-1219. Sequence homology to the 11p15 locus will indicate that PRO533 is useful for the treatment of Osher syndrome and atrophia areata.
[872] As described above, fibroblast growth factors can act on cells in mitotic and non-mitotic ways. These factors include, but are not limited to, granulosa cells, adrenocortical cells, chondrocytes, myoblasts, corneal cells and vascular endothelial cells (bovine or human), vascular smooth muscle cells, lens, retina and prostate epithelial cells, dendritic cells, astrocytes, The myelin sheep and osteoblasts, as well as various normal diploid mesenchymal-derived and neuron-derived cells.
[873] Non-mitotic actions of fibroblast growth factors include: cell migration (chemotaxis) to the wound site, initiation of new angiogenesis (angiogenesis), regulation of neuronal regeneration and survival (neurotrophic), regulation of endocrine function, Promoting and inhibiting protein expression and production of extracellular matrix and cell survival (Baird, A. and Bohlen, P. Handbook of Exp. Phrmacol. 95 (1): 369-418 (1990)]. This property is the basis for the use of fibroblast growth factor in therapeutic methods to promote wound healing, nerve repair, and vascularization. For example, fibroblast growth factor has been shown to minimize myocardial damage in heart disease and surgery (U.S. Patent No. 4,378,437).
[874] The PRO245 polypeptide and the nucleic acid encoding it can be used to identify other novel transmembrane tyrosine kinase proteins using probes based on the PRO245 nucleotide sequence as they are sequence homologous to the transmembrane protein tyrosine kinase protein and the nucleic acid encoding it. The soluble forms of PRO245 polypeptides can be used as antagonists of membrane bound PRO245 activity both in vitro and in vivo. The PRO245 polypeptides can be used in screening assays designed to identify agonists or antagonists of native PRO245 polypeptides, which can take the form of any conventional cell type or biochemical binding assays. Moreover, PRO245 polypeptides can function as molecular markers of tissues in which they are specifically expressed.
[875] PRO220, PRO221 and PRO227 all have a lucine rich repeat. PRO220 and PRO221 are also homologous to the SLIT and leucine rich repeat proteins. These proteins are therefore useful in the assays described in the literature, where SLIT and leucine rich repeat proteins are used. In relation to the SLIT protein, PRO227 can be used in assays to measure the effect of PRO227 on neurodegenerative diseases. PRO227 is also homologous to human glycoprotein V. In the case of PRO227, this polypeptide is used for analysis to measure the effect on hemorrhage, blood clotting, tissue repair and scarring.
[876] The PRO266 polypeptide can be used in the assay to determine whether it is involved in neurodegenerative diseases.
[877] The PRO269 polypeptide and a portion thereof may affect the activity of thrombin and therefore may be useful for in vivo treatment as well as in vitro applications. In addition, PRO269 polypeptides and portions thereof may have therapeutic use as antithrombotic agents with a reduced risk of bleeding relative to heparin. The peptide is particularly preferred because it is homologous to thrombomodulin.
[878] The PRO287 polypeptide and a portion thereof may also be useful for in vivo therapy as well as in vitro as it affects the activity of the bone morphogenetic protein " BMP1 " / procollagen C-proteinase (PCP). Also PRO287 polypeptides and portions thereof may be of therapeutic use in wound healing and tissue repair. Peptides that are homologous to the procollagen C-proteinase enhancer protein and its precursor can also be used to induce bone and / or cartilage formation and are of particular interest to academia and the medical community.
[879] Therapeutic indications that require the PRO214 polypeptide include disorders associated with the preservation and maintenance of gastric mucosa and acute, chronic mucosal lesions (e. G., Small bowel colitis, Sollinger Ellison syndrome, gastric ulcer and congenital microvilli atrophy), skin diseases involving abnormal keratinocyte differentiation For example, there are epithelial cancers such as psoriasis, lung cancer, cancer of the vulva, and repair of glioma.
[880] A study of the production and analysis of mice lacking TGF-giant family members is described in Matzuk, Trends in Endocrinol. and Metabol. , 6 : 120-127 (1995).
[881] Herein, PRO317 polypeptides and PRO317-specific antibodies, inhibitors, agonists, receptors or analogs thereof are useful for treating PRO317-related disorders. Thus, for example, they can be used to regulate endometrial hemorrhagic angiogenesis and can also affect renal tissue. Endometrial bleeding can occur as abnormal bleeding in gynecological diseases such as endometrial cancer. Therefore, the composition of the present invention can be used to diagnose or treat abnormal bleeding state in the endometrium, so that hysterectomy can be avoided or its necessity can be alleviated. The molecules of the present invention may also be used in the field of angiogenesis, such as antitumor indications, where antibodies against vascular endothelial growth factor are used, or vice versa, in the ischemic indications where blood and endothelial cell growth factors are used.
[882] The bioactive compositions of PRO317 or agonists or antagonists thereof are useful for the treatment of mammalian species that determine the maximum tolerated dose and the appropriate treatment as determined by any of a variety of methodologies, including clinical studies on normal human subjects, Dose. ≪ / RTI > The bioactive agent may also be compounded with various well-established compounds or compositions that improve the pharmaceutical properties, such as half life, or stability. Therapeutic bioactive compositions may be delivered via intravenous infusion into the bloodstream or other effective means that may be used to treat problems in the kidney, uterus, endometrium, blood vessels or related tissues (e. G., The heart or reproductive tract) .
[883] The dosage and administration of PRO317, PRO317 agonist or PRO317 antagonist in a pharmaceutical composition can be determined by those skilled in the pharmaceutical or pharmacodynamic arts [Mordenti and Rescigno, Pharmaceutical Research, 9 : 17- 25 (1992), Morenti et al. , Pharmaceutical Research, 8 : 1351-1359 (1991), and Mordenti and Chappell, "The use of interspecies scaling in toxicokinetics " in Toxicokinetics and New Drug Development , Yacobi et al. (eds) (Pergamon Press: NY, 1989), pp. 42-96)]. The therapeutically effective amount of PRO317, PRO317 agonist, or PRO317 antagonist will depend, for example, on the therapeutic purpose, the route of administration, and the condition of the mammal. Therapeutic practitioners will therefore need to modify the route of administration and titrate doses if necessary to achieve optimal therapeutic effects. A typical daily dose can be from about 10 ng / kg to about 100 mg / kg or more per kilogram of mammalian body weight per day, preferably from about 1 g / kg / day to 10 mg / kg / day. Typically, a clinician may administer a PRO317, a PRO317 agonist or a PRO317 antagonist until a dose is reached that achieves the effect necessary for the treatment of the disorders mentioned above.
[884] The PRO317 or PRO317 agonist or PRO317 antagonist may achieve the desired pharmacological effect by administration alone or in combination with each other. PRO317 itself or an agonist or antagonist of PRO317 will provide different effects when administered therapeutically. Such a compound for treatment is formulated in a non-toxic inert pharmaceutically acceptable aqueous carrier medium having a pH of from about 5 to 8, preferably from 6 to 8, and the pH is adjusted to the nature of the PRO317, agonist, ≪ / RTI > The properties of the therapeutic compound include the solubility, half-life, and antigenicity / immunogenicity of the molecule, and other properties may be helpful in determining an effective carrier.
[885] PRO317 or PRO317 agonists or PRO317 antagonists may be formulated with topical creams and gels, transmucosal sprays and aerosols, transdermal patches and dressings, injectable intravenous and lavage preparations, and orally administered solutions and pills, especially gastric acid ≪ RTI ID = 0.0 > and / or < / RTI > enzyme-resistant agents. The specific dosage and exact dosage and route of administration will be determined by the attending physician and will vary according to each particular situation.
[886] The determination of such administration is made by considering a number of variables such as the condition to be treated, the type of mammal to be treated, the compound to be administered, the pharmacokinetic properties of the particular treatment compound, and the like. Other factors to consider include the patient's disease state (eg, symptom), age, weight, sex, diet, timing of administration, drug combination, response sensitivity, and tolerance / response to treatment. (Eg, PRO317 encapsulated in liposomes or PEGylated PRO317 or PRO317 polymer entities, eg, poly lactic acid based entities) acting at long intervals may be administered at intervals of 3 to 4 days, or at weekly intervals, depending on the half- Once, or once every two weeks.
[887] The normal dosage may be about 10 ng / kg to 100 mg / kg or more, preferably about 1 g / kg to 10 mg / kg, per kg of mammalian body weight per day, depending on the route of administration. Specific dosage and delivery instructions are provided, for example, in U.S. Patent Nos. 4,657,760, 5,206,344, or 5,225,212. It is anticipated that different agents will be effective depending on the type of treatment compound and the type of disorder. For example, uterine targeting may require delivery differently from other organs or tissues, such as heart tissue.
[888] Microencapsulation of the PRO317 polypeptide is contemplated when sustained release of a PRO317 polypeptide is required for an agent having release characteristics suitable for the treatment of a disease or disorder in need of administration of a PRO317 polypeptide. Microencapsulation of recombinant proteins for delayed release has been successfully performed using human growth hormone (rhGH), interferon (rhIFN), interferon-2 and MN rgp120 [Johnson et al ., Nat. Med. , ≪ / RTI > 2 : 795-799 (1996); Yasuda, Biomed. Ther. , ≪ / RTI > 27 : 1221-1223 (1993); Hora et al., Bio / Technology, 8 : 755-758 (1990); (Plenum Press: New York, 1995), pp. 445-454, for example, in Cleland, "Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems," in Vaccine Design: The Subunit and Adjuvant Approach , Powell and Newman, eds . 439-462; WO 97/03692, WO 96/40072, WO 96/07399 and U.S. Patent No. 5,654,010).
[889] If the condition or disease of the uterus, endometrial tissue or other reproductive or cardiac tissues is treated with a PRO317 or PRO317 agonist when the expression of PRO317 is reduced in the disease state and the expression of PRO317 is increased in the disease state, It is anticipated that the antibody or other PRO317 antagonist may cause treatable damage. Such conditions or diseases may be specifically diagnosed through the above probing for physiological and pathological problems affecting the functioning of the organ.
[890] The PRO317, PRO317 agonist, or PRO317 antagonist may be administered with other biologically active agents, which may be administered separately or jointly administered as the same agent to treat a common indication that they are needed. For example, it is believed that both PRO317 and EBAF-1 can be administered together for the same qualitative biological effect. EBAF-1 may also be administered with an antagonist of PRO317, for example an anti-PRO 317 antibody, if they exhibit the opposite effect. In addition, PRO317 may be administered with VEGF in patients with coronary ischemia where necessary, and if necessary, with anti-VEGF, if necessary, with PRO317 antagonist if necessary, VEGF in coronary ischemia, anti- Can be administered together. Such administration will be in an amount effective to treat such disorder.
[891] Native PRO301 (SEQ ID NO: 119) has a Blast value of 246 and 30% homology with A33_HUMAN, the A33 antigen precursor at residues 24-282 of Figure 44. The A33 antigen precursor is a tumor specific antigen, as described in the Background section above, and is therefore a recognizable marker and therapeutic target for the diagnosis and treatment of colon cancer. Expression of tumor-specific antigens is often accompanied by the development of neoplastic tissue disorders. Native PRO301 (SEQ ID NO: 119) and A33_HUMAN also show A33_HUMAN and Blast value 245, homology 30% in residues 21-282 of FIG. 44, varying depending on how the space is inserted in the compared sequence. Native PRO301 (SEQ ID NO: 119) also has a Blast value of 165 and a homology of 29% with human Coxsackie and the adenoviral receptor protein HS46KDA_1 (also known as cell surface protein HCAR) at residues 60-255 of Figure 44. This region of PRO301 also has a similar Blast value and homology to HSU90716_1. Since the expression of these proteins is usually accompanied by virus infection, a therapeutic agent for preventing such infection can be devised. As described in the Background section, expression of viral receptors is often associated with neoplastic tumors.
[892] Therapeutic uses of the PRO234 polypeptides of the present invention include treatment of leukocyte with endothelial cell interactions or leukocyte replacement during an inflammatory reaction. Examples include asthma, rheumatoid arthritis, psoriasis and multiple sclerosis.
[893] Since the PRO231 polypeptide and the nucleic acid encoding it are sequence homologous to the putative acid phosphatase and the nucleic acid encoding the same, probes based on the PRO231 nucleotide sequence can be used to detect other novel phosphatase proteins. The soluble form of the PRO231 polypeptide can be used both in vitro and in vivo as an antagonist of membrane bound PRO231 activity. PRO231 polypeptides can be used in screening assays to identify agonists or antagonists of native PRO231 polypeptides, which can take the form of any conventional cell type or biochemical binding assay. Moreover, PRO231 polypeptides can function as molecular markers for tissues in which this polypeptide is specifically expressed.
[894] A PRO229 polypeptide can be fused with a peptide of interest to determine whether the fusion peptide has an increased half-life over that peptide of interest. Accordingly, PRO229 polypeptides can be used to increase the half-life of peptides of interest. Portions of PRO229 that cause an increase in half-life are also aspects of the present invention.
[895] PRO238 can be used to analyze the ability to reduce oxygen and other substrates such as acetyl-CoA, in particular, the ability of PRO238 to produce oxygen free radicals. This is done using the assay described above. PRO238 can also be used to analyze candidate compounds that block, reduce or reverse their ability to reduce. This can be done by performing a parallel analysis on the same analytical system except that the candidate compound is added to the analyzer containing PRO238 and the substrate to be reduced and the candidate compound is not added.
[896] Since the PRO233 polypeptide and portions thereof are homologous to the reductase, they may be useful for in vivo treatment and for a variety of other uses. The identification of novel reductase proteins and molecules associated therewith is commonly associated with many human disorders, including inflammatory diseases, organ failure, atherosclerosis, cardiac insufficiency, infertility, congenital defects, premature aging, AIDS, cancer, diabetic complications and mutations . Since oxygen free radicals and antioxidants appear to play an important role in many disease processes, the discovery of new reductase proteins and reductase-like molecules is particularly important in that such proteins can serve as potential therapeutic agents for a variety of human disorders It is important. Such polypeptides may also play an important role in biotechnology and medical research as well as in industrial applications. As a result, interest in academia and the medical community exists for new molecules such as PRO233.
[897] Since the PRO223 polypeptide of the present invention exhibits serine carboxypeptidase activity, it can be used for in vivo therapeutic purposes as well as in vitro. Those skilled in the art will know how to use PRO233 for such applications.
[898] The PRO235 polypeptides and portions thereof may be involved in cell attachment, and thus are useful for in vivo therapeutic applications, as well as in a variety of in vitro applications. Also PRO235 polypeptides and portions thereof may have therapeutic use in disease states in which cell adhesion is involved. Due to the physiological importance of the cell attachment mechanism in vivo, efforts are now being made to find new natural proteins involved in cell attachment. Thus, peptides that are homologous to flexin are of particular interest to academia and the medical community.
[899] Because the PRO236 and PRO262 polypeptides disclosed herein are homologous to a number of known galactosidase proteins, it is believed that by producing an active drug from a galactosylated prodrug (e.g., prodrug-D-galactosyl-5-fluorouridine The PRO236 and PRO262 polypeptides of the present invention can be used in the conjugation of a monoclonal antibody with a polypeptide that specifically kills the tumor. The PRO236 and PRO262 polypeptides disclosed herein may have a variety of uses in vivo and in vitro, and such uses will be similar or identical to the applications for which the galactosidase protein is currently used. Those skilled in the art will know how to use the PRO236 and PRO262 polypeptides for such use.
[900] The PRO239 polypeptide and portions thereof will be homologous to densin and therefore will be useful for in vivo therapeutic applications as well as in vitro applications. The PRO239 polypeptide and a portion thereof may also be used for therapy in disease states associated with synaptic mechanisms, regeneration or cell adhesion. Because of the physiological importance of synaptic processes, regeneration, and attachment mechanisms in vivo, efforts are underway to find new natural proteins involved in synaptic organization and cell adhesion. Thus, peptides with homology to tansin are of particular interest to academia and the medical community.
[901] The PRO260 polypeptides described herein can be used in assays to determine their relevance to fucosidases. In particular, PRO260 polypeptides can be used in assays to measure their ability to remove fucose or other sugar residues from proteoglycans. The PRO260 polypeptide can also be used in assays to determine whether there is a functional or site similarity with a fucosidase. PRO260 polypeptides can also be used to regulate systems in which they are essential.
[902] PRO263 can be used in assays in which CD44 antigen is commonly used to measure the activity of PRO263 on the activity of CD44. The results can be used accordingly.
[903] The PRO270 polypeptides and portions thereof may affect the redox state and therefore may be useful for in vitro as well as in vivo therapeutic applications. More specifically, PRO270 polypeptides can affect the expression of many different genes thought to be involved in oxidative stress-related conditions such as AIDS, cancer, atherosclerosis, diabetic complications and stroke and inflammation. In addition, PRO270 polypeptides and portions thereof may also affect the expression of genes involved in apoptosis. Thus, peptides that are homologous to thioredoxin are of particular interest to academia and the medical community.
[904] Because PRO272 polypeptides and portions thereof are capable of binding to calcium, they can have many in vivo therapeutic applications as well as in vitro applications. Thus, peptides that are homologous to reticulo calvin are particularly preferred. Those skilled in the art will know how to use PRO272 polypeptides and portions thereof for such purposes.
[905] Since PRO294 polypeptides and portions thereof are homologous to collagen, they may also be useful in a variety of in vivo therapeutic applications and in a variety of other applications. Identification of new collagen and collagen-like molecules may be associated with many human disorders. Therefore, finding new collagen and collagen-like molecules is especially important because these proteins can serve as potential treatments for a variety of human disorders. Such polypeptides may also play an important role in biotechnological and medical research as well as in many industrial applications. Because of the high use of collagen, there is considerable interest in polypeptides that are homologous to collagen molecules.
[906] Since PRO295 polypeptides and portions thereof are homologous to integrins, they may be useful in many other applications as well as in vivo therapeutic purposes. Identification of new integrins and integrin-like molecules may be associated with many human disorders, such as modulating cell binding or cellular activity of the immune system. Therefore, finding new integrin and integrin-like molecules is especially important because these proteins can function as potential therapeutic agents for a variety of human disorders. Such polypeptides may also have important functions in biotechnology and medical research as well as in a variety of industrial applications. As a result, there is special interest in academia and the medical community for new molecules such as PRO295.
[907] This polypeptide can be used in all applications where the known NLRR-1 and NLRR-2 polypeptides are used, since the PRO293 polypeptide is clear to be a leucine-rich repeat-domain polypeptide homolog. Since the activity between these peptides can be compared, their activity can be applied accordingly.
[908] The PRO247 polypeptides described herein can be used in assays in which densin is used to determine the activity of PRO247 relative to tentin or other such proteins. For this, the results can be used for diagnostic and / or therapeutic applications of PRO247.
[909] The present invention, PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides have protease activity and can therefore be used for therapeutic purposes in vivo and in vitro. Those skilled in the art will know how to use the PRO302, PRO303, PRO304, PRO307 and PRO343 polypeptides of the invention for this purpose.
[910] PRO328 polypeptides and portions thereof that are homologous to GLIP and CRISP may be useful for a variety of other applications as well as for in vivo therapeutic purposes. The identification of novel GLIP and CRISP-like molecules may be associated with many human disorders that are overexpressed or associated with transcriptional regulation in human tumors. Therefore, finding new GLIP and CRISP-like molecules is especially important because these proteins can function as potential therapeutic agents for a variety of human disorders. Such polypeptides may also have important functions in biotechnology and medical research as well as in a variety of industrial applications. As a result, there is special interest in academia and the medical community for new molecules such as PRO328.
[911] PRO335, PRO331 or PRO326 are used in competitive assays with LIG-1, ALS and decolin to determine their relative activity. The results can be used accordingly. PRO335, PRO331 or PRO326 are used in the analysis in which LIG-1 is used to determine if the same effect occurs.
[912] PRO332 has a GAG repeats (GKEK) at amino acid positions 625- 628 in Figure 108 (SEQ ID NO: 310). Losses in such repeats can be accompanied by human disease. Thus, PRO332 can be used by gene therapy, that is, by introducing genes containing the correct GKEK sequence motifs to treat such disease states.
[913] Other uses for PRO334 include determining the relative activity of PRO334 against fibril or pibulin using fibrillin and pibulin in assays using it. In particular, PRO334 can be used for assays that require a mechanism conferred by epithelial growth factor repeat.
[914] Native PRO346 (SEQ ID NO: 320) has a Blast value of 230 between amino acid residues 21 and 343, corresponding to a homology of 27 to 27% with residues 35 to 1040 of CGM6_HUMAN, the protozoan cgm6 precursor. This homology region encompasses almost all but the two N-terminal extracellular domain residues, for example, immunoglobulin macrophage homology to several transmembrane residues (340-343) and residues 148 to 339 of PRO346 . The cancer antigens precursor is a tumor-specific antigen as described in the Background section, and thus is a recognizable marker and therapeutic target for the diagnosis and treatment of colon cancer. Expression of tumor antigens often involves the progression of neoplastic tissue disorders. Native PRO346 (SEQ ID NO: 320) and human marine antigen CEA-d, P_W06874, have a Blast value of 224 and a homology of 28% at residues 2 to 343 and 67 to 342, respectively. This homology encompasses all extracellular domain residues (residues 2 to 18) and multiple transmembrane residues (340-343) of native PRO346 except for the initiation methionine.
[915] Because the PRO268 polypeptide has the activity of a protein disulfide isomerase, it can be used in a variety of applications where protein disulfide isomerase activity is desired, for example, by promoting proper disulfide bond formation of a recombinantly produced protein to yield a correctly folded protein Height will be useful for applications and so on. Those skilled in the art will know how to use this PRO268 polypeptide for this purpose.
[916] The PRO330 polypeptide of the present invention has biological activity associated with the prolyl 4-hydroxyslase alpha subunit protein and can thus be used for therapeutic purposes in vitro and in vivo. Those skilled in the art will know how to use PRO330 polypeptides for this purpose.
[917] F. Anti-PRO antibody
[918] The present invention further provides an anti-PRO antibody. Examples of antibodies include polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies.
[919] 1. polyclonal antibody
[920] The anti-PRO antibody may comprise a polyclonal antibody. Methods for producing polyclonal antibodies are known to those skilled in the art. Polyclonal antibodies can be produced in mammals, for example, by injection of one or more immunizing agents and, if desired, adjuvants. In general, the immunizing agent and / or adjuvant will be injected into the mammal by subcutaneous or intraperitoneal injection. The immunizing agent may comprise a PRO polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the immunized mammal. Examples of such immunogenic proteins include, but are not limited to, keyhole limpet hemocyanin, serum albumin, sorbitol globulin, and soybean trypsin inhibitor. Examples of adjuvants that may be used include Freud's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose decolinomicolate). Immunization methods may be selected by those skilled in the art without undue experimentation.
[921] 2. Monoclonal antibody
[922] The anti-PRO antibody may alternatively be a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma methods as described in Kohler and Milstein, Nature , 256 : 495 (1975). In the hybridoma method, a mouse, hamster or other appropriate host animal is usually immunized with an immunizing agent to induce a lymphocyte capable of producing or producing an antibody that specifically binds to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.
[923] The immunizing agent will generally comprise a PRO polypeptide or a fusion protein thereof. In general, peripheral blood lymphocytes (" PBL ") are used when cells of human origin are desired, but spleen cells or lymph nodes are used when a non-human mammalian source is desired. Subsequently, a suitable fusion agent, such as polyethylene glycol, is used to fuse lymphocytes and immortalized cell lines to form hybridoma cells (Goding, Monoclonal Antibodies: Principle and Practice , Academic Press, (1986) pp. 59-103]. Immortalized cell lines are generally transformed mammalian cells, particularly myeloma cells of rodent, bovine, and human origin. Generally, a rat or mouse myeloma cell line is used. The hybridoma cells may preferably be cultured in a suitable culture medium containing one or more substances that inhibit the proliferation or survival of unfused, immortalized cells. For example, if the parental cell lacks hypoxanthine guanine phospholibosyl transferase (HGPRT or HPRT) enzyme, the hybridoma culture medium (" HAT medium ") typically contains hypoxanthine, aminopterin, and thymidine These substances inhibit the proliferation of HGPRT-deficient cells.
[924] Preferred immortalized cell lines are those that efficiently fuse, remain stable to high levels of antibody production by the selected antibody-producing cells, and susceptible to media such as HAT medium. A more preferred immortalized cell line is, for example, a murine myeloma cell line available from the Salk Institute Cell Distribution Center (San Diego, CA) and the American Type Culture Collection (Manassas, Va., USA) . In addition, human myeloma cell lines and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133 : 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications , Marcel Dekker, Inc., New York, (1987) pp. 51-63].
[925] The culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed against PRO. Preferably, the binding specificity of the monoclonal antibody produced by the hybridoma cells is determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) . Such techniques and analyzes are well known in the art. The binding affinity of the monoclonal antibody is determined, for example, by the Scatchard analysis (Munson and Pollard, Anal. Biochem. , 107 : 220 (1980).
[926] After identifying the desired hybridoma cells, the clones were subcloned by limiting dilution procedures and cultured by standard methods (Goding, supra ). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. Alternatively, the hybridoma cells may be cultivated in vivo as multiple tumors in a mammal.
[927] The monoclonal antibody secreted by the subclone may be cultured by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography Can be isolated and purified from a medium or a plurality of liquids.
[928] In addition, monoclonal antibodies can be prepared by recombinant DNA methods such as those described in U.S. Patent No. 4,816,567. The DNA encoding the monoclonal antibody of the present invention can be readily obtained by using a conventional method (for example, by using an oligonucleotide probe capable of specifically binding to a gene encoding a heavy chain and a light chain of a mouse antibody) Can be isolated and sequenced. The hybridoma cells of the present invention serve as a desirable source of such DNA. Once isolated, the DNA is placed into expression vectors and transfected into expression vectors into host cells such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, Monoclonal antibodies can be synthesized from host cells. In addition, DNA, for example, replacing the homologous murine sequences portion Province in the coding sequence for human heavy and light chain constant domain, or (U.S. Patent No. 4,816,567; and the literature, such as Morrison (Morrison)), or a non-immunoglobulin polypeptide Can be modified by covalently attaching all or part of the coding sequence to the immunoglobulin coding sequence. Such non-immunoglobulin polypeptides may substitute for the constant domain of an antibody of the invention, or substitute for a variable domain of an antibody-binding site of an antibody of the invention to generate a chimeric dipeptide antibody.
[929] The antibody may be a monovalent antibody. Methods for making monovalent antibodies are known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. To prevent heavy chain cross-linking, the heavy chain is typically truncated at any point in the Fc region. Alternatively, the related cysteine residue is substituted or deleted with another amino acid residue to prevent cross-linking.
[930] In addition, in vitro methods are suitable for the production of antibodies. The degradation of antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using techniques commonly known in the art.
[931] 3. Human and humanized antibodies
[932] The anti-PRO antibodies of the present invention may also include humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine) antibodies include chimeric immunoglobulins, immunoglobulin chains comprising minimal sequence derived from non-human immunoglobulin or fragments thereof (e.g., Fv, Fab, Fab ' , F (ab ') _2, or other antigen-binding small sequences of antibodies). Humanized antibodies are human immunoglobulins (CDRs) in which the residue of the complementarity determining region (CDR) of the receptor is replaced with a residue from the CDR of a non-human species (donor antibody) such as a mouse, rat or rabbit having the desired specificity, (Acceptor antibody). In some cases, the Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may comprise residues that are not found in the recipient antibody or in the CDR or framework sequences introduced. In general, a humanized antibody will comprise substantially all of one or more, typically two or more, variable domains, wherein all or substantially all of the CDR regions correspond to regions of non-human immunoglobulin, and all or substantially all of the FR regions Corresponds to the region of the human immunoglobulin consensus sequence. In addition, the humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fc), generally at least a portion of a human immunoglobulin region (Jones et al. , Nature , 321 : 522-525 (1986); Riechmann et al. , Nature , 332 : 323-329 (1988); And Presta, Curr. Op. Struct. Biol. 2 : 593-596 (1992)].
[933] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced thereto from a non-human source. These non-human amino acid residues are often referred to as " import " residues and are typically obtained from the " import " Humanization can be carried out essentially as described by Winter et al. (Jones et al. , Nature , 321 : 522-525 (1986)) by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence; Riechmann et al. , Nature , 332 : 323-327 (1988); Can be performed according to Verhoeyen et al. Science , 239 : 1534-1536 (1988). Thus, such " humanized " antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) in which substantially less intact human variable domains are replaced by corresponding sequences from non-human species. In fact, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues have been replaced with residues from similar sites in rodent antibodies.
[934] Human antibodies can also be prepared using a variety of techniques known in the art including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. , 227 : 381 (1991); Marks et al ., J. Mol. Bio. , 222 : 581 (1991)]. Cole et al. And Boerner et al. Also can be used in the manufacture of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy , Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. , 147 (1) : 86-95 (1991)]. Similarly, human antibodies can be produced by introducing human immunoglobulin loci into transgenic animals, e. G., Mice, wherein the internal immunoglobulin genes are partially or fully functional. After antigen administration, human antibody production was observed, which was very similar to that observed in humans in all respects including gene rearrangement, assembly and antibody listing. These are described, for example, in US 5,545,807, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661,016, and scientific publications, Marks et al. Bio / Technology 10 , 779-783 (1992); Lonberg et al. , Nature 368 , 856-859 (1994), Morrison, Nature 368 , 812-13 (1994); Fishwild et al. , Nature Biotechnology 14 , 845-51 (1996); Neuberger, Nature Biotechnology 14 , 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).
[935] 4. Bispecific antibodies
[936] Bispecific antibodies are monoclonal, preferably human or humanized, antibodies having binding specificity for two or more different antigens. In this case, one of the binding specificities is for PRO, and the other is for any other antigen, preferably a cell surface protein or receptor or receptor subunit.
[937] Methods for producing bispecific antibodies are known in the art. Typically, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain / light- chain-heavy chain pairs, wherein the two heavy chains have different specificities (Millstein and Kuello Cuello, Nature , 305 : 537-539 (1983)]. Due to the random classification of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is generally carried out by an affinity chromatography step. Similar methods are described in WO 93/08829 (published May 13, 1993) and Traunecker et al. ( EMBO J. , 10: 3655-3659 (1991)).
[938] The variable domain (antibody-antigen binding site) of the antibody with the desired binding specificity can be fused to the immunoglobulin constant domain sequence. This fusion is preferably fused with immunoglobulin heavy chain constant domains comprising at least a portion of the hinge, CH2 and CH3 regions. It is preferred that the first heavy chain constant region (CHl) comprising the site necessary for light chain binding is present in at least one of the fusions. The immunoglobulin heavy chain fusions, and optionally the DNA encoding the immunoglobulin light chain, are inserted into separate expression vectors and cotransfected into a suitable host organism. For further details on producing bispecific antibodies see, for example, Suresh et al. , Methods in Enzymology , 121 : 210 (1986).
[939] According to another method disclosed in WO 96/27011, the interface between a pair of antibody molecules can be manipulated to maximize the percentage of heterodimers recovered from the recombinant cell culture. A preferred interface comprises at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a small amino acid side chain (e.g., alanine or threonine) a complementary " cavity " of the same or similar size to the larger side chain was generated at the interface of the second antibody molecule. This provides a mechanism to increase the yield of the heterodimer above the yield of other undesired end-products such as homodimers.
[940] Bispecific antibodies can be prepared from full-length antibodies or antibody fragments (e. G., F (ab ') ₂ bispecific antibodies). Techniques for generating bispecific antibodies from antibody fragments are described in the literature. For example, bispecific antibodies can be prepared using chemical linkages. Brennan et al., Science 229: 81 (1985), describe a method of generating F (ab ') ₂ fragments by truncation of intact antibodies by protein hydrolysis. These fragments are reduced in the presence of sodium arsenite, a dithiol complex, to stabilize nearby dithiols and prevent disulfide formation in the bonsai. The resulting Fab 'fragment was then converted to a thionitrobenzoate (TNB) derivative. Thereafter, one of the Fab'-TNB derivatives was re-converted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as a material for selective immobilization of the enzyme.
[941] this. Fab 'fragments were directly recovered from the E. coli and chemically coupled to form bispecific antibodies. Shalaby et al ., J. Exp. Med. 175: 217-225 (1992)) discloses a fully humanized bispecific antibody F (ab ') ₂ molecule. Each Fab 'fragment was secreted separately from E. coli and ligated by the in vitro chemical method indicated to form a bispecific antibody. The bispecific antibodies thus formed were capable of binding to ErbB2 receptor overexpressing cells and normal human T cells, as well as initiating the lytic activity of human cytotoxic lymphocytes against human breast cancer targets.
[942] In addition, several techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have been disclosed. For example, bispecific antibodies have been prepared using leucine zippers [Kostelny et al ., J. Immunol . 148 (5): 1547-1553 (1992)). By gene fusion, leucine zipper peptides from the Fos and Jun proteins were ligated to the Fab 'portion of two different antibodies. The hinge region of the antibody homodimer was reduced to form monomers and then reoxidized to form antibody heterodimers. This method can also be used as a method for producing antibody homodimers. Hollinger et al ., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) provides another mechanism for making bispecific antibody fragments. This fragment contains a heavy chain variable domain (V _H ) linked to the light chain variable domain (V _L ) by the linker, which is too short to pair the two domains to the same chain. Therefore, V _H and V _L domains of one fragment are formed complementary to the V _H and V _L domain pairs and of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments using short chain Fv (sFv) dimers has been reported [Gruber et al., J. Immunol. 152: 5368 (1994)]. Antibodies having a binding affinity greater than or equal to 1 are also contemplated. For example, triple specific antibodies can be made [Tutt et al., J. Immunol. 147: 60 (1991)].
[943] Exemplary bispecific antibodies may bind to two different epitopes of the PRO polypeptide provided herein. Alternatively, the anti-PRO polypeptide arm may be conjugated to a leukocyte-initiating molecule, such as a T-cell receptor molecule (e. G., CD2, CD3, CD28 Or B7) or an arm that binds an Fc receptor (Fc [gamma] R) of IgG, such as Fc [gamma] RI (CD64), Fc [gamma] RII (CD32) and Fc [gamma] RIII (CD16). Bispecific antibodies can also be used to localize cytotoxic agents to cells that express a particular PRO polypeptide. These antibodies have arms that combine with PRO-binding arms and cytotoxic agents or radionuclide chelators such as EOTUBE, DPTA, DOTA or TETA. Another bispecific antibody of interest binds to the PRO polypeptide and further binds to the tissue factor (TF).
[944] 5. Heterozygous antibodies
[945] Heterozygous antibodies are also included within the scope of the present invention. A heterozygous antibody consists of two covalently linked antibodies. Such antibodies are useful, for example, for targeting immune system cells to undesired cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089 Respectively. It is believed that the heterologous antibody can be prepared in vitro using methods known in the art of synthetic protein chemistry, including methods using cross-linking agents. For example, immunotoxins can be prepared by forming disulfide exchange reactions or thioether linkages. Reagents suitable for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and reagents disclosed in U.S. Patent No. 4,676,980.
[946] 6. Operation of effector function
[947] It may be desirable to modify the antibody of the invention to function as an effector, for example, to increase the effect of the antibody in the treatment of cancer. For example, a cysteine residue can be introduced into the Fc region to form interchain disulfide bonds within this region. The homodimeric antibody thus generated enhances the internalization capacity and enhances complement-mediated cell death and antibody-dependent cellular cytotoxicity (ADCC) [Caron et al ., J. Exp Med. , 176: 1191-1195 (1992) and Shopes, J. Immunol. , 148: 2918-2922 (1992)]. Homotypic dimeric antibodies with increased anti-tumor activity can also be prepared using the heterobifunctional crosslinking described in Wolff et al., Cancer Research , 53 : 2560-2565 (1993). Alternatively, antibodies with dual Fc regions can be engineered to increase complement fungi and ADCC capabilities (see Stevenson et al., Anti-Cancer Drug Design , 3: 219-230 (1989) ].
[948] 7. Immune complexes
[949] The present invention also relates to a method of treating or preventing a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active bacterial, fungal, plant or animal toxin, or a fragment thereof) or a radioactive isotope (i.e., ≪ RTI ID = 0.0 > and / or < / RTI >
[950] Chemotherapeutic agents useful for the production of the immunoconjugate are described above. Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), lysine A chain, Aleurites fordii protein, Dianthin protein, Phytolaca americana protein (PAPI, PAPII and PAP-S), bitter melon (momordica charantia) inhibitor, Cicin, sapaonaria officinalis inhibitor, gelonin, mitogelin, restricotisin, penomycin, enomycin, and tricothecenes. Several radionuclides can be used in the preparation of radiolabeled antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re. Various bifunctional protein-linking agents such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (For example, dimethyl adipimidate HCL), active esters (e.g., disuccinimidylsuberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., (P-diazonium benzoyl) -ethylenediamine), a diisocyanate (e.g., toluene 2,6-diisocyanate), and a bis-diazonium derivative (e.g., A bis-active fluorine compound (e.g., 1,5-difluoro-2,4-dinitrobenzene) can be used to make a complex of an antibody and a cytotoxic agent. For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science , 238 : 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriamine pentaacetic acid (MX-DTPA) is a typical chelating agent linking a radioactive nucleotide and an antibody (see WO 94/11026 do).
[951] In another embodiment, an antibody and a " receptor " (e.g., streptavidin) used for tumor preliminary targeting can be linked, wherein the antibody- receptor complex is administered to the subject, Unbound complexes are removed and a " ligand " (e.g., avidin) linked to a cytotoxic agent (e. G., A radionucleotide) is administered.
[952] 8. Immune liposome
[953] The antibodies disclosed herein may also be formulated as immuno-liposomes. Liposomes containing this antibody can be prepared by methods known in the art, for example, as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad. Sci. USA, 77: 4030 (1980); And the methods described in U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Patent No. 5,013,556.
[954] Particularly useful liposomes can be produced by reverse phase evaporation using lipid compositions containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposome was extruded through a filter having a predetermined pore size to obtain a liposome having a predetermined diameter. Martin et al ., J. Biol. Chem. , 257 : 286-288 (1982)), a Fab 'fragment of an antibody of the present invention can be linked to a liposome via a disulfide-interchange reaction. Chemotherapeutic agents (e. G., Doxorubicin) are optionally included in liposomes (Gabizon et al ., J. National Cancer Inst. , 81 (19): 1484 (1989)].
[955] 9. Antibody pharmaceutical composition
[956] To treat various diseases, antibodies that specifically bind to the PRO polypeptides identified herein and other molecules identified by the screening assays described above may be administered in the form of pharmaceutical compositions.
[957] Where the PRO polypeptide is within the cell and all of the antibody is used as an inhibitor, an internalizing antibody is preferred. However, lipofection or liposomes can also be used to deliver an antibody or antibody fragment into a cell. When an antibody fragment is used, a minimal inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable-region sequence of the antibody, a peptide molecule with the ability to bind to a target protein sequence can be designed. Such peptides can be chemically synthesized and / or produced by recombinant DNA techniques (see, for example, Marasco et al ., Proc. Natl. Acad. Sci. USA , 90: 7889-7893 (1993)]. In addition, the formulations herein may comprise one or more active compounds, preferably compounds with complementary activity that do not adversely affect one another, as necessary for the particular condition being treated. Alternatively, or additionally, the composition may comprise a substance that enhances its function, for example, a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a proliferation inhibitor. Such molecules are suitably combined in an amount effective for the intended purpose.
[958] The active ingredient may be formulated, for example, in a colloidal drug delivery system (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively, for example by coacervation techniques or interfacial polymerization, May be encapsulated in microcapsules prepared by hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules. This technique is described in Remington's, Pharmaceutical Sciences , supra.
[959] Preparations used for intravenous administration should be sterilized. This is easily accomplished by filtration through a sterile filter membrane.
[960] Sustained release formulations may also be prepared. Suitable examples of sustained-release preparations include molded articles of solid hydrophobic polymers containing the antibody, such as semipermeable matrices in the form of films or microcapsules. Examples of sustained release matrices are polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (U.S. Patent No. 3,773,919), L- And lactic acid-glycolic acid copolymers such as LUPRON DEPOT (registered trademark) (lactic acid-glycolic acid copolymer and looprolide acetate), non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer, Possible microspheres), and poly-D - (-) - 3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid release molecules for more than 100 days, but some hydrogels release proteins for shorter periods of time. If the encapsulated antibody remains in the body for a long period of time, he or she may be denatured or aggregated as a result of exposure to moisture at 37 ° C, thereby reducing biological activity and altering immunogenicity. can do. For example, if it is found that the cohesion mechanism is SS bond formation in the bonsai through thio-disulfide interchange, it is contemplated that modifications of the sulfhydryl moiety, lyophilization from an acidic solution, control of moisture content, Stabilization can be achieved by the development of a matrix composition.
[961] G. Uses of anti-PRO antibodies
[962] The anti-PRO antibodies of the present invention have a variety of utility. For example, anti-PRO antibodies can be used for diagnostic assays for PRO, for example, detection of its expression in certain cells, tissues or serum. A variety of diagnostic analytical techniques known in the art can be used, such as competitive binding assays performed on heterologous or homologous forms, direct or indirect sandwich assays and immunoprecipitation assays (see Zola, Monoclonal Antibodies: A Manual of Techniques , CRC Press , Inc. (1987) pp. 147-158]. Antibodies used in diagnostic assays can be labeled with detectable moieties. The detectable moiety should be capable of generating a detectable signal, either directly or indirectly. For example, the detectable moiety may be a radioactive isotope such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I, a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, Luciferin, or enzymes such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Hunter et al. , Nature , 144 : 945 (1962); David et al., Biochemistry , 13 : 1014 (1974); Pain et al ., J. Immunol. Meth. , ≪ / RTI > 40 : 219 (1981); And Nygren, J. Histochem. and Cytochem. , 30 : 407 (1982)), as well as methods known in the art for binding antibodies to detectable moieties.
[963] In addition, anti-PRO antibodies are useful for affinity purification of PRO from recombinant cell culture fluids or natural sources. In this process, the antibody to PRO is immobilized on a support suitable for Sephadex resin or filter paper using methods known in the art. The immobilized antibody is then contacted with a sample containing PRO to be purified and then washed with a suitable solvent to substantially remove all substances in the sample except the PRO polypeptide bound to the immobilized antibody. Finally, the support is washed with another suitable solvent to release the PRO polypeptide from the antibody.
[964] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.
[965] All patents and documents cited in this specification are hereby incorporated by reference in their entirety.
[966] Commercial reagents mentioned in the examples were used according to manufacturer's instructions unless otherwise indicated. In the following examples and throughout the specification, the source of cells identified by ATCC Accession Number is the American Type Culture Collection, < RTI ID = 0.0 >
[967] Example 1: Extracellular domain homology screening to identify novel polypeptides and cDNA encoding them
[968] The extracellular domain (ECD) sequence of about 950 known secreted proteins from the Swiss-Prot public database (including the secretory signal sequence in the presence of the secretory signal sequence) Respectively. The EST database can be stored in a public database (e.g., Dayhoff, GenBank) and a proprietary database (e.g., LIFESEQ ™ (Incyte Pharmaceuticals, Palo Alto, Calif.)) A search for a comparison of the EFT sequence with the six-frame translation of the EST sequence was performed using the computer program WU-BLAST-2 (Altschul et al., Methods in Enzymology 266: 460-480 (1996) . Comparators representing 70 (or in some cases, over 90) blast scores that do not code known proteins were pooled, and the program "phrap" (Phil Green, University of Washington, Seattle, Washington) And assembled into consensus DNA sequences.
[969] Consensus DNA sequences were assembled against other EST sequences identified using this extracellular domain homology screen. Also, consensus DNA sequences were extended using repeated cycles of BLAST and phrap to extend the consensus sequences as long as possible, often (though not always), using the EST sequence sources discussed above.
[970] Then, based on the consensus sequence obtained as described above, oligonucleotides were synthesized, used to confirm the cDNA library containing the desired sequence by PCR, and used as a probe for isolating clones of the full-length coding sequence for PRO polypeptide Respectively. The forward (.f) and reverse (.r) PCR primers generally range from 20 to 30 nucleotides and are often designed to provide PCR products of about 100 to 1000 bp in length. The length of the probe (.p) sequence is typically 40-55 bp. In some cases, when the consensus sequence is greater than about 1 to 1.5 kbp, the oligonucleotide is further synthesized. To screen multiple libraries to obtain full-length clone circles, DNAs from these libraries were screened using PCR primer pairs amplified by PCR according to Ausubel et al. ( Current Protocols in Molecular Biology ). A positive library was then used to isolate clones encoding the desired gene using probe oligonucleotides and one of the primer pairs.
[971] The cDNA library used to isolate cDNA clones was prepared by standard methods using commercially available reagents, such as reagents purchased from Invitrogen (San Diego, Calif.). CDNA was primed with oligo dT containing the NotI site and ligated to the SalI adapter at one end only with a phosphorylated SalI adapter followed by cleavage with NotI, followed by appropriate classification according to size by gel electrophoresis, followed by appropriate cloning vector A vector such as pRKB or pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI site; (See Science , 253 : 1278-1280 (1991) by Holmes et al.).
[972] Example 2: Isolation of cDNA clones encoding PRO211 and PRO217
[973] Consensus DNA sequences were assembled as described in Example 1 above and were referred to as DNA 28730 and DNA 28760, respectively. Based on these consensus sequences, oligonucleotides were synthesized, used to identify cDNA libraries containing the desired sequences by PCR, and used as probes to isolate clones of full-length coding sequences for PRO211 and PRO217 polypeptides. The library used to isolate DNA32292-1131 and DNA33094-1131 was a fetal lung library.
[974] The sequence of the entire cDNA clone was confirmed. The entire nucleotide sequence of PRO211 (DNA32292-1131) and PRO217 (UNQ191) is shown in FIG. 1 (SEQ ID NO: 1) and FIG. 3 (SEQ ID NO: 3), respectively. The predicted polypeptides are 353 and 379 amino acids in length, respectively, and the respective molecular weights are about 38,190 and 41,520 daltons.
[975] The oligonucleotide sequences used in the above method are as follows.
[976] 28730.p (OLI 516) (SEQ ID NO: 5)
[977] 5'-AGGGAGCACGGACAGTGTGCAGATGTGGACGAGTGCTCACTAGCA-3 '
[978] 28730.f (OLI 517) (SEQ ID NO: 6)
[979] 5'-AGAGTGTATCTCTGGCTACGC-3 '
[980] 28730.r (OLI 518) (SEQ ID NO: 7)
[981] 5'-TAAGTCCGGCACATTACAGGTC-3 '
[982] 28760.p (OLI 617) (SEQ ID NO: 8)
[983] 5'-CCCACGATGTATGAATGGTGGACTTTGTGTGACTCCTGGTTTCTGCATC-3 '
[984] 28760.f (OLI 618) (SEQ ID NO: 9)
[985] 5'-AAAGACGCATCTGCGAGTGTCC-3 '
[986] 28760.r (OLI 619) (SEQ ID NO: 10)
[987] 5'-TGCTGATTTCACACTGCTCTCCC-3 '
[988] Example 3: Isolation of cDNA clones encoding human PRO230
[989] The consensus DNA sequence, as described in Example 1 above, was assembled for another identified EST sequence and was referred to herein as DNA30857. Genentech-owned EST was used for the consensus assembly. This EST is referred to as DNA20088 and its nucleotide sequence is shown in Fig. 7 (SEQ ID NO: 13).
[990] Based on the DNA30857 consensus sequence, oligonucleotides were synthesized, used to identify the cDNA library containing the desired sequence by PCR, and used as a probe to isolate clones of the full-length coding sequence for the PRO230 polypeptide.
[991] A pair of PCR primers (forward and reverse) were synthesized.
[992] The forward PCR primer 5'-TTCGAGGCCTCTGAGAAGTGGCCC-3 '(SEQ ID NO: 14)
[993] Reverse PCR primer 5'-GGCGGTATCTCTCTGGCCTCCC-3 '(SEQ ID NO: 15)
[994] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 30857 sequence having the following nucleotide sequence.
[995] Hybridization probe
[996] 5'-TTCTCCACAGCAGCTGTGGCATCCGATCGTGTCTCAATCCATTCTCTGGG-3 '(SEQ ID NO: 16)
[997] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO230 gene using either probe oligonucleotides and PCR primers.
[998] The RNA for constructing the cDNA library was isolated from human fetal lung tissue. DNA sequence analysis of the isolated clones as described above yielded a full-length DNA sequence for PRO230 (referred to herein as DNA33223-1136) and a derived protein sequence for PRO230.
[999] The entire nucleotide sequence of DNA33223-1136 is shown in Figure 5 (SEQ ID NO: 11). Clone DNA33223-1136 contains a single open reading frame with an open translation initiation site at nucleotide positions 100-103 and terminating at a stop codon at nucleotide positions 1501-1503 (Figure 5, SEQ ID NO: 11). The expected polypeptide precursor has an amino acid length of 467 (Figure 6).
[1000] Example 4: Isolation of cDNA clones encoding human PRO232
[1001] The consensus DNA sequence, as described in Example 1 above, was assembled for another identified EST sequence and was referred to herein as DNA30935. Based on the DNA30935 consensus sequence, oligonucleotides were synthesized and used to identify the cDNA library containing the desired sequence by PCR and used as a probe to isolate clones of the full-length coding sequence for the PRO232 polypeptide.
[1002] A pair of PCR primers (forward and reverse) were synthesized.
[1003] The forward PCR primer 5'-TGCTGTGCTACTCCTGCAAAGCCC-3 '(SEQ ID NO: 19)
[1004] Reverse PCR primer 5'-TGCACAAGTCGGTGTCACAGCACG-3 '(SEQ ID NO: 20)
[1005] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 30935 sequence having the following nucleotide sequence.
[1006] Hybridization probe
[1007] 5'-AGCAACGAGGACTGCCTGCAGGTGGAGAACTGCACCCAGCTGGG-3 '(SEQ ID NO: 21)
[1008] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO232 gene using either probe oligonucleotides and PCR primers.
[1009] The RNA for constructing the cDNA library was isolated from human fetal kidney tissue.
[1010] DNA sequence analysis of the isolated clones as described above yielded a full-length DNA sequence for PRO232 (referred to herein as DNA34435-1140) and a derived protein sequence for PRO232.
[1011] The entire nucleotide sequence of DNA34435-1140 is shown in Figure 8 (SEQ ID NO: 17). Clone DNA34435-1140 contains a single open reading frame with an open translation initiation site at nucleotide positions 17-19 and terminating at a stop codon at nucleotide positions 359-361 (Figure 8, SEQ ID NO: 17). The predicted polypeptide precursor has an amino acid length of 114 (Figure 9). Clone DNA34435-1140 was deposited with the ATCC on September 16, 1997, and the ATCC deposit number is ATCC No. 209250.
[1012] Amino acid sequence analysis of the full-length PRO232 indicates that the PRO232 is 35% sequence identical to the stem cell surface antibody of Gallus gallus.
[1013] Example 5: Isolation of cDNA clones encoding PRO187
[1014] (EGF), which is also known as androgen-inducible growth factor (FGF-8), by searching the proprietary expression sequence tag (EST) DNA database (LIFESE ^™ , Incyte Pharmaceuticals, Palo Alto, Calif. # 843193). MRNA was isolated from human fetal lung tissue using reagents and protocols purchased from Invitrogen (Fast Track 2, San Diego, Calif.). The cDNA library used to isolate cDNA clones was constructed by standard methods using commercially available reagents (e.g., Invitrogen, Life Technologies, Gaithersburg, MD, San Diego, CA). CDNA was primed with oligo dT containing the NotI site, bound to the SmI adapter with a phosphorylated SalI adapter at one end only, and then cleaved with NotI and appropriately separated according to the size by gel electrophoresis. Then, (Super Script Plasmid System) and protocol, which are commercially available from Githersburg, Md., And cloned into the cloning vector pRK5D in the determined direction. The double-stranded cDNA was separated to a size of 1000 bp or more, and the SalI / NotI linker-bound cDNA was cloned into a vector digested with XhoI / NotI. pRK5D is a cloning vector with the sp6 transcription initiation site followed by the SfiI restriction site in front of the XhoI / NotI cDNA cloning site.
[1015] Several libraries constructed from various tissues were screened by PCR amplification using the following oligonucleotide probes.
[1016] IN843193.f (OLI 315) (SEQ ID NO: 24)
[1017] 5'-CAGTACGTGAGGGACCAGGGCGCCATGA-3 '
[1018] IN843193.r (OLI 317) (SEQ ID NO: 25)
[1019] 5'-CCGGTGACCTGCACGTGCTTGCCA-3 '
[1020] Next, positive libraries were used to isolate clones encoding PRO187 using either the oligonucleotide probe or the oligonucleotide probe.
[1021] IN843193.p (OLI 316) (SEQ ID NO: 26)
[1022] 5'-GCGGATCTGCCGCCTGCTCANCTGGTCGGTCATGGCGCCCT-3 '
[1023] The sequence of the entire cDNA clone was confirmed. The entire nucleotide sequence of PRO187 (DNA27864-1155) is shown in FIG. 10 (SEQ ID NO: 22).
[1024] Clone DNA27864-1155 contains a single open reading frame with a pronounced translation initiation site at nucleotide position 1 (Fig. 10; SEQ ID NO: 22). The expected polypeptide precursor has an amino acid length of 205. Clone DNA27864-1155 was deposited with the ATCC (name: DNA27864-1155) and the ATCC deposit number is ATCC No. 209375.
[1025] Based on the BLAST and FastA sequence alignment analysis (using the ALIGN computer program) of the full-length sequence, the PRO187 polypeptide exhibits 74% amino acid sequence identity (BLAST score 310) with human fibroblast growth factor-8 (androgen- .
[1026] Example 6: Isolation of cDNA clones encoding PRO265
[1027] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA33679. Based on the DNA33679 consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use as a probe to isolate clones of the full-length coding sequence of PRO265.
[1028] PCR primer pairs (two forward and one reverse) were synthesized.
[1029] Forward PCR primer A: 5'-CGGTCTACCTGTATGGCAACC-3 '(SEQ ID NO: 29)
[1030] Omni-directional PCR primer B: 5'-GCAGGACAACCAGATAAACCAC-3 '(SEQ ID NO: 30)
[1031] The reverse PCR primer 5'-ACGCAGATTTGAGAAGGCTGTC-3 '(SEQ ID NO: 30)
[1032] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 33679 sequence having the following nucleotide sequence.
[1033] Hybridization probe
[1034] 5'-TTCACGGGCTGCTCTTGCCCAGCTCTTGAAGCTTGAAGAGCTGCAC-3 '(SEQ ID NO: 32)
[1035] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO265 gene using either probe oligonucleotides and PCR primers.
[1036] The RNA for constructing the cDNA library was isolated from a human fetal brain library.
[1037] DNA sequence analysis of the isolated clones as described above yielded a full-length DNA sequence for PRO265 [referred to herein as DNA36350-1158] (SEQ ID NO: 27) and a derived protein sequence for PRO265.
[1038] The entire nucleotide sequence of DNA36350-1158 is shown in Figure 12 (SEQ ID NO: 27). Clone DNA 36350-1158 contains a single open reading frame with an open translation initiation site at nucleotide positions 352-354 and terminating at a stop codon at nucleotide positions 2332-2334 (Figure 12). The predicted polypeptide precursor has an amino acid length of 660 (Figure 13). Clone DNA 36350-1158 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209378.
[1039] Amino acid sequence analysis of the full-length PRO265 polypeptide indicates that a portion of the PRO265 polypeptide is highly homologous to the pibromodulin and the pibromodulin precursor, indicating that PRO265 is a novel member of the leucine rich repeat subtype, It may be related to lean.
[1040] Example 7: Isolation of cDNA clones encoding human PRO219
[1041] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA28729. Based on the DNA28729 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone the full-length coding sequence of PRO219.
[1042] A pair of PCR primers (forward and reverse) were synthesized.
[1043] The omnidirectional PCR primer 5'-GTGACCCTGGTTGTGAATACTCC-3 '(SEQ ID NO: 35)
[1044] The reverse PCR primer 5'-ACAGCCATGGTCTATAGCTTGG-3 '(SEQ ID NO: 36)
[1045] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 28729 sequence having the following nucleotide sequence.
[1046] Hybridization probe
[1047] 5'-GCCTGTCAGTGTCCTGAGGGACACGTGCTCCGCAGCGATGGGAAG-3 '(SEQ ID NO: 37)
[1048] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO219 gene using either probe oligonucleotides and PCR primers.
[1049] The RNA for constructing the cDNA library was isolated from human fetal kidney tissue.
[1050] DNA sequence analysis of the isolated clones as described above yielded a full-length DNA sequence for PRO219 (referred to herein as DNA32290-1164) (SEQ ID NO: 33) and a derived protein sequence for PRO219.
[1051] The entire nucleotide sequence of DNA32290-1164 is shown in Figure 14 (SEQ ID NO: 33). Clone DNA 32290-1164 contains a single open reading frame with an open translation initiation site at nucleotide positions 204-206 and terminating at a stop codon at nucleotide positions 2949-2951 (Figure 14). The predicted polypeptide precursor has an amino acid length of 915 (Figure 15). Clone DNA 32290-1164 was deposited with the ATCC and ATCC accession number is ATCC No. 209384.
[1052] Amino acid sequence analysis of the full-length PRO219 polypeptide indicates that a portion of the PRO219 polypeptide is highly homologous to the mouse and human matreillin-2 precursor polypeptide.
[1053] Example 8: Isolation of cDNA clones encoding human PRO246
[1054] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA30955. Based on the DNA30955 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone the full-length coding sequence of PRO246.
[1055] A pair of PCR primers (forward and reverse) were synthesized.
[1056] The forward PCR primer 5'-AGGGTCTCCAGGAGAAAGACTC-3 '(SEQ ID NO: 40)
[1057] Reverse PCR primer 5'-ATTGTGGGCCTTGCAGACATAGAC-3 '(SEQ ID NO: 41)
[1058] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 30955 sequence having the following nucleotide sequence.
[1059] Hybridization probe
[1060] 5'-GGCCACAGCATCAAAACCTTAGAACTCAATGTACTGGTTCCTCCAGCTCC-3 '(SEQ ID NO: 42)
[1061] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO246 gene using either probe oligonucleotides and PCR primers.
[1062] The RNA for constructing the cDNA library has been isolated from human fetal liver tissue. DNA sequence analysis of isolated clones as described above yielded a full-length DNA sequence for PRO246 (referred to herein as DNA35639-1172) (SEQ ID NO: 38) and a derived protein sequence for PRO246.
[1063] The entire nucleotide sequence of DNA35639-1172 is shown in FIG. 16 (SEQ ID NO: 38). Clone DNA35639-1172 contains a single open reading frame with an open translation initiation site at nucleotide positions 126-128 and terminating at a stop codon at nucleotide positions 1296-1298 (Figure 16). The predicted polypeptide precursor has an amino acid length of 390 (Figure 17). Clone DNA 35639-1172 was deposited with the ATCC and ATCC deposit number ATCC 209396.
[1064] Amino acid sequence analysis of the full-length PRO246 polypeptide indicates that the PRO246 polypeptide is highly homologous to the human cell surface protein HCAR, suggesting that PRO246 may be a novel cell surface viral receptor.
[1065] Example 9: Isolation of cDNA clones encoding human PRO228
[1066] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA28758. Genentech-owned EST was used for the consensus assembly. This EST is shown in Figure 20 (SEQ ID NO: 50) and is referred to herein as DNA21951. Based on the DNA28758 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence of PRO228.
[1067] PCR primers (forward and reverse) were synthesized.
[1068] The forward PCR primer 5'-GGTAATGAGCTCCATTACAG-3 '(SEQ ID NO: 51)
[1069] The forward PCR primer 5'-GGAGTAGAAAGCGCATGG-3 '(SEQ ID NO: 52)
[1070] The forward PCR primer 5'-CACCTGATACCATGAATGGCAG-3 '(SEQ ID NO: 53)
[1071] Reverse PCR primer 5'-CGAGCTCGAATTAATTCG-3 '(SEQ ID NO: 54)
[1072] Reverse PCR primer 5'-GGATCTCCTGAGCTCAGG-3 '(SEQ ID NO: 55)
[1073] Reverse PCR primer 5'-CCTAGTTGAGTGATCCTTGTAAG-3 '(SEQ ID NO: 56)
[1074] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 28757 sequence having the following nucleotide sequence.
[1075] Hybridization probe
[1076] 5'-ATGAGACCCACACCTCATGCCGCTGTAATCACCTGACACATTTTGCAATT-3 '(SEQ ID NO: 57)
[1077] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO228 gene using either probe oligonucleotides and PCR primers.
[1078] The RNA for constructing the cDNA library was isolated from human fetal kidney tissue.
[1079] DNA sequence analysis of isolated clones as described above yielded a full-length DNA sequence for PRO228 (referred to herein as DNA33092-1202) (SEQ ID NO: 48) and a derived protein sequence for PRO228.
[1080] The entire nucleotide sequence of DNA33092-1202 is shown in Figure 18 (SEQ ID NO: 48). Clone DNA33092-1202 contains a single open reading frame terminating at a stop codon following the nucleotide position 2093 of Figure 48 with an obvious translation initiation site at nucleotide positions 24-26 of Figure 48. [ The predicted polypeptide precursor has an amino acid length of 690 (Figure 19). Clone DNA 33092-1202 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209420.
[1081] Amino acid sequence analysis of the full-length PRO228 polypeptide shows that a portion of the PRO228 polypeptide is highly homologous to the secretin-related proteins CD97 and EMR1 as well as to the secretory member ratrophilin, which may be a new member of the secretin-related protein PRO228 .
[1082] Example 10: Isolation of cDNA clones encoding human PRO533
[1083] The murine fibroblast growth factor (FGF-15), the EST sequence member AF007268, was used to search for a variety of public EST databases (e.g., GenBank, DayHope, etc.). A search for comparing the six frame translations of the ECD protein sequence and the EST sequence was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in Enzymology, 266: 460-480 (1996); http: //blast.wust1/edu/blast/README.html]]. The above search hints were likely to be GenBank EST AA220994 identified as precursor 937230 of Stratagene NT2 neuron.
[1084] Based on the GenBank EST AA220994 sequence, 1) oligonucleotides were synthesized by PCR to confirm the cDNA library containing the desired sequence and 2) as probes for isolating clones of the full-length coding sequence. The forward and reverse PCR primers can be in the range of 20-30 nucleotides (typically about 24r) and are constructed to obtain PCR products of 100-1000 bp in length. The probe sequence is typically 40-55 bp in length (typically about 50). To obtain full-length clone circles and screen several libraries for their libraries, the DNA of the library was screened through PCR amplification using a PCR primer pair (Ausubel et al., Current Protocols in Molecular Biology). Next, positive libraries were used to isolate clones encoding the desired gene using either probe oligonucleotides and PCR primers.
[1085] To screen multiple libraries to obtain full-length clone sources, the library DNAs were screened by PCR amplification using the PCR primer pairs identified below. Thereafter, positive libraries were used to isolate clones encoding the PRO533 gene using either probe oligonucleotides and PCR primers.
[1086] The RNA for constructing the cDNA library was isolated from the human fetal retina. The cDNA library used to isolate cDNA clones was constructed by standard methods using commercially available reagents (e.g. Invitrogen, Clontech, San Diego, CA). The cDNA was primed with oligo dT containing the NotI site, bound to the SmI adapter with a phosphorylated SalI adapter at one end only, cleaved with NotI, suitably separated according to size by gel electrophoresis, A suitable cloning vector with a NotI site [such as pRKB or pRK5B; pRK5B is a precursor of pRK5D that does not contain the SfiI site and has been cloned in the determined orientation in Holmes et al., Science, 253: 1278-1280 (1991)).
[1087] The entire sequence of the cDNA clone was confirmed. The full-length nucleotide sequence of PRO533 is shown in Figure 21 (SEQ ID NO: 58). Clone DNA 49435-1219 contains a single open reading frame with a translational initiation site apparent at nucleotide positions 459-461 (Figure 21, SEQ ID NO: 58). The predicted polypeptide precursor has an amino acid length of 216. Clone DNA47412-1219 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209480.
[1088] Based on the BLAST-2 and FastA sequence alignment analysis of the full-length sequence, PRO533 shows amino acid sequence identity (53%) with fibroblast growth factor.
[1089] The oligonucleotide sequence used in the above method is as follows.
[1090] FGF15 forward direction: 5'-ATCCGCCCAGATGGCTACAATGTGTA-3 '(SEQ ID NO: 60)
[1091] FGF 15. Probe: 5'-GCCTCCCGGTCTCCCTGAGCAGTGCCAAACAGCGGCAGTGTA-3 '(SEQ ID NO: 61)
[1092] FGF 15. Reverse direction: 5'-CCAGTCCGGTGACAAGCCCAAA-3 '(SEQ ID NO: 62)
[1093] Example 11: Isolation of cDNA clones encoding human PRO245
[1094] The consensus DNA sequence, as described in Example 1 above, was assembled for another identified EST sequence and referred to herein as DNA30954. Based on the DNA30954 consensus sequence, oligonucleotides were synthesized and used to confirm the cDNA library containing the desired sequence by PCR and used as a probe to isolate a clone of the full-length coding sequence of PRO245.
[1095] A pair of PCR primers (forward and reverse) were synthesized.
[1096] The forward PCR primer 5'-ATCGTTGTGAAGTTAGTGCCCC-3 '(SEQ ID NO: 65)
[1097] The reverse PCR primer 5'-ACCTGCGATATCCAACAGAATTG-3 '(SEQ ID NO: 66)
[1098] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 30954 sequence having the following nucleotide sequence.
[1099] Hybridization probe
[1100] 5'-GGAAGAGGATACAGTCACTCTGGAAGTATTAGTGGCTCCAGCAGTTCC-3 '(SEQ ID NO: 67)
[1101] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO245 gene using either probe oligonucleotides and PCR primers.
[1102] The RNA for constructing the cDNA library has been isolated from human fetal liver tissue. DNA sequence analysis of isolated clones as described above yielded a full-length DNA sequence for PRO245 (referred to herein as DNA35638-1141) and a derived protein sequence for PRO245.
[1103] The entire nucleotide sequence of DNA35638-1141 is shown in Figure 23 (SEQ ID NO: 63). Clone DNA 35638-1141 contains a single open reading frame with an open translation initiation site at nucleotide positions 89-91 and terminating at a stop codon at nucleotide positions 1025-1027 (Figure 23, SEQ ID 63). The predicted polypeptide precursor has an amino acid length of 312 (Figure 24). Clone DNA35638-1141 was deposited with the ATCC on September 16, 1997, and ATCC deposit number ATCC No. 209265.
[1104] Amino acid sequence analysis of the full-length PRO245 shows that PRO245 is a 60% amino acid sequence identity with human c-myb protein, so PRO245 can be a new member of the transmembrane protein receptor tyrosine kinase family.
[1105] Example 12: Isolation of cDNA clones encoding human PRO220, PRO221 and PRO227
[1106] (a) PRO220
[1107] The consensus DNA sequence, as described in Example 1 above, was assembled for another identified EST sequence and was referred to herein as DNA28749. Based on the DNA28749 consensus sequence, oligonucleotides were synthesized for use as probes to identify cDNA libraries containing the desired sequences by PCR and to isolate clones of the full-length coding sequence for PRO220.
[1108] PCR primers (forward and reverse) were synthesized.
[1109] Omni-directional PCR primer 5'-TCACCTGGAGCCTTTATTGGCC-3 '(SEQ ID NO: 74)
[1110] The reverse PCR primer 5'-ATACCAGCTATAACCAGGCTGCG-3 '(SEQ ID NO: 75)
[1111] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 28749 sequence having the following nucleotide sequence.
[1112] Hybridization probe
[1113] 5'-CAACAGTAAGTGGTTTGATGCTCTTCCAAATCTAGAGATTCTGATGATTGGG-3 '(SEQ ID NO: 76)
[1114] The library DNA was screened by PCR amplification using the identified PCR primer pairs to screen multiple libraries to obtain full length clone circles. The PRO220 gene was then coded using either the probe oligonucleotide and PCR primers A positive library was used to isolate clones.
[1115] The RNA for constructing the cDNA library was isolated from human fetal lung tissue.
[1116] DNA sequence analysis of the clones isolated as described above yielded a full-length DNA sequence for PRO220 (referred to herein as DNA32298-1132) and a derived protein sequence for PRO220.
[1117] The entire nucleotide sequence of DNA32298-1132 is shown in Figure 25 (SEQ ID NO: 68). Clone DNA 32298-1132 contains a single open reading frame with an open translation initiation site at nucleotide positions 480-482 and terminating at a stop codon at nucleotide positions 2604-2606 (Figure 25). The predicted polypeptide precursor has an amino acid length of 708 (Figure 26). Clone DNA32298-1132 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209257.
[1118] Amino acid sequence analysis of the full-length PRO220 shows that the PRO220 is homologous to a member of the leucine-rich repeat protein major group, including the lucine-rich repeat protein 1 and the lucine-rich repeat protein 1 of the neuron.
[1119] (b) PRO221
[1120] The consensus DNA sequence, as described in Example 1 above, was assembled for another identified EST sequence and was referred to herein as DNA28756. Based on the DNA28756 consensus sequence, an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and isolate a clone of the full-length coding sequence for the PRO221 polypeptide.
[1121] A pair of PCR primers (forward and reverse) were synthesized.
[1122] The forward PCR primer 5'-CCATGTGTCTCCTCCTACAAAG-3 '(SEQ ID NO: 77)
[1123] Reverse PCR primer 5'-GGGAATAGATGTGATCTGATTGG-3 '(SEQ ID NO: 78)
[1124] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 28756 sequence having the following nucleotide sequence.
[1125] Hybridization probe
[1126] 5'-CACCTGTAGCAATGCAAATCTCAAGGAAATACCTAGAGATCTTCCTCCTG-3 '(SEQ ID NO: 79)
[1127] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Subsequently, positive libraries were used to isolate clones encoding the PRO221 gene using either probe oligonucleotides and PCR primers.
[1128] The RNA for constructing the cDNA library was isolated from human fetal lung tissue. DNA sequence analysis of isolated clones as described above yielded a full-length DNA sequence for PRO221 (referred to herein as DNA33089-1132) and a derived protein sequence for PRO221.
[1129] The entire nucleotide sequence of DNA33089-1132 is shown in Figure 27 (SEQ ID NO: 70). Clone DNA33089-1132 contains a single open reading frame terminating at a stop codon at the nucleotide position 956-958 with an obvious translation initiation site at nucleotide positions 179-181 (Figure 27). The predicted polypeptide precursor has an amino acid length of 259 (Figure 28). Clone DNA33089-1132 was deposited with the ATCC and the ATCC accession number is ATCC No. 209262.
[1130] Amino acid sequence analysis of the full-length PRO221 shows that the PRO221 is homologous to a member of the leucine-rich repeat repoprotein macropeptide including the SLIT protein.
[1131] (c) PRO227
[1132] The consensus DNA sequence, as described in Example 1 above, was assembled for another identified EST sequence and referred to herein as DNA28740. Based on the DNA28740 consensus sequence, oligonucleotides were synthesized for use as probes for identifying cDNA libraries containing desired sequences and isolating clones of full-length coding sequences for PRO227 polypeptides.
[1133] A pair of PCR primers (forward and reverse) were synthesized.
[1134] The forward PCR primer 5'-AGCAACCGCCTGAAGCTCATCC-3 '(SEQ ID NO: 80)
[1135] Reverse PCR primer 5'-AAGGCGCGGTGAAAAGATGTAGACG-3 '(SEQ ID NO: 81)
[1136] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 28740 sequence having the following nucleotide sequence.
[1137] Hybridization probe
[1138] 5'-GACTACATGTTTCAGGACCTGTACAACCTCAAGTCACTGGAGGTTGGCGA-3 '(SEQ ID NO: 82)
[1139] The library DNA was screened by PCR amplification using the identified PCR primer pairs to screen multiple libraries to obtain full length clone circles. The PRO227 gene was then coded using either probe oligonucleotides and PCR primers A positive library was used to isolate clones.
[1140] The RNA for constructing the cDNA library was isolated from human fetal lung tissue. DNA sequence analysis of the isolated clones as described above yielded a full-length DNA sequence for PRO227 (referred to herein as DNA33786-1132) and a derived protein sequence for PRO227.
[1141] The entire nucleotide sequence of DNA33786-1132 is shown in Figure 29 (SEQ ID NO: 72). Clone DNA 33786-1132 contains a single open reading frame terminating at a stop codon at the nucleotide positions 1893-1895, with a pronounced translation initiation site at nucleotide positions 33-35 (Figure 29). The predicted polypeptide precursor has an amino acid length of 620 (Figure 30). PRO227 is believed to have a transmembrane domain. Clone DNA 33786-1132 was deposited with the ATCC and the ATCC accession number is ATCC No. 209253.
[1142] Amino acid sequence analysis of the full-length PRO227 shows that the PRO227 is homologous to a member of the leucine-rich repeat subpopulation macromolecule including the platelet glycoprotein V precursor and the human glycoprotein V.
[1143] Example 13: Isolation of cDNA clones encoding human PRO258
[1144] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA28746.
[1145] Based on the DNA28746 consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use as a probe to isolate clones of the full-length coding sequence of PRO258.
[1146] PCR primers (forward and reverse) were synthesized.
[1147] The forward PCR primer 5'-GCTAGGAATTCCACAGAAGCCC-3 '(SEQ ID NO: 85)
[1148] The reverse PCR primer 5'-AACCTGGAATGTCACCGAGCTG-3 '(SEQ ID NO: 86)
[1149] Reverse PCR primer 5'-CCTAGCACAGTGACGAGGGACTTGGC-3 '(SEQ ID NO: 87)
[1150] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 28740 sequence having the following nucleotide sequence.
[1151] Hybridization probe
[1152] 5'-AAGACACAGCCACCCTAAACTGTCAGTCTTCTGGGAGCAAGCCTGCAGCC-3 '(SEQ ID NO: 88)
[1153] 5'-GCCCTGGCAGACGAGGGCGAGTACACCTGCTCAATCTTCACTATGCCTGT-3 '(SEQ ID NO: 89)
[1154] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO258 gene using either probe oligonucleotides and PCR primers.
[1155] The RNA for constructing the cDNA library was isolated from human fetal lung tissue. DNA sequence analysis of isolated clones as described above yielded the full-length DNA sequence for PRO258 (referred to herein as DNA35918-1174) (SEQ ID NO: 83) and the deduced protein sequence for PRO258.
[1156] The entire nucleotide sequence of DNA 35918-1174 is shown in Figure 31. Clone DNA 35918-1174 has a translational initiation site at the nucleotide positions 147-149 of Figure 83 and an apparent translational initiation site at the stop codon following the nucleotide position 1340 of Figure 83 Lt; / RTI > frame (FIG. 31). The predicted polypeptide precursor has an amino acid length of 398 (Figure 32). Clone DNA 35918-1174 was deposited with the ATCC and ATCC accession number is ATCC No. 209402.
[1157] Amino acid sequence analysis of the full-length PRO258 polypeptide shows that a portion of the PRO258 polypeptide is highly homologous to the CRTAM and poliovirus receptors and contains the Ig domain, indicating that PRO258 is a new member of the Ig large group.
[1158] Example 14: Isolation of cDNA clones encoding human PRO266
[1159] Expression sequence for ESTs homologous to SLIT The sequence tag database was searched to identify a single EST sequence referred to herein as T73996. Based on the T73996 EST sequence 1) oligonucleotides were synthesized for use as probes to identify cDNA libraries containing the desired sequences by PCR and 2) to clone the full-length coding sequence of PRO266.
[1160] A pair of PCR primers (forward and reverse) were synthesized.
[1161] The forward PCR primer 5'-GTTGGATCTGGGCAACAATAAC-3 '(SEQ ID NO: 92)
[1162] Reverse PCR primer 5'-ATTGTTGTGCAGGCTGAGTTTAAG-3 '(SEQ ID NO: 93)
[1163] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared.
[1164] Hybridization probe
[1165] 5'-GGTGGCTATACATGGATAGCAATTACCTGGACACGCTGTCCCGGG-3 '(SEQ ID NO: 94)
[1166] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO266 gene using either probe oligonucleotides and PCR primers.
[1167] The RNA for constructing the cDNA library was isolated from human fetal brain tissue. DNA sequence analysis of isolated clones as described above yielded the full-length DNA sequence for PRO266 (referred to herein as DNA 37150-1178) (SEQ ID NO: 90) and the deduced protein sequence for PRO266.
[1168] The entire nucleotide sequence of DNA37150-1178 is shown in Figure 33 (SEQ ID NO: 90). Clone DNA 37150-1178 contains a single open reading frame terminating at a stop codon following the nucleotide position 2254 of Figure 90 with an obvious translation initiation site at nucleotide positions 167-169 of Figure 90. The predicted polypeptide precursor has an amino acid length of 696 (Figure 34). Clone DNA 37150-1178 was deposited with the ATCC and ATCC accession number is ATCC No. 209401.
[1169] Amino acid sequence analysis of the full-length PRO266 polypeptide shows that a portion of the PRO266 polypeptide is highly homologous to the SLIT protein, indicating that PRO266 may be a novel lucine-rich repeat protein.
[1170] Example 15: Isolation of cDNA clones encoding human PRO269
[1171] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA35705. Based on the DNA35705 consensus sequence, 1) oligonucleotides were synthesized to confirm the cDNA library containing the desired sequence by PCR and 2) to use as a probe to isolate clones of the full-length coding sequence of PRO269.
[1172] The forward and reverse PCR primers were synthesized.
[1173] An omnidirectional PCR primer (.f1) 5'-TGGAAGGAGATGCGATGCCACCTG-3 '(SEQ ID NO: 97)
[1174] An omnidirectional PCR primer (.f2) 5'-TGACCAGTGGGGAAGGACAG-3 '(SEQ ID NO: 98)
[1175] An omnidirectional PCR primer (.f3) 5'-ACAGAGCAGAGGGTGCCTTG-3 '(SEQ ID NO: 99)
[1176] Reverse PCR primer (.r1) 5'-TCAGGGACAAGTGGTGTCTCTCCC-3 '(SEQ ID NO: 100)
[1177] Reverse PCR primer (.r2) 5'-TCAGGGAAGGAGTGTGCAGTTCTG-3 '(SEQ ID NO: 101)
[1178] In addition, a synthetic oligonucleotide hybridization probe was prepared from the consensus DNA 35705 sequence having the following nucleotide sequence.
[1179] Hybridization probe
[1180] 5'-ACAGCTCCCGATCTCAGTTACTTGCATCGCGGACGAAATCGGCGCTCGCT-3 '(SEQ ID NO: 102)
[1181] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO269 gene using either probe oligonucleotides and PCR primers.
[1182] The RNA for constructing the cDNA library was isolated from human fetal kidney tissue. DNA sequence analysis of isolated clones as described above yielded a full-length DNA sequence for PRO269 (referred to herein as DNA38260-1180) (SEQ ID NO: 95) and a derived protein sequence for PRO269.
[1183] The entire nucleotide sequence of DNA38260-1180 is shown in Figure 35 (SEQ ID NO: 95). Clone DNA 38260-1180 contains a single open reading frame with an open translation initiation site at nucleotide positions 314-316 and terminating at a stop codon at nucleotide positions 1784-1786 (Figure 35: SEQ ID NO: 95). The expected polypeptide precursor has an amino acid length of 490 (Figure 36). Clone DNA38260-1180 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209397.
[1184] Amino acid sequence analysis of the full-length PRO269 shows that a portion of the PRO269 has substantial homology with the thrombomodulin protein, meaning that PRO269 can contain more than one thrombomodulin-like domain.
[1185] Example 16: Isolation of cDNA clones encoding human PRO287
[1186] The consensus DNA sequence encoding PRO287 as described in Example 1 above was assembled for another identified EST sequence and referred to herein as DNA28728. Based on the DNA28728 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence of PRO287.
[1187] A pair of PCR primers (forward and reverse) were synthesized.
[1188] The forward PCR primer 5'-CCGATTCATAGACCTCGAGAGT-3 '(SEQ ID NO: 105)
[1189] Reverse PCR primer 5'-GTCAAGGAGTCCTCCACAATAC-3 '(SEQ ID NO: 106)
[1190] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 28728 sequence.
[1191] Hybridization probe
[1192] 5'-GTGTACAATGGCCATGCCAATGGCCAGCGCATTGGCCGCTTCTGT-3 '(SEQ ID NO: 107)
[1193] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO287 gene using either probe oligonucleotides and PCR primers.
[1194] The RNA for constructing the cDNA library was isolated from human fetal kidney tissue.
[1195] DNA sequence analysis of isolated clones as described above yielded a full-length DNA sequence for PRO287 (referred to herein as DNA39969-1185, SEQ ID NO: 103) and a derived protein sequence for PRO287.
[1196] The entire nucleotide sequence of DNA39969-1185 is shown in Figure 37 (SEQ ID NO: 103). Clone DNA39969-1185 contains a single open reading frame with an open translation initiation site at nucleotide positions 307-309 and terminating at a stop codon at nucleotide positions 1552-1554 (Figure 37, SEQ ID NO: 103). The expected polypeptide precursor has an amino acid length of 415 (Figure 38). Clone DNA39969-1185 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209400.
[1197] Amino acid sequence analysis of the full-length PRO287 shows that the PRO287 may contain more than one procollagen C-proteinase enhancer protein precursor or a procollagen C-proteinase enhancer protein-like domain. Based on the BLAST and FastA sequence alignment analysis of the full-length sequence, PRO287 shows nucleic acid sequence identity (47 and 54%, respectively) with the procollagen C-proteinase enhancer protein precursor and the procollagen C-proteinase enhancer protein .
[1198] Example 17: Isolation of cDNA clones encoding human PRO214
[1199] Consensus DNA sequences were assembled against other identified EST sequences using phrap as described in Example 1 above. This consensus DNA sequence was referred to herein as DNA28744. Based on this consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use it as a probe to isolate clones of the full-length coding sequence.
[1200] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO214 gene using either probe oligonucleotides and PCR primers.
[1201] The RNA for constructing the cDNA library was isolated from human fetal lung tissue.
[1202] The entire sequence of the cDNA clone was confirmed. The full-length nucleotide sequence of DNA32286-1191 is shown in Figure 39 (SEQ ID NO: 108). DNA 32286-1191 contains a single open reading frame with a pronounced translation initiation site at nucleotide position 103 (Figure 39, SEQ ID 108). The predicted polypeptide precursor has an amino acid length of 420 (SEQ ID NO: 109).
[1203] Full-length BLAST and FastA Based on sequence alignment analysis, PRO214 polypeptides show amino acid sequence identity (49% and 38%, respectively) with HT protein and / or phyborine.
[1204] The oligonucleotide sequences used in the above method are as follows.
[1205] 28744.p (OLI555) 5'-CCTGGCTATCAGCAGGTGGGCTCCAAGTGTCTCGATGTGGATGAGTGTGA-3 '(SEQ ID NO: 110)
[1206] 28744.f (OLI556) 5'-ATTCTGCGTGAACACTGAGGGC-3 '(SEQ ID NO: 111)
[1207] 28744.r (OLI557) 5'-ATCTGCTTGTAGCCCTCGGCAC-3 '(SEQ ID NO: 112)
[1208] Example 18: Isolation of cDNA clones encoding human PRO317
[1209] The consensus DNA sequence was assembled using phrap as described in Example 1 above, and this consensus DNA sequence was referred to herein as DNA28722. Based on this consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use it as a probe to isolate clones of the full-length coding sequence. The forward and reverse PCRs synthesized for this purpose are as follows.
[1210] 5'-AGGACTGCCATAACTTGCCTG (OLI489) (SEQ ID NO: 115)
[1211] 5'-ATAGGAGTTGAAGCAGCGCTGC (OLI490) (SEQ ID NO: 116)
[1212] The probes synthesized for this purpose are as follows.
[1213] 5'-TGTGTGGACATAGACGAGTGCCGCTACCGCTACTGCCAGCACCGC (OLI488) (SEQ ID NO: 117)
[1214] The mRNA for constructing the cDNA library was isolated from human fetal kidney tissue.
[1215] In order to screen multiple libraries to obtain full-length clone circles, amplified by PCR according to Ausubel et al. (Current Protocols in Molecular Biology (1989)) using the pair of PCR primers identified above, DNA was screened. Thereafter, positive libraries were used to isolate clones containing the PRO317 gene using one of the identified probe oligonucleotides and PCR primers.
[1216] The entire sequence of the cDNA clone was confirmed. The entire nucleotide sequence of DNA33461-1199 (encoding PRO317) is shown in Figure 41 (SEQ ID NO: 113). Clone DNA 33461-1199 contains a single open reading frame with a translational initiation site apparent at nucleotide positions 68-70 (Figure 41, SEQ ID NO: 113). The predicted polypeptide precursor has an amino acid length of 366 (SEQ ID NO: 114). There is one N-linked glycosylation site predicted at amino acid residue 160. The clone DNA33461-1199 was deposited with the ATCC and the ATCC deposit number is ATCC 299367.
[1217] Based on the BLAST ^™ and FastA ^™ sequence alignment analysis of the full length PRO317 sequence (using the ALIGN ^™ computer program), PRO317 has the highest amino acid sequence identity (92%) with EBAF-1. In addition, the above analysis shows that there is significant homology between human PRO317 and mouse LEFTY protein. The C-terminus of the PRO317 protein contains a number of conserved sequences consistent with the expected pattern of members of the TGF-giant family.
[1218] In situ expression analysis of human tissues performed as described below clearly demonstrated that the PRO317 polypeptide is highly expressed in pancreatic tissues.
[1219] Example 19: Isolation of cDNA clones encoding human PRO301
[1220] A consensus DNA sequence, referred to herein as DNA35936, was assembled using phrap as described in Example 1 above. Based on this consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use it as a probe to isolate clones of the full-length coding sequence.
[1221] To screen multiple libraries to obtain full-length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified below. Thereafter, positive libraries were used to isolate clones encoding the PRO301 gene using either probe oligonucleotides and PCR primers.
[1222] The RNA for constructing the cDNA library was isolated from human fetal kidney.
[1223] The entire sequence of the cDNA clone was confirmed. The full-length nucleotide sequence of native sequence PRO301 is shown in Figure 43 (SEQ ID NO: 118). Clone DNA 40628-1216 contains a single open reading frame with a pronounced translation initiation site at nucleotide positions 52-54 (Figure 43: SEQ ID 118). The predicted polypeptide precursor has an amino acid length of 299, a predicted molecular weight of 32,583 daltons and a pI of 8.29. Clone DNA 40628-1216 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209432.
[1224] Full-length BLAST and FastA Based on sequence alignment analysis, PRO301 shows amino acid sequence identity with the A33 antibody precursor (30%), and with cockasaki and adenovirus receptor protein (29%).
[1225] The oligonucleotide sequences used in the above method are as follows.
[1226] OLI2162 (35936.f1) 5'-TCGCGGAGCTGTGTTCTGTTTCCC-3 '(SEQ ID NO: 120)
[1227] OLI2163 (35936.p1)
[1228] 5'-TGATCGCGATGGGGACAAAGGCGCAAGCTCGAGAGGAAACTGTTGTGCCT-3 '(SEQ ID NO: 121)
[1229] OLI2164 (35936.f2) 5'-ACACCTGGTTCAAAGATGGG-3 '(SEQ ID NO: 122)
[1230] OLI2165 (35936.r1) 5'-TAGGAAGAGTTGCTGAAGGCACGG-3 '(SEQ ID NO: 123)
[1231] OLI2166 (35936.f3) 5'-TTGCCTTACTCAGGTGCTAC-3 '(SEQ ID NO: 124)
[1232] OLI2167 (35936.r2) 5'-ACTCAGCAGTGGTAGGAAAG (SEQ ID NO: 125)
[1233] Example 20: Isolation of cDNA clones encoding human PRO224
[1234] The consensus DNA sequence encoding PRO287 as described in Example 1 above was assembled for another identified EST sequence and referred to herein as DNA30845. Based on the DNA30845 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence of PRO224.
[1235] A pair of PCR primers (forward and reverse) were synthesized.
[1236] The forward PCR primer 5'-AAGTTCCAGTGCCGCACCAGTGGC-3 '(SEQ ID NO: 128)
[1237] Reverse PCR primer 5'-TTGGTTCCACAGCCGAGCTCGTCG-3 '(SEQ ID NO: 129)
[1238] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA30845 sequence.
[1239] Hybridization probe
[1240] 5'-GAGGAGGAGTGCAGGATTGAGCCATGTACCCAGAAAGGGCAATGCCCACC-3 '(SEQ ID NO: 130)
[1241] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO224 gene using either probe oligonucleotides and PCR primers.
[1242] The RNA for constructing the cDNA library has been isolated from human fetal liver tissue.
[1243] DNA sequence analysis of the clones isolated as described above yielded a full-length DNA sequence for PRO224 (referred to herein as DNA33221-1133) and a derived protein sequence for PRO224.
[1244] The entire nucleotide sequence of DNA33221-1133 is shown in Figure 45 (SEQ ID NO: 126). Clone DNA33221-1133 contains a single open reading frame with an open translation initiation site at nucleotide positions 33-35 and terminating at a stop codon at nucleotide positions 879-899 (Figure 45, SEQ ID 126). The transmembrane domain starts at nucleotide position 777. The expected polypeptide precursor has an amino acid length of 282 (Figure 46). Clone DNA33221-1133 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209263.
[1245] Amino acid sequence analysis of the full-length PRO224 shows that the PRO224 is homologous to the very low density lipoprotein receptor, the apolipoprotein E receptor and the chicken ovary receptor P95. Based on the BLAST and FastA sequence alignment analysis of the full-length sequence, PRO224 shows amino acid identity within a range of 28% to 45% of the protein and overall identity within the range of 33% to 39% with the protein.
[1246] Example 21: Isolation of cDNA clones encoding human PRO222
[1247] The consensus DNA sequence, as described in Example 1 above, was assembled for another identified EST sequence and was referred to herein as DNA28771. Based on the DNA28771 consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use as a probe to isolate clones of the full-length coding sequence of PRO222.
[1248] A pair of PCR primers (forward and reverse) were synthesized.
[1249] The forward PCR primer 5'-ATCTCCTATCGCTGCTTTCCCGG-3 '(SEQ ID NO: 133)
[1250] Reverse PCR primer 5'-AGCCAGGATCGCAGTAAAACTCC-3 '(SEQ ID NO: 134)
[1251] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 28771 sequence.
[1252] Hybridization probe
[1253] 5'-ATTTAAACTTGATGGGTCTGCGTATCTTGAGTGCTTACAAAACCTTATCT-3 '(SEQ ID NO: 135)
[1254] The library DNA was screened by PCR amplification using the PCR primer pair identified above to screen multiple libraries to obtain full length clone circles. The PRO222 gene was then coded using either probe oligonucleotide and PCR primers A positive library was used to isolate clones.
[1255] The RNA for constructing the cDNA library was isolated from human fetal kidney tissue.
[1256] DNA sequence analysis of isolated clones as described above yielded a full-length DNA sequence for PRO222 (referred to herein as DNA33107-1135) and a derived protein sequence for PRO222.
[1257] The entire nucleotide sequence of DNA33107-1135 is shown in Figure 47 (SEQ ID NO: 131). Clone DNA 33107-1135 contains a single open reading frame with an open translation initiation site at nucleotide positions 159-161 and terminating at a stop codon at nucleotide positions 1629-1631 (Figure 47, SEQ ID 131). The predicted polypeptide precursor has an amino acid length of 490 (Figure 48). Clone DNA 33107-1135 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209251.
[1258] Based on the BLAST and FastA sequence alignment analysis of the full-length sequence, PRO222 is expressed in mouse complement factor h precursor (25-26%), complement receptor (27-29%), mouse complement C3b receptor type 2 elongate precursor (25-47% And amino acid sequence identity with the human hypothetical protein kiaa0247 (40%).
[1259] Example 22: Isolation of cDNA clones encoding human PRO234
[1260] Consensus DNA (DNA 30926) sequence was assembled using phrap as described in Example 1 above. Based on this consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use it as a probe to isolate clones of the full-length coding sequence.
[1261] RNA for constructing a cDNA library can be isolated from a tissue or cell line using a standard isolation protocol (e.g., Ausubel et al. Current Protocols in Molecular Biology) Clontech). The cDNA library used to isolate cDNA clones was constructed using standard methods (e.g., Ausubel et al.) using commercially available reagents (e.g., Invitrogen). This library was derived from the brain tissue of the fetus at 22 weeks of age.
[1262] The entire sequence of the cDNA clone was confirmed. The entire nucleotide sequence of PRO234 is shown in Figure 49 (SEQ ID NO: 136). The predicted polypeptide precursor has an amino acid length of 382 and a calculated molecular weight of about 43.1 kDa.
[1263] The oligonucleotide sequence used in the above method is as follows.
[1264] 30926.p (OLI826) (SEQ ID NO: 138): 5'-GTTCATTGAAAACCTCTTGCCATCTGATGGTGACTTCTGGATTGGGCTCA-3 '
[1265] 30926.f (OLI827) (SEQ ID NO: 139): 5'-AAGCCAAAGAAGCCTGCAGGAGGG-3 '
[1266] 30926.r (OLI828) (SEQ ID NO: 140): 5'-CAGTCCAAGCATAAAGGTCCTGGC-3 '
[1267] Example 23: Isolation of cDNA clones encoding human PRO231
[1268] The consensus DNA sequence, as described in Example 1 above, was assembled for another identified EST sequence and referred to herein as DNA30933. Based on the DNA30933 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone the full-length coding sequence of PRO231.
[1269] Three PCR primers (two forward primers and one reverse primer) were synthesized.
[1270] Omni-directional PCR primer 1 5'-CCAACTACCAAAGCTGCTGGAGCC-3 '(SEQ ID NO: 143)
[1271] Forward PCR primer 2 5'-GCAGCTCTATTACCACGGGAAGGA-3 '(SEQ ID NO: 144)
[1272] Reverse PCR primer 5'-TCCTTCCCGTGGTAATAGAGCTGC-3 '(SEQ ID NO: 145)
[1273] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA30933 sequence.
[1274] Hybridization probe
[1275] 5'-GGCAGAGAACCAGAGGCCGGAGGAGACTGCCTCTTTACAGCCAGG-3 '(SEQ ID NO: 146)
[1276] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO231 gene using either probe oligonucleotides and PCR primers.
[1277] The RNA for constructing the cDNA library has been isolated from human fetal liver tissue.
[1278] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO231 (referred to herein as DNA34434-1139) and the derived protein sequence for PRO231.
[1279] The entire nucleotide sequence of DNA34434-1139 is shown in Figure 51 (SEQ ID NO: 141). Clone DNA34434-1139 contains a single open reading frame with an open translation initiation site at nucleotide positions 173-175 and terminating at a stop codon at nucleotide positions 1457-1459 (Figure 51, SEQ ID NO: 141). The predicted polypeptide precursor has an amino acid length of 428 (Figure 52). Clone DNA34434-1139 was deposited with the ATCC on September 16, 1997, and the ATCC deposit number is ATCC No. 209252.
[1280] Amino acid sequence analysis of the full-length PRO231 shows that the PRO231 has 30% and 31% amino acid identity with the human and rat prostate phosphorylase precursor protein, respectively.
[1281] Example 24: Isolation of cDNA clones encoding human PRO229
[1282] Consensus DNA sequences were assembled against other identified EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA28762. Based on the DNA28762 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence for PRO229.
[1283] A pair of PCR primers (forward and reverse) were synthesized.
[1284] The forward PCR primer 5'-TTCAGCTCATCACCTTCACCTGCC-3 '(SEQ ID NO: 149)
[1285] Reverse PCR primer 5'-GGCTCATACAAAATACCACTAGGG-3 '(SEQ ID NO: 150)
[1286] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 28762 sequence.
[1287] Hybridization probe
[1288] 5'-GGGCCTCCACCGCTGTGAAGGGCGGGTGGAGGTGGAACAGAAAGGCCAGT-3 '(SEQ ID NO: 151)
[1289] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO229 gene using either probe oligonucleotides and PCR primers.
[1290] The RNA for constructing the cDNA library has been isolated from human fetal liver tissue.
[1291] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence for PRO229 (referred to herein as DNA 33100-1159, SEQ ID NO: 147) and the deduced protein sequence for PRO229.
[1292] The entire nucleotide sequence of DNA 33100-1159 is shown in Figure 53 (SEQ ID NO: 147). Clone DNA 33100-1159 contains a single open reading frame with an open translation initiation site at nucleotide positions 98-100 and terminating at a stop codon at nucleotide positions 1139-1141 (Figure 53). The predicted polypeptide precursor has an amino acid length of 347 (Figure 54). Clone DNA 33100-1159 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209377.
[1293] Amino acid sequence analysis of the full-length PRO229 polypeptide shows that a portion of the PRO2229 polypeptide is highly homologous to the antigen wc1.1, M130 antigen and CD6.
[1294] Example 25: Isolation of cDNA clones encoding human PRO238
[1295] Consensus DNA sequences were assembled against other identified EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA30908. Based on the DNA30908 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence for PRO238.
[1296] PCR primers (forward and reverse) were synthesized.
[1297] Omni-directional PCR primer 1 5'-GGTGCTAAACTGGTGCTCTGTGGC-3 '(SEQ ID NO: 154)
[1298] Omni-directional PCR primer 2 5'-CAGGGCAAGATGAGCATTCC-3 '(SEQ ID NO: 155)
[1299] Reverse PCR primer 5'-TCATACTGTTCCATCTCGGCACGC-3 '(SEQ ID NO: 156)
[1300] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 30908 sequence.
[1301] Hybridization probe
[1302] 5'-AATGGTGGGGCCCTAGAAGAGCTCATCAGAGAACTCACCGCTTCTCATGC-3 '(SEQ ID NO: 157)
[1303] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO238 gene using either probe oligonucleotides and PCR primers.
[1304] The RNA for constructing the cDNA library has been isolated from human fetal liver tissue.
[1305] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence for PRO238 and the derived protein sequence for PRO238.
[1306] The entire nucleotide sequence of DNA35600-1162 is shown in Figure 55 (SEQ ID NO: 152). Clone DNA 35600-1162 contains a single open reading frame with an open translation initiation site at nucleotide positions 134-136 and terminating in front of the stop codon at nucleotide positions 1064-1066 (Figure 55). The expected polypeptide precursor has an amino acid length of 310 (Figure 56). Clone DNA35600-1162 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209370.
[1307] Amino acid sequence analysis of the full-length PRO238 polypeptide shows that a portion of the PRO238 polypeptide has significant homology with the reductase, particularly the oxydorodic agent, suggesting that PRO238 may be a novel reductase.
[1308] Example 26: Isolation of cDNA clones encoding PRO233
[1309] Using the extracellular domain (ECD) sequence (including the secretory signal sequence in the presence of the secretory signal sequence) of about 950 known secretory proteins from the Swiss-Prot common protein database, The tag (EST) was used to search for data bottlenecks. EST databases include public EST databases (eg, GenBank) and proprietary databases (LIFESE ^™ , Incyte Pharmaceuticals, Palo Alto, Calif.). A search to compare six frame translations for ECD protein sequences and EST sequences was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in Enzymology 266: 460-480 (1996)]. A comparative sequence that does not code a known protein and records a BLAST score of 70 (or in some cases 90) or more is collected and analyzed using the consensus DNA sequence (University of Washington, Seattle Philly Green, Washington; http: // boaeman. mbt.washington.edu/phrap.docs/phrap.html).
[1310] Expression sequence tags (ESTs) were identified in the EST database and consensus DNA sequences were assembled against other EST sequences using phrap. This consensus sequence was referred to herein as DNA30945. Based on the DNA30945 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence for PRO233.
[1311] The forward and reverse PCR primers were synthesized.
[1312] The forward PCR primer 5'-GGTGAAGGCAGAAATTGGAGATG-3 '(SEQ ID NO: 160)
[1313] Reverse PCR primer 5'-ATCCCATGCATCAGCCTGTTTACC-3 '(SEQ ID NO: 161)
[1314] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA30945 sequence.
[1315] Hybridization probe
[1316] 5'-GCTGGTGTAGTCTATACATCAGATTTGTTTGCTACACAAGATCCTCAG-3 '(SEQ ID NO: 162)
[1317] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Subsequently, positive libraries were used to isolate clones encoding the PRO233 gene using probe oligonucleotides.
[1318] The RNA for constructing the cDNA library was isolated from human fetal brain tissue.
[1319] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO233 (referred to herein as DNA34436-1238, SEQ ID NO: 158) and the derived protein sequence for PRO233.
[1320] The entire nucleotide sequence of DNA34436-1238 is shown in Figure 57 (SEQ ID NO: 158). Clone DNA34436-1238 contains a single open reading frame with an open translation initiation site at nucleotide positions 101-103 and terminating at a stop codon at nucleotide positions 1001-1003 (Figure 57). The predicted polypeptide precursor has an amino acid length of 300 (Figure 58). The full length PRO233 protein shown in Figure 58 has an estimated molecular weight of about 32,964 daltons and a pi of about 9.52. The clone DNA34436-1238 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209523.
[1321] Amino acid sequence analysis of the full-length PRO233 polypeptide shows that a portion of the PRO233 polypeptide is highly homologous to the reductase protein, suggesting that PRO233 may be a novel reductase.
[1322] Example 27: Isolation of cDNA clones encoding human PRO223
[1323] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA30836. Based on the DNA30836 consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use as a probe to isolate clones of the full-length coding sequence for PRO223.
[1324] PCR primer pairs (one forward primer and two reverse primers) were synthesized.
[1325] The forward PCR primer 5'-TTCCATGCCACCTAAGGGAGACTC-3 '(SEQ ID NO: 165)
[1326] Reverse PCR primer 1 5'-TGGATGAGGTGTGCAATGGCTGGC-3 '(SEQ ID NO: 166)
[1327] Reverse PCR primer 2 5'-AGCTCTCAGAGGCTGGTCATAGGG-3 '(SEQ ID NO: 167)
[1328] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 30836 sequence.
[1329] Hybridization probe
[1330] 5'-GTCGGCCCTTTCCCAGGACTGAACATGAAGAGTTATGCCGGCTTCCTCAC-3 '(SEQ ID NO: 168)
[1331] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO223 gene using either probe oligonucleotides and PCR primers.
[1332] The RNA for constructing the cDNA library has been isolated from human fetal liver tissue.
[1333] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO223 (referred to herein as DNA 33206-1165, SEQ ID NO: 163) and the deduced protein sequence for PRO223.
[1334] The entire nucleotide sequence of DNA33206-1165 is shown in Figure 59 (SEQ ID NO: 163). Clone DNA 33206-1165 contains a single open reading frame with an open translation initiation site at nucleotide positions 97-99 and terminating at a stop codon at nucleotide positions 1525-1527 (Figure 59). The expected polypeptide precursor has an amino acid length of 476 (Figure 60). Clone DNA 33206-1165 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209372.
[1335] Amino acid sequence analysis of the full-length PRO223 polypeptide shows that the PRO223 polypeptide is highly homologous to various serine carboxy peptidase proteins suggesting that PRO223 may be a novel serine carboxypeptidase.
[1336] Example 28: Isolation of cDNA clones encoding human PRO235
[1337] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA30927. Based on the DNA30927 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence for PRO235.
[1338] A pair of PCR primers (forward and reverse) were synthesized.
[1339] The forward PCR primer 5'-TGGAATACCGCCTCCTGCAG-3 '(SEQ ID NO: 171)
[1340] Reverse PCR primer 5'-CTTCTGCCCTTTGGAGAAGATGGC-3 '(SEQ ID NO: 172)
[1341] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 30927 sequence.
[1342] Hybridization probe
[1343] 5'-GGACTCACTGGCCCAGGCCTTCAATATCACCAGCCAGGACGAT-3 '(SEQ ID NO: 173)
[1344] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO235 gene using either probe oligonucleotides and PCR primers.
[1345] The RNA for constructing the cDNA library has been isolated from human fetal liver tissue.
[1346] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO235 (referred to herein as DNA35558-1167, SEQ ID169) and the derived protein sequence for PRO235.
[1347] The entire nucleotide sequence of DNA35558-1167 is shown in Figure 61 (SEQ ID NO: 169). Clone DNA 35558-1167 contains a single open reading frame with an open translation initiation site at nucleotide positions 667-669 and terminating at a stop codon at nucleotide positions 2323-2325 (Figure 61). The predicted polypeptide precursor has an amino acid length of 552 (Figure 62). Clone DNA 35558-1167 was deposited with the ATCC and the ATCC accession number is ATCC No. 209374.
[1348] Analysis of the amino acid sequence of the full-length PRO235 polypeptide shows that a portion of the PRO235 polypeptide is highly homologous to human, mouse and genopus flexin protein, suggesting that PRO235 may be a novel flexin protein.
[1349] Example 29: Isolation of cDNA clones encoding human PRO236 and human PRO262
[1350] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA30901 and DNA30847. Based on the DNA30901 and DNA30847 consensus sequences, 1) oligonucleotides were synthesized for use as probes to identify cDNA libraries containing the desired sequences by PCR and 2) to isolate clones of full-length coding sequences for PRO236 and PRO262, respectively .
[1351] Based on the DNA30901 consensus sequence, a pair of PCR primers (forward and reverse) were synthesized.
[1352] The forward PCR primer 5'-TGGCTACTCCAAGACCCTGGCATG-3 '(SEQ ID NO: 178)
[1353] Reverse PCR primer 5'-TGGACAAATCCCCTTGCTCAGCCC-3 '(SEQ ID NO: 179)
[1354] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 30901 sequence.
[1355] Hybridization probe
[1356] 5'-GGGCTTCACCGAAGCAGTGGACCTTTATTTTGACCACCTGATGTCCAGGG-3 '(SEQ ID NO: 180)
[1357] Based on the DNA30847 consensus sequence, a pair of PCR primers (forward and reverse) were synthesized.
[1358] The forward PCR primer 5'-CCAGCTATGACTATGATGCACC-3 '(SEQ ID NO: 181)
[1359] Reverse PCR primer 5'-TGGCACCCAGAATGGTGTTGGCTC-3 '(SEQ ID NO: 182)
[1360] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 30847 sequence.
[1361] Hybridization probe
[1362] 5'-CGAGATGTCATCAGCAAGTTCCAGGAAGTTCCTTTGGGACCTTTACCTCC-3 '(SEQ ID NO: 183)
[1363] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO236 and PRO262 genes using either probe oligonucleotides and PCR primers.
[1364] The RNA for constructing the cDNA library for PRO236 was isolated from human fetal lung tissue and the RNA for constructing the cDNA library for PRO262 was isolated from human fetal liver tissue.
[1365] (Referred to herein as DNA35599-1168, SEQ ID NO: 174), an inducible protein sequence for PRO236, a full-length DNA sequence for PRO262 (herein referred to as DNA36992- 1168, SEQ ID NO: 176) and derived protein sequences for PRO262.
[1366] The entire nucleotide sequence of DNA35599-1168 is shown in Figure 63 (SEQ ID NO: 174). Clone DNA 35599-1168 contains a single open reading frame with an open translation initiation site at nucleotide positions 69-71 and terminating at a stop codon at nucleotide positions 1977-1979 (Figure 64). The predicted polypeptide precursor has an amino acid length of 636 (Figure 64). Clone DNA35599-1168 was deposited with the ATCC and ATCC accession number is ATCC No. 209373.
[1367] The entire nucleotide sequence of DNA36992-1168 is shown in Figure 65 (SEQ ID NO: 176). Clone DNA 36992-1168 contains a single open reading frame with an open translation initiation site at nucleotide positions 240-242 and terminating at a stop codon at nucleotide positions 2202-2204 (Figure 65). The predicted polypeptide precursor has an amino acid length of 654 (Figure 66). The clone DNA36992-1168 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209382.
[1368] Analysis of the amino acid sequences of the full-length PRO236 and PRO262 polypeptides shows that a portion of these polypeptides are highly homologous to the -Galactosidase protein from a variety of sources, indicating that PRO236 and PRO262 bind to the novel -Galactosidase phase It implies that it may be a fuselage.
[1369] Example 30: Isolation of cDNA clones encoding human PRO239
[1370] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA30909. Based on the DNA30909 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence for PRO239.
[1371] A pair of PCR primers (forward and reverse) were synthesized.
[1372] Omni-directional PCR primer 5'-CCTCCCTCTATTACCCATGTC-3 '(SEQ ID NO: 186)
[1373] Reverse PCR primer 5'-GACCAACTTTCTCTGGGAGTGAGG-3 '(SEQ ID NO: 187)
[1374] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 30909 sequence.
[1375] Hybridization probe
[1376] 5'-GTCACTTTATTTCTCTAACAACAAGCTCGAATCCTTACCAGTGGCAG-3 '(SEQ ID NO: 188)
[1377] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Subsequently, positive libraries were used to isolate clones encoding the PRO239 gene using either probe oligonucleotides and PCR primers.
[1378] The RNA for constructing the cDNA library was isolated from human fetal lung tissue.
[1379] DNA sequence analysis of the isolated clones as described above yielded a full-length DNA sequence for PRO239 (referred to herein as DNA34407-1169, SEQ ID NO: 184) and a derived protein sequence for PRO239.
[1380] The entire nucleotide sequence of DNA34407-1169 is shown in Figure 67 (SEQ ID NO: 184). Clone DNA34407-1169 contains a single open reading frame with an open translation initiation site at nucleotide positions 72-74 and terminating at a stop codon at nucleotide positions 1575-1577 (Figure 67). The predicted polypeptide precursor has an amino acid length of 501 (Figure 68). Clone DNA34407-1169 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209383.
[1381] Analysis of the amino acid sequence of the full-length PRO239 polypeptide shows that a portion of the PRO239 polypeptide has substantial homology with the tansin protein, suggesting that PRO239 may be a novel molecule of the tansin family.
[1382] Example 31: Isolation of cDNA clones encoding human PRO257
[1383] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA28731. Based on the DNA28731 consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR, and 2) use as a probe to isolate clones of the full-length coding sequence for PRO257.
[1384] A pair of PCR primers (forward and reverse) were synthesized.
[1385] Omni-directional PCR primer 5'-TCTCTATTCCAAACTGTGGCG-3 '(SEQ ID NO: 191)
[1386] Reverse PCR primer 5'-TTTGATGACGATTCGAAGGTGG-3 '(SEQ ID NO: 192)
[1387] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 28731 sequence.
[1388] Hybridization probe
[1389] 5'-GGAAGGATCCTTCACCAGCCCCAATTACCCAAAGCCGCATCCTGAGC-3 '(SEQ ID NO: 193)
[1390] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO257 gene using either probe oligonucleotides and PCR primers.
[1391] The RNA for constructing the cDNA library was isolated from human fetal kidney tissue.
[1392] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence for PRO257 (referred to herein as DNA35841-1173, SEQ ID NO: 189) and the deduced protein sequence for PRO257.
[1393] The entire nucleotide sequence of DNA35841-1173 is shown in Figure 69 (SEQ ID NO: 189). Clone DNA 35841-1173 contains a single open reading frame with an open translation initiation site at nucleotide position 964-966 and terminating at a stop codon at nucleotide position 2785-2787 (Figure 69). The predicted polypeptide precursor has an amino acid length of 607 (Figure 70). Clone DNA 35841-1173 was deposited with the ATCC and ATCC accession number is ATCC No. 209403.
[1394] Analysis of the amino acid sequence of the full-length PRO257 polypeptide shows that a portion of the PRO257 polypeptide is highly homologous to the Ebnerin protein, suggesting that PRO257 may be a novel protein member associated with Ebnerine.
[1395] Example 32: Isolation of cDNA clones encoding human PRO260
[1396] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA30834. Based on the DNA30834 consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use as a probe to isolate clones of the full-length coding sequence for PRO260.
[1397] PCR primers (one forward primer and two reverse primers) were synthesized.
[1398] Forward PCR primer : 5'-TGGTTTGACCAGGCCAAGTTCGG-3 '(SEQ ID NO: 196)
[1399] Reverse PCR primer A: 5'-GGATTCATCCTCAAGGAAGAGCGG-3 '(SEQ ID NO: 197)
[1400] Reverse PCR primer B: 5'-AACTTGCAGCATCAGCCACTCTGC-3 '(SEQ ID NO: 198)
[1401] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 30834 sequence.
[1402] Hybridization probe
[1403] 5'-TTCCGTGCCCAGCTTCGGTAGCGAGTGGTTCTGGTGGTATTGGCA-3 '(SEQ ID NO: 199)
[1404] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO260 gene using either probe oligonucleotides and PCR primers.
[1405] The RNA for constructing the cDNA library was isolated from human fetal kidney tissue.
[1406] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO260 (referred to herein as DNA33470-1175, SEQ ID NO: 194) and the derived protein sequence for PRO260.
[1407] The entire nucleotide sequence of DNA33470-1175 is shown in Figure 71 (SEQ ID NO: 194). Clone DNA 33470-1175 contains a single open reading frame with an open translation initiation site at nucleotide positions 67-69 and terminating at a stop codon at nucleotide positions 1468-1470 (Figure 71). The predicted polypeptide precursor has an amino acid length of 467 (Figure 72). Clone DNA 33470-1175 was deposited with the ATCC and ATCC deposit number ATCC 209398.
[1408] Analysis of the amino acid sequence of the full-length PRO260 polypeptide shows that a portion of the PRO260 polypeptide is highly homologous to the alpha-1-fucosidase precursor, suggesting that PRO260 may be a novel fucosidase.
[1409] Example 33: Isolation of cDNA clones encoding human PRO263
[1410] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA30914. Based on the DNA30914 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence for PRO263.
[1411] PCR primers (two forward primers and one reverse primer) were synthesized.
[1412] Omni-directional PCR primer 1: 5'-GAGCTTTCCATCCAGGTGTCATGC-3 '(SEQ ID NO: 202)
[1413] Forward PCR primer 2: 5'-GTCAGTGACAGTACCTACTCGG-3 '(SEQ ID NO: 203)
[1414] Reverse PCR primer : 5'-TGGAGCAGGAGGAGTAGTAGTAGG-3 '(SEQ ID NO: 204)
[1415] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA30914 sequence.
[1416] Hybridization probe
[1417] 5'-AGGAGGCCTGTAGGCTGCTGGGACTAAGTTTGGCCGGCAAGGACCAAGTT-3 '(SEQ ID NO: 205)
[1418] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Subsequently, positive libraries were used to isolate clones encoding the PRO263 gene using either probe oligonucleotides and PCR primers.
[1419] The RNA for constructing the cDNA library has been isolated from human fetal liver tissue.
[1420] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO263 (referred to herein as DNA34431-1177, SEQ ID NO: 200) and the derived protein sequence for PRO263.
[1421] The entire nucleotide sequence of DNA34431-1177 is shown in Figure 73 (SEQ ID NO: 200). Clone DNA34431-1177 contains a single open reading frame with an open translation initiation site at nucleotide positions 160-162 of SEQ ID NO: 200 and terminating at a stop codon following nucleotide positions 1126-1128 of SEQ ID NO: 200 (FIG. 73). The predicted polypeptide precursor has an amino acid length of 322 (Figure 74). Clone DNA34431-1177 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209399.
[1422] Analysis of the amino acid sequence of the full-length PRO263 polypeptide shows that a portion of the PRO263 polypeptide is highly homologous to the CD44 antigen suggesting that PRO263 may be a novel cell surface attachment molecule.
[1423] Example 34: Isolation of cDNA clones encoding human PRO270
[1424] Consensus DNA sequences were assembled for other identified EST sequences as described in Example 1, and this consensus sequence was referred to herein as DNA 35712. Based on the consensus sequence of DNA 35712, 1) a cDNA library containing the desired sequence Were confirmed by PCR and 2) oligonucleotides were synthesized for use as probes to isolate clones of the full-length coding sequence for PRO270. The forward and reverse PCR primers were synthesized.
[1425] Omni-directional PCR primer (.f1) 5'-GCTTGGATATTCGCATGGGCCTAC-3 '(SEQ ID NO: 208)
[1426] An omnidirectional PCR primer (.f2) 5'-TGGAGACAATATCCCTGAGG-3 '(SEQ ID NO: 209)
[1427] Reverse PCR primer (.r1) 5'-AACAGTTGGCCACAGCATGGCAGG-3 '(SEQ ID NO: 210)
[1428] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 35712 sequence.
[1429] Hybridization probe
[1430] 5'-CCATTGATGAGGAACTAGAACGGGACAAGAGGGTCACTTGGATTGTGGAG-3 '(SEQ ID NO: 211)
[1431] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO270 gene using either probe oligonucleotides and PCR primers.
[1432] The RNA for constructing the cDNA library was isolated from human fetal lung tissue.
[1433] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence for PRO270 (referred to herein as DNA39510-1181, SEQ ID NO: 206) and the derived protein sequence for PRO270.
[1434] The entire nucleotide sequence of DNA39510-1181 is shown in Figure 75 (SEQ ID NO: 206). Clone DNA39510-1181 contains a single open reading frame with an open translation initiation site at nucleotide positions 3-5 and terminating at a stop codon at nucleotide positions 891-893 (Figure 75, SEQ ID NO: 206). The predicted polypeptide precursor has an amino acid length of 296 (Figure 76). Clone DNA39510-1181 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209392.
[1435] Analysis of the amino acid sequence of the full-length PRO270 polypeptide shows that a portion of the PRO270 polypeptide has substantial homology with the thioredoxin-protein, suggesting that the PRO270 protein may be a new member of the thioredoxin family.
[1436] Example 35: Isolation of cDNA clones encoding human PRO271
[1437] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA35737. Based on the DNA35737 consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR and 2) use as a probe to isolate clones of the full-length coding sequence for PRO271.
[1438] The forward and reverse PCR primers were synthesized.
[1439] Forward PCR primer 1: 5'-TGCTTCGCTACTGCCCTC-3 '(SEQ ID NO: 214)
[1440] Omni-directional PCR primer 2: 5'-TTCCCTTGTGGGTTGGAG-3 '(SEQ ID NO: 215)
[1441] Omni-directional PCR primer 3: 5'-AGGGCTGGAAGCCAGTTC-3 '(SEQ ID NO: 216)
[1442] Reverse PCR primer 1: 5'-AGCCAGTGAGGAAATGCG-3 '(SEQ ID NO: 217)
[1443] Reverse PCR primer 2: 5'-TGTCCAAAGTACACACACCTGAGG-3 '(SEQ ID NO: 218)
[1444] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 35737 sequence.
[1445] Hybridization probe
[1446] 5'-GATGCCACGATCGCCAAGGTGGGACAGCTCTTTGCCGCCTGGAAG-3 '(SEQ ID NO: 219)
[1447] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO271 gene using either probe oligonucleotides and PCR primers.
[1448] The RNA for constructing the cDNA library was isolated from human fetal brain tissue.
[1449] DNA sequence analysis of the clones isolated as described above yielded a full-length DNA sequence for PRO271 (referred to herein as DNA39423-1182, SEQ ID NO: 212) and a derived protein sequence for PRO271.
[1450] The entire nucleotide sequence of DNA39423-1182 is shown in Figure 77 (SEQ ID NO: 212). Clone DNA39423-1182 contains a single open reading frame with an open translation initiation site at nucleotide positions 101-103 and terminating at a stop codon at nucleotide positions 1181-1183 (Figure 77). The predicted polypeptide precursor has an amino acid length of 360 (Figure 78). The clone DNA39423-1182 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209387.
[1451] Analysis of the amino acid sequence of the full-length PRO271 polypeptide shows that the PRO271 polypeptide is highly homologous to the proteoglycan link protein, suggesting that PRO271 may be a link protein homolog.
[1452] Example 36: Isolation of cDNA clones encoding human PRO272
[1453] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA36460. Based on the DNA36460 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence for PRO272.
[1454] The forward and reverse PCR primers were synthesized.
[1455] An omnidirectional PCR primer (.f1) 5'-CGCAGGCCCTCATGGCCAGG-3 '(SEQ ID NO: 222)
[1456] Omni-directional PCR primer (.f2) 5'-GAAATCCTGGGTAATTGG-3 '(SEQ ID NO: 223)
[1457] Reverse PCR primer 5'-GTGCGCGGTGCTCACAGCTCATC-3 '(SEQ ID NO: 224)
[1458] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA36460 sequence.
[1459] Hybridization probe
[1460] 5'-CCCCCCTGAGCGACGCTCCCCCATGATGACGCCCACGGGAACTTC-3 '(SEQ ID NO: 225)
[1461] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO272 gene using either probe oligonucleotides and PCR primers.
[1462] The RNA for constructing the cDNA library was isolated from human fetal lung tissue.
[1463] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO272 (referred to herein as DNA40620-1183, SEQ ID NO: 220) and the derived protein sequence for PRO272.
[1464] The entire nucleotide sequence of DNA40620-1183 is shown in Figure 79 (SEQ ID NO: 220). Clone DNA 40620-1183 contains a single open reading frame with an open translation initiation site at nucleotide positions 35-37 and terminating at a stop codon at nucleotide positions 1019-1021 (Figure 79). The predicted polypeptide precursor has an amino acid length of 328 (Figure 80). Clone DNA 40620-1183 was deposited with the ATCC and ATCC deposit number ATCC 209388.
[1465] Analysis of the amino acid sequence of the full-length PRO272 polypeptide shows that a portion of the PRO272 polypeptide is highly homologous to each of the human and mouse reticulo-calvin proteins, suggesting that PRO272 may be a novel reticulo-calvin protein.
[1466] Example 37: Isolation of cDNA clones encoding human PRO294
[1467] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA35731. Based on the DNA35731 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the desired sequence by PCR and 2) to clone a full-length coding sequence for PRO294.
[1468] The forward and reverse PCR primers were synthesized.
[1469] An omnidirectional PCR primer (.f1) 5'-TGGTCTCGCACACCGATC-3 '(SEQ ID NO: 228)
[1470] An omnidirectional PCR primer (.f2) 5'-CTGCTGTCCACAGGGGAG-3 '(SEQ ID NO: 229)
[1471] An omnidirectional PCR primer (.f3) 5'-CCTTGAAGCATACTGCTC-3 '(SEQ ID NO: 230)
[1472] Omni-directional PCR primer (.f4) 5'-GAGATAGCAATTTCCGCC-3 '(SEQ ID NO: 231)
[1473] Reverse PCR primer (.r1) 5'-TTCCTCAAGAGGGCAGCC-3 '(SEQ ID NO: 232)
[1474] Reverse PCR primer (.r2) 5'-CTTGGCACCAATGTCCGAGATTTC-3 '(SEQ ID NO: 233)
[1475] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 35731 sequence.
[1476] Hybridization probe
[1477] 5'-GCTCTGAGGAAGGTGACGCGCGGGGCCTCCGAACCCTTGGCCTTG-3 '(SEQ ID NO: 234)
[1478] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO294 gene using either probe oligonucleotides and PCR primers.
[1479] The RNA for constructing the cDNA library was isolated from human fetal brain tissue.
[1480] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO294 (referred to herein as DNA40604-1187, SEQ ID NO: 226) and the derived protein sequence for PRO294.
[1481] The entire nucleotide sequence of DNA40604-1187 is shown in Figure 81 (SEQ ID NO: 226). Clone DNA 40604-1187 contains a single open reading frame with an open translation initiation site at nucleotide positions 396-398 and terminating at a stop codon at nucleotide positions 2046-2048 (Figure 81). The predicted polypeptide precursor has an amino acid length of 550 (Figure 82). Clone DNA 40604-1187 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209394.
[1482] Analysis of the amino acid sequence of the full-length PRO294 polypeptide shows that a portion of the PRO294 polypeptide is highly homologous to a portion of various collagen proteins, suggesting that PRO294 may be a collagen-like molecule.
[1483] Example 38: Isolation of cDNA clones encoding human PRO295
[1484] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA35814. Based on the DNA35814 consensus sequence, 1) oligonucleotides were synthesized to confirm the cDNA library containing the desired sequence by PCR and 2) to use as a probe to isolate clones of the full-length coding sequence for PRO295.
[1485] The forward and reverse PCR primers were synthesized.
[1486] Omni-directional PCR primer (, f1) 5'-GCAGAGCGGAGATGCAGCGGCTTG-3 '(SEQ ID NO: 238)
[1487] Omni-directional PCR primer (.f2) 5'-CCCAGCATGTACTGCCAG-3 '(SEQ ID NO: 239)
[1488] An omnidirectional PCR primer (.f3) 5'-TTGGCAGCTTCATGGAGG-3 '(SEQ ID NO: 240)
[1489] Omni-directional PCR primer (.f4) 5'-CCTGGGCAAAAATGCAAC-3 '(SEQ ID NO: 241)
[1490] Reverse PCR primer (.r1) 5'-CTCCAGCTCCTGGCGCACCTCCTC-3 '(SEQ ID NO: 242)
[1491] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 35814 sequence.
[1492] Hybridization probe
[1493] 5'-GGCTCTCAGCTACCGCGCAGGAGCGAGGCCACCCTCAATGAGATG-3 '(SEQ ID NO: 243)
[1494] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Subsequently, positive libraries were used to isolate clones encoding the PRO295 gene using either probe oligonucleotides and PCR primers.
[1495] The RNA for constructing the cDNA library was isolated from human fetal lung tissue.
[1496] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO295 (referred to herein as DNA38268-1188, SEQ ID NO: 235) and the derived protein sequence for PRO295.
[1497] The entire nucleotide sequence of DNA38268-1188 is shown in Figure 83 (SEQ ID NO: 235). Clone DNA 38268-1188 contains a single open reading frame with an open translation initiation site at nucleotide positions 153-155 and terminating at a stop codon at nucleotide positions 1202-1204 (Figure 83). The predicted polypeptide precursor has an amino acid length of 350 (Figure 84). Clone DNA 38268-1188 was deposited with the ATCC and the ATCC deposit number is ATCC No. 209421.
[1498] Analysis of the amino acid sequence of the full-length PRO295 polypeptide shows that a portion of the PRO295 polypeptide has significant homology with the integrin protein, suggesting that PRO295 may be a novel integrin.
[1499] Example 39: Isolation of cDNA clones encoding human PRO293
[1500] Using the extracellular domain (ECD) sequence (including the secretory signal sequence in the presence of the secretory signal sequence) of about 950 known secretory proteins from the Swiss-Prot common protein database, Tag (EST) database. EST databases include public EST databases (eg, GenBank) and proprietary databases (LIFESE ^™ , Incyte Pharmaceuticals, Palo Alto, Calif.). A search to compare six frame translations of ECD protein sequences and EST sequences was performed using the computer program BLAST or BLAST2 [Altschul et al., Methods in Enzymology 266: 460-480 (1996)] . A comparative sequence that does not code a known protein and records a BLAST score of 70 (or in some cases 90) or more is collected and analyzed using the consensus DNA sequence (University of Washington, Seattle Philly Green, Washington; http: // boaeman. mbt.washington.edu/phrap.docs/phrap.html).
[1501] Based on the expression tag sequence identified herein as T08294 and identified in the assay, 1) a cDNA library containing the desired sequence is identified by PCR and 2) used as a probe to isolate clones of the full-length coding sequence for PRO293 Oligonucleotides were synthesized.
[1502] A pair of PCR primers (forward and reverse) were synthesized.
[1503] The forward PCR primer 5'-AACAAGGTAAGATGCCATCCTG-3 '(SEQ ID NO: 246)
[1504] Reverse PCR primer 5'-AAACTTGTCGATGGAGACCAGCTC-3 '(SEQ ID NO: 247)
[1505] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the expression sequence tag.
[1506] Hybridization probe
[1507] 5'-AGGGGCTGCAAAGCCTGGAGAGCCTCTCCTTCTATGACAACCAGC-3 '(SEQ ID NO: 248)
[1508] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO293 gene using either probe oligonucleotides and PCR primers.
[1509] The RNA for constructing the cDNA library was isolated from human fetal brain tissue.
[1510] DNA sequence analysis of the isolated clones as described above yielded the full-length DNA sequence for PRO293 (referred to herein as DNA37151-1193, SEQ ID NO: 244) and the derived protein sequence for PRO293.
[1511] The entire nucleotide sequence of DNA37151-1193 is shown in Figure 85 (SEQ ID NO: 244). Clone DNA 37151-1193 contains a single open reading frame terminating at the stop codon following the nucleotide position 3019 of SEQ ID NO: 244 (Fig. 85) with an obvious translation initiation site at nucleotide positions 881-883 of SEQ ID NO: 244 (Fig. 85). The predicted polypeptide precursor has an amino acid length of 713 (Figure 86). Clone DNA 37151-1193 was deposited with the ATCC and ATCC accession number is ATCC No. 209393.
[1512] Amino acid sequence analysis of the full-length PRO293 polypeptide shows that a portion of the PRO293 polypeptide has substantial homology with the NLRR protein, suggesting that PRO293 may be a novel NLRR protein.
[1513] Example 40: Isolation of cDNA clones encoding human PRO247
[1514] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA33480. Based on the DNA33480 consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the desired sequence by PCR, and 2) use as a probe to isolate clones of the full-length coding sequence for PRO247.
[1515] A pair of PCR primers (forward and reverse) were synthesized.
[1516] The forward PCR primer 5'-CAACAATGAGGGCACCAAGC-3 '(SEQ ID NO: 251)
[1517] Reverse PCR primer 5'-GATGGCTAGGTTCTGGAGGTTCTG-3 '(SEQ ID NO: 252)
[1518] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the DNA33480 expression sequence tag.
[1519] Hybridization probe
[1520] 5'-CAACCTGCAGGAGATTGACCTCAAGGACAACAACCTCAAGACCATCG-3 '(SEQ ID NO: 253)
[1521] To screen multiple libraries to obtain full length clone sources, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Thereafter, positive libraries were used to isolate clones encoding the PRO247 gene using either probe oligonucleotides and PCR primers.
[1522] The RNA for constructing the cDNA library was isolated from human fetal brain tissue.
[1523] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence for PRO247 (referred to herein as DNA35673-1201, SEQ ID NO: 249) and the derived protein sequence for PRO247.
[1524] The entire nucleotide sequence of DNA35673-1201 is shown in Figure 89 (SEQ ID NO: 249). Clone DNA 35673-1201 contains a single open reading frame with an open translation initiation site at nucleotide positions 80-82 of SEQ ID NO: 249 and terminating at a stop codon following nucleotide position 1717 of SEQ ID NO: 249 (FIG. 89). The predicted polypeptide precursor has an amino acid length of 546 (Figure 88). Clone DNA 35673-1201 was deposited with the ATCC and ATCC deposit number ATCC No. 209418.
[1525] Analysis of the amino acid sequence of the full-length PRO247 polypeptide shows that a portion of the PRO247 polypeptide is highly homologous to the tacrine molecule and KIAA0231 suggesting that PRO247 may be a novel protein with a lucin-rich repeat.
[1526] Example 41 : Isolation of cDNA clones encoding human PRO302, PRO303, PRO304, PRO307 and PRO343
[1527] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. These consensus sequences were referred to herein as DNA35953, DNA35955, DNA35958, DNA37160 and DNA30895. Based on the DNA35953 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR, and 2) use as probes to isolate clones of the full-length coding sequence of PRO302.
[1528] PCR primers (forward and reverse) were synthesized:
[1529] Omni-directional PCR primer 1 5'-GTCCGCAAGGATGCCTACATGTTC-3 '(SEQ ID NO: 264)
[1530] Forward PCR primer 2 5'-GCAGAGGTGTCTAAGGTTG-3 '(SEQ ID NO: 265)
[1531] Reverse PCR primer 5'-AGCTCTAGACCAATGCCAGCTTCC-3 '(SEQ ID NO: 266)
[1532] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was prepared from the consensus DNA 35953 sequence.
[1533] Hybridization probe
[1534] 5'-GCCACCAACTCCTGCAAGAACTTCTCAGAACTGCCCCTGGTCATG-3 '(SEQ ID NO: 267)
[1535] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO302 gene were isolated using a positive library using either probe oligonucleotides and PCR primers.
[1536] RNA for cDNA library preparation was isolated from human fetal kidney tissue (LIB228).
[1537] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 254) of PRO302 (herein referred to as DNA40370-1217) and the protein sequence of PRO302 derived therefrom.
[1538] The entire nucleotide sequence of DNA40370-1217 is shown in Figure 89 (SEQ ID NO: 254). Clone DNA 40370-1217 contains a single open reading frame with an open translation initiation site at nucleotide positions 34 to 36 and terminating at a stop codon at nucleotide positions 1390 to 1392 (Figure 89). The predicted polypeptide precursor has a length of 452 amino acids (Figure 90). Several unique features of the PRO302 protein are shown in FIG. Clone DNA 40370-1217 was deposited with the ATCC on Nov. 21, 1997 as ATCC Deposit No. 209485.
[1539] Based on the DNA35955 consensus sequence, 1) an oligonucleotide was synthesized for use as a probe to identify a cDNA library containing the sequence of interest by PCR and 2) to clone a full-length coding sequence of PRO303.
[1540] A pair of PCR primers (forward and reverse) were synthesized:
[1541] The forward PCR primer 5'-GGGGAATTCACCCTATGACATTGCC-3 '(SEQ ID NO: 268)
[1542] Reverse PCR primer 5'-GAATGCCCTGCAAGCATCAACTGG-3 '(SEQ ID NO: 269)
[1543] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus DNA 35955 sequence.
[1544] Hybridization probe
[1545] 5'-GCACCTGTCACCTACACTAAACACATCCAGCCCATCTGTCTCCAGGCCTC-3 '(SEQ ID NO: 270)
[1546] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO303 gene were isolated using a positive library using either probe oligonucleotides and PCR primers.
[1547] RNA for cDNA library construction was isolated from human fetal lung tissue (LIB25).
[1548] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 256) of PRO303 (herein referred to as DNA42251-1217) and the protein sequence of PRO303 derived therefrom.
[1549] The entire nucleotide sequence of DNA42551-1217 is shown in Figure 91 (SEQ ID NO: 256). Clone DNA42551-1217 contains a single open reading frame with an open translation initiation site at nucleotide positions 20 to 22 and terminating at a stop codon at nucleotide positions 962 to 964 (Figure 91). The predicted polypeptide precursor has a length of 314 amino acids (Figure 92). Several unique features of the PRO303 protein are shown in FIG. Clone DNA42551-1217 was deposited with ATCC on Nov. 21, 1997 as ATCC Deposit No. 209483.
[1550] Based on the DNA35958 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR, and 2) use as probes to isolate clones of the full-length coding sequence of PRO304.
[1551] A pair of PCR primers (forward and reverse) were synthesized:
[1552] Omni-directional PCR primer 1 5'-GCGGAAGGGCAGAATGGGACTCCAAG-3 '(SEQ ID NO: 271)
[1553] Forward PCR primer 2 5'-CAGCCCTGCCACATGTGC-3 '(SEQ ID NO: 272)
[1554] Forward PCR primer 3 5'-TACTGGGTGGTCAGCAAC-3 '(SEQ ID NO: 273)
[1555] Reverse PCR primer 5'-GGCGAAGAGCAGGGTGAGACCCCG-3 '(SEQ ID NO: 274)
[1556] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus DNA 35958 sequence.
[1557] Hybridization probe
[1558] 5'-GCCCTCATCCTCTCTGGCAAATGCAGTTACAGCCCGGAGCCCGAC-3 '(SEQ ID NO: 275)
[1559] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO304 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1560] RNA for cDNA library production was isolated from 22-week human fetal brain tissue (LIB153).
[1561] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 258) of PRO304 (herein referred to as DNA39520-1217) and the protein sequence of PRO304 derived therefrom.
[1562] The entire nucleotide sequence of DNA39520-1217 is shown in Figure 93 (SEQ ID NO: 258). Clone DNA39520-1217 contains a single open reading frame with an open translation initiation site at nucleotide positions 34-36 and terminating at a stop codon at nucleotide positions 1702-1704 (Figure 93). The predicted polypeptide precursor has a length of 556 amino acids (Figure 94). Several unique features of the PRO304 protein are shown in FIG. Clone DNA39520-1217 was deposited with the ATCC on Nov. 21, 1997 as ATCC Deposit No. 209482.
[1563] Based on the DNA37160 consensus sequence, 1) oligonucleotides were synthesized to confirm the cDNA library containing the target sequence by PCR and 2) as a probe to isolate clones of the full-length coding sequence of PRO307.
[1564] PCR primer pairs (forward and reverse) were synthesized:
[1565] Omni-directional PCR primer 1 5'-GGGCAGGGATTCCAGGGCTCC-3 '(SEQ ID NO: 276)
[1566] Forward PCR primer 2 5'-GGCTATGACAGCAGGTTC-3 '(SEQ ID NO: 277)
[1567] Omni-directional PCR primer 3 5'-TGACAATGACCGACCAGG-3 '(SEQ ID NO: 278)
[1568] Reverse PCR primer 5'-GCATCGCATTGCTGGTAGAGCAAG-3 '(SEQ ID NO: 279)
[1569] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus DNA 37160 sequence.
[1570] Hybridization probe
[1571] 5'-TTACAGTGCCCCCTGGAAACCCACTTGGCCTGCATACCGCCTCCC-3 '(SEQ ID NO: 280)
[1572] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO307 gene were isolated using a positive library using either probe oligonucleotides and PCR primers.
[1573] The RNA for cDNA library preparation was isolated from human fetal liver tissue (LIB229).
[1574] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 260) of PRO307 (herein referred to as DNA41225-1217) and the protein sequence of PRO307 derived therefrom.
[1575] The entire nucleotide sequence of DNA41225-1217 is shown in Figure 95 (SEQ ID NO: 260). Clone DNA41225-1217 contains a single open reading frame with an open translation initiation site at nucleotide positions 92 to 94 and terminating at a stop codon at nucleotide positions 1241 to 1243 (Figure 95). The predicted polypeptide precursor has a length of 383 amino acids (Figure 96). Several unique features of the PRO307 protein are shown in FIG. Clone DNA41225-1217 was deposited with the ATCC on Nov. 21, 1997 as ATCC Deposit No. 209491.
[1576] Based on the DNA30895 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR and 2) use as probes to isolate clones of the full length coding sequence of PRO343.
[1577] A pair of PCR primers (forward and reverse) were synthesized:
[1578] Forward PCR primer 5'-CGTCTCGAGCGCTCCATACAGTTCCCTTGCCCCA-3 '(SEQ ID NO: 281)
[1579] Reverse PCR primer
[1580] 5'-TGGAGGGGGAGCGGGATGCTTGTCTGGGCGACTCCGGGGGCCCCCTCATGTGCCAGGTGGA-3 '(SEQ ID NO: 282)
[1581] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus DNA 30895 sequence.
[1582] Hybridization probe
[1583] 5'-CCCTCAGACCCTGCAGAAGCTGAAGGTTCCTATCATCGAC
[1584] TCGGAAGTCTGCAGCCATCTGTACTGGCGGGGAGCAGGACAGGGACCCATCACTGAGGACATGCTGTGTGCCGGCTACT-3 '(SEQ ID NO: 283)
[1585] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO343 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1586] RNA for cDNA library construction was isolated from human fetal lung tissue (LIB26).
[1587] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 262) of PRO343 (herein referred to as DNA43318-1217) and the protein sequence of PRO343 derived therefrom.
[1588] The entire nucleotide sequence of DNA43318-1217 is shown in FIG. 97 (SEQ ID NO: 262). Clone DNA43318-1217 contains a single open reading frame with an open translation initiation site at nucleotide positions 53 to 55 and terminating at a stop codon at nucleotide positions 1004 to 1006 (Figure 97). The predicted polypeptide precursor has a length of 317 amino acids (Figure 98). Several unique features of the PRO343 protein are shown in FIG. Clone DNA43318-1217 was deposited with ATCC on Nov. 21, 1997 as ATCC Deposit No. 209481.
[1589] Example 42 : Isolation of cDNA clones encoding human PRO328
[1590] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA35615. Based on the DNA35615 consensus sequence, 1) an oligonucleotide was synthesized to confirm the cDNA library containing the target sequence by PCR and 2) as a probe for isolating clones of the full-length coding sequence of PRO328.
[1591] An omnidirectional and reverse PCR primer was synthesized:
[1592] Forward PCR primer 5'-TCCTGCAGTTTCCTGATGC-3 '(SEQ ID NO: 286)
[1593] Reverse PCR primer 5'-CTCATATTGCACACCAGTAATTCG-3 '(SEQ ID NO: 287)
[1594] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus DNA 35615 sequence.
[1595] Hybridization probe
[1596] 5'-ATGAGGAGAAACGTTTGATGGTGGAGCTGCACAACCTCTACCGGG-3 '(SEQ ID NO: 288)
[1597] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. Clones encoding the PRO328 gene were isolated using a positive library using either probe oligonucleotides and PCR primers.
[1598] RNA for cDNA library construction was isolated from human fetal kidney tissue.
[1599] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 284) of PRO328 (herein referred to as DNA40587-1231) and the protein sequence of PRO328 derived therefrom.
[1600] The entire nucleotide sequence of DNA40587-1231 is shown in Figure 99 (SEQ ID NO: 284). Clone DNA 40587-1231 contains a single open reading frame with an open translation initiation site at nucleotide positions 15 to 17 and terminating at a stop codon at nucleotide positions 1404 to 1406 (Figure 99). The predicted polypeptide precursor has a length of 463 amino acids (Figure 100). Clone DNA 40587-1231 was deposited with the ATCC as ATCC Deposit No. 209438.
[1601] Amino acid sequence analysis of the full-length PRO328 polypeptide indicates that a part of it has significant homology to the human myeloproliferative protein and cystine-rich secretory proteins, indicating that PRO328 can be a novel mycloxacin protein or a cysteine-rich secretory protein.
[1602] Example 43 : Isolation of cDNA clones encoding human PRO335, PRO331 or PRO326 clones
[1603] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA36685. Based on the DNA36685 consensus sequence and the Insight EST sequence No. 2228990, 1) a cDNA library containing the sequence of the target was identified by PCR and 2) a probe for isolating a clone of the full length coding sequence of PRO335, PRO331 or PRO 326 Lt; RTI ID = 0.0 > oligonucleotides < / RTI >
[1604] The forward and reverse PCR primers for PRO335 were synthesized:
[1605] The forward PCR primer 5'-GGAACCGAATCTCAGCTA-3 '(SEQ ID NO: 295)
[1606] The forward PCR primer 5'-CCTAAACTGAACTGGACCA-3 '(SEQ ID NO: 296)
[1607] The forward PCR primer 5'-GGCTGGAGACACTGAACCT-3 '(SEQ ID NO: 297)
[1608] The forward PCR primer 5'-ACAGCTGCACAGCTCAGAACAGTG-3 '(SEQ ID NO: 298)
[1609] Reverse PCR primer 5'-CATTCCCAGTATAAAAATTTTC-3 '(SEQ ID NO: 299)
[1610] Reverse PCR primer 5'-GGGTCTTGGTGAATGAGG-3 '(SEQ ID NO: 300)
[1611] Reverse PCR primer 5'-GTGCCTCTCGGTTACCACCAATGG-3 '(SEQ ID NO: 301)
[1612] In addition, a synthetic oligonucleotide hybridization probe for PRO335 measurement having the following nucleotide sequence was prepared.
[1613] Hybridization probe
[1614] 5'-GCGGCCACTGTTGGACCGAACTGTAACCAAGGGAGAAACAGCCGTCCTAC-3 '(SEQ ID NO: 302)
[1615] The forward and reverse PCR primers for PRO331 were synthesized:
[1616] The forward PCR primer 5'-GCCTTTGACAACCTTCAGTCACTAGTGG-3 '(SEQ ID NO: 303)
[1617] Reverse PCR primer 5'-CCCCATGTGTCCATGACTGTTCCC-3 '(SEQ ID NO: 304)
[1618] In addition, a synthetic oligonucleotide hybridization probe for PRO331 measurement with the following nucleotide sequence was prepared:
[1619] Hybridization probe
[1620] 5'-TACTGCCTCATGACCTCTTCACTCCCTTGCATCATCTTAGAGCGG-3 '(SEQ ID NO: 305)
[1621] The forward and reverse PCR primers for PRO326 were synthesized:
[1622] The forward PCR primer 5'-ACTCCAAGGAAATCGGATCCGTTC-3 '(SEQ ID NO: 306)
[1623] Reverse PCR primer 5'-TTAGCAGCTGAGGATGGGCACAAC-3 '(SEQ ID NO: 307)
[1624] In addition, a synthetic oligonucleotide hybridization probe for PRO331 measurement with the following nucleotide sequence was prepared:
[1625] Hybridization probe
[1626] 5'-GCCTTCACTGGTTTGGATGCATTGGAGCATCTAGACCTGAGTGACAACGC-3 '(SEQ ID NO: 308)
[1627] In order to screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO335, PRO331 or PRO326 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1628] RNA for cDNA library construction was isolated from human fetal kidney tissues (PRO335 and PRO326) and human fetal brain (PRO331).
[1629] The full-length DNA sequence of PRO335, PRO331 or PRO326 (referred to herein as SEQ ID NOS: 289, 291 and 293 respectively, see Figures 103, 105 and 107, respectively) and DNA sequences derived therefrom Protein sequences of PRO335, PRO331 or PRO326 (see Figs. 104, 106 and 108, respectively SEQ ID NOS: 290, 292 and 294, respectively) were obtained.
[1630] The entire nucleotide sequence shown in Figures 103, 105 and 107 was deposited with the ATCC on June 2, 1998, November 7, 1997, and November 21, 1997, respectively.
[1631] Amino acid sequence analysis of the full length PRO335, PRO331 or PRO326 polypeptides showed that some of the PRO335, PRO331 or PRO326 polypeptides could be a novel LIG-1-related protein, suggesting that some of them show significant homology with the LIG-1 protein.
[1632] Example 44 : Isolation of cDNA clones encoding human PRO332
[1633] Based on the ECD homology survey performed as described in Example 1, a consensus DNA sequence referred to herein as DNA36688 was assembled. Based on the DNA36688 consensus sequence, cDNA libraries containing the sequence of interest were identified by PCR and oligonucleotides were synthesized for use as probes to isolate clones of the full-length coding sequence of PRO332.
[1634] A pair of PCR primers (forward and reverse) were synthesized:
[1635] 5'-GCATTGGCCGCGAGACTTTGCC-3 '(SEQ ID NO: 311)
[1636] 5'-GCGGCCACGGTCCTTGGAAATG-3 '(SEQ ID NO: 312)
[1637] The probe was synthesized.
[1638] 5'-TGGAGGAGCTCAACCTCAGCTACAACCGCATCACCAGCCCACAGG-3 '(SEQ ID NO: 313)
[1639] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO332 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1640] The RNA for cDNA library preparation was isolated from human fetal liver tissue (LIB229).
[1641] DNA sequence analysis of the isolated clone as described above yielded the full-length DNA sequence of DNA 40982-1235 and the protein sequence of PRO332 derived therefrom.
[1642] The entire nucleotide sequence of DNA40982-1235 is shown in Figure 109 (SEQ ID NO: 309). Clone DNA 40982-1235 contains a single open reading frame with a pronounced translation initiation site at nucleotide positions 342 to 344 (Figure 109). The predicted polypeptide precursor has a length of 642 amino acids and has an estimated molecular weight of 72,067 (pI: 6.60). Clone DNA 40982-1235 was deposited with the ATCC and assigned ATCC Accession No. 209433.
[1643] Based on the BLAST and FastA sequence alignment analysis of the full-length sequence, PRO332 can be obtained, for example, from several classes of fibridolandrin and fibromodulin precursor sequences (FMOD BOVIN, FMOD CHICK, FMOD RAT, FMOD MOUSE, FMOD HUMAN, P R36773), the osteo modulin sequence (AB000114 1, AB007848 1), decolin sequence (CFU83141 1, OCU03394 1, P R42266, P R42267, P R42260, P R89439), keratan sulfate proteoglycan (BTU48360 1, AF022890 1), corneal proteoglycan (AF022256 1), and bone / cartilage proteoglycans and proteoglycan precursors (PGS1 BOVIN, PGS2 MOUSE, PGS2 ≪ / RTI > HUMAN) with about 30-40% amino acid sequence identity to the known proteoglycan sequence species.
[1644] Example 45 : Isolation of cDNA clones encoding human PRO334
[1645] Consensus DNA sequences were assembled for other EST sequences using phrap as described in Example 1. [ Based on the consensus sequence, oligonucleotides were synthesized to 1) identify a cDNA library containing the sequence of interest by PCR, and 2) use as a probe to isolate clones of the full-length coding sequence of PRO334.
[1646] The forward and reverse PCR primers for PRO334 were synthesized:
[1647] The forward PCR primer 5'-GATGGTTCCTGCTCAAGTGCCCTG-3 '(SEQ ID NO: 316)
[1648] Reverse PCR primer 5'-TTGCACTTGTAGGACCCACGTACG-3 '(SEQ ID NO: 317)
[1649] In addition, a synthetic oligonucleotide hybridization probe for PRO334 measurement having the following nucleotide sequence was prepared.
[1650] Hybridization probe
[1651] 5'-CTGATGGGAGGACCTGTGTAGATGTTGATGAATGTGCTACAGGAAGAGCC-3 '(SEQ ID NO: 318)
[1652] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO334 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1653] The human fetal kidney cDNA library used to isolate cDNA clones was prepared by standard methods using reagents available from Invitrogen (San Diego, CA, USA).
[1654] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 314) of PRO334 (herein referred to as DNA41379-1236) and the protein sequence of PRO334 derived therefrom.
[1655] The entire nucleotide sequence of DNA41379-1236 (also known as UNQ295) is shown in Figure 109 (SEQ ID NO: 314). Clone DNA41379-1236 contains a single open reading frame with an open translation initiation site at nucleotide positions 203-205 and terminating at a stop codon at nucleotide positions 1730-1732 (Figure 109). The predicted polypeptide precursor has a length of 509 amino acids (Figure 110). Clone DNA41379-1236 was deposited with ATCC as ATCC Deposit No. 209488.
[1656] Amino acid sequence analysis of the full-length PRO334 polypeptide showed that the PRO334 could be a new member of the EGF protein family by suggesting that some of it has significant homology with the pibuline and fibriline proteins.
[1657] Example 46 : Isolation of cDNA clones encoding human PRO346
[1658] The consensus DNA sequence was confirmed using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA38240. Based on the DNA38240 consensus sequence, oligonucleotides were synthesized to 1) identify cDNA libraries containing the sequence of interest by PCR, and 2) use as probes to isolate clones of the full-length coding sequence of PRO346.
[1659] RNA for cDNA library production was isolated from human fetal liver. The cDNA library used to isolate cDNA clones was prepared by standard methods using commercially available reagents (e.g., Invitrogen, Clontech, San Diego, CA). The cDNA was primed with oligo dT with the NotI site, ligated to the SalI hemi kinase adapter at the blunt end, cleaved with NotI, classified into appropriate sizes by gel electrophoresis, and cloned into a suitable cloning vector [e.g., pRKB pRKD; pRK5B is a precursor of pRK5D that does not contain the SfiI site; Were cloned in the predetermined orientation at the unique Xho I and Not I sites in Holmes et al., Science , 253 : 1278-1280 (1991)).
[1660] cDNA was sequenced as a whole. The entire nucleotide sequence of DNA44167-1243 is shown in Figure 111 (SEQ ID NO: 319). Clone DNA44167-1243 contains a single open reading frame with a pronounced translation initiation site at nucleotide positions 64-66 (Figure 113, SEQ ID 319). The expected polypeptide precursor has a length of 450 amino acids. Clone DNA44167-1243 was deposited with ATCC as ATCC Accession No. 209434 (herein referred to as DNA44167-1243).
[1661] Based on the BLAST, BLAST-2 and FastA sequence alignment analysis of the full-length sequence (using the ALIGN computer program), PRO346 showed amino acid sequence identity to the carcinoma fetal antigen (28%).
[1662] The following oligonucleotides have been used in the above method:
[1663] OLI2691 (38240.f1)
[1664] 5'-GATCCTGTCACAAAGCCAGTGGTGC-3 '(SEQ ID NO: 321)
[1665] OLI 2693 (38240.r1)
[1666] 5'-CACTGACAGGGTTCCTCACCCAGG-3 '(SEQ ID NO: 322)
[1667] OLI2692 (38240.p1)
[1668] 5'-CTCCCTCTGGGCTGTGGAGTATGTGGGGAACATGACCCTGACATG-3 '(SEQ ID NO: 323)
[1669] Example 47 : Isolation of cDNA clones encoding human PRO268
[1670] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. These consensus sequences were referred to herein as DNA35698. Based on the DNA35698 consensus sequence, 1) an oligonucleotide was synthesized by PCR to confirm the cDNA library containing the target sequence and 2) as a probe for isolating clones of the full-length coding sequence of PRO268.
[1671] An omnidirectional and reverse PCR primer was synthesized:
[1672] Omni-directional PCR primer 1 5'-TGAGGTGGGCAAGCGGCGAAATG-3 '(SEQ ID NO: 326)
[1673] Forward PCR primer 2 5'-TATGTGGATCAGGACGTGCC-3 '(SEQ ID NO: 327)
[1674] The forward PCR primer 3 5'-TGCAGGGTTCAGTCTAGATTG-3 '(SEQ ID NO: 328)
[1675] Reverse PCR primer 5'-TTGAAGGACAAAGGCAATCTGCCAC-3 '(SEQ ID NO: 329)
[1676] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was obtained from the consensus DNA 35698 sequence.
[1677] Hybridization probe
[1678] 5'-GGAGTCTTGCAGTTCCCCTGGCAGTCCTGGTGCTGTTGCTTTGGG-3 '(SEQ ID NO: 330)
[1679] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO268 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1680] RNA for cDNA library construction was isolated from human fetal lung tissue.
[1681] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 324) of PRO268 [herein referred to as DNA39427-1179] and the protein sequence of PRO268 derived therefrom.
[1682] The entire nucleotide sequence of DNA39427-1179 is shown in Figure 113 (SEQ ID NO: 324). Clone DNA39427-1179 contains a single open reading frame with an open translation initiation site at nucleotide positions 13 to 15 and terminating at a stop codon at nucleotide positions 853 to 855 (Figure 113). The predicted polypeptide precursor has a length of 280 amino acids (Figure 114). Clone DNA39427-1179 was deposited with ATCC as ATCC Deposit No. 209395.
[1683] Amino acid sequence analysis of the full-length PRO268 polypeptide showed that PRO268 could be a novel protein disulfide isomerase by suggesting that some of it has considerable homology with protein disulfide isomerase.
[1684] Example 48 : Isolation of cDNA clones encoding human PRO330
[1685] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. These consensus sequences were referred to herein as DNA35730. Based on the DNA35730 consensus sequence, oligonucleotides were synthesized for 1) identification of cDNA libraries containing the sequence of interest by PCR and 2) use as probes for isolating clones of the full-length coding sequence of PRO330.
[1686] An omnidirectional and reverse PCR primer was synthesized:
[1687] Omni-directional PCR primer 1 5'-CCAGGCACAATTTCCAGA-3 '(SEQ ID NO: 333)
[1688] Forward PCR primer 2 5'-GGACCCTTCTGTGTGCCAG-3 '(SEQ ID NO: 334)
[1689] Reverse PCR primer 1 5'-GGTCTCAAGAACTCCTGTC-3 '(SEQ ID NO: 335)
[1690] Reverse PCR primer 2 5'-ACACTCAGCATTGCCTGGTACTTG-3 '(SEQ ID NO: 336)
[1691] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus sequence.
[1692] Hybridization probe
[1693] 5'-GGGCACATGACTGACCTGATTTATGCAGAGAAAGAGCTGGTGCAG-3 '(SEQ ID NO: 337)
[1694] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO330 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1695] RNA for cDNA library production was isolated from human fetal liver tissue.
[1696] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 331) of PRO330 (herein referred to as DNA40603-1232) and the protein sequence of PRO330 derived therefrom.
[1697] The entire nucleotide sequence of DNA40603-1232 is shown in Figure 115 (SEQ ID NO: 331). Clone DNA 40603-1232 contains a single open reading frame with an open translation initiation site at nucleotide positions 167 to 169 and terminating at a stop codon at nucleotide positions 1766 to 1768 (Figure 115). The predicted polypeptide precursor has a length of 533 amino acids (Figure 116). Clone DNA 40603-1232 was deposited with the ATCC on November 21, 1997 as ATCC Deposit No. 209486.
[1698] Amino acid sequence analysis of the full-length PRO330 polypeptide shows that PRO330 may be a prolyl 4-hydroxylase alpha subunit polypeptide by suggesting that some of it has substantial homology with the mouse prolyl 4-hydroxylase alpha subunit protein Respectively.
[1699] Example 49 : Isolation of cDNA clones encoding human PRO310
[1700] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. This consensus sequence was referred to herein as DNA40553. Based on the DNA40553 consensus sequence, 1) an oligonucleotide was synthesized by PCR to confirm the cDNA library containing the target sequence, and 2) as a probe for isolating clones of the full-length coding sequence of PRO310.
[1701] An omnidirectional and reverse PCR primer was synthesized:
[1702] Omni-directional PCR primer 1 5'-TCCCCAAGCCGTTCTAGACGCGG-3 '(SEQ ID NO: 342)
[1703] Forward PCR primer 2 5'-CTGGTTCTTCCTTGCACG-3 '(SEQ ID NO: 343)
[1704] Reverse PCR primer 5'-GCCCAAATGCCCTAAGGCGGTATACCCC-3 '(SEQ ID NO: 344)
[1705] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus sequence.
[1706] Hybridization probe
[1707] 5'-GGGTGTGATGCTTGGAAGCATTTTCTGTGCTTTGATCACTATGCTAGGAC-3 '(SEQ ID NO: 345)
[1708] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO310 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1709] RNA for cDNA library production was isolated from human fetal liver tissue.
[1710] DNA sequence analysis of the clones isolated as described above yielded the full-length DNA sequence (SEQ ID NO: 340) of PRO310 (herein referred to as DNA43046-1225) and the protein sequence of PRO310 (SEQ ID NO: 341) derived therefrom.
[1711] The entire nucleotide sequence of DNA43046-1225 is shown in Figure 119 (SEQ ID NO: 340). Clone DNA43046-1225 contains a single open reading frame with an open translation initiation site at nucleotide positions 81 to 83 and terminating at a stop codon at nucleotide positions 1035 to 1037 (Figure 119). The predicted polypeptide precursor has a length of 318 amino acids (Figure 120) and has an estimated molecular weight of 36,382 daltons. Clone DNA43046-1225 was deposited with the ATCC as ATCC Deposit No. 209484.
[1712] Amino acid sequence analysis of the full-length PRO310 polypeptide suggested that a part of it has homology to C. elegans and its vicinity, indicating that PRO310 may be involved in its growth.
[1713] Example 50 : Isolation of cDNA clones encoding human PRO339
[1714] EST was confirmed by irradiating an expression sequence tag (EST) DNA database (LIFESEQ (registered trademark, Insight Pharmaceuticals, Palo Alto, Calif., USA). Insight clones and consensus sequences were generated using phrap as described in Example 1 above.
[1715] Based on the assembled consensus sequence, forward and reverse PCR primers were synthesized:
[1716] Forward PCR primer 1 5'-GGGATGCAGGTGGTGTCTCATGGGG-3 '(SEQ ID NO: 346)
[1717] Forward PCR primer 2 5'-CCCTCATGTACCGGCTCC-3 '(SEQ ID NO: 347)
[1718] Forward PCR primer 3 5'-GTGTGACACAGCGTGGGC-3 '(SEQ ID NO: 43)
[1719] Omni-directional PCR primer 4 5'-GACCGGCAGGCTTCTGCG-3 '(SEQ ID NO: 44)
[1720] Reverse PCR primer 1 5'-CAGCAGCTTCAGCCACCAGGAGTGG-3 '(SEQ ID NO: 45)
[1721] Reverse PCR primer 2 5'-CTGAGCCGTGGGCTGCAGTCTCGC-3 '(SEQ ID NO: 46)
[1722] In addition, a synthetic oligonucleotide hybridization probe having the following nucleotide sequence was constructed from the consensus sequence.
[1723] Hybridization probe
[1724] 5'-CCGACTACGACTGGTTCTTCATCATGCAGGATGACACATATGTGC-3 '(SEQ ID NO: 47)
[1725] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO339 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1726] RNA for cDNA library production was isolated from human fetal liver tissue.
[1727] The cDNA clones were sequenced as a whole. The entire nucleotide sequence of DNA43466-1225 is shown in Figure 117 (SEQ ID NO: 338). Clone DNA43466-1225 contains a single open reading frame with an open translation initiation site at nucleotide positions 333 to 335 and terminating at a stop codon at nucleotide positions 2649 to 2651 (Figure 117, SEQ ID NO: 338). The predicted polypeptide precursor has an amino acid length of 772 and an estimated molecular weight of approximately 86,226 daltons. Clone DNA43466-1225 was deposited with the ATCC as ATCC Deposit No. 209490.
[1728] Based on the BLAST and FastA sequence alignment analysis of the full-length sequence (using the ALIGN computer program), PRO339 has homology to the collagen-type polymer sequence as well as to C. elegans and its vicinity, and PRO339 is involved in tissue growth or development Respectively.
[1729] Example 51 : Isolation of cDNA clones encoding human PRO244
[1730] Consensus DNA sequences were assembled against other EST sequences using phrap as described in Example 1 above. Based on this consensus sequence, oligonucleotides were synthesized to confirm the cDNA library containing the target sequence by PCR and to use as a probe to isolate clones of the full-length coding sequence of PRO244.
[1731] A pair of PCR primers (forward and reverse) were synthesized:
[1732] 5'-TTCAGCTTCTGGGATGTAGGG-3 '(30923.f1) (SEQ ID NO: 378)
[1733] 5'-TATTCCTACCATTTCACAAATCCG-3 '(30923.r1) (SEQ ID NO: 379)
[1734] The probe was also synthesized:
[1735] 5'-GGAGGACTGTGCCACCATGAGAGACTCTTCAAACCCAAGGCAAAATTGG-3 '(30923.p1) (SEQ ID NO: 380)
[1736] To screen several libraries for the source of the full-length clone, the DNA of the library was screened by PCR amplification using the PCR primer pairs identified above. A clone encoding the PRO244 gene was isolated using a positive library using either probe oligonucleotides and PCR primers.
[1737] RNA for cDNA library construction was isolated from human fetal kidney library. DNA sequence analysis of the isolated clones as described above yielded full-length DNA sequences and PRO244 protein sequences derived therefrom.
[1738] The entire nucleotide sequence of PRO244 is shown in Figure 121 (SEQ ID NO: 376). Clone DNA 35668-1171 contains a single open reading frame with a pronounced translation initiation site at nucleotide positions 106-108 (Figure 121). The predicted polypeptide precursor has a length of 219 amino acids. Clone DNA 35668-1171 was deposited with the ATCC as ATCC Deposit No. 209371 (referred to herein as DNA35663-1171). The protein has the cystophosphatomic domain (aa 1-20), the transmembrane domain (aa 21-46) and the extracellular domain (aa 47-219) and has a C-lectin domain at aa 55-286.
[1739] Based on the BLAST and FastA sequence alignment analysis of the full-length sequence, PRO244 was found to contain the following sequences: hepatic lectin fibrin (43%), HIC hp 120 -linked C-type lectin (42%), macrophage lectin 2 (HUMHML2-1, 41% Significant amino acid identity with PR32188 (44%).
[1740] Example 52 : Use of a PRO polypeptide encoding a nucleic acid as a hybridization probe
[1741] The following method describes the use of a nucleotide sequence encoding a PRO polypeptide as a hybridization probe.
[1742] As described herein, DNA comprising the coding sequence of the PRO polypeptide of interest can be used as a probe, or it can be used as a probe in a human tissue cDNA library or a human tissue genomic library in the presence of homologous DNA (e. G., Encoding a native variant of a PRO polypeptide Were used for the preparation of probes for screening.
[1743] Hybridization and washing of filters containing these library DNAs were performed under the following stringent conditions. The radiolabeled PRO polypeptide-encoding nucleic acid-derived probe for the filter was diluted in 50% formamide, 5 x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2 x Denhardt's solution And 10% dextran sulfate at 42 < 0 > C for 20 hours. The filters were washed at 42 [deg.] C in an aqueous solution of 0.1 x SSC and 0.1% SDS.
[1744] DNA having the desired degree of sequence identity with the DNA encoding the full length native sequence PRO polypeptide was then identified by standard methods known in the art.
[1745] Example 53: a. Expression of PRO polypeptides in E. coli
[1746] In this embodiment, Described is a method for producing a non-glycosylated form of a target PRO polypeptide by recombinant expression in E. coli.
[1747] First, the selected PCR primer was used to amplify the DNA sequence encoding the desired PRO polypeptide. The primer should contain a restriction enzyme site corresponding to the restriction enzyme site on the selected expression vector. A variety of expression vectors may be used. Examples of suitable vectors include pBR322 [which contains ampicillin and the tetracycline resistance gene. Bolivar et al., Gene , 2 : 95 (1977)], which is derived from E. coli. This vector was digested with restriction enzymes and dephosphorylated. The PCR amplified sequence was then ligated to the vector. The vector preferably comprises an antibiotic resistance gene, a trp promoter, a polyhis leader (including the first six STII codons, a poly-his sequence and an enterokinase cleavage site), a specific PRO polypeptide coding region, a lambda transcription terminator and an argU gene Lt; / RTI > coding sequence.
[1748] Subsequently, the ligation mixture was used to select it according to the method described in Sambrook et al. The E. coli strain was transformed. After identifying transformants capable of growing on LB plates, colonies with antibiotic resistance were selected. Plasmid DNA was isolated and confirmed by restriction analysis and DNA sequencing.
[1749] Selected clones could be grown overnight in liquid culture media such as LB broth supplemented with antibiotics. This culture was used to inoculate larger scale cultures. The cells were then incubated to the desired optical density, during which the expression promoter was activated.
[1750] After culturing the cells for several hours or more, cells were recovered by centrifugation. The cell pellet obtained by centrifugation was dissolved using various reagents known in the art, and the solubilized PRO polypeptide was purified under the condition of tightly binding the protein using a metal chelating column.
[1751] PRO187, PRO317, PRO301, PRO224 and PRO238 were prepared using the following method. Lt; RTI ID = 0.0 > poly-His-tagged < / RTI > First, DNA encoding PRO187, PRO317, PRO301, PRO224 or PRO238 was amplified using the selected PCR primers. Primers included restriction enzyme sites corresponding to restriction enzyme sites on selected expression vectors and other useful sequences that provided efficient and reliable translation initiation, rapid purification in metal chelate columns, and proteolytic cleavage using enterokinase. The PCR-amplified, poly-His tagged sequence was then ligated to the expression vector and the E. coli host, strain 52 (W3110 fuhA (tonA) lon galE rpoHts (htpRts) clpP (lacIq) First, the transformant was shake-cultured at 30 DEG C in an LB containing 50 mg / ml of carbenicillin until an OD600 reached 3 to 5. Then, the culture was added to the CRAP medium (water 500 3.57 g of (NH ₄ ) ₂ SO ₄ , 0.71 g of sodium citrate · 2H ₂ O, 1.07 g of KCl, 5.36 g of Difco yeast extract and 5.36 g of Sheffield hycase SF as well as 110 mM MPOS ), 0.55% (w / v) glucose and 7 mM MgSO ₄ ) and incubated for about 20 to 30 hours at 30 ° C. The samples were taken and analyzed by SDS-PAGE analysis And the bulk culture was centrifuged to pellet the cells. The cell pellet was purified Until refolding was kept frozen.
[1752] 0.5 to 1 L fermentation broth. The coli paste (6 to 10 g pellet) was resuspended in 10 volumes (w / v) of 7 M guanidine, 20 mM Tris buffer (pH 8). Solid sodium sulphate and sodium sulphate were added to give final concentrations of 0.1 M and 0.02 M, respectively, and the solution was stirred overnight at 4 ° C. At this stage, a denatured protein with all cysteine residues blocked by sulfation was produced. The solution was centrifuged at 40,000 rpm for 30 minutes with a Beckman ultracentrifuge. The supernatant was diluted with 3-5 volumes of metal chelating column buffer (6 M guanidine, 20 mM Tris, pH 7.4) and clarified by filtration through a 0.22 micron filter. The purified extract was loaded onto a 5 ml Qiagen Ni-NTA metal chelate column equilibrated with metal chelate column buffer. The column was washed with additional buffer (pH 7.4) containing 50 mM imidazole (Calbiochem, Utrol grade). The protein was eluted with a buffer containing 250 mM imidazole. Fractions containing the desired protein were pooled and stored at 4 < 0 > C. The protein concentration was measured at 280 nm absorbance using the extinction coefficient calculated based on the amino acid sequence.
[1753] Samples were slowly diluted in freshly prepared refolding buffer consisting of 20 mM Tris (pH 8.6), 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA to refold the proteins. The refolding volume was determined such that the final protein concentration was 50-100 占 퐂 / ml. The refolding solution was gently stirred at 4 DEG C for 12 to 36 hours. TFA was added to terminate the refolding reaction and the final concentration was 0.4% (about pH 3). Before further protein purification, the solution was filtered through a 0.22 micron filter and acetonitrile was added to give a final concentration of 2-10%. The refolded protein was chromatographed on a Poros R1 / H reverse phase column using a mobile buffer of 0.1% TFA with a gradient of acetonitrile concentration gradient from 10% to 80%. The aliquots of the fraction showing the A280 absorbance were analyzed on SDS polyacrylamide gel and fractions containing homogeneous refolded proteins were collected. In general, properly refolded proteins of most proteins have hydrophobic interiors that are protected from interaction with reverse phase resins and are eluted at the lowest concentration of acetonitrile because they are the most dense. Aggregates are usually eluted at high acetonitrile concentrations. The reverse phase step not only separates the desired form of the protein from the misfolded form of the protein, but also removes the endotoxin from the sample.
[1754] Fractions containing each of the desired folded PRO187, PRO317, PRO301, PRO224 and PRO238 proteins were pooled and the acetonitrile was removed by slowly adding a stream of nitrogen towards the solution. Proteins were prepared in 20 mM Hepes (pH 6.8) containing 0.14 M sodium chloride and 4% mannitol by gel filtration using G25 superfine (Pharmacia) resin equilibrated with dialysis or preparation buffer and sterile filtered.
[1755] Example 54 : Expression of PRO polypeptides in mammalian cells
[1756] This example describes a method for producing a glycolated form of a target PRO polypeptide by recombinant expression in mammalian cells.
[1757] The vector pRK5 (1989, 3.15, published EP 307,247) was used as an expression vector. Optionally, the PRO polypeptide-encoding DNA was ligated to pRK5 using the selected restriction enzyme using the ligation method described in Sambrook et al., Supra, to insert the PRO polypeptide DNA. The resulting vector was named pRK5-PRO polypeptide.
[1758] In one embodiment, 293 cells can be selected as host cells. Human 293 cells (ATCC CCL 1573) were cultured to confluence in tissue culture plates in medium such as fetal calf serum and DMEM supplemented with nutrients and / or antibiotics. Approximately 10 μg of pRK5-PRO polypeptide DNA was mixed with approximately 1 μg of DNA encoding the VA RNA gene (Thimmappaya et al., Cell , 31 : 543 (1982)), and 500 μl of 1 mM Tris -HCl, 0.1 mM EDTA and 0.227 M CaCl ₂ . 500 쨉 l of 50 mM HEPES (pH 7.35), 280 mM NaCl, and 1.5 mM NaPO ₄ were added dropwise to the mixture to form a precipitate at 25 째 C for 10 minutes. The precipitate was suspended and added to 293 cells and allowed to stand at 37 DEG C for 4 hours. The culture medium was aspirated off and 2 ml of 20% glycerol in PBS was added for 30 seconds. Subsequently, 293 cells were washed with serum-free medium, fresh medium was added, and cells were incubated for about 5 days.
[1759] After about 24 hours of transfection, the culture medium was removed and replaced with culture medium (alone) or with culture medium containing 200 μCi / ml ³⁵ S-cysteine and 200 μCi / μl ³⁵ S-methionine. After 12 hours of incubation, the conditioned medium was collected, concentrated on a rotary filter and loaded onto 15% SDS gel. The treated gel was dried and exposed to film for a period of time to confirm the presence of the PRO polypeptide. The culture containing the transfected cells was further incubated (in serum-free medium) and the medium was tested with the selected biochemical assay.
[1760] Alternatively, Somparyrac et al ., Proc. Natl. Acad. Sci. , 12 : 7575 (1981), the PRO polypeptide may be transiently introduced into 293 cells. 293 cells were cultured to a maximum density in a spinner flask and 700 ㎍ of pRK5-PRO polypeptide DNA was added. The cells were first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate was incubated in the cell pellet for 4 hours. Cells were treated with 20% glycerol for 90 seconds, washed with tissue culture medium and re-inserted into tissue culture medium, 5 ug / ml bovine insulin and 0.1 μg / ml bovine transferrin spinner flask. After about 4 days, the conditioned media was centrifuged and filtered to remove cells and lysates. Samples containing the expressed PRO polypeptides were concentrated and purified by any selected method, e. G., Dialysis and / or column chromatography.
[1761] In another embodiment, the PRO polypeptide can be expressed in CHO cells. The reagent, a known pRK5-PRO polypeptide, for example by using a CaPO ₄ or DEAE- dextran can be transfected into CHO cells. As mentioned above, cell cultures were incubated and the medium was replaced with culture medium (alone) or medium containing radioactive label such as ³⁵ S-methionine. After confirming the presence of the PRO polypeptide, the culture medium can be replaced with a serum-free medium. Preferably, the culture medium is incubated for about 6 days, and then the conditioned medium is recovered. The medium containing the expressed PRO polypeptide could then be concentrated and purified by any selected method.
[1762] In addition, epitope tagged PRO polypeptides could be expressed in host CHO cells. The PRO polypeptide is subcloned from the pRK5 vector. The subclone insert is fused to the baculovirus expression vector in frame with a selected epitope tag such as a poly-his tag by PCR. The poly-his tagged PRO polypeptide insert is subcloned into an SV40 inducible vector containing a selection marker such as DHFR to select a stable clone. Finally, CHO cells can be transfected with an SV40 inducible vector (as above). May be labeled in the same manner as described above to confirm expression. The culture medium containing the expressed poly-His-tagged PRO polypeptide is then concentrated and purified by any selected method, such as Ni ²⁺ -chelate affinity chromatography.
[1763] PRO211, PRO217, PRO230, PRO219, PRO245, PRO221, PRO258, PRO301, PRO224, PRO222, PRO234, PRO229, PRO223, PRO328 and PRO332 were successfully expressed in CHO cells by both transient expression and stable expression methods. PRO232, PRO265, PRO246, PRO228, PRO227, PRO220, PRO266, PRO269, PRO287, PRO214, PRO231, PRO233, PRO238, PRO244, PRO235, PRO236, PRO262, PRO239, PRO257, PRO260, PRO263, PRO270, PRO294, PRO295, PRO293, PRO247, PRO303, and PRO268 were successfully transiently expressed in CHO cells.
[1764] And stably expressed in CHO cells using the following method. A protein is defined as an IgG construct (immunoadhesin) in which the coding sequence of the soluble form of each protein (e. G., Extracellular domain) is fused to an IgGl constant region sequence comprising the CH2 and CH2 domains as hinge moieties and (Or) poly-His tagged form.
[1765] Following PCR amplification, each DNA was subcloned into a CHO expression vector using standard methods described in Ausubel et al., Current Protocols of Molecular Biology , Unit 3.16, John Wiley and Sons (1997). The CHO expression vector is constructed so as to have a restriction site suitable for 5 'and 3' of the DNA of interest so that cDNA can be conveniently shuttled. The vector used for expression in CHO cells is described by Lucas et al ., Nucl. Acids Res. 24 : 9, 1774-1779 (1996), and the cDNA and dihydrofolate reductase (DHFR) were expressed using the SV40 early promoter / enhancer. DHFR expression allowed selection of plasmids that remained stable after transfection.
[1766] 12 μg of the target plasmid DNA was mixed with approximately 10 million copies of commercially available transfection reagent Superfect (registered trademark) Qiagen, Dosper (registered trademark) or Fugene (registered trademark) (Boehringer Mannheim) Of CHO cells. Cells were cultured according to the method described in Lucas et al., Supra. Subsequently, approximately 3 x 10 < ^-7 > cells were frozen in ampoules according to the following method to cultivate and produce the cells.
[1767] Plasmid DNA-containing ampoules were dissolved in a water bath and mixed by vortexing. The contents were pipetted into a centrifuge tube containing 10 ml of the medium and centrifuged at 1000 rpm for 5 minutes. The supernatant was inhaled and the cells resuspended in 10 ml of selective medium (0.2 [mu] m filtered PS20 containing 0.2% demineralized, 5% fetal bovine serum). Cells were dispensed into 100 ml Spinner containing 90 ml of selection medium. After 1-2 days, the cells were transferred to 250 ml Spinner filled with 150 ml of selective culture medium and incubated at 37 [deg.] C. After 2 to 3 days were inoculated to 3 × 10 ⁵ cells / ㎖ to 250 ㎖, 500 ㎖ and 2000 ㎖ spin too. The cell culture medium was centrifuged, replaced with fresh medium and resuspended in the production medium. Any suitable CHO medium can be used, and in fact the production medium described in U.S. Patent No. 5,122,469 (June 16, 1992) was used. Lt; ⁶ > cells / ml in a 3 L production spinner. Cell number and pH were measured at day 0. On the first day, samples were taken from the spinner and spraying of the filtered air started. On the second day, samples were taken on a spinner and the temperature was changed to 33 ° C and 30 ml of 500 g / l glucose and 0.6 ml of 10% anti-foam (eg 35% polydimethylsiloxane emulsion, Dow Corning 365 medicament grade emulsion ). During the entire production process, the pH should be maintained at about 7.2. After 10 days or when the survival rate dropped to less than 70%, the cell culture was recovered by centrifugation and filtered through a 0.22 mu m filter. The filtrate was stored at 4 [deg.] C or immediately loaded onto the column for purification.
[1768] For poly-his tagged constructs, proteins were purified using Ni-NTA column (Qiagen). Imidazole was added to the conditioned medium at a concentration of 5 mM before purification. The conditioned media was pumped to a 6 ml Ni-NTA column equilibrated with 20 mM Hepes buffer (pH 7.4) containing 0.3 M NaCl and 5 mM imidazole at 4 캜 at a flow rate of 4-5 ml / min. After loading, the column was washed with additional equilibration buffer and the protein was eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then removed from the buffer in 25 ml of a G25 supernatant (Pharmacia) column in Storage Buffer (pH 6.8) containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol and stored at -80 ° C.
[1769] The immunoadhesin (including Fc) construct was purified from the conditioned medium as follows. The conditioned media was pumped into a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer (pH 6.8). After loading, the column was washed thoroughly with equilibration buffer and eluted with 100 mM citric acid (pH 3.5). The eluted protein was collected in 1 ml fractions and immediately neutralized in a tube containing 275 의 of 1 M Tris buffer (pH 9). The highly purified protein was then cleared of salts with the storage buffer in the manner described above for the case of poly-His tagged proteins. The homogeneity was evaluated by N-terminal amino acid sequence analysis performed with SDS-polyacrylamide gel and Edman degradation.
[1770] PRO211, PRO217, PRO230, PRO232, PRO187, PRO265, PRO219, PRO246, PRO228, PRO533, PRO245, PRO221, PRO227, PRO220, PRO258, PRO266, PRO269, PRO287, PRO214, PRO317, PRO301, PRO224, PRO222, PRO229, PRO233, PRO238, PRO223, PRO235, PRO236, PRO262, PRO239, PRO257, PRO260, PRO263, PRO270, PRO271, PRO272, PRO294, PRO295, PRO293, PRO247, PRO304, PRO302, PRO307, PRO303, PRO343, PRO331, PRO332, PRO334, PRO346, PRO268, PRO330, PRO310 and PRO339 were also successfully transiently expressed in COS cells.
[1771] Example 55 Expression of PRO Polypeptides in Yeast
[1772] The following is a description of the recombinant expression of the desired PRO polypeptide in yeast.
[1773] First, yeast expression vectors were prepared for intracellular production or secretion of PRO polypeptides from the ADH2 / GAPDH promoter. The PRO polypeptide, the selected signal polypeptides and the DNA encoding the promoter were inserted into the appropriate restriction enzyme sites of the plasmid selected for intracellular expression of the PRO polypeptide. In the case of secretion, the DNA encoding the PRO polypeptide can be cloned into the selected plasmid, along with the DNA encoding the ADH2 / GAPDH promoter, the yeast alpha-factor secretion signal / leader sequence and the linker sequence (if necessary) for expression of the PRO polypeptide have.
[1774] Yeast cells, such as yeast strain AB110, can then be transformed with the above expression plasmids and cultured in selected fermentation media. The transformed yeast supernatant can be precipitated with 10% trichloroacetic acid, separated by SDS-PAGE and then stained with Coomassie blue to analyze the gel.
[1775] Thereafter, the yeast cells are removed from the fermentation medium by centrifugation and the medium can be concentrated using a selected cartridge filter to isolate and purify the recombinant PRO polypeptide. The concentrate containing the PRO polypeptide can be further purified using a selected column chromatography resin.
[1776] Example 56 Expression of PRO Polypeptides in Baculovirus-Infected Insect Cells
[1777] The following is a description of the recombinant expression of PRO polypeptides in baculovirus-infected insect cells.
[1778] Purpose The PRO polypeptide was fused upstream of the epitope tag included in the baculovirus expression vector. The epitope tag includes a poly-his tag and an immunoglobulin tag (such as the Fc region of IgG). Several plasmids can be used, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). In short, the desired portion of the PRO polypeptide or PRO polypeptide (eg, the sequence encoding the extracellular domain of the transmembrane protein) was amplified by PCR using primers complementary to the 5 'and 3' regions. The 5 ' primer may comprise a restriction enzyme site adjacent (selected) on both sides. The product was then digested with the selected restriction enzymes and subcloned into an expression vector.
[1779] Recombinant baculovirus was transformed with the above plasmid and BaculoGold (R) virus DNA (Pharmingen) using lipofectin (purchased from GIBCO-BRL) into Spodoptera frugiperda ("Sf9" ) Cells (ATCC CRL 1711). After incubation at 28 ° C for 4 to 5 days, the released virus was recovered and used for subsequent amplification. Viral infections and protein expression were performed according to O'Reilley et al. [Baculovirus Expression vectors: A laboratory Manual, Oxford: Oxford University Press (1994)].
[1780] The expressed poly-His tagged PRO polypeptide can then be purified, for example, by Ni ²⁺ -chiral affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells according to Rupert et al., Nature, 362: 175-179 (1993). In short, to clean the Sf9 cell sonication buffer on (25 ㎖ Hepes, pH 7.9; 12.5 mM MgCl 2; 0.1 mM EDTA; 10% glycerol;; 0.1% NP-40 0.4 M KCl) was resuspended placed on ice 20 seconds And then ultrasonicated twice. The supernatant was removed by centrifugation and the supernatant was diluted 50-fold with loading buffer (50 mM phosphoric acid, 300 mM NaCl, 10% glycerol, pH 7.8) and filtered through a 0.45 [mu] m filter. A Ni ²⁺ -NTA agarose column (commercially available from Quiagen) was prepared in a 5 ml fill volume, washed with 25 ml water and equilibrated with 25 ml loading buffer. The filtered cell extract was loaded onto the column at 0.5 ml per minute. The column was washed with loading buffer to A ₂₈₀ baseline, at which time fraction recovery was initiated. Next, the column was washed with secondary washing buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH 6.0) to elute nonspecifically bound proteins. Again, to reach the A ₂₈₀ baseline, the column was developed using a 0-500 mM imidazole gradient in the secondary wash buffer. 1 ml fractions were collected and analyzed by SDS-PAGE and silver staining, or western blotting with Ni ²⁺ -NTA (Qiagen) coupled to alkaline phosphatase. Fractions containing eluted His ₁₀ tagged PRO polypeptides were pooled and dialyzed against loading buffer.
[1781] Alternatively, purification of IgG tagged (or Fc-tagged) PRO polypeptides could be performed using known chromatographic techniques, such as protein A or protein G column chromatography.
[1782] PRO211, PRO217, PRO230, PRO187, PRO265, PRO246, PRO228, PRO533, PRO245, PRO221, PRO220, PRO258, PRO266, PRO269, PRO287, PRO214, PRO301, PRO224, PRO222, PRO234, PRO272, PRO294, PRO295, PRO328, PRO326, PRO331, PRO334, PRO346 and PRO310 were successfully expressed in baculovirus-infected Sf9 or high5 insect cells. In practice, the reaction was carried out on a scale of 0.5 to 2 L, but could easily be scaled up (for example, 8 L). The protein was expressed as an IgG construct (immunohedhedin) and / or as a poly-His tagged form fused to an IgGl constant region sequence containing the CH2 and CH3 domains which are the hinge portion of the protein.
[1783] After PCR amplification, each coding sequence was subcloned into a baculovirus expression vector (pb.PH.IgG for IgG fusion and pb.PH.His.c for poly-His tagged protein) and ligated with lipofectin (Gibco BRL ) And vector were co-transfected with Baculov gold (TM) baculovirus DNA (Pharmingen) into 105 Spodoptera frugiperda (" Sf9 ") cells (ATCC CRL 1711). pb.PH.IgG and pb.PH.His are variants of the commercial baculovirus expression vector pVL1393 (Pharmingen) with a modified polylinker region containing the His or Fc tag sequence. Cells were cultured in Hink's TNM-FH medium (Hyclone) supplemented with 10% FBS. The cells were incubated at 28 [deg.] C for 5 days. The supernatant was collected and then used for initial viral amplification by infecting Sf9 cells with approximately 10 multi-infectious doses (MOI) in Hink's TNM-FH medium supplemented with 10% FBS. The cells were incubated at 28 [deg.] C for 3 days. The supernatant was collected and the histidine-tagged protein was bound to Ni-NTA beads (Qiagen) or IgG-tagged proteins in bulk by adding 1 ml of the supernatant to 25 ml of protein-A Sepharose CL-4B beads (Pharmacia) Expression of the constructs in baculovirus expression vectors was assessed by SDS-PAGE analysis as compared to standard proteins at known concentrations by staining.
[1784] The first viral amplification supernatant was used to infect Spinner's culture (500 ml) of Sf9 cells cultured in ESF-921 medium (Expression System LLC) at an MOI of about 0.1. Cells were incubated at 28 ° C for 3 days. The supernatant was collected and filtered. Batch binding and SDS-PAGE analysis were repeated as needed until expression in spinner culture was confirmed.
[1785] The conditioned medium (0.5-3 L) from the transfected cells was recovered by centrifugation, the cells were removed and filtered through a 0.22 micron filter. The poly-His-tagged construct was purified using a Ni-NTA column (Qiagen). Prior to purification, imidazole was added to the conditioned medium to a concentration of 5 mM. The conditioned media was pumped to a 6 ml Ni-NTA column equilibrated with 20 mM Hepes (pH 7.4) buffer containing 0.3 M NaCl and 5 mM imidazole at 4 ° C at a flow rate of 4-5 ml / min. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The highly purified protein was then removed from the buffer in a buffer (pH 6.8) containing 10 mM Hepes, 0.14 M NaCl and 4% mannitol in a 25 ml G25 supernatant (Pharmacia) column and stored at -80 ° C .
[1786] The immunoadhesin (including Fc) structure of the protein was purified from the conditioned medium as follows. The conditioned media was pumped into a 5 ml protein A column (Pharmacia) equilibrated with 20 mM sodium phosphate buffer (pH 6.8). After loading, the column was washed thoroughly with equilibration buffer and eluted with 100 mM citric acid (pH 3.5). The eluted protein was recovered in 1 ml fractions and immediately put in a tube containing 275 ml of 1 M Tris buffer (pH 9) and neutralized. The highly purified protein was then removed with the storage buffer in the same manner as described above for the poly-His tagged protein. Homogeneity of the protein was confirmed by N-terminal amino acid sequence analysis performed by SDS-polyacrylamide gel (PEG) electrophoresis and Edman degradation.
[1787] Example 57 : Preparation of Antibodies Binding to PRO Polypeptides
[1788] This example describes a method for producing a monoclonal antibody capable of specifically binding to a PRO polypeptide.
[1789] Techniques for producing monoclonal antibodies are known in the art and are described, for example, in Goding, supra. Immunogens that may be used include purified PRO polypeptides, fusion proteins comprising PRO polypeptides, and cells expressing recombinant PRO polypeptides on the cell surface. One skilled in the art can select an immunogen without undue experimentation.
[1790] A mouse, for example Balb / c, was immunized by subcutaneous or intraperitoneal injection of PRO polypeptide immunogen in an amount of 1 to 100 [mu] g in a complete adjuvant solution. Alternatively, the immunogen was emulsified in MPL-TDM supplement (Ribi Immunochemical Research, Hamilton, Mont.) And injected into the hindpaw of the animal. The immunized mice were then further antigen challenged with additional immunizations emulsified in the selected adjuvant after 10-12 days. Thereafter, the mice can be further boosted with additional immunization for several weeks. Blood was drawn from the orbital area and serum samples were periodically taken from the mice and the anti-PRO polypeptide antibody was detected by ELISA assay.
[1791] Once the appropriate antibody titer was detected, the PRO polypeptide was injected intravenously into the animal " positive " to the antibody. After 3 to 4 days, mice were sacrificed to recover spleen cells. The spleen cells were then fused (using 35% polyethylene glycol) with the selected mouse myeloma cell line, for example P3X63AgU.1, available from ATCC number CRL 1597. Fusions produce hybridoma cells that can be plated in 96-well tissue culture plates containing HAT (hypoxanthine, aminopterin and thymidine) medium that inhibits the proliferation of non-fused cells, myeloma hybrids and spleen cell hybrids Respectively.
[1792] The reactivity of the hybridoma cells to PRO polypeptides can be screened by ELISA. Methods for determining " positive " hybridoma cells that secrete the desired monoclonal antibody of the PRO polypeptide are well known to those of skill in the art.
[1793] Positive hybridoma cells can be injected intraperitoneally to produce ascites containing Balb / c mice containing anti-PRO polypeptide monoclonal antibodies. Alternatively, the hybridoma cells can be cultured in tissue culture flasks or roller bottles. Monoclonal antibodies produced in the ascites can be purified using ammonium sulfate precipitation followed by gel exclusion chromatography. Alternatively, affinity chromatography based on protein A or protein G binding of the antibody may be used.
[1794] Example 58 : Chimeric PRO polypeptide
[1795] The PRO polypeptide may be expressed as a chimeric protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitating domains include, but are not limited to, metal chelate peptides such as histidine-tryptophan modules that can be purified on immobilized metals, protein A domains that can be purified on immobilized immunoglobulin, and FLAGS TM kidney / affinity purification systems , ≪ / RTI > Seattle, Washington, USA). Including cleavable linker sequences, such as the factor XA or enterokinase (Invitrogen, San Diego, Calif., USA) between the purification domain and the PRO polypeptide sequence, may be useful to facilitate expression of the DNA encoding the PRO polypeptide.
[1796] Example 59 Purification of PRO Polypeptides Using Specific Antibodies
[1797] Natural or recombinant PRO polypeptides can be purified by a variety of standard techniques in the field of protein purification. For example, a pro-PRO polypeptide, a mature PRO polypeptide or a pre-PRO polypeptide is purified by immunoaffinity chromatography using an antibody specific for the target PRO polypeptide. Generally, the immunoaffinity column is made by coupling the anti-PRO polypeptide antibody to the activated chromatographic resin by covalent attachment.
[1798] Polyclonal immunoglobulins were prepared from immune sera by precipitation with ammonium sulfate or by purification on Immobilized Protein A (Pharmacia LKB Biotechnology, Pittsburgh, New Jersey, USA). Similarly, monoclonal antibodies were prepared from murine duplicates by ammonium sulfate precipitation or chromatography on immobilized protein A. Partially purified immunoglobulins were attached by covalent attachment to a chromatographic resin such as CnBr-activated SEPHAROSE (R) (Pharmacia LKB Biotechnology). The antibody was coupled to the resin, the resin was blocked, and the derived resin was washed according to the manufacturer's instructions.
[1799] Such an immunoaffinity column is used to purify the PRO polypeptide by preparing a fraction containing the PRO polypeptide in soluble form from the cell. By solubilization of whole cells by differential addition or by other methods well known in the art or by solubilization of the cell fraction obtained by differential centrifugation. Alternatively, the soluble PRO polypeptide comprising the signal sequence may be secreted in a useful amount into the medium in which the cells are cultured.
[1800] The soluble PRO polypeptide-containing preparation is passed through an immunoaffinity column and the column is washed under conditions permitting preferential absorption of the PRO polypeptide (e. G., High ion concentration buffer in the presence of detergent). The column is then eluted under conditions that disrupt antibody / PRO polypeptide binding (e. G., A low pH buffer, such as about pH 2 to 3, or a high concentration of urea or chaotrope such as thiocyanate ions) PRO polypeptides were recovered.
[1801] Example 60 : Drug screening
[1802] The present invention is particularly useful for screening compounds with any of a variety of drug screening techniques using PRO polypeptides or binding fragments thereof. The PRO polypeptide or fragment used in this test may be free in solution, adhered to a solid support, retained on a cell surface, or located within a cell. One method of drug screening utilizes eukaryotic or prokaryotic host cells that are stably transformed with recombinant nucleic acids expressing PRO polypeptides or fragments thereof. The drug was screened against the transformed cells by competitive binding assay. The cells can be used for standard binding assays, whether viable or immobilized. For example, the formation of a complex between the PRO polypeptide or fragment and the test substance can be measured. Alternatively, a decrease in complex formation between the PRO polypeptide and its target cell or target receptor induced by the test substance may be examined.
[1803] Thus, the present invention provides a method for screening for a drug or any other substance that may affect a PRO polypeptide-related disease or disorder. These methods include contacting the substance with a PRO polypeptide or a fragment thereof, and determining the presence or absence of a complex between (i) the substance and the PRO polypeptide or fragment, or (ii) the presence of the PRO polypeptide or fragment and And analyzing the presence or absence of the complex. In such competitive binding assays, PRO polypeptides or fragments are typically labeled. After appropriate incubation, the free PRO polypeptide or fragment is separated from that present in bound form, and the amount of free or uncomplexed label is a measure of the ability of a particular substance to bind to or inhibit the PRO polypeptide / cell complex .
[1804] Another drug screening technique is described in detail in WO84 / 03564 (published Aug. 13, 1984) as a large-volume screening method for compounds with appropriate binding affinity for polypeptides. In short, a number of different small peptide test compounds are synthesized on solid phase substrates such as plastic pins or some other surface. Like the PRO polypeptide, the peptide test compound is reacted with the PRO polypeptide and washed. Bound PRO polypeptides are detected by methods well known in the art. The purified PRO polypeptides may also be coated directly on the plates used in the above-described drug screening techniques. In addition, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
[1805] Also included in the invention is the use of competitive drug screening assays in which neutralizing antibodies capable of binding PRO polypeptides specifically compete with test compounds for binding to PRO polypeptides or fragments thereof. In this manner, antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with a PRO polypeptide.
[1806] Example 61 : Reasonable drug design
[1807] The goal of rational drug design is to produce structural analogs of the target biologically active polypeptide (i. E., PRO polypeptide) or small molecules that interact with them, for example, agonists, antagonists or inhibitors. Any of these examples can be used to produce a drug that is a more active or more stable form of a PRO polypeptide, or a drug that enhances or inhibits the function of a PRO polypeptide in vivo [Hodgson, Bio / Technology , 9 : 19-21 (1991)].
[1808] In one method, the three-dimensional structure of the PRO polypeptide or PRO polypeptide-inhibitor complex is determined by x-ray crystallography, computer modeling or most typically by a combination of the two methods. In order to identify the structure of the molecule and to determine the active site, both the morphology and charge of the PRO polypeptide should be identified. Although less frequent, useful information about the structure of PRO polypeptides can be obtained by modeling based on the structure of homologous proteins. In both cases, the relevant structural information is used to design a cognate PRO polypeptide-like molecule or identify an effective inhibitor. Useful examples of rational drug design include molecules that have enhanced activity or stability, such as those shown in Braxton and Wells , Biochemistry, 31 : 7796-7801 (1992), or Athauda et al. J. Biochem. , 113 : 742-746 (1993)], which act as inhibitors, agonists or antagonists of natural peptides.
[1809] In addition, the target-specific antibody selected by functional analysis may be isolated as described above, and then its crystal structure may be analyzed. In principle, this method provides a pharmacore that can be the basis of subsequent drug design. The protein crystallization method may be omitted by producing an anti-individual specific antibody (anti-id) against a functional pharmacologically active antibody. On a mirror image, the binding site of the anti-id is thought to be the homolog of the original receptor. The peptides can then be identified and isolated from the chemically or biologically produced peptide banks using anti-id. Subsequently, the isolated peptide is used as a pharmacore.
[1810] With the present invention, a sufficient amount of the PRO polypeptide has become available to perform analytical studies such as x-ray crystallization. In addition, the amino acid sequence information of the PRO polypeptides provided herein will provide guidance for using computer modeling techniques in place of or in addition to x-ray crystallization techniques.
[1811] Example 62 Diagnostic Tests Using PRO317 Polypeptide-Specific Antibodies
[1812] Certain anti-PRO 317 polypeptide antibodies are useful in the diagnosis of gynecological or ischemic diseases characterized by pre-onset symptoms, and a difference in the amount or distribution of a chronic or acute disease such as PRO317. PRO317 has been shown to be expressed in the human kidney and will therefore be associated with an abnormality or etiology affecting this organ. In addition, since it is closely related to EBAF-1, it is likely to affect the endometrium and other genital tissues. In addition, due to the library source of a particular EST, PRO317 appears to be a regulator of angiogenesis, since it is involved in the formation of blood vessels.
[1813] Diagnostic tests for PRO317 include methods using antibodies and labels to detect PRO317 in human body fluids, tissues, or extracts of such tissues. Polypeptides and antibodies of the invention can be used without modification or modification. Often, polypeptides and antibodies are labeled by covalent or non-covalent association with a material that provides a detectable signal. A wide variety of labeling and bonding techniques are known and reported in the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents and magnetic particles. The patent documents describing the use of the label include U.S. Patent Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and 4,366,241. Also, recombinant immunoglobulins can be produced as shown in U.S. Patent No. 4,816,567.
[1814] Various protocols for measuring soluble or membrane bound PRO317 using polyclonal or monoclonal antibodies specific for PRO317 are known in the art. Examples include enzyme dependent immunoassay (ELISA), radioimmunoassay (RIA), radioactive receptor assay (RRA), and fluorescence activated cell sorting (FACS). Two-site monoclonal-based immunoassays using monoclonal antibodies reactive to two non-interfering epitopes on PRO317 are preferred, and competitive binding assays may be used. These assays are described in Maddox et al . J Exp. Med. , 158 : 1211 (1983).
[1815] Example 63 : Identification of PRO317 receptor
[1816] Purified PRO317 is useful for characterizing and purifying specific cell surface receptors and other binding molecules. Cells that respond to PRO317 by metabolic changes or other specific responses can express the receptor for PRO317. Such receptors include, but are not limited to, receptors associated with and activated by tyrosine and serine / threonine kinases. For a review of known receptors for the TGF-giant family, see Kolodziejczyk and Hall, supra. Candidate receptors for these large families are classified into two main groups called type I and type II receptors. Both are serine / threonine kinases. After being activated by appropriate ligands, the Type I and Type II receptors physically interact to form hetero- oligomers and then ultimately regulate gene transcription and expression by activating intracellular signaling cascades. TGF- also binds to proteoglycans anchored to a membrane lacking the typical kinase activity of a signaling molecule, a third type of receptor, to type III.
[1817] The PRO317 receptor or other PRO317-binding molecule can be identified by interaction with radiolabeled PRO317. Radioactive labels can be incorporated into PRO317 by a variety of methods known in the art. A preferred embodiment is to label the major amino group of PRO317 with a ¹²⁵ I Bolton-Hunter reagent (Bolton and Hunter, Biochem. J. , 133 : 529 (1973)), Without secondary loss of biological activity (Hebert et al ., J. Biol. Chem. , 266 : 18989 (1991) and McColl et al., J. Immunol. , ≪ / RTI > 150 : 4550-4555 (1993)). The receptor-bearing cells were incubated with labeled PRO317. The cells were then washed to remove unbound PRO317 and receptor-bound PRO317 was quantitated. Values for the number and affinity of the receptors were calculated using data obtained using various concentrations of PRO317.
[1818] The labeled PRO317 is useful as a reagent for the purification of its specific receptor. In one embodiment of affinity purification, PRO317 is covalently bound to the chromatography column. The receptor-bearing cells were extracted and the extract passed through a column. The receptor binds to the column by its biological affinity for PRO317. The receptor was recovered from the column and the N-terminal protein was sequenced. Next, a degenerate oligonucleotide probe was designed to clone the receptor gene using the amino acid sequence.
[1819] Alternatively, mRNA was obtained from receptor-bearing cells to generate cDNA libraries. Cell populations were transfected with this library and cells expressing the receptor were screened using fluorescently labeled PRO317. This receptor is identified by recovering from highly labeled cells and sequencing the recombinant DNA.
[1820] Alternatively, the antibody is made against the surface of a receptor-bearing cell, particularly a monoclonal antibody. Monoclonal antibodies are screened to identify monoclonal antibodies that inhibit the binding of the labeled PRO317. This monoclonal antibody is then used for affinity purification or expression of the receptor.
[1821] Soluble receptors or other soluble binding molecules are identified in a similar manner. The labeled PRO317 is incubated with an uterine extract or other suitable material. After incubation, PRO317 complexes of a size greater than the purified PRO317 are identified by size sorting techniques such as size-exclusion chromatography or density gradient centrifugation and purified by methods known in the art. N-terminal sequencing is performed on the soluble receptor or binding protein to obtain sufficient information for cloning if the soluble protein is known, for database identification, or if the soluble protein is unknown.
[1822] Example 64 : Measurement of PRO317-induced cell response
[1823] The biological activity of PRO317 is measured, for example, by binding PRO317 receptor to PRO317 of the present invention. Test compounds as antagonists were screened for their ability to block binding of PRO317 to the receptor. The test compound as an agonist of PRO317 can be prepared by the method described in Sadick et al. , Analytical Biochemistry, 235 : 207-214 (1996), in which the activation of receptor tyrosine kinases is activated by immune capture of the activating receptor and ligand- Using the KIRA-ELISA assay, which was monitored by quantitative analysis of induced phosphorylation levels, we screened for the ability to bind to the PRO317 receptor and affect the same physiological events as PRO317. This assay is suitable for monitoring PRO317-induced receptor activation by using PRO317 receptor-specific antibodies that capture activated receptors. These techniques can also be applied to other PRO polypeptides described herein.
[1824] Example 65 : Use of PRO224 in compound screening
[1825] PRO224 was expressed in cells capable of expressing PRO224 with membrane protein removed. Low density lipoprotein with detectable label was added to the cells and incubated for a sufficient time for endocytosis. The cells were washed. Cells were then analyzed for labeling in cells bound to the membrane after cell lysis. Low density lipoproteins in the cells were detected to determine if PRO224 belonged to the low density lipoprotein receptor protein family. Subsequently, proteins identified to belong to the above group were used to screen for compounds affecting these receptors, particularly affecting the uptake of cholesterol through these receptors.
[1826] Example 66 : Ability of PRO polypeptides to inhibit endothelial cell proliferation, proliferation promoted by vascular endothelial growth factor (VEGF)
[1827] Various PRO polypeptide abilities to inhibit VEGF-stimulated endothelial cell proliferation were tested. Specifically, bovine adrenocortical capillary endothelial (ACE) cells (up to 12-14 times subculture from the primary culture) were cultured in DMEM supplemented with 3 ng / ml VEGF, 10% calf serum, 2 mM glutamine, Plates were plated in 96-well microtiter plates (Amersham Life Science) at a density of 500 cells / well per 100 [mu] l in x pen / strept and fungizone. Controls were plated in the same way except that they did not contain VEGF. A test sample of the target PRO polypeptide was added in a volume of 100 [mu] l to a final volume of 200 [mu] l. Cells were incubated at 37 DEG C for 6-7 days. The medium was aspirated and the cells were washed once with PBS. Acid phosphatase reaction mixture (100 L, 0.1 M sodium acetate, pH 5.5, 0.1% Triton-100, 10 mM p-nitrophenyl phosphate). After incubation at 37 [deg.] C for 2 hours, 10 [mu] l 1N NaOH was added to quench the reaction. The OD at 405 nm was measured with a microtiter plate reader. Cells were cultured in the absence, cell alone, cell + FGF (5 ng / ml), cells + VEGF (3 ng / ml), cells + VEGF (3 ng / ml) + TGF- + VEGF (3 ng / ml) + LIF (5 ng / ml). (TGF-beta at a concentration of 1 ng / ml is known to block 70-90% of the cell proliferation promoted by VEGF).
[1828] The inhibition rate (%) of cell proliferation promoted by VEGF (3 ng / ml) was determined by measuring the acid phosphatase activity by the OD value at 405 nm (1) for non-promoted cells and (2) And TGF-beta inhibition in the control group. The results, as shown in Table 2 below, show the utility of PRO polypeptides in the treatment of cancer, particularly inhibition of tumor angiogenesis. The numerical value (relative inhibition rate) shown in Table 2 was calculated by calculating the inhibition rate (%) of VEGF-induced proliferation by PRO polypeptide in comparison with non-promoted cells, and this percentage was evaluated as 70 to 90% (%) Obtained by 1 ng / ml of TGF- [beta], which is known to block.
[1829] PRO namePRO concentrationRelative inhibition rate PRO2110.01%99.0 PRO2110.01%1.09 PRO2110.1%0.95 PRO2110.1%67.0 PRO2111.0%0.27 PRO2111.0%20.0 PRO2170.01%1.06 PRO2170.1%0.84 PRO2171.0%0.39 PRO2172.5 μM0.2 PRO21725 nM0.88 PRO217250 nM0.58 PRO1870.01%0.91 PRO1870.1%0.82 PRO1871.0%0.44 PRO2195.7 [mu] M0.61 PRO21957 nM1.09 PRO219570 nM0.97 PRO2460.01%1.04 PRO2460.1%1.0 PRO2461.0%0.49 PRO2280.01%0.99 PRO2280.1%0.93 PRO2281.0%0.57
[1830] PRO2280.01%0.95 PRO2280.01%0.98 PRO2280.1%0.77 PRO2280.1%0.88 PRO2281.0%0.16 PRO2281.0%0.48 PRO2450.01%0.76 PRO2450.1%0.35 PRO2451.0%0.11 PRO2450.48 nM1.03 PRO2454.8 nM0.95 PRO24548 nM0.49 PRO2210.01%1.03 PRO2210.01%1.06 PRO2210.1%0.82 PRO2210.1%0.93 PRO2211.0%0.31 PRO2211.0%0.43 PRO2580.01%0.98 PRO2580.01%1.06 PRO2580.1%0.95 PRO2580.1%1.02 PRO2581.0%0.6 PRO2581.0%0.69 PRO3017.0 μM1.02 PRO30170 μM0.88 PRO301700 μM0.44 PRO3010.01%0.92 PRO3010.1%0.85 PRO3011.0%0.68 PRO2240.01%101.0 PRO2240.1%65.0 PRO2241.0%23.0 PRO2720.01%0.95 PRO2720.1%0.57 PRO2721.0%0.18 PRO3280.01%0.98 PRO3280.1%0.96 PRO3281.0%0.6 PRO3310.01%0.88 PRO3310.1%0.82 PRO3311.0%0.56
[1831] Example 67 : Retinal neuron survival
[1832] The following examples demonstrate that PRO220 polypeptides have the effect of increasing the survival rate of retinal neuronal cells.
[1833] Seven-day-old Sprague-Dawley rat pups (mixed group: glia and retinal neurons) were sacrificed by CO ₂ anesthesia, and eyes were sterilized. Neuronal retinas were excised from the pigment epithelium and other ocular tissues and then dissolved in single cell suspensions using 0.25% trypsin in Ca ²⁺ , Mg ^{2+ -free} PBS. After retinas were incubated at 37 ° C for 7-10 minutes, 1 ml soybean trypsin inhibitor was added to trypsinize. Cells were plated at 100,000 cells per well in 96 well plates in DMEM / F12 supplemented with N2 with or without specific test PRO polypeptide. All experimental cells were incubated at 37 ° C in a 5% CO ₂ atmosphere saturated with water. After 2-3 days of culture, the cells were stained with calcein AM, fixed with 4% paraformaldehyde, and stained with DAPI to determine the total number of cells. Whole cells (fluorescence) were quantitatively analyzed with a 20 × objective lens magnification using a CCD camera and NIH imaging software for Mackintosh. In the well, the field of view is randomly selected.
[1834] The effect of varying concentrations of PRO220 polypeptides is shown in Table 3, where% survival is the total number of calcein AM-positive cells from days 2 to 3 of culture to the total number of DAPI- Respectively. A survival rate of more than 30% is considered to be positive.
[1835] PRO namePRO concentrationSurvival rate (%) PRO2200.01%2.4% PRO2200.01%4.1% PRO2200.1%3.0% PRO2200.1%3.1% PRO2201.0%72.4% PRO2201.0%42.1%
[1836] Example 68 : Liver photoreceptor survival
[1837] This example demonstrates the efficacy of PRO220 polypeptides to increase the survival rate of stromal photoreceptor cells.
[1838] Sprague-Dawley rat pups (mixed group: glia and retinal neuronal cell type) of 7 days old were sacrificed by CO ₂ anesthesia, and eyes were sterilized. The neural retina was excised from the pigment epithelium and other ocular tissues and then dissolved in a single cell suspension using 0.25% trypsin in Ca ²⁺ , Mg ^{2+ -free} PBS. After retinas were incubated at 37 ° C for 7-10 minutes, 1 ml soybean trypsin inhibitor was added to trypsinize. Cells were plated at 100,000 cells per well in 96 well plates in DMEM / F12 supplemented with N2, with or without specific test PRO polypeptides. All experimental cells were incubated at 37 ° C in a 5% CO ₂ atmosphere saturated with water. After 2-3 days of incubation, the cells were fixed with 4% paraformaldehyde and stained using CellTracker Green CMFDA. The photoreceptor cells were detected by indirect immunofluorescence using Rho 4D2 (multiple or IgG 1: 100), a monoclonal antibody of visual rhodopsin. The results are reported as the survival rate (%) divided by the total number of rhodopsin-positive cells in the culture medium 2 to 3 days after the culture, when the total number of calcein / cell tracker-rhodopsin positive cells in the culture medium was 2 to 3 days after the culture. Whole cells (fluorescence) were quantitatively analyzed at 20 × objective magnification using a CCD camera and NIH imaging software for Mackintosh. In the wells, the field of view was randomly selected.
[1839] The effects of various concentrations of PRO220 polypeptide are shown in Table 4 below. A survival rate of more than 10% is considered to be positive.
[1840] PRO namePRO concentrationSurvival rate (%) PRO2200.01%0.0% PRO2200.1%0.0% PRO2202.0%0.0% PRO22010%0.0% PRO22020%66.9% PRO2201.0%56.9%
[1841] Example 69 : Induction of endothelial cell apoptosis
[1842] The ability of RPO228 polypeptides to induce apoptosis in endothelial cells was tested in human venous umbilical vein endothelial cells (HUVEC, Cell Systems) using a 96 well format in 0% serum medium supplemented with 100 ng / ml VEGF HUVEC cells are easily removed from the plating surface, so pipetting in the well should always be as smooth as possible).
[1843] The medium was aspirated and the cells were washed once with PBS. 5 ml of 1 x trypsin was added to the cells in the T-175 flask and left until the cells were liberated from the plate (about 5-10 minutes). 5 ml of the culture medium was added to stop the trypsin treatment. The cells were spun at 1000 rpm for 5 min at 4 < 0 > C. The medium was aspirated and cells were resuspended in 10% serum media (Cell Systems) supplemented with 1 x penn / strep.
[1844] Cells were cultured in a 96-well microtiter plate (Amersham Life Science, Cystostar-T scintillation microtiter plate, Sigma-Aldrich) at a density of 2 × 10 ⁴ cells per well in a total volume of 100 μl in 10% serum (CSG- RPNQ160, sterile, tissue-culture treated, individual packaging). PRO228 polypeptides were added to dilutions of 1%, 0.33% and 0.11%, respectively. Cell-free wells were used as blanks, and wells containing only cells were used as negative controls. As a positive control, 50 [mu] l of a 1: 3 passage dilution was used as a 3x parent solution of astragalus spores. Apoptosis was detected by measuring the ability of the PRO228 polypeptide to induce apoptosis using Annexin V, an element of calcium and phospholipid binding proteins.
[1845] 0.2 ml of a stock solution of annexin V-biotin (100 쨉 g / ml) was diluted (1:25 dilution) in 4.6 ml of 2x Ca2 ⁺ binding buffer and 2.5% BSA. 50 [mu] l of the diluted Annexin V-biotin solution was added to each well (excluding the control) to a final concentration of 1.0 [mu] g / ml. Samples were incubated with Annexin-Biotin for 10-15 minutes prior to the direct addition of ³⁵ S-streptavidin. ³⁵ S-streptavidin was diluted with 2 x Ca ²⁺ binding buffer, 2.5% BSA and added to all wells to a final concentration of 3 x 10 ⁴ (cpm / well). The plate was then sealed, centrifuged at 1000 rpm for 15 minutes, and placed in an orbital shaker for 2 hours. 1450 microbeta trilux (Microbeta Trilux, Wallac). The results are shown in Table 5 below, and the background excess value (%) represents the percentage of the total amount per minute in excess of the negative control. An excess background value of 30% or more is considered positive.
[1846] PRO namePRO concentrationBackground Value Excess (%) PRO2280.11%0.7% PRO2280.11%47.6% PRO2280.33%92.2% PRO2280.33%123.7% PRO2281.0%51.4% PRO2281.0%95.3%
[1847] Example 70 : PDB12 cell inhibition
[1848] The following examples demonstrate that several PRO polypeptides have the effect of inhibiting protein production by PDB12 pancreatic duct cells.
[1849] PDB12 pancreatic duct cells are plated in fibronectin coated 96-well plates at 1.5 x 10 ³ cells per well in 100 μl / 180 μl of culture medium. 100 占 퐇 of a culture medium containing a PRO polypeptide test sample or a negative control lacking the PRO polypeptide was added to the wells to make a final volume of 200 占 퐇. Controls include culture media containing proteins that appeared to be inactive in this assay. Cells were incubated at 37 [deg.] C for 4 days. Subsequently, 20 μl of Allama blue dye (Alamar Blue Dye, AB) was added to each well and fluorescence readings were measured with a microtiter plate reader by excitation at 530 nm and release at 590 nm after 4 hours of AB addition . The standard used is bovine pituitary extract (BPE) free and various concentrations of BPE containing cells. Buffer or unknown CM controls were run twice in each 96-well plate.
[1850] The results of the analysis are shown in Table 6 below and the percent reduction of protein production was determined by comparing the protein concentration produced by the negative control cells (calculated as Allaam Blue dye) and the protein produced by the PRO polypeptide- Concentration (calculated as Alamar blue dye). A percent reduction in protein production of 25% or greater relative to negative control cells is considered positive.
[1851] PRO namePRO concentrationDecrease in protein production (%) PRO2110.1%0.0% PRO2110.01%0.6% PRO2111.0%59.7% PRO2872.0%22.3% PRO28710%18.2% PRO28750%67.5% PRO2872.0%45.53% PRO28710%57.3% PRO28750%52.24% PRO3012.0%0.0% PRO30110%59.8% PRO30150%65.6% PRO2932.0%0.0% PRO29310%40.4% PRO29350%56.7%
[1852] Example 71 : Promotion of adult cardiac hypertrophy
[1853] This assay is designed to measure the ability of various PRO polypeptides to stimulate adult cardiac hypertrophy.
[1854] Newly isolated ventricular myocytes from adult Sprague-Dawley rats (250 g) were plated at 2000 cells / well in a volume of 180 [mu] l. Cells were isolated and plated on day 1, and a PRO polypeptide-containing test sample or culture medium alone (negative control) (20 쨉 l volume) was added on day 2, and cells were fixed on day 5 and stained. After the staining, the cell size of the untreated cells was 0.0 compared to the control cells, and the cells of which the growth was moderately increased to 1.0 as compared with the control cells, and the cells whose growth was greatly increased as compared with the control cells 2.0, respectively. In this assay, growth increases compared to negative control cells were considered positive. The results are shown in Table 7 below.
[1855] PRO namePRO concentrationGrowth increase score PRO28720%1.0 PRO28720%1.0 PRO30120%1.0 PRO30120%1.0 PRO29320%1.0 PRO29320%1.0 PRO30320%1.0 PRO30320%1.0
[1856] Example 72 : PDB12 cell proliferation
[1857] This example demonstrates that several PRO polypeptides are effective in inducing the proliferation of PDB12 pancreatic duct cells.
[1858] PDB12 pancreatic duct cells were plated 100 ㎕ / 1.5 per well in 180 of culture medium ㎕ play × 10 ³ cells on fibronectin coated 96 well plates. 100 [mu] l culture medium containing PRO polypeptide test sample or negative control lacking PRO polypeptide was added to the wells to a final volume of 200 [mu] l. In this assay, the control group contains a culture medium containing the protein indicated to be inactive. Cells were incubated at 37 [deg.] C for 4 days. Subsequently, 20 μl of Allama Blue dye (AB) was added to each well and fluorescence readings were measured with a microtiter plate reader after 4 hours of AB excitation at 530 nm and emission at 590 nm after AB addition. The standard used is bovine pituitary extract (BPE) free and various concentrations of BPE containing cells. Buffer, or culture medium containing only unknown control, was run in 96-well plates twice.
[1859] The results from the above analysis are shown in Table 8 below and the percent increase in protein production was calculated by multiplying the protein concentration produced by the negative control cells (calculated as Allaam Blue dye) and the protein produced by the PRO polypeptide- And the protein concentration (calculated as Alamar blue dye). Positive rate of 25% or more protein production increase (%) compared to negative control cells was considered.
[1860] PRO namePRO concentrationProtein production growth rate (%) PRO3012.0%44.0% PRO30110%67.4% PRO30150%185.8% PRO3032.0%27.9% PRO30310%174.9% PRO30350%193.1%
[1861] Example 73 : Enhancement of neonatal cardiac hypertrophy induced by PRO224
[1862] This assay was designed to measure the ability of PRO224 polypeptides to promote neonatal cardiac hypertrophy.
[1863] Myocardial cells were obtained from day Harlan Sprague-Dawley rats. On day 1, cells (180 μl at 7.5 × 10 ⁴ / ml, serum <0.1%, freshly isolated) were added to 96-well plates previously coated with DMEM / F12 + 4% FCS. On day 1, a test sample (20 [mu] l / well) containing the test PRO224 polypeptide or culture broth (negative control) was added directly to the wells. Then, PGF (20 [mu] l / well) was added to the final concentration of 10 ^-6 M on the second day. Cells were stained on day 4 and scored by eyes on day 5, where cells with no increase in size compared to negative control were counted as 0.0, cells with intermediate size increased to 1.0 as compared to negative control, Cells showing a larger size increase compared to negative control were 2.0. The results are shown in Table 9.
[1864] PRO namePRO concentrationGrowth increase score PRO2240.01%0.0 PRO2240.1%0.0 PRO2241.0%1.0
[1865] Example 74 : In situ hybridization
[1866] In situ hybridization is a powerful general-purpose technique for the detection and localization of nucleic acid sequences in a cell or tissue sample. For example, it is useful in identifying gene expression sites, analyzing tissue distribution of transcription, identifying and locating virus infections, tracking changes in specific mRNA synthesis, and assisting in chromosomal mapping.
[1867] In situ hybridization was performed using a PCR-generated ³³ P-labeled riboprobe, according to the protocol of the optimal version of Lu and Gillett, Cell Vision 1: 169-176 (1994) Respectively. In brief, formalin-fixed and paraffin-embedded human tissues were dissected, paraffin was removed, proteins were removed with Proteinase K (20 g / ml) for 15 min at 37 ° C, And further in situ hybridization as described in Gillett, supra. The [ ^33- P] UTP-labeled antisense riboprobe was generated from the PCR product and hybridized overnight at 55 ° C. The slide was immersed in a Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
[1868] ³³ P-riboprobe synthesis
[1869] (125 mCi) of ³³ P-UTP (Amersham BF 1002, SA < 2000 Ci / mmol). The following ingredients were added to each tube containing dry ³³ P-UTP:
[1870] 2.0 [mu] l 5x Transfer buffer
[1871] 1.0 [mu] l DTT (100 mM)
[1872] ㎕ 2.0 NTP mix (2.5 mM: 10 μ; 10 mM of GTP, CTP and ATP + 10 H ₂ O, respectively ㎕)
[1873] 1.0 [mu] l UTP (50 [mu] M)
[1874] 1.0 [mu] l Rnasin
[1875] 1.0 [mu] l DNA template (1 [mu] g)
[1876] 1.0 ㎕ H ₂ O
[1877] 1.0 [mu] l RNA polymerase (usually the PCR product T3 = AS, T7 = S)
[1878] The tubes were incubated at 37 [deg.] C for 1 hour. After adding 1.0 [mu] L of RQ1 DNase, it was incubated at 37 [deg.] C for 15 minutes. 90 [mu] l TE (10 mM Tris pH 7.6 / 1 mM EDTA pH 8.0) was added and the mixture was pipetted onto DE81 paper. The remaining solution was loaded into a Microcon-50 microfiltration unit and spun using program 10 (6 min). The filtration device was reversed with a second tube and rotated (3 min) using program 2. After the final round of rotation, 100 [mu] l TE was added. One μl of the final product was pipetted onto DE81 paper and counted in 6 ml of Biofluor II.
[1879] The probe was run on a TBE / urea gel. 1-3 μl of probe or 5 μl of RNA Mrk III was added to 3 μl of loading buffer. The gel was placed on ice immediately after heating for 3 minutes in a heating block at 95 ° C. The wells of the gel were washed, the sample was loaded and run for 45 minutes at 180-250 volts. The gel was wrapped in saran wrap and exposed to XAR film with a sensitizer in a -70 &lt; 0 &gt; C refrigerator overnight for 1 hour to overnight.
[1880] ³³ P-hybridization
[1881] A. Pretreatment of frozen sections
[1882] The slide was removed from the freezer and placed in an aluminum tray and thawed at room temperature for 5 minutes. The tray was placed in a 55 ° C incubator for 5 minutes to reduce condensation. Slides were fixed in 4% paraformaldehyde on ice in a fume hood for 10 min and washed with 0.5 x SSC for 5 min at room temperature (20 x SSC 25 ml + SQ H ₂ O 975 ml). Proteins were removed in 0.5 μg / ml Proteinase K for 10 min at 37 ° C (12.5 μl of 10 mg / ml stock solution in 250 ml of pre-warmed RNase-free RNAse buffer), and the sections were washed with 0.5 × SSC at room temperature And washed for 10 minutes. The sections were dehydrated in 70%, 95% and 100% ethanol for 2 minutes each.
[1883] B. Pretreatment of paraffin-embedded sections
[1884] The paraffin on the slide was removed, placed in SQ H ₂ O and rinsed twice with 2 x SSC for 5 minutes each at room temperature. The sections were incubated with 20 [mu] g / ml Proteinase K (500 [mu] l of 10 mg / ml in 250 ml RNase-free RNase buffer; Rnase buffer, 37 [deg.] C, 30 min) -formalin tissue. Then, it was washed with 0.5 x SSC, and dehydration was performed as described above.
[1885] C. Prehybridization
[1886] The slides were placed in plastic boxes divided into compartments saturated with Box buffer (4 x SSC, 50% formamide). Covering the tissue with 50 ㎕ hybridization buffer (3.75 g dextran sulfate _{+ 6 ㎖ SQ H 2 O)} , vortexed, and 2 minutes while the cap loosened while heated to electromagnetic waves. After ice-cooling, 18.75 mL of formamide, 3.75 mL of 20 x SSC and 9 mL of SQ H ₂ O were added and the tissue was well vortexed and incubated at 42 ° C for 1-4 hrs.
[1887] D. Hybridization
[1888] 1.0 占¹⁰⁶ cpm of probe per slide and 1.0 占 퐇 of tRNA (50 mg / ml stock solution) were heated at 95 占 폚 for 3 minutes. Slides were ice-cooled and 48 [mu] l of hybridization buffer per slide was added. After vortexing, ³³ P mix were added to 50 ㎕ on the slide 50 ㎕ pre-mixed cargo. Slides were incubated overnight at 55 ° C.
[1889] E. Wash
[1890] After 2 x 10 minutes of washing with 2 x SSC, EDTA (20 x SSC 400 ml + 0.25 M EDTA 16 ml, V _f = 4 l) at room temperature, RNase A treatment (30 minutes in 10 ml of Rnase buffer mg / ml 500 占 퐇 = 20 占 퐂 / ml). The slides were washed with 2 x SSC, EDTA for 2 x 10 minutes at room temperature. The stringent washing conditions were: 0.1 x SSC, EDTA (20 ml 20 x SSC + 16 ml EDTA, V _f = 4 l) at 55 캜 for 2 hours.
[1891] F. oligonucleotides
[1892] In situ analysis was performed on the various DNA sequences disclosed herein. The oligonucleotides used in these analyzes are as follows.
[1893] (1) DNA33094-1131 (PRO217)
[1894] p1 5'-GGATTCTAATACGACTCACTATAGGGCTCAGAAAAGCGCAACAGAGAA-3 '(SEQ ID NO: 348)
[1895] p2 5'-CTATGAAATTAACCCTCACTAAAGGGATGTCTTCCATGCCAACCTTC-3 '(SEQ ID NO: 349)
[1896] (2) DNA33223-1136 (PRO230)
[1897] p1 5'-GGATTCTAATACGACTCACTATAGGGCGGCGATGTCCACTGGGGCTAC-3 '(SEQ ID NO: 350)
[1898] p2 5'-CTATGAAATTAACCCTCACTAAAGGGACGAGGAAGATGGGCGGATGGT-3 '(SEQ ID NO: 351)
[1899] (3) DNA34435-1140 (PRO232)
[1900] p1 5'-GGATTCTAATACGACTCACTATAGGGCACCCACGCGTCCGGCTGCTT-3 '(SEQ ID NO: 352)
[1901] p2 5'-CTATGAAATTAACCCTCACTAAAGGGACGGGGGACACCACGGACCAGA-3 '(SEQ ID NO: 353)
[1902] (4) DNA35639-1172 (PRO246)
[1903] p1 5'-GGATTCTAATACGACTCACTATAGGGCTTGCTGCGGTTTTTGTTCCTG-3 '(SEQ ID NO: 354)
[1904] p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGCTGCCGATCCCACTGGTATT-3 '(SEQ ID NO: 355)
[1905] (5) DNA49435-1219 (PRO533)
[1906] p1 5'-GGATTCTAATACGACTCACTATAGGGCGGATCCTGGCCGGCCTCTG-3 '(SEQ ID NO: 356)
[1907] p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGCCCGGGCATGGTCTCAGTTA-3 '(SEQ ID NO: 357)
[1908] (6) DNA35638-1141 (PRO245)
[1909] p1 5'-GGATTCTAATACGACTCACTATAGGGCGGGAAGATGGCGAGGAGGAG-3 '(SEQ ID NO: 358)
[1910] p2 5'-CTATGAAATTAACCCTCACTAAAGGGACCAAGGCCACAAACGGAAATC-3 '(SEQ ID NO: 359)
[1911] (7) DNA33089-1132 (PRO221)
[1912] p1 5'-GGATTCTAATACGACTCACTATAGGGCTGTGCTTTCATTCTGCCAGTA-3 '(SEQ ID NO: 360)
[1913] p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGGGTACAATTAAGGGGTGGAT-3 '(SEQ ID NO: 361)
[1914] (8) DNA35918-1174 (PRO258)
[1915] p1 5'-GGATTCTAATACGACTCACTATAGGGCCCGCCTCGCTCCTGCTCCTG-3 '(SEQ ID NO: 362)
[1916] p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGGATTGCCGCGACCCTCACAG-3 '(SEQ ID NO: 363)
[1917] (9) DNA32286-1191 (PRO214)
[1918] p1 5'-GGATTCTAATACGACTCACTATAGGGCCCCTCCTGCCTTCCCTGTCC-3 '(SEQ ID NO: 364)
[1919] p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGTGGTGGCCGCGATTATCTGC-3 '(SEQ ID NO: 365)
[1920] (10) DNA33221-1133 (PRO224)
[1921] p1 5'-GGATTCTAATACGACTCACTATAGGGCGCAGCGATGGCAGCGATGAGG-3 '(SEQ ID NO: 366)
[1922] p2 5'-CTATGAAATTAACCCTCACTAAAGGGACAGACGGGCAGAGGGAGTG-3 '(SEQ ID NO: 367)
[1923] (11) DNA35557-1137 (PRO234)
[1924] p1 5'-GGATTCTAATACGACTCACTATAGGGCCAGGAGGCGTGAGGAGAAAC-3 '(SEQ ID NO: 368)
[1925] p2 5'-CTATGAAATTAACCCTCACTAAAGGGAAAGACATGTCATCGGGAGTGG-3 '(SEQ ID NO: 369)
[1926] (12) DNA33100-1159 (PRO229)
[1927] p1 5'-GGATTCTAATACGACTCACTATAGGGCCGGGTGGAGGTGGAACAGAAA-3 '(SEQ ID NO: 370)
[1928] p2 5'-CTATGAAATTAACCCTCACTAAAGGGACACAGACAGAGCCCCATACGC-3 '(SEQ ID NO: 371)
[1929] (13) DNA34431-1177 (PRO263)
[1930] p1 5'-GGATTCTAATACGACTCACTATAGGGCCAGGGAAATCCGGATGTCTC-3 '(SEQ ID NO: 372)
[1931] p2 5'-CTATGAAATTAACCCTCACTAAAGGGAGTAAGGGGATGCCACCGAGTA-3 '(SEQ ID NO: 373)
[1932] (14) DNA38268-1188 (PRO295)
[1933] p1 5'-GGATTCTAATACGACTCACTATAGGGCCAGCTACCCGCAGGAGGAGG-3 '(SEQ ID NO: 374)
[1934] p2 5'-CTATGAAATTAACCCTCACTAAAGGGATCCCAGGTGATGAGGTCCAGA-3 '(SEQ ID NO: 375)
[1935] G. Results
[1936] In situ analysis was performed on the various DNA sequences disclosed herein. The results of these analyzes are as follows.
[1937] (1) DNA33094-1131 (PRO217)
[1938] The biological function is not clear because the expression pattern is very apparent. Human embryos were expressed in the gastrointestinal tract, the respiratory cartilage, the basal respiratory epithelium, the osteoblasts, the outer smooth muscle layer of the tendons and gonads, the optic nerve head, and the developing dermal layer. In adults, chimpanzee tongue was expressed in epidermis, prostate and bladder basal epithelial cells / muscle epithelial cells. In addition, it was expressed in lung lung lining cells of the adult lung, erectile tissues of the penis, and mesenchymal cells near the cerebral cortex (usually, glial cells). In the case of the kidney, the expression only appeared in the disease in cells surrounding the thyroid gland.
[1939] Tissue: Placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall , Pelvis and not.
[1940] Adult human tissues examined include : kidney (normal and end), adrenal gland, myocardium, aorta, spleen, lymph node, gallbladder, pancreas, lung, skin, eye (including retina), prostate, bladder, liver .
[1941] Non-human primate tissues examined :
[1942] (a) Chimpanzee tissue : salivary glands, stomach, thyroid, parathyroid, skin, thymus, ovary, lymph node.
[1943] (b) Rhesus monkey tissue : cortex, hippocampus, cerebellum, penis.
[1944] (2) DNA33223-1136 (PRO230)
[1945] The intercept represents a strong signal related to the arteries and veins of the fetus. In the artery, the signal appears to be restricted to smooth muscle / pericardial cells. Signals are also found in capillaries and glomeruli. Whether endothelial cells express the mRNA is not clear. Expression is also observed in epithelial cells of the fetal lens. Expression is also strongly expressed in the villi of placental trophoblasts, which are located between nutrient cells and fibroblast-like cells that express HGF and produce non-specific tissues. In adults, there is no evidence of expression, and the walls of the aorta and most vessels appear to be negative. However, expression was evident in the normal prostate, and in the vascular channels of the epithelium surrounding the gallbladder. Definitive expression was observed in the blood vessels of soft tissue sarcoma and kidney cell carcinoma. In short, it is a molecule that exhibits relatively specific angiogenesis in the fetal as well as some adult organs. Expression was also observed in the fetal lens and adult gallbladder.
[1946] In the second screening, vascular expression was observed in fetal blocks similar to those observed above. Expression was found in vascular smooth muscle rather than endothelium. In addition, expression has been found in the smooth muscle of the growing esophagus, as previously reported, and the molecule is not vascular specific. Expression was tested in four lung cancer types and four breast cancer types. Actual expression was observed in 3/4 or more lung cancers and in vascular smooth muscle of 2/4 or more breast cancers. In addition, in the case of one breast cancer, expression was found in stromal cells around the tumor (usually fibroblasts) in the development of nonspecific tissue. No endothelial cell expression was observed in this study.
[1947] (3) DNA34435-1140 (PRO232)
[1948] Expression level was high in prostate epithelium and bladder epithelium, and expression level was low in bronchial epithelium. High background values / low levels of expression were observed in many areas, especially bone, blood, chondrosarcoma, adult heart and fetal liver. This level of signal is thought to represent the background value, in part, when this level of signal is observed in the blood. All other tissues were negative.
[1949] Tissue : Placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall , Pelvis, testicles and not.
[1950] Adult human tissues examined : kidney (normal and end), adrenal, spleen, lymph node, pancreas, lung, eye (including retina), bladder, liver (normal, cirrhosis, acute dysfunction).
[1951] Non-human primate tissues examined :
[1952] Chimpanzee organization : Adrenal glands
[1953] Rhesus monkey tissue : cerebral cortex, hippocampus
[1954] In the second screening, expression was observed in the prostatic epithelium, the superficial layer of urinary incontinence of the bladder, the urinary epithelium surrounding the kidney pelvis, and the urinary tract of the ureter (once in two trials). The urethra of the rhesus monkey was negative; It is not clear whether this represents a lack of expression by the urethra or a result of the probe not cross-reacting with rhesus monkeys. Detection in the prostate and bladder was performed using an isotope detection technique and is similar to that described above. Expression of mRNA to the antigen is not specific to the prostate epithelium. The antigen may serve as a useful marker for urinary incontinence-induced tissue. Expression in the epidermal and posterior mitotic cells of the urinary tract epithelium is also difficult to express specific branch cell markers and is expected to be expressed specifically in the basal epithelium.
[1955] (4) DNA35639-1172 (PRO246)
[1956] Expression was strongly expressed in the fetal vascular endothelium, including the tissues of the CNS. Expression was low in the adult vasculature, including the CNS. It is not clear whether the expression level in tumor vascular endothelium is high. Signals also appeared in bone marrow stroma and adult spleen, but it is not clear whether they are cell-associated, and are usually associated with nonspecific background values at these sites.
[1957] Tissue : Placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall , Pelvis, testicles and not.
[1958] Adult tissues examined : kidney (normal and end), adrenal, spleen, lymph node, pancreas, lung, eye (including retina), bladder, liver (normal, cirrhosis, acute insufficiency).
[1959] Non-human primate tissues examined :
[1960] Chimpanzee organization : Adrenal glands
[1961] Rhesus monkey tissue : cerebral cortex, hippocampus
[1962] (5) DNA49435-1219 (PRO533)
[1963] Expression is moderate in fetal neurons in the fetal brain. Expression occurs inside the fetal retina and may occur in the growth lens. Expression occurs in fetal skin, cartilage, small intestine, placental villi and umbilical cord. In adult tissues, expression is very high in the gallbladder epithelium. Expression is weak in adult kidney, stomach, and intestinal epithelium. Expression is low in many types of cells in many tissues, and this can be related to the stickiness of the probe, so this data should be interpreted with caution.
[1964] Tissue : Placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall , Pelvis, testicles and not.
[1965] Adult tissues examined : kidney (normal and end), adrenal, spleen, lymph node, pancreas, lung, eye (including retina), bladder, liver (normal, cirrhosis, acute insufficiency).
[1966] Non-human primate tissues examined :
[1967] Chimpanzee organization : Adrenal glands
[1968] Rhesus monkey tissue : cerebral cortex, hippocampus, cerebellum.
[1969] (6) DNA35638-1141 (PRO245)
[1970] Expression is observed in the endothelium surrounding the fetal and placental vessels. Endothelial expression was restricted to these tissue blocks. Expression was also observed in the placental intermediate cells. All other tissues were negative.
[1971] Ectopic , umbilical, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, Pelvis and not.
[1972] Adult tissues examined : liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cortex (rm), hippocampus (rm), cerebellum (rm) (Chimpanzee), ovary (chimpanzee), and chondrosarcoma. Acetaminophen-induced liver injury and cirrhosis.
[1973] (7) DNA33089-1132 (PRO221)
[1974] Expression was observed to be specific in fetal cerebral white matter and gray matter, and spinal cord neurons. The probe appears to cross-react with the rat. Expression was low in the cerebellar neurons of adult rhesus monkeys. All other tissues were negative.
[1975] Ectopic , umbilical, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, Pelvis and not.
[1976] Adult tissues examined : liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cortex (rm), hippocampus (rm), cerebellum (rm) Bell, intestine, intestinal carcinoma and chondrosarcoma. Acetaminophen-induced liver injury and cirrhosis.
[1977] (8) DNA35918-1174 (PRO258)
[1978] Expression was high in the nervous system. In the rhesus monkey brain, expression was observed in the cortex, hippocampus, and cerebellar neurons. Expression was found within spinal neurons, growing brain and fetal retina in fetal spinal cord. Expression was seen not only in the growing rhythm and autonomic ganglia, but also in the small intestine. Expression was seen in ganglion cells of adult prostate. In rats, expression was higher in the growing hindbrain and spinal cord. Expression was higher in interstitial cells of placental villi. All other tissues were negative.
[1979] Ectopic , umbilical, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, Pelvis and not.
[1980] Adult tissues examined include liver, kidney, kidney cell carcinoma, adrenal gland, aorta, spleen, lymph node, pancreas, lung, myocardium, skin, cortex (rm), hippocampus (rm), cerebellum (rm) , Gastric carcinoma, bowel, intestinal carcinoma, thyroid (chimpanzee), parathyroid (chimpanzee) ovary (chimpanzee) and chondrosarcoma. Acetaminophen induced liver injury and cirrhosis.
[1981] (9) DNA32286-1191 (PRO214)
[1982] Fetal tissue: Expression was low in the whole mesenchyme. Expression was moderate in placental stromal cells and thyroid gland. Expression was low in cortical neurons. Adult tissue: All were negative.
[1983] Ectopic , umbilical, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, Pelvis and not.
[1984] Adult tissues examined : liver, kidney, adrenal gland, myocardium, aorta, spleen, lymph node, pancreas, lung and skin.
[1985] (10) DNA33221-1133 (PRO224)
[1986] Expression is confined to the vascular endothelium of the fetal spleen, adult spleen, fetal liver, adult thyroid, and adult lymph nodes (chimpanzee). The other expression site is the growing spinal ganglion. All other tissues were negative.
[1987] Tissue : Placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall , Pelvis and not.
[1988] Adult tissues examined include : kidney (normal and end), adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, eye (including retina), bladder, liver (normal, cirrhosis, acute dysfunction).
[1989] Non-human primate tissues examined :
[1990] Chimpanzee tissue : salivary glands, stomach, thyroid, parathyroid, skin, thymus, ovary, lymph node.
[1991] Rhesus monkey tissue : cerebral cortex, hippocampus, cerebellum, penis.
[1992] (11) DNA35557-1137 (PRO234)
[1993] Expression occurs specifically in motor neurons that grow on the abdominal side of the fetal spinal cord (develop into the abdominal projections of the spinal cord). All other tissues were negative. This may allow spinal motor neurons to grow, differentiate, and / or develop.
[1994] Ectopic , umbilical, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, Pelvis and not.
[1995] Adult tissues examined : liver, kidney, adrenal, myocardium, aorta, spleen, lymph node, pancreas, lung, skin, cortex (rm), hippocampus (rm), cerebellum (rm) Bell, intestine, intestinal carcinoma and chondrosarcoma. Acetaminophen induced liver injury and cirrhosis.
[1996] (12) DNA33100-1159 (PRO229)
[1997] Expression was high (in actual human macrophages) in mononuclear cells (macrophages) in fetal and adult spleen, liver, lymph nodes and adult thymus. Expression was highest in the spleen. Positioning and homology are in general consistent with their roles as scavenger receptors on the reticuloendothelial cells. Expression was also observed in placental mononuclear cells.
[1998] Tissue : Placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall , Pelvis and not.
[1999] Adult tissues examined include : kidney (normal and end), adrenal gland, myocardium, aorta, spleen, lymph node, gallbladder, pancreas, lung, skin, eye (including retina), prostate, bladder, liver (normal, cirrhosis, acute dysfunction).
[2000] Non-human primate tissues examined :
[2001] Chimpanzee tissue : salivary glands, stomach, thyroid, parathyroid, skin, thymus, ovary, lymph node.
[2002] Rhesus monkey tissue : cerebral cortex, hippocampus, cerebellum, penis.
[2003] (13) DNA34431-1177 (PRO263)
[2004] Expression has been extensively observed in human fetal tissues and mononuclear cells, usually macrophage +/- lymphocytes. Cell distribution follows the morphology of the blood vessels in many tissues. Expression was high in epithelial cells of fetal adrenal cortex. All adult tissues were found to be negative.
[2005] Ectopic , umbilical, liver, kidney, adrenal, thyroid, lung, heart, large vessels, esophagus, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, Pelvis and not.
[2006] Adult tissues examined : liver, kidney, adrenal, spleen, lymph node, pancreas, lung, skin, cerebral cortex (rm), hippocampus (rm), cerebellum (rm), bladder, stomach, intestine and intestinal carcinoma. Acetaminophen induced liver injury and cirrhosis.
[2007] A second screening demonstrated that expression occurs in epileptic mononuclear cells, usually tissue.
[2008] (14) DNA38268-1188 (PRO295)
[2009] Expression was observed at high levels in the ganglion cells of the human fetal spinal ganglion and large neurons of the proximal lobes of the growing spinal cord. In adult cases, chimpanzee adrenal medulla (neurons), rhesus monkey brain neurons (hippocampal [+++] and cerebral cortex) and neurons of ganglia of normal adult prostate (sections containing ganglion cells, Is not thought to be confined to the prostate). All other tissues were negative.
[2010] The placenta, umbilical cord, liver, kidney, adrenal, thyroid, lung, large blood vessels, stomach, small intestine, spleen, thymus, pancreas, brain, eye, spinal cord, body wall, pelvis, testicles And not.
[2011] Adult tissues examined : kidney (normal and end), adrenal, spleen, lymph node, pancreas, lung, eye (including retina), bladder, liver (normal, cirrhosis, acute insufficiency).
[2012] Non-human primate tissues examined :
[2013] Chimpanzee organization : adrenal.
[2014] Rhesus monkey tissue : cerebral cortex, hippocampus, cerebellum.
[2015]
[2016]
[2017]
[2018]
[2019]
[2020]
[2021]
[2022]
[2023]
[2024]
[2025]
[2026]
[2027]
[2028]
[2029]
[2030]
[2031]
[2032]
[2033]
[2034]
[2035]
[2036] Deposit of a substance
[2037] The following materials are deposited with the American Type Culture Collection (ATCC), 12301 Rockville Park Drive, Maryland, USA.
[2038] matter ATCC Accession Number Date of deposit
[2039] DNA32292-1131 209258 September 16, 1997
[2040] DNA33094-1131 209256 September 16, 1997
[2041] DNA33223-1136 209264 September 16, 1997
[2042] DNA34435-1140 209250 September 16, 1997
[2043] DNA27864-1155 209375 October 16, 1997
[2044] DNA36350-1158 209378 October 16, 1997
[2045] DNA32290-1164 209384 October 16, 1997
[2046] DNA35639-1172 209396 October 17, 1997
[2047] DNA33092-1202 209420 October 28, 1997
[2048] DNA49435-1219 209480 November 21, 1997
[2049] DNA35638-1141 209265 September 16, 1997
[2050] DNA32298-1132 209257 September 16, 1997
[2051] DNA33089-1132 209262 September 16, 1997
[2052] DNA33786-1132 209253 September 16, 1997
[2053] DNA 35918-1174 209402 October 17, 1997
[2054] DNA37150-1178 209401 October 17, 1997
[2055] DNA38260-1180 209397 October 17, 1997
[2056] DNA39969-1185 209400 October 17, 1997
[2057] DNA32286-1191 209385 October 16, 1997
[2058] DNA33461-1199 209367 October 15, 1997
[2059] DNA40628-1216 209432 November 7, 1997
[2060] DNA33221-1133 209263 September 16, 1997
[2061] DNA33107-1135 209251 September 16, 1997
[2062] DNA35557-1137 209255 September 16, 1997
[2063] DNA34434-1139 209252 September 16, 1997
[2064] DNA 33100-1159 209373 October 16, 1997
[2065] DNA35600-1162 209370 October 16, 1997
[2066] DNA34436-1238 209523 Dec. 10, 1997
[2067] DNA 33206-1165 209372 October 16, 1997
[2068] DNA35558-1167 209374 October 16, 1997
[2069] DNA35599-1168 209373 October 16, 1997
[2070] DNA36992-1168 209382 October 16, 1997
[2071] DNA34407-1169 209383 October 16, 1997
[2072] DNA35841-1173 209403 October 17, 1997
[2073] DNA33470-1175 209398 October 17, 1997
[2074] DNA34431-1177 209399 October 17, 1997
[2075] DNA39510-1181 209392 October 17, 1997
[2076] DNA39423-1182 209387 October 17, 1997
[2077] DNA40620-1183 209388 October 17, 1997
[2078] DNA40604-1187 209394 October 17, 1997
[2079] DNA38268-1188 209421 October 28, 1997
[2080] DNA37151-1193 209393 October 17, 1997
[2081] DNA35673-1201 209418 October 28, 1997
[2082] DNA40370-1217 209485 November 21, 1997
[2083] DNA42551-1217 209483 November 21, 1997
[2084] DNA39520-1217 209482 November 21, 1997
[2085] DNA41225-1217 209491 November 21, 1997
[2086] DNA43318-1217 209481 November 21, 1997
[2087] DNA40587-1231 209438 November 7, 1997
[2088] DNA41338-1234 209927 June 2, 1998
[2089] DNA 40981-1234 209439 November 7, 1997
[2090] DNA37140-1234 209489 November 21, 1997
[2091] DNA40982-1235 209433 November 7, 1997
[2092] DNA41379-1236 209488 November 21, 1997
[2093] DNA44167-1243 209434 November 7, 1997
[2094] DNA39427-1179 209395 October 17, 1997
[2095] DNA40603-1232 209486 Nov. 21, 1997
[2096] DNA43466-1225 209490 November 21, 1997
[2097] DNA43046-1225 209484 November 21, 1997
[2098] DNA35668-1171 209371 October 16, 1997
[2099] These depositions were made under the provisions of the Budapest Treaty on the International Recognition of Microorganism Deposit under the Patent Procedure and the Budapest Treaty (the Budapest Treaty). This ensures the maintenance of the survival culture of the deposit for 30 years from the date of deposit. The deposit will be sold under the agreement of the Budapest Treaty with ATCC, under the agreement between Genenciek and the ATCC, which will be granted at the time of granting the relevant US patent or at the time of publication of the US or foreign patent application, (C) to ensure a permanent and unrestricted sale of the progeny of water, and to the United States Patent and Trademark Office in accordance with the provisions of 35 USC § 122 and the United States Patent and Trademark Office Regulations therefor, including 37 CFR §1.14, in particular 886 OG 638 We guarantee the sale of the progeny to those who decide to have the right to do so.
[2100] The assignee of the present application has agreed that if the culture of the deposit is killed, lost or destroyed when cultivated under suitable conditions, the notification will promptly replace the substance with another identical substance. The sale of the deposited material shall not be construed as permitting the implementation of the invention in violation of the rights granted by the national government under its patent law.
[2101] The foregoing specification is believed to be sufficient to enable those skilled in the art to practice the invention. It is to be understood that the present invention is not intended to be limited to the scope of the deposited structures, and any functionally equivalent structures are within the scope of the present invention, as the deposited embodiments are intended as an illustration of certain aspects of the invention. The deposit of a substance here does not mean that the description contained herein is inappropriate to carry out any aspect including the best mode of the present invention and is intended to limit the scope of the claims to the specific description presented in the specification It should not be interpreted. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing detailed description, which is within the scope of the appended claims.

权利要求:
Claims (18)
[1" claim-type="Currently amended] (SEQ ID NO: 2), 4 (SEQ ID NO: 4), 6 (SEQ ID NO: 12), 9 (SEQ ID NO: 39), 19 (SEQ ID NO: 49), 22 (SEQ ID NO: 59), 24 (SEQ ID NO: 84), FIG. 34 (SEQ ID NO: 91), FIG. 36 (SEQ ID NO: 96), FIG. (SEQ ID NO: 127), FIG. 48, SEQ ID NO: 137, FIG. 52, SEQ ID NO: 64, SEQ ID NO: 175, SEQ ID NO: 175, SEQ ID NO: 169, SEQ ID NO: (SEQ ID NO: 207), FIG. 78, SEQ ID NO: 213, FIG. 80, SEQ ID NO: 221, SEQ ID NO: , 90 (SEQ ID NO: 255), 92 (SEQ ID NO: 257), 94 (SEQ ID NO: 259), 96 (SEQ ID NO: 285), Figure 102 (SEQ ID NO: 290), Figure 104 (SEQ ID NO: 292), Figure 106 (SEQ ID NO: 294), Figure 108 (SEQ ID NO: 332), Figure 118 (SEQ ID NO: 339), Figure 120 (SEQ ID NO: 341) and Figure 122 (SEQ ID NO: 377), and a nucleotide sequence encoding a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: An isolated nucleic acid having greater than 80% sequence identity.
[2" claim-type="Currently amended] 2. The method of claim 1, wherein the nucleotide sequence is at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: (SEQ ID NO: 33), FIG. 16, SEQ ID NO: 38, FIG. 18, SEQ ID NO: (SEQ ID NO: 72), FIG. 31, SEQ ID NO: 90, SEQ ID NO: 90, SEQ ID NO: 49 (SEQ ID NO: 136), 51 (SEQ ID NO: 141), FIG. 53 (SEQ ID NO: 147), FIG. 55 69 (SEQ ID NO: 158), FIG. 59 (SEQ ID NO: 163), FIG. 61 (SEQ ID NO: 169), FIG. (SEQ ID NO: 194), Figure 73 (SEQ ID NO: 200), Figure 75 (SEQ ID NO: 206), Figure 77 SEQ ID NO: 244), Figure 87 (SEQ ID NO: 249), Figure 89 (SEQ ID NO: 254), Figure 91 (SEQ ID NO: 256), Figure 93 (SEQ ID NO: 260), 97 (SEQ ID NO: 262), 99 (SEQ ID NO: 284), 101 117 (SEQ ID NO: 338), FIG. 119 (SEQ ID NO: 340) and FIG. 121 (SEQ ID NO: 376) &Lt; / RTI &gt; wherein the nucleic acid comprises a nucleotide sequence selected from the group consisting of: &lt; RTI ID = 0.0 &gt;
[3" claim-type="Currently amended] 2. The method of claim 1, wherein the nucleotide sequence is at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: (SEQ ID NO: 33), FIG. 16, SEQ ID NO: 38, FIG. 18, SEQ ID NO: (SEQ ID NO: 72), FIG. 31, SEQ ID NO: 90, SEQ ID NO: 90, SEQ ID NO: 49 (SEQ ID NO: 136), 51 (SEQ ID NO: 141), FIG. 53 (SEQ ID NO: 147), FIG. 55 69 (SEQ ID NO: 158), FIG. 59 (SEQ ID NO: 163), FIG. 61 (SEQ ID NO: 169), FIG. (SEQ ID NO: 194), Figure 73 (SEQ ID NO: 200), Figure 75 (SEQ ID NO: 206), Figure 77 SEQ ID NO: 244), Figure 87 (SEQ ID NO: 249), Figure 89 (SEQ ID NO: 254), Figure 91 (SEQ ID NO: 256), Figure 93 (SEQ ID NO: 260), 97 (SEQ ID NO: 262), 99 (SEQ ID NO: 284), 101 117 (SEQ ID NO: 338), FIG. 119 (SEQ ID NO: 340) and FIG. 121 (SEQ ID NO: 376) Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt;
[4" claim-type="Currently amended] ATCC Accession No. 209258, ATCC Accession No. 209256, ATCC Accession No. 209264, ATCC Accession No. 209250, ATCC Accession No. 209375, ATCC Accession No. 209378, ATCC Accession No. 209384, ATCC Accession Nos. ATCC Accession No. 209,062, ATCC Accession No. 209480, ATCC Accession No. 209265, ATCC Accession No. 209257, ATCC Accession No. 209262, ATCC Accession No. 209253, ATCC Accession No. , ATCC Accession No. 209402, ATCC Accession No. 209401, ATCC Accession No. 209397, ATCC Accession No. 209400, ATCC Accession No. 209385, ATCC Accession No. 209367, ATCC Accession No. 209432, ATCC Accession No. , ATCC Accession No. 209251, ATCC Accession No. 209255, ATCC Accession No. 209252, ATCC Accession No. 209373, ATCC Accession No. 209370, ATCC Accession No. 209523, ATCC Accession No. 209372, ATCC Accession No. 209374, ATCC Accession No. 209373, ATCC Accession No. 209382, ATCC Deposit No. 209383, ATCC Accession No. 209403, ATCC Accession No. 209398, ATCC Accession No. 209399, ATCC Accession No. 209392, ATCC Accession No. 209387, ATCC Accession No. 209388, ATCC Accession No. 209394, ATCC Accession No. 209421, ATCC Accession No. 209393, ATCC Accession No. 209418, ATCC Accession No. 209485, ATCC Accession No. 209483, ATCC Accession No. ATCC Accession No. 209481, ATCC Accession No. 209481, ATCC Accession No. 209438, ATCC Accession No. 209927, ATCC Accession No. 209439, ATCC Accession No. 209489, ATCC Accession No. 209433 , ATCC Accession No. 209488, ATCC Accession No. 209434, ATCC Accession No. 209395, ATCC Accession No. 209486, ATCC Accession No. 209490, ATCC Accession No. 209484 or ATCC Accession No. 209371 An isolated nucleic acid comprising a full-length coding sequence of the deposited DNA.
[5" claim-type="Currently amended] A vector comprising the nucleic acid of claim 1.
[6" claim-type="Currently amended] 6. The vector of claim 5, operatively linked to a regulatory sequence recognized by a host cell transformed with the vector.
[7" claim-type="Currently amended] A host cell comprising the vector of claim 5.
[8" claim-type="Currently amended] 8. The host cell according to claim 7, which is a CHO cell.
[9" claim-type="Currently amended] 8. The method according to claim 7, Host cells.
[10" claim-type="Currently amended] 8. The host cell according to claim 7, which is a yeast cell.
[11" claim-type="Currently amended] 8. A method of producing a PRO polypeptide, comprising culturing the host cell of claim 7 under conditions suitable for expression of the PRO polypeptide and recovery of the PRO polypeptide from the cell culture.
[12" claim-type="Currently amended] (SEQ ID NO: 2), 4 (SEQ ID NO: 4), 6 (SEQ ID NO: 12), 9 (SEQ ID NO: 39), 19 (SEQ ID NO: 49), 22 (SEQ ID NO: 59), 24 (SEQ ID NO: 84), FIG. 34 (SEQ ID NO: 91), FIG. 36 (SEQ ID NO: 96), FIG. (SEQ ID NO: 127), FIG. 48, SEQ ID NO: 137, FIG. 52, SEQ ID NO: 64, SEQ ID NO: 175, SEQ ID NO: 175, SEQ ID NO: 169, SEQ ID NO: (SEQ ID NO: 207), FIG. 78, SEQ ID NO: 213, FIG. 80, SEQ ID NO: 221, SEQ ID NO: , 90 (SEQ ID NO: 255), 92 (SEQ ID NO: 257), 94 (SEQ ID NO: 259), 96 (SEQ ID NO: 285), Figure 102 (SEQ ID NO: 290), Figure 104 (SEQ ID NO: 292), Figure 106 (SEQ ID NO: 294), Figure 108 (SEQ ID NO: 332), Figure 118 (SEQ ID NO: 339), Figure 120 (SEQ ID NO: 341) and Figure 122 Natural sequence PRO polypeptide.
[13" claim-type="Currently amended] ATCC Accession No. 209258, ATCC Accession No. 209256, ATCC Accession No. 209264, ATCC Accession No. 209250, ATCC Accession No. 209375, ATCC Accession No. 209378, ATCC Accession No. 209384, ATCC Accession Nos. ATCC Accession No. 209,062, ATCC Accession No. 209480, ATCC Accession No. 209265, ATCC Accession No. 209257, ATCC Accession No. 209262, ATCC Accession No. 209253, ATCC Accession No. , ATCC Accession No. 209402, ATCC Accession No. 209401, ATCC Accession No. 209397, ATCC Accession No. 209400, ATCC Accession No. 209385, ATCC Accession No. 209367, ATCC Accession No. 209432, ATCC Accession No. , ATCC Accession No. 209251, ATCC Accession No. 209255, ATCC Accession No. 209252, ATCC Accession No. 209373, ATCC Accession No. 209370, ATCC Accession No. 209523, ATCC Accession No. 209372, ATCC Accession No. 209374, ATCC Accession No. 209373, ATCC Accession No. 209382, ATCC Deposit No. 209383, ATCC Accession No. 209403, ATCC Accession No. 209398, ATCC Accession No. 209399, ATCC Accession No. 209392, ATCC Accession No. 209387, ATCC Accession No. 209388, ATCC Accession No. 209394, ATCC Accession No. 209421, ATCC Accession No. 209393, ATCC Accession No. 209418, ATCC Accession No. 209485, ATCC Accession No. 209483, ATCC Accession No. ATCC Accession No. 209481, ATCC Accession No. 209481, ATCC Accession No. 209438, ATCC Accession No. 209927, ATCC Accession No. 209439, ATCC Accession No. 209489, ATCC Accession No. 209433 , ATCC Accession No. 209488, ATCC Accession No. 209434, ATCC Accession No. 209395, ATCC Accession No. 209486, ATCC Accession No. 209490, ATCC Accession No. 209484 or ATCC Accession No. 209371 The amino acid sequence encoded by the deposited nucleotide The isolated PRO polypeptide having at least 80% sequence identity.
[14" claim-type="Currently amended] A chimeric molecule comprising a polypeptide according to claim 12 fused to a heterologous amino acid sequence.
[15" claim-type="Currently amended] 15. The chimeric molecule of claim 14, wherein the heterologous amino acid sequence is an epitope tag sequence.
[16" claim-type="Currently amended] 15. The chimeric molecule of claim 14, wherein the heterologous amino acid sequence is an Fc region of an immunoglobulin.
[17" claim-type="Currently amended] 14. An antibody that specifically binds a PRO polypeptide according to claim 12.
[18" claim-type="Currently amended] 18. The antibody of claim 17, wherein said antibody is a monoclonal antibody.

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同族专利:

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公开号 | 公开日

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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:
1998-09-16|Priority to PCT/US1998/019330

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1998-09-16|Priority to USPCT/US98/19330

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1999-09-15|Application filed by 제넨테크, 인크.

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1999-09-15|Priority to PCT/US1999/021090

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2001-09-15|Publication of KR20010087359A

优先权:

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申请号 | 申请日 | 专利标题

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PCT/US1998/019330|WO1999014328A2|1997-09-17|1998-09-16|Secreted and transmembrane polypeptides and nucleic acids encoding the same|

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USPCT/US98/19330|1998-09-16|

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PCT/US1999/021090|WO2000015796A2|1998-09-16|1999-09-15|Secreted and transmembrane polypeptides and nucleic acids encoding the same|