New dendritic cell co-stimulatory molecules

专利摘要:
B7-DC, a novel interstimulatory protein molecule, is described as a member of the B7 family, with its encoded DNA and an expression vector comprising this DNA. B7-DC protein, fragments, mixed polypeptides / proteins and other functional derivatives and transformed cells expressing B7-DC are useful in vaccine compositions and composition methods. Compositions and methods of composition are found with respect to inducing potential T cells that mediate responses that may be reflected in anti-tumor and anti viral immunity.
公开号:KR20030028466A
申请号:KR1020027014537
申请日:2001-04-27
公开日:2003-04-08
发明作者:파돌드류엠.；즈치야하루오；고르스키케빈에스.；쳉슈-와이
申请人:더 존스 홉킨스 유니버시티；
IPC主号:

专利说明:

New dendritic cell stimulating molecule {NEW DENDRITIC CELL CO-STIMULATORY MOLECULES}
[2] The development of T lymphocyte responses is a complex process involving cell-cell interactions and the generation of soluble modulators (cytokines or lymphokines). The reaction is regulated by a number of T cell surface molecules and other "accessory" surface molecules that act as "receptors" comprising T cell receptor (TCR) aggregates, and a number of cell surface " Differentiation antigens "are first captured by monoclonal antibodies (" CD molecules ").
[3] Selective activity of all lymphocytes is characterized by two signals; It is believed to require not only antigen specific signals or clone signals, but also second antigen non-specific signals (Janeway, C., Cold Spring Harbor Symp. Quant. Biol. 54: 1-14 (1989)). When lymphocytes have captured antigens alone without co-stimulation by so-called co-stimulatory molecules such as B7 described below, lymphocytes are also known as the inactivation of clones called "anergy" (Schwartz, R. Science 248: 1349 (1990)) or “apoptosis (programmed cell death)”; If co-signal stimulation is provided, lymphocytes will have a specific clonal proliferative response to the stimulating antigen. Without co-stimulation no amplification of any signal of the immune response to a given antigen occurs (June et al., Immunology Today 15: 321-331, 1994; Chen et al., Immunology Today 14: 483- 486; Townsend, SE and Allison, JP (1993) Science 259: 368-370).
[4] The quality and effect of the immune response is highly dependent on the type of antigen presenting cells (APCs) that process and present antigens against T cells. Density of peptide antigen / major histocampatibility complex (MHC) ligand capable of binding T cell receptor, and membrane binding co-stimulation signal by antigen-expressing cells upon soluble and / or T cell binding and activation The provision of is important. For these reasons, the tactics of immunological treatment began to focus on providing appropriate co-stimulatory molecules that enhance (a) the target antigen for the appropriate antigen-presenting cell type and (b) T cell activity.
[5] Antigen presenting cells that provide the signals required for activation of T cells include mononuclear leukocytes / macrophages, B lymphocytes, and most important dendritic cells (DCs). In the past, activated macrophages were considered to be important antigen expressing cells that initiate T cell responses in vivo. This notion is based on the ability to effectively remove antigens and to treat and display surfaces. More recently, interest has shifted to dendritic cells as the main initiator of antigens in particular T cell responses in vivo. Dendritic cells have a distinct appearance from the activated macrophages and are divided into other subtypes that can initiate distinct immune responses. A characteristic feature of dendritic cells is that they have a nearly 100-fold greater effect than macrophages on activating natural T cells in vitro. Today, the explanation for this effect is based on quantitative differences in molecules known to be important for antigen presentation. The present invention is based on the discovery of significant quantitative differences.
[6] The first signal in antigen presentation is initiated by the interaction of T cell receptors with antigens expressing the contents of the Group II major histocompatibility complex (class II MHC) molecules on antigen fused cells (Allen, Immunol. Today 8 : 270 (1987)). Co-stimulatory signals arrive from other molecules characterized by the B7 family, which are also expressed on antigen expressing cells, namely B7.1, B7.2 and possibly B7.3.
[7] The two proteins expressed on the surface of T cells are most characterized as ligands or corresponding-receptors for co-signal molecules such as B7. CD28 is an immunoglobulin (Ig) superfamily found in the most mature human T cells that act on T cell activity (Aruffo and Seed, Proc. Natl. Acad. Sco. 84: 8573-8577 (1987)). Is a homodimer protein. CD28 is structurally expressed on dormant T cells and increases after activity. After signaling through the T cell receptor, the ligand of CD28 induces the proliferation of T cells and the secretion of IL-2 (Linsley, PS, et al. (1991) J. Exp. Med. 173, 721-). 731; Gimmi, CD, et al. (1991) Proc. Natl. Acad. Sci. USA. 88, 6575-6579; Thompson, CB, et al. (1989) Proc. Natl. Acd. Sci. USA. 86, 1333-1337; Jun, CH, et al. (1990) Immunol. Today. 11, 211-6; Harding, FA, et al. (1992) Nature.356, 607-609. ). CD28 mediates cell-cell contact (“intercellural adhesion”) and the formation of antigen-independent intercellular interactions essential for immune responses (Springer et al. Ann. Rev. Immunol. 5 : 223-252 (1987).
[8] CTLA4 is a T cell molecule homologous to CD28, but is not expressed on the remaining T cells and exhibits the following T cell activation (Brunet, J. F. et al., (1987) Nature 328, 267-270). CTLA-4 is originally identified by differential screening of murine soluble T cell cDNA libraries (Brunet et al. Supra). The role of CTLA-4 as a second receptor for B7 has been described by Linsley et al. (1991) J Exp. Med. 174: 561-569, where B7 has a higher affinity for CTLA4 than CD28. It is shown that it has. Freeman et al. (1993) Science 262: 907-909 discloses CTLA-4 in mice lacking B7. Ligands of CTLA-4 are disclosed by Lenschow et al. (1993) Proc. Nat'l. Acad. Sci. 90: 11054-11058.
[9] Th cells secrete growth, and in the region of Th-B cells, differentially-induced cytokines such as IL-2, IL-4 and IL-6 are contacted in a targeted manner, which exists only as an antibody of Th cells. Helps to confirm activation of B cells and prevents activation of Bystander B cells.
[10] CD28 and CTLA-4 interact with an inter-stimulatory molecule, commonly known as B7. B7 was generally described as a B cell activating antibody because it was found on B cells and called B7 / BB-1 (Linsley et al. Proc. Nat'l. Acad. Sci. USA 87: 5031-). 5035 (1990), hereinafter, this molecule will be referred to as B7, B7-1 or B7.1). B7 and more recently described B7 homologues are also members of the Ig superfamily. Compared to CD28 and CTLA-4, B7 contains two external cellular Ig domains, which are the N-terminal variable domain followed by the constant domain.
[11] B-7 family members are generally represented on APCs and, as is known, have decisive importance in the activation of original T cells. These family members include B7-1 (= B7, or CD80) and B7 (or CD86). References describing B7-1 are described in Schwartz, RH Cell 71: 1065-1068, 1992; Chen, L. et al. Cell 71: 1093-1102; Freeman, GJ et al. J. Immunol 143: 2714-2722 , 1989; and Freeman, GJ et al. J. Exp. Med. 174: 625-631, 1991). References describing B7-2 are in Freeman, G. J. et al. Science 262: 909-911 813-960, 1993. In addition, murine B7-1 and B7-2, and human B7-1 and B7-1 are both described (Freeman, G. J. et al., 1989, supra; 1991, supra; and 1993, supra). Activated human lymphocytes express CTLA4 / CD28 bound to the inhibitory-receptors of B7-2 and B7-3, both of which can transmit costimulatory signals to T cells via CD28 or CTLA4.
[12] B7-2 is expressed about 24 hours following stimulation with anti-Ig or anti-MHC class II mAbs by B cells. B7-2 induces detectable IL-2 transcription and T cell proliferation. From about 48 to 72 hours after activation, B cells inhibited third CTLA4, identified by B7-1 and mAb BB-1 (Yokochi, T, et al. (1982) J. Immunol. 128, 823-827). It expresses both receptors, which are called B7-3. B7-3 is also expressed on B7-negative activated B cells and can co-stimulate T cell proliferation without detectable IL-2 production, indicating that B7-1 and B7-3 molecules have been separated . B7-3 is expressed on a broad variety of cells, including activated B cells, activated white blood cells, dendritic cells, Langerhans cells, and keratinocytes. After 72 hours of B cell activation, the expression of B7-1 and B7-3 begins to slow down. The presence of these CTLA4 / CD28 bound to inhibitor-receptors on the surface of activated B lymphocytes indicates that T cell interstimulation is mediated in part by periodic expression of these molecules following B cell activation.
[13] B7: The importance of the CD28 / CTLA4 Mutual Stimulus Pathway is self-evident. There is a direct relationship between increased T cell activation and increased B7 expression (Razi-wolf et al., Proc. Natl. Acad. Sci. USA, 89: 4210-4214 (1992)). T cells lack immunity, where they encounter peptide antibodies on cells lacking an interstimulatory ligand bound to the CD28 blockcade of the interstimulatory pathway, resulting in the development of antibodies that are particularly acceptable to murine and human systems. (Harding et al., Supra; Lenschow, DJ et al. (1992) Science. 257, 789-792; Turka, LA et al. (1992) Proc. Natl. Acad. Sci. USA. 89. 11102-11105; Gimmi, CD et al. (1993) Proc. Natl. Acad. Sci USA 90, 6586-6590; Bousiotis, V. et al. (1993) J. Exp. Med. 178, 1753-1763). Expression of B7 by negative murine tumor cells induces T-cell mediated specific immunity accompanied by tumor ablation and long lasting protection against tumor challenge (Chen, L, et al. (1992) Cell 71 : 1093-1102; Yownsend et al., Supra; Baskar, S, et al. (1993) Proc. Natl. Acad. Sci. 90,5687-5690.). Therefore, manipulation of B7: CD28 / CTLA4 pathways offers great potential to stimulate or suppress immune responses in humans.
[14] Interactivation between CD28 and B7 was characterized using genetic mixing of the extracellular portion of B7 or CD28 with Ig Cγ1 chains (Linsley et al. (1991) J Exp. Med. 173: 721-730 (1991). )). When B7Ig mixed proteins are immobilized, or when B7 is expressed on the surface of cells, such as infected CHO cells, they co-stimulate T cell proliferation. T cell stimulation with B7 + CHO cells also specifically stimulates transcription levels for IL-2.
[15] U. S. Patent No. 5521288 describes a method of modulating an immune response by contacting a CD28 positive T cell with fragments encoded by a portion of the DNA encoding B7, in particular the external cellular domain (ECD) of B7. It is about. Immune responses were also modulated by derivatives of B7, a mixed protein structure comprising at least B7 ECD and portions of other proteins, such as human IgCγ1 domains of varying solubility, at the valence and / or affinity of B7. Tie. For example, DNA encoding amino acid residues starting from 1-215 of B7-ECD may be combined with DNA encoding amino acid residues of sequences related to the hinge, CH2 and CH3 regions of human IgCγ1 and incorporated into the B7Ig mixed protein. Form the encoded DNA mixture. Also disclosed are methods of addressing immune system diseases mediated by T cells in a manner in which the B7 or B7Ig mixed protein is reacted with the T cells by binding to the CD28 receptor. T cell proliferation in transplantation versus host disease is avoided by reacting CD28 + T cells with B7Ig mixed protein bound with B7 antibody or immunosuppressant.
[16] US Pat. No. 5,586,10 discloses tumor cells that are tuned to express one or more T cell interstimulatory molecules, including B7-2 and B7-3. One embodiment additionally includes expression of B7. Modulation is by infection with nucleic acid encoding B7-2, B7-3 or B7 proteins. Tumor cells are also adjusted in a genetically similar manner. Tumor cells adjusted as described above are useful for treating tumor patients, preventing metastatic spread or preventing tumor recurrence. This recording discloses a method of specifically inducing a CD4 + T cell response to tumors.
[17] U. S. Patent No. 5942607 discloses isolated nucleic acids encoding novel CTLA4 / CD28 ligands that stimulate T cell activation. In one embodiment, the isolated nucleic acid is encoded by B7-2. Also disclosed are nucleic acids comprising at least a portion of a disclosed B7-2 sequence of total length. According to this technique, nucleic acid sequences are integrated into variable expression vectors, which can detect the synthesis of related proteins or peptides in various host cells, including mammalian and insect cells. Also disclosed are infected host cells encoded by these nucleic acid sequences and infected to produce proteins or peptides separated to include at least a portion of the B7-2 sequence.
[18] Dong H et al., Nat Med 19995: 1365-1399, which describe a third member of the B7 family, indicate that B7-H1 is not bound to either CD28, CTLA4 or Inducible CO-Stimulator (ICOS). Indicates. B7-H1 ligation is mutually stimulated in the T cell response to polyclonal stimulants and homologous antigens, preferably to the production of interleukin-10. IL-2, a small amount produced, was required for the effect of B7-H1 inter-stimulation.
[19] The study defined an inter-stimulatory molecule that may be associated with a previously unknown negative regulation of the immune response delivered to cells. The same study (Wang S et al., Blood . 2000; 96: 2808-2813) describes a new human B7-like gene designated B7-H2, a recognized expression on the surface of unfinished DCs derived from monocytes. did. Soluble B7-H2 and Ig-fused proteins, B7-H2Ig, are bound to activated T cells rather than idle. This binding is inhibited by the soluble form of ICOS (ICOSIg) but not CTLA4Ig. ICOCIg stains CHO cells infected with the B7-H2 gene. By using the suboptimal interconnection of CD3 as a stimulant, it was confirmed that the mutual stimulation of T cell proliferation by B7-H2Ig is dose-dependent and correlated with the secretion of IL-2. On the other hand, optimal CD3 ligation was found to preferentially stimulate IL-10 products. The authors conclude that B7-H2 is a putative ligand for ICOS T cell molecules.
[20] Swallow MM et al ., Immunity, 1999, 11: 423-432 reported that the replication of the new gene b7h is similar to the B7 molecule expressed in APCs. B7h mutually stimulates proliferation of purified T cells by acting as a different receptor than CD28 or CTLA-4. Surprisingly, although B7h was expressed as unstimulated B cells, its expression was induced in nonlymphoid cells (3T3 cells; undeveloped fibroblasts) treated with TNFα, and mice treated with LPS, a potent activator of TNFα. It is regulated by the nonlymphoid tissue of. This study defined new interstimulating ligands of T cells and suggested that induction of B7h by TNFα may directly increase self-perception in inflammation.
[21] Yoshinaga SK et al., Nature , 1999, 402: 827-832 described a novel receptor ligand pair that is mediated by rats. The receptor associated with CD28 is a murine homologue of the human protein ICOS and is expressed in activated T cells and T cells in resting memory. This ligand is an analog of the B7 molecule designated B7-related protein-1 (B7RP-1). B7RP-1 is a type of transmembrane protein that is equivalent to 20% and 19% amino acids in rat B7.1 (CD80) and B7.2 (CD86), respectively. This homologue is meaningful by sharing 27% identical proteins with B7.1 and B7.2 (Frreeman, GJ et al ., J. Exp. Med. 178: 2185-2192 (1993)). This homologue contains cysteines that are important for Ig loop formation at conserved sites (residing at 62, 138, 185 and 242 from initial methionine). Overall length, the relative position of the transmembrane region of B7RP-1 is similar to that of the B7 molecule (Greenfield, EA et al., Crit. Rev. Immunol. 18: 389-418 (1998)). B7RP-1 is shown to be expressed in B cells and macropages. ICOS and B7RP-1 did not interact with proteins in the CD28-B7 pathway, and B7RP-1 mutually stimulated T cells independently of CD28. Transgenic mice representing lysed proteins of the Fc portion of B7RP-1 and Ig (“B7-RP1-Fc”) had lymphoid proliferation in the spleen, lymph node and Peyer patches. The mutual stimulatory activity of B7RP-1 in vivo was found by explaining enhanced delayed-type hypersensitivity, which was previously sensitive to antigens treated with B7RP-1-Fc when challenged by antigen. The authors conclude that ICOS and B7RP-1 define novel receptor ligand pairs that are structurally related to CD28-B7 and related to adaptive immune responses.
[22] Yoshinaga SK et al ., Int Immunol, 2000 Oct, 12: 1439-1447, published the mutual stimulation of human T cells through the interaction of human B7RP-1 with ICOS. This ligand receptor pair interacted with an off rate K _D with about 33 nM and t _(1/2) greater than 10 minutes. TNFα differentially regulated the expression of human B7RP-1 on B cells, monosite and DC. TNFα enhanced B7RP-1 expression on B cells and monosite and limited expression on DC. Human B7RP-1-Fc protein, ie, cells expressed by membrane-bound B7RP-1, mutually stimulated proliferation of T cells in vivo. Special cytokines such as IFNγ and IL-10 were induced by B7RP-1 mutual stimulants. Although IL-2 levels did not increase significantly, B7RP-1 induced IL-2 dependent mutual stimulants. This study defined human orthologs for rat B7RP-1 and characterized human interaction with ICOS.
[23] PD-1 is an immune-limiting receptor expressed by activated T, B and myeloid cells. Mice lacking PD-1 showed multiple forms of autoimmunity due to a loss of peripheral tolerance. Freeman, GJ et al. , J. Exp. Med. 192: 1027-1034 (2000) reported that the ligand of PL-1 (PL-L1) is a member of the B7 gene family. Binding of PD-1 by PD-L1 resulted in a limitation of TCR-delivered lymphocyte proliferation. In addition, PD-1 signaling limited the suboptimal level of CD28-delivered cross-stimulation. PD-L1 is expressed by APCs (human monosites stimulated by INFγ and activated by DCs). Moreover, PD-L1 has been shown to appear in the heart and lungs. The authors described that the relative magnitude of the limited PD-L1 signal and the interstimulatory B7-1 / B7-2 signal in APCs determined the range of T cell proliferation, tolerance and autoimmunity thresholds. The presence of PD-L1 in nonlymphoid tissue can contribute to the immune response of the site of inflammation.
[24] References in this document are intended as admissions, and therefore none of the foregoing may be appropriate prior art.
[1] The present invention in the fields of biochemistry and medicine relates to novel proteins that are selectively expressed on the surface of dendritic cells and can be used as cell surface molecules or soluble substances in vaccine compositions that stimulate an immune response.
[138] 1 is a diagram depicting a map of hB7-DC located on human chromosome 9p24. HB7-DC maps to BAC clone RPCI-11.2.
[139] 2 shows B7-DC expressed differently between DCs and macropages. B7-DC mRNA distribution in bone marrow DCs, spleen DCs, macropages, macropage lines and tissues was assessed by realistic northern blot analysis using 0.5 μg / lane purified DNA in 1% agarose gel. It became. G3PDH was used as a modulator. J774A1, Raw264.7, Pu5-1.8 and WEHI cells are macropage stems.
[140] 3 shows a virtual Northern blot B7-DC representation of human DCs. Lane 1 shows human DCs incubated with GM-CSF + Flt-3L, lane 2 shows human placenta, and lane 3 shows human DCs incubated with GM-CSF + IL4. Oligonucleotides from the 5 'and 3' UTRs of human B7-DC were used to construct PCR-DNA probes used for virtual Northern analysis to analyze total RNA of human DCs. β-actin was used as a modulator to ensure the quality of mRNA.
[141] 4 shows a cytological flow showing surface protein synthesis of B7-DCs in mature BM-DCs. On day 9 murine BM-DCs were either Fc-blocked or contaminated with regulatory antibodies or B7-DC antiserum.
[142] The specificity of the binding was demonstrated by adding B7-DC-Ig to compete with anti-B7-CD binding with the surface of DCs.
[143] 5 shows that B7-DC binds to PD-1 rather than CTLA-4 or CD28.
[144] 293T cells were transiently infected with cells by pCAGGS-B7.1opCAGGS-B7-DC. Infected ones are stained by binding molecules of PD-1 or PD-1 and CTLA-4-Ig according to secondary antibodies labeled with PE.
[145] Figure 6 (left and right panels) shows the mutual stimulation of T cells proliferated by Anti-CD3 and B7-DC-Ig.
[146] Left graph: Purified T cells (CD4 + CD8) are B7.1-Ig (◆), B7-DC with anti-CD3 (mAb 2C11) and their fixed concentration (0.1 μg / ml). Cultured in Wells precoated by Ig (•) or Isotype control (▲). The results represent one representative experiment out of three. Cells are incubated for 72 hours, labeled with H-Thymidine and counted per minute (CPM; Count per minutes).
[147] Graph on the right: Purified CD8 T cells were fixed B7.1-Ig (◆), B7-DC-Ig (●) or Cultured in Wells precoated by Isotype control (▲). The results represent one representative experiment of the two. Cells are incubated for 72 hours, labeled with H-Thymidine and counted per minute (CPM; Count per minutes).
[148] 7 shows the mutual stimulation of the proliferative response of T cells with antigenic properties. RENCA cells were IFN treated for 72 hours to induce MHC class-2 synthesis and incubated in 12.5 μg / ml HA110-120 peptide.
[149] Transgenic T-cells with purified HA + IE ^d characteristics were either soluble in B7.1-Ig (◆), B7-DC-Ig (●), or isotopically controlled (▲). Were added to each other to increase gradually. Cells were incubated for 48 hours and labeled with H-Thymidine and counted per minute, and the results represent one of three representative experiments.
[150] FIG. 8 shows cytoplasmic secretions (liquid) of T cells stimulated by B7-DC.
[151] Top panel: Purified T cells were fixed B7.1-Ig (◆), B7- with fixed concentrations of 0.1 μg / ml and Anti-CD3 at a concentration of 0.12 μg / ml as shown on the left of FIG. Cultured in Wells precoated by DC-Ig (•) or Isotype control (▲). The results represent one representative experiment out of three.
[152] Lower panel: ν-IFN treated RENCA cells are loaded into 12.5 μg / ml HA (110-120) peptide. The HA (110-120) peptide has T cells by the purified HA + IE ^d characteristic transgenic T cells and the above-mentioned symbols of 2 µg / ml and are soluble B7.1-Ig, B7-DC-Ig or the same standard. Sample controls were incubated in conjunction with each other. The results represent one representative experiment, and the supernatants were collected after 24 and 48 hours of incubation and tested for potency of Lymphokines directed using ELICA.
[153] 9 shows that the antibody properties of B7-DC-Ig proliferated significantly after mutual stimulation in vivo.
[154] After an adaptive transfer of HA to cells by 2.5 * 10 ⁶ TCR transgenes, three groups of mice were immunized (Immunizeds.c), and the hind limbs of the mice were HA peptides (110-). 120) or incomplete IFA (Freund's adjuvant) or separately mixed B7-DC-Ig + IFA or IFA and isotype control antibodies were prescribed.
[155] Draining lymph nodes were harvested exactly 7 days. 1.5 × 10 ⁵ LN cells were incubated in HA peptides for 48 hours and radiotreated with ¹ μCi [ ³ H] thymidine for a limited time of 12 hours.
[25] In order to define genes encoding specific costimulatory molecules of new dendritic cells (DCs) for T cell activation, the inventors of the present invention deducted between DC and activated macrophage. subtracted) cDNA library screened. The approach through the subtraction of cDNA defines a gene expressed by DC but not by activated macrophages. The use of this approach has led to the discovery of new DC-specific genes that are useful for enhancing the potential of vaccines that rely on T cell activation. The present application relates to such genes.
[26] According to the present invention, a new code sequence called "B7-DC", which is in the DC library and not in the activated macrophage library, has been identified. The B7-DC gene is a component of the B7 group of genes encoding interstimulatory molecules. B7-DC is the first component of the B7 family with receptor properties that differ from DC-specific expression. The product of this gene plays an important role in coordinating the unique properties of DCs to stimulate T cells. Functional analysis shows that B7-DC is more activated than B7-1 in stimulating IFN _γ products by T cells. Accordingly, B7-DC DNA and polypeptides are useful for synthesis or methods that enhance the efficacy of vaccine synthesis at the cellular and molecular levels, both with and without antigen-specific. Do.
[27] In one embodiment, an isolated nucleic acid molecule is provided that encodes a mammalian protein called B7-DC that is selectively expressed in dendritic cells as compared to activated macrophages. The nucleic acid molecule preferably comprises a nucleotide sequence selected from the sequence SEQ ID NO: 1 (human) or SEQ ID NO: 5 (rodent). The present invention also supervises isolated nucleic acids that hybridize the nucleic acid molecules under stringent hybridization conditions. Preferred stringent conditions are about 45 ° C. 6X sodium chloride / sodium citrate (sodium) followed by a wash of about 50 ° C. 0.2X sodium chloride / sodium citrate (SSC). latent period in chloride / sodium citrate (SSC). The nucleic acid molecule preferably comprises the nucleotide sequence SEQ ID NO: 1. Such preferred nucleic acid molecules encode a protein having an amino acid selected from SEQ ID NO: 2 and SEQ ID NO4, or of SEQ ID NO: 2 having a biologically active fragment, homologue or other function. Encode derivatives.
[28] In a preferred embodiment, the nucleic acid molecule encodes an extracellular range of the B7-DC protein that encodes a co-stimulatory homolog, fragment or derivative having their other functions.
[29] In another embodiment, the nucleic acid molecule encoding the B7-DC fusion protein is
[30] (a) a first nucleic acid sequence encoding a first polypeptide that is all or part of a B7-CD protein (preferably SEQ ID NO: 2 or SEQ ID NO: 4)
[31] (b) optionally, a linker nucleic acid sequence fused to a structure of the first nucleic acid sequence and encoding the linker peptide; And
[32] (c) a second nucleic acid sequence linked to said first nucleic acid sequence or linker nucleic acid sequence structure and encoding a second polypeptide.
[33] It includes.
[34] The second polypeptide consists of a range of one or more Ig heavy chain constant regions, preferably two C ranges of human IgG, moreover IgG1.
[35] Also,
[36] (a) a promoter and
[37] (b) Optionally, there is provided an expression clinic comprising any of said nucleic acid molecules effectively linked with additional regulatory sequences that regulate the expression of the nucleic acid of the eukaryotic cell.
[38] The expression hospital may be a plasmid or viral hospital. These clinics are self-replicating RNA replicons (DNA-transcription or RNA), suicide RNA hospital DNA viruses (adenovirus, vaccina virus, etc.) and RNA cultured in packaged cell lines. Virion.
[39] The pathogenic DNA or RNA is polymerized with gold particles that are gun-mediated and introduced into the host, or polymerized with other polymers, for example, release into cells and tissues of desired portions. By controlled release formula to enhance
[40] In addition, hospital combinations
[41] (a) a first recombinant expression response in which a sequence constituting a nucleic acid encoding an antigen from which an immune response is induced is polymerized into a sequence of the antigen
[42] (b) a second recombinant expression hospital incorporated into a sequence further constituting a nucleic acid sequence or nucleotide sequence encoding a polypeptide interstimulator to at least one of a B7-DC polypeptide or a biologically active fragment, homologue, or other functional derivative.
[43] In addition, the fuming pathogens can mutually infect or sensitize host cells as a result of the mutual expression of proteins and interstimulatory polypeptides, fragments, homologs or derivatives.
[44] In a variation of this embodiment, (i) promote the spread of the expressed product (antigen), and (ii) display of antigens on APCs in the presence of nucleic acid (nucleic acid) is expressed ( increase the display, and (iii) promote the display of antigen in APCs of the host into which re-presentation (cross-priming) and the vector is introduced. Providing a third nuclear sequence that encodes a target protein. The target protein nucleic acid coding may be solubilized with a nucleic acid encoding an antigen or a co-stimulator or both. The acids of the first and second hospitals contain nucleic acids. In one embodiment, the hospital synthesis combines three antigen encoding nucleic acids with the mutual stimulator encoding nucleic acid (preferably B7-DC) and the "target" protein encoding nucleic acid in a single fusion construct.
[45] The present invention includes cells that have been transformed or infected with any of the nucleic acid molecules or expression paths described above. Preferably, the cell is an eukaryotic cell, preferably a mammalian cell, more preferably a human cell. The cells can be dendritic crystalline cells or their progenitors. In another embodiment, the cell is a tumor cell, preferably a malignant tumor cell that withstands an antigen, such as an antigen for a tumor in a host, or an unresponsive antigen in response to an immune response request.
[46] Preferred embodiments include exogenous nucleic acid molecules or biologically active fragments, homologs or the like encoding a mammalian B7-DC protein (preferably SEQ ID NO: 2 or SEQ ID NO: 4). Isolated mammalian tumor cells infected with other functional derivatives, when the protein, fragment, homologue or derivative is expressed by tumor cells, the tumor cells are contacted with the following T cells.
[47] (i) B7-DC protein, fragment, homologue or derivative bound to T cells; And
[48] (ii) tumor cells that mutually stimulate T cells to provide and hide or proliferate cytoplasmic division.
[49] The present invention also relates to a polypeptide which is selectively expressed in dendritic crystal cells with respect to active macrophages and has the following functional properties.
[50] (i) bound as a binding partner on T cells
[51] (ii) mutual stimulation to provide and hide or proliferate cellular division.
[52] In addition, the present invention includes biologically active fragments, derivatives or derivatives of other functions of the polypeptide.
[53] The polypeptide, fragment, homologue or functional derivative is preferably encoded by a nucleic acid molecule having a sequence of SEQ ID NO: 1 or SEQ ID NO: 5 or an equivalent of a fragment, homologue or nucleic acid molecule. Preferably, the polypeptide has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
[54] Peptides of such polypeptides or biologically active fragments, homologs or other functional derivatives may be provided by a preformal representation of one of the nucleic acids described above.
[55] Preferably, the polypeptide comprises the extracellular range of the B7-DC protein as follows.
[56] (a) amino acid residues 26-221 of SEQ ID NO: 2 (human) or
[57] (b) amino acid residues 26-221 of SEQ ID NO: 4 (mouse).
[58] Such polypeptides may essentially constitute the extracellular range of B7-DC.
[59] In addition, the present invention
[60] (i) fused directly to a second polypeptide, or
[61] (ii) optionally, fused to a linker peptide sequence that can be fused to a second polypeptide
[62] B7-DC fusion polypeptides having a first fusion comprising all or a portion of the B7-DC protein.
[63] In addition, said B7-DC fusion protein is preferably fused to a second polypeptide in one or more ranges of Ig heavy chain constant region having amino acid sequences corresponding to the range C _H 2 and C _H 3 hinges of the human immunoglobulin Cr1 chain. do.
[64] In one embodiment of the above fusion protein, the first fusion partner is a B7-DC protein in the extracellular range of the total length sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
[65] Preferably, the fusion protein binds to the binding partner on the T cells and mutually stimulates the T cells in a state of sufficient stimulation with the T cell receptor.
[66] In addition, the present invention is a two- or three-molecule fusion protein that is a two- or three-molecule of the fusion protein. Preferably, the chains are connected side by side longitudinally via a bisulfide bond or other interchain covalent bonds.
[67] In a preferred bimolecular fusion protein, the bimolecular body consists of the covalent binding of Cys residues in the C _H region of two Ig heavy chains which are identical Cys residues which are bisulfides linked in a bimolecularized IgH chain.
[68] The compacted protein of the present invention may comprise multiple bonds of two or more consecutive repeats of a second fusion partner that connects end to end vertically directly to a linker sequence between one or more monomers.
[69] The present invention also provides specific antibodies against epitopes of the B7-DC protein, which epitopes do not exist in known members of the B7 family of proteins. The epitope may be a linear or conformational epopope of a polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4. Preferably, the antibody is a monoclonal antibody derived from a single cell, more preferably an antibody derived from a single cell of human or humanized (via engineering).
[70] The present invention also provides a method of using the above-described antibody to standardize or quantify cells expressed as B7-DC polypeptide on the surface of the cell population. The configuration is as follows.
[71] (a) antibodies bind to cells called epitopes (antigenic determinants) and contact the cells of the population with the antibody described above;
[72] (b) Quantify or estimate the number of cells to which the antibody is linked.
[73] Another method provides for isolating cells called B7-DC polypeptides from their population. It is composed as follows.
[74] (a) antibodies bind to cells called epitopes (antigenic determinants) and contact the cells of the population with the antibody described above;
[75] (b) Quantify or detect polypeptides or fragments linked to antibodies.
[76] The present invention also provides a method for quantifying or detecting a B7-DC polypeptide, fragment or homologue in a sample. It consists of the following steps:
[77] (a) The antibody is linked to any polypeptide or fragment that withstands the epitope and contacts the sample with the antibody of claim 43.
[78] (b) Quantify or detect polypeptides or fragments linked to antibodies.
[79] The invention also provides a method of increasing or inducing a B7-DC polypeptide in an antigen presenting cell or precursor cell that increases the ability of cells to stimulate T cells in vitro or in vivo in front of a stimulant suitable for T cell receptors. Indicates. This receptor consists of transferring or infecting antigen presenting cells or precursor cells with the mediators expressed above, and the B7-DC polypeptide is induced or increased on cells. Antigen present cells are rather dendritic cells and precursor cells are dendritic cell precursors.
[80] The present invention provides methods for stimulating immune responses using polypeptide stimulation aids as well as stimulation aid elements of cells. One method for increasing the T cell response of a mammalian patient to antigenic stimulation is, with the antigenic stimulant, rather than the cancer cells, where the cells are effective to increase the T cell response to antigenic stimulation. It consists of administering cells of. The above is rather realized by the injection of antigen and stimulatory auxiliary elements.
[81] A method for increasing mammalian T cell response to antigen stimulation in combination with a cancer-associated antigen can be achieved by administering cancer cells that are effective to increase T cell response to cancer antigen stimulation, as described above, where the cancer cells appear as antigens. It constitutes an effective amount of cancer cells.
[82] A method for increasing the T cell response of a mammalian patient to antigenic stimulation may include the above-mentioned fusion polypeptide or protein, or the above-mentioned agent, together with an antigenic stimulant, where the administration of the polypeptide is effective to increase the T cell response to antigenic stimulation. Effective doses of polypeptides, fragments, homologues or functional derivatives.
[83] The invention also provides for the administration of an effective amount of the above-described antibody, where it is effective for the administration of the antibody to prevent stimulation of the T cells, to abolish the antibody response of the T cells, and eventually to inhibit the response of the T cells. To inhibit mammalian T cell responses to antibody stimulation. These methods are particularly useful for dealing with patients with tissue transplantation to facilitate transplantation or to inhibit transplant rejection. In the case of autoantibodies, this method prevents or eliminates autoimmune reactions and their pathological sequelae.
[84] The present invention provides a therapy with T cells that perform ex vivo stimulation with the elements of this invention. One method of increasing the immune response of a mammalian patient to antibody stimulation consists of:
[85] (a) obtaining T cells from the patient, from an immunologically compatible donor to the patient, or from an immunologically acceptable cultured cell line;
[86] (b) contacting the T cells ex vivo with an effective amount of cells described above where the contact is effective to increase the response of the T cells to antigenic stimulation; And,
[87] (c) Administration of the T cells of step (b) to the patient, resulting in an increase in the patient's immune response.
[88] In other antibodies, methods of increasing the immune response of a mammalian patient to antigenic stimulation consist of:
[89] (a) obtaining T cells from the patient, thereby obtaining T cells from an immunologically compatible donor to the patient, or from an immunologically acceptable cultured cell line;
[90] (b) a T cell with an effective amount of the fusion polypeptide described above, where (i) the polypeptide, fragment, homologue, or functional derivative described above is effective to increase the response of the T cells to antibody stimulation. Contact ex vivo; And,
[91] (c) Administration of the T cells of step (b) to the patient, resulting in an increase in the patient's immune response.
[92] In addition, the vaccine elements in the documentation provided consist of:
[93] (a) the cells described above represented by (i) a B7-DC fusion polypeptide or a protein, (ii) represented by a B7-DC polypeptide, a fragment, a homolog, or a functional derivative. ;
[94] (b) In general, where cells express themselves as antigens (for cancer cells that are resistant to cancer antigens), an additional source of antigen for which an immune response is required, even though it may not be required for cell-based vaccines ( source);
[95] (c) optionally, a general immunostimulator or adjuvant; And
[96] (d) A pharmaceutically or immunologically acceptable excipient or carrier for (a), (b), and (C).
[97] A method of inducing or elevating an immune response to an antigen in a mammalian patient is by administering to the patient an effective amount of the aforementioned vaccine components.
[98] In addition, co-stimulatory complexes used as antigens or vaccines comprise:
[99] (a) with a B7-DC polypeptide (preferably SEQ ID NO: 2 or SEQ ID NO: 4), fragment, homologue or functional derivative thereof, or B7-DC mixed polypeptide
[100] (b) Pharmaceutically and immunologically appropriate excipients or carriers.
[101] A method of increasing the immune response to an antigen or vaccine in a mammalian subject, comprising a combination of the antigen or vaccine and a method of administering the co-stimulatory complex described above to the subject.
[102] In a method of stimulating a systemic immune response to a tumor in a subject by administering genetically modified tumor cells to the subject, the tumor cells are
[103] (a) is derived from a tumor of an individual, and
[104] (b) genetically modified by in vitro induction of the aforementioned B7-DC nucleic acid,
[105] In stimulation of a systemic immune response, protein synthesis results in a costimulatory signal in the subject in which the dosing acts directly on the tumor.
[106] Tumors that have been properly treated or properly treated to prevent growth after dosing.
[107] Individuals who have undergone chemotherapy to reduce tumors or have undergone radiation or surgical resection prior to administering the therapeutic composition described above.
[108] Methods for inducing anti-tumor responses in mammals with positive-antigens against tumors include the following.
[109] (a) preparing a tumor cell or tumor cell stem as follows:
[110] (i) tumor cells for protein synthesis of antigens shared with mammalian tumors;
[111] (ii) tumor cells infected with the aforementioned B7-DC encoding nucleic acid vector, which, when protein synthesized, induces a T-cell response to the antigen of the tumor;
[112] (iii) tumor cells conditionally irradiated prior to step (b);
[113] (b) sufficiently administering to the subject a cell that proteolyses the antigen and the B7-DC molecule;
[114] In the method described above, the semi-tumor reaction is characterized as follows.
[115] (A) a reduction of at least 50% in the sum of the maximum vertical magnifications of all measurable damages;
[116] (B) evidence of no new damage, and
[117] (C) Existing damage no longer develops.
[118] In addition, a method of reducing the growth of the basic tumor or the regrowth of the tumor in a mammal that retains the tumor,
[119] (a) preparing a tumor cell or tumor cell stem as follows:
[120] (i) tumor cells for protein synthesis of antigens shared with mammalian tumors;
[121] (ii) tumor cells infected with the aforementioned B7-DC encoding nucleic acid vector, which, when protein synthesized, induces a T-cell response to the antigen of the tumor;
[122] (iii) tumor cells conditionally irradiated prior to step (b);
[123] (b) sufficiently administering to the subject a cell that proteolyses the antigen and the B7-DC molecule;
[124] It is made, including, through this method induces a system immune response to a particular melanoma tumor antigen.
[125] In mammals, a method of preventing recurrent growth of benign-antigen tumors,
[126] (a) preparing a tumor cell or tumor cell stem as follows:
[127] (i) tumor cells for protein synthesis of antigens shared with mammalian tumors;
[128] (ii) when synthesized, induces cells to co-stimulate T-cell responses to the antigens of the tumor and is infected with the aforementioned B7-DC encoding nucleic acid vectors;
[129] (iii) tumor cells conditionally irradiated prior to step (b);
[130] (b) sufficiently administering to the subject a cell that proteolyses the antigen and the B7-DC molecule;
[131] It comprises a, and through such a method induces a system immune response to a specific tumor antigen of the mammal, the immune response prevents tumor regrowth.
[132] Another embodiment of the present invention is directed to a method of providing a co-stimulatory signal in the vicinity of an antigen locally injected into a mammalian subject in order to increase the local response of inflammation or an immune response resulting in a systemic immune status against the antigen. The method consists of administering the following to the topical part of an individual.
[133] (a) cells synthesizing a sufficient amount of costimulatory to B7-DC polypeptides, fragments, homologs or functional derivatives thereof as described above;
[134] (b) antigen
[135] This common stimulus in physical proximity to the antigen increases the local development of the immune response and results in system immune status.
[136] In the above method, the antigen is suitably a tumor antigen and is a tumor antigen administered in (b) in the form of tumor cells or in the form of an antigenic substance of cytoplasmic inhibition.
[137] Tumor cells may also be cells that protein synthesize B7-DC polypeptides, fragments, homologs or derivatives of (a).
[156] Example
[157] The inventors have new proteins and nucleic acids that are now identified, which serve as the basis for improved immunotherapeutic compositions and methods. Humans and Murine form proteins in different forms of interstimulation, the B7-DCs found and described here.
[158] The DNA encoding for human BC-DC is shown below as Nucleotide sequence SEQ NO: 1.
[159]
[160] The DNA encoding for human BC-DC is as amino acid sequence SEQ ID NO: 2 (preferred sequence, annotated with modified cell membrane region and cytoplasmic tail).
[161]
[162] The extracellular region of this protein is from residue P ²⁶ to residue W ²²¹ .
[163] DNA replication comprises the murine B7-DC incoding coding sequence with the following nucleotide sequence SEQ ID NO: 3.
[164] The coding sequence (Triplets, underlined) begins with nucleotides starting with bold atg , the methionine codons, and completes with bold tags indicating stops of codons (nucleotides 951-953).
[165]
[166] SEQ ID NO: 5 is the coding order portion of SEQ ID NO: 3. The murine B7-DC protein encoded in the coding region of SEQ ID NO: 3 (ie SEQ ID NO: 5) has the amino acid sequence of SEQ ID NO: 4 shown below. (A leading sequence, annotated with modified cell membrane regions and cytoplasmic tails.)
[167]
[168] The extracellular region of this protein is from residue P ²⁶ to residue W ²²¹ . The complete DNA sequence of murine B7-DC (originally "butyrophilin-like protein" or "Btdc") is Ac. No. AF142780.2 of Genbank.
[169] Basic molecular research
[170] This study is illustrated in detail. The inventor uses a PCR select study that blends two important properties. First and foremost, the PCR reaction requires only a small amount of RNA. This technique follows the use of highly purified fruit DCs that have given practical freedom of contaminated microphages or parental cells or other potentially contaminated cells. Such highly refined open DCs have been known to be difficult to obtain large numbers. Secondly, the PCR selection process was very small, allowing for the duplication of specifically expressed genes.
[171] In order to assess genes finely expressed by DCs involved in cytoplasmic copies, activated microphages, and to assess genes associated with DC-specific functions, the present inventors applied cDNA subtraction studies. They use a modified PCR-based representative differential analysis (RDA) combined with suppressor PCR (PCR Select ™).
[172] General recombination DNA method.
[173] Basic texts teach the general method of molecular biology, including the following reference books:
[174] Sambrook, J et al. Molecular Cloning: A Laboratory Manual, 2nd Edition Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989; Ausubel, FM et al. Current Protocols in Moelcular Biology, Vol. 2, Wiley-Interscience, New York, (current edition); Kiregler, Gene Transfer and Expression: A Laboratory Manual (1990); Glover, DM, dd, DNA Cloning: A Practical Approach, Vol. I & II, IRL press, 1985; Albers, B, et al., Molecular Biology of the Cell, 2nd Edition, Garland Publishing, Inc., New York, NY (1989); Watson, JD et al., Recombinant DNA, 2 , Scientific American Books, New York, 1992, and Old, RW et al., Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2nd edition, University of California Press, Berkeley, CA (1981).
[175] Unless otherwise specified, particular nucleic acid sequences encompass conservative alternative modifications and complementary sequences. The term nucleic acid is synonymous with polynucleotide and is intended to be a gene, a cDNA molecule, an mRNA molecule, an oligonucleotide, and the following equivalents (expressed below). The size of the nucleic acid is referred to as kilobase (kb) or base pairs (bp). This judgment is derived from agarose or polyacrylamide gel electrophoresis (PAGE) from defined nucleic acid sequences by users or publications. Protein size is stated as molecular weight or length (number of amino residues) in kilodaltons (kDa). Protein size is from PAGE, from an assumed amino acid sequence based on a nucleic acid sequence or a known amino acid sequence encoded from the sequence. Is measured.
[176] In particular, cDNA molecules encoding amiroic acid sequences corresponding to B7-DC or fragments or derivatives can be synthesized by polymerase chain reaction (PCR) (see US 4,683,202) using primers derived from the protein sequences identified herein. have. This cDNA sequence can be assembled into eukaryotic (matured) or prokaryotic expression vectors. The resulting vector can then be used directly for the synthesis of fragments or derivatives by B7-DCs or appropriate host cells such as COS or CHO cells.
[177] This invention includes an isolated nucleic acid having a nucleotide sequence encoding a new B7-DC, fragment or equivalent thereof. The term nucleic acid as used herein is intended to include fragments or equivalents thereof. The nucleic acid sequence of the present invention may be DNA or RAN. Suitable nucleic acids are cDNAs encoding human B7-DC having the sequence SEQ ID NO: 1 or an equivalent thereof.
[178] Preferably the nucleic acid of the invention is a cDNA molecule encoding at least a portion of B7-DC. This cDNA can be made from mature DCs or mRNA extracted from other cells naturally synthesized with this protein. Nucleic acid sequences encoding B7-DC can be obtained from DC genomic DNA. Thus, DNAs encoding B7-DCs can be cloned from cDNA or cloned in genomic libraries in conformity with known protocols.
[179] CDNA nucleotide sequences encoding B7-DC can be obtained by separating total mRNA from appropriate cell lines. Double standard cDNA is prepared from total mRNA. cDNA can be inserted into a viral vector using an appropriate plasmid, bacteriophage, or one of well known techniques.
[180] In reference to nucleotide sequences, the term equivalent is intended to include sequences encoding structurally homologous and / or functional equivalent proteins. For example, the natural polymorphism of the B7-DC nucleotide sequence (particularly the third base of the codon) is summarized as a silent mutation without changing the amino acid sequence. However, polymorphisms involved in amino acid sequence changes in B7-DC exist in humans (or other mammals).
[181] It will be appreciated in the art that this allelic variant with one or more nucleotides (more than about 3-4% of the total coding sequence) will be found in humans that will be the basis for natural allelic changes. Any, allelic variant and resulting nucleic acid and polypeptide polymorphisms in the DNA encoding B7-DC are within the scope of the present invention.
[182] Furthermore, it may be one or more naturally occurring isoforms or a family member of the related and immunologically correlated B7-DC proteins described herein. Such isoforms or family members are defined as proteins that share the function of an amino acid sequence similar to B7-DC even if they are encoded by genes elsewhere.
[183] Fragment of nucleic acid
[184] Fragments of nucleic acid sequences are defined as one nucleotide sequence with fewer nucleotides than the nucleotide sequence encoding the full length of the B7-DC protein. The invention encompasses nucleic acid fragments, wherein the nucleic acid fragments encode a polypeptide, which (1) retains the ability of B7-DC to bind its natural ligand in T cells, and (2) (cytokines Retains the ability to increase or inhibit activated T cells (depending on how they are synthesized) that have modulated an activated immune response (as measured by T cell proliferation by T cells that have received production and / or first activation signals) .
[185] For example, a nucleic acid fragment as intended herein encodes a B7-DC polypeptide, which binds the surface of T cells to a receptor that has not yet been identified (but does not appear as CD28 or CTLA-4), Has the ability to deliver simultaneous stimulus signals to the lymphocytes. By other regions, the nucleic acid fragments presented are those that hybridize with nucleic acids of other animal species and are therefore useful for screening analytes that detect new proteins that are "interrelated" with B7-DC.
[186] In general, nucleic acid sequences encoding fragments of B7-DC polypeptides consist of nucleotides from sequences encoding mature proteins. In some instances, however, it is desirable to include all or part of the leading sequence portion of the nucleic acid. In addition, the nucleic acid sequences of the invention may include linker sequences, natural or improved restriction endonuclease sites and other sequences useful for manipulations involved in the replication, synthesis or purification of the encoded protein or fragments. Improvements to these and other nucleic acid sequences are described herein or are well known in the art.
[187] In one embodiment, the DNA encoding the amino acid sequence corresponding to the ECD of B7-DC and comprising the amino acids at positions 26-221 is selected from human Ig Cr1. Wow The amino acid sequence corresponding to the phosphorus hinge is bound to DNA using PCR to form a construct synthesized with a B7-DC-Ig mixed protein. Analogous DNA molecules encoding B7-Ig mixed proteins are known from US Pat. No. 5,521,288 and deposited accession number 68627 in the American Type Culture Collection in Rockville, Md.
[188] Techniques for combining and synthesizing DNA encoding B7-DC and soluble B7-DC mixed proteins, oligonucleotide synthesis, PCR, cell transformation, vector construction, system synthesis, and the like are well established. Such general techniques are well described in standard resource materials for specific conditions and procedures.
[189] In other embodiments, the DNA encoding a domain or fragment of B7-DC is mixed with a nucleic acid encoding an extra majority or all of the other B7 family proteins such as B7.1, B7.2 or B7.3. The complete DNA sequence of human B7.1 (CD80) has genbank accession number X60958, accession number for the rat (mouse) sequence is X60958, and accession number for the rat (rat) sequence is U05593. The total cDNA sequence of human B72 (CD86) is Genbank Accession Number L25259 and accession number for the rat (mouse) sequence is L25606.
[190] Synthetic vector and host cell
[191] The invention includes synthetic vectors comprising nucleic acid sequences encoding B7-DC polypeptides operably linked to at least one regular sequence. By "operably linked" is meant that the coding sequence is linked to a regular sequence in a manner that allows the synthesis of the coding sequence. Known regular sequences are chosen to indicate the synthesis of the desired protein in the appropriate host cell. Thus, the term "regular sequence" includes promoters, enhancers and other synthetic control elements. Such regular sequences are described, for example, in Goeddel, Gene Expression Technology. Methods in Enzymology, vol. 185, Academic Press, San Diego, Calif. (1990).
[192] Those skilled in the art understand that the particular design of the synthetic vector of this invention depends on such considerations as the shape of the transfected host cell and / or the protein being synthesized.
[193] The synthetic vectors presented include the full range of nucleic acid molecules encoding various examples of B7-DCs: full-length proteins and functional derivatives thereof, including (eg, limited to) polypeptide fragments, variants, mixed proteins, and the like. Therefore, in one embodiment vector synthesis comprises nucleic acid encoding at least a portion of B7-DC, such as ECD itself or ECD mixed with other proteins.
[194] Such synthetic vectors are used for the transfection of host cells for the synthesis of DNA and for the cultivation of encoded proteins, including mixed proteins or peptides. It will be appreciated that genetically improved cells synthesizing the B7-DC polypeptide can temporarily synthesize exogenous DNA for sufficient time for cells useful for explicit purposes. Therefore, if a cell behaves as an immunogen with enhanced interstimulatory capacity in vivo or ex vivo, the length of time that synthesis is required or the cell survives may require the cell to perform its and / or interstimulatory functions as its immunogen. Is the time required. For example, in the case of transduced tumor cells synthesizing the B7-DC of the present invention, the synthesis of B7-DC can be as little as 6 hours, hopefully 24 hours, more preferably at least 2-4 days Can be. Synthesis can of course be stable (ie during the survival of the cell). Appropriate synthesis vectors and regular elements (e.g., inducible or constitutive promoters) mentioned below are chosen to match the required stability of the synthesis.
[195] The present invention provides a method for culturing B7-DC protein, fragments and derivatives. For example, host cells transfected with a nucleic acid vector encoding at least a portion of the B7-DC protein are cultured under appropriate conditions for the synthesis of the B7-DC polypeptide.
[196] Host cells can also be transfected into one or more synthetic vectors comprising DNA encoding at least a B7-DC protein portion and DNA encoding at least a second protein, such that the host cells comprise two proteins. Generate mixed polypeptides.
[197] When the recombiner synthesis vector comprises DNA encoding a portion of B7-DC and DNA encoding other proteins such as human IgCr1, the resulting mixed protein may be altered in availability, binding flexibility and / or valency.
[198] Prokaryotic or eukaryotic host cells that have been modified or infected to express B7-DC or cognate or functional derivatives are within the scope of the invention. For example, B7-DC will be expressed in bacterial cells, such as mammalian cells or host cells, such as E. coli, insect cells (baculovirus), yeast or Chinese hamster ovary cells (CHO). Other suitable host cells may be found in Goeddel, (1990) supra and are well known to those skilled in the art.
[199] Synthesis in echaotic cells leads to partial or complete glycosylation of recombinant proteins and / or related intrachain or interchain sulfite bonds.
[200] Examples of vectors for synthesis in yeast S. cerevisiae include pYepSecl (Baldari etall., (1987) EMBO J. 6: 229-234), pMFa (Kurjan et al. (1982) cell 30: 933-943), pJRY88 ( Schultz et al., (1987) Gene 54: 113-123), and pYES2 (Invitrogen Corporation, San Diego, Calif). Useful Baculovirus vectors for protein synthesis in cultured insect cells (SF 9 cells) include pAc series (Smith et al., (1983) Mol. Cell Biol. 3: 2156-2165) and pVL series (Lucklow, VA, and Summers, MD, (1989) Virology 170: 31-39. In general, COS cells (Gluzman, Y., (1981) Cell 23: 175-182) are used in combination with vectors such as pCDM 8 (Aruffo A. and B., supra) for transient amplification / synthesis in mammalian cells. do. Furthermore, CHO (dhfr-negative VHO) cells are used with vectors such as pMT2PC (Kaufman et al. (1987), EMBO J. 6; 187-195) for stable amplification / synthesis in mammalian cells. NSO myeloma cell line (glutamine synthesis synthetic system) is available from Celltech Ltd.
[201] Often, in the fusion synthetic vector, the cleavage site of proteolysis is introduced at the junction of the reporter group and the target protein to give the possibility of separation of consecutive target proteins from the reporter group for purification of the fusion protein. Enzymes of cleavage and proteolysis for their recognition sequences include factors Xa, thrombin and entrokinase.
[202] Typical fusion synthetic vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.), Wherein each of glutathione S-transferase, maltose E binding protein, or Protein A is fused to a target recombinant protein. And pRIT5 (Pharmacia, Piscataway, NJ).
[203] Inducible nonsynthetic vectors include pTrc (Amann et al., (1988) Gene 69: 301-315) and pET 11d (Studier et al., Gene Expression Technlolgy: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene synthesis relies on host PNA polymerase transcription from a hybrid trp-lac mixed catalyst in pTrc, while the synthesis of target genes injected into pET 11d is modulated by PNA polymerase (T7 gn1) of intersynthesized virus Depends on transcription from the T7 gn10-lacO mixed catalyst. The polymerase of this virus is provided by host strain BL21 (DE3) or HMS174 (DE3) from the resident λ propagation hide under the transcriptional control of the lacUV 5 catalyst.
[204] One embodiment of this invention is a transfected cell that synthesizes de novo novel B7-DCs. In the case of cells of already synthesized B7-DC, such as mature DC, the transfected cells are synthesized at an increased amount of B7-DC protein or fragment thereof at the cell surface.
[205] For example, tumor cells, such as sarcoma, melanoma, leukemia, lymphoma, carcinoma or neuroblastoma, may be B7-DC gene proteins. Transfection (infection of isolated nucleic acid cells) with a gene protein expression vector that directs synthesis is performed. Such infected tumor cells can be used as immunogens to promote therapeutic antitumor immunity as described below.
[206] Vector generation
[207] Generation of suitable vectors containing the desired coding and control sequences uses standard ligation and restriction techniques that are well known in the art.
[208] Isolated plasmids, DNA sequences, or synthesized oligonucleotides are isolated, cut, and re-ligated into the desired form.
[209] DNA sequences forming the vector are available from a number of sources. Backbone vectors and control systems are commonly found on the available 'host' vectors used for the bulk of the DNA sequence in production. For proper coding order, initial generation is probably a matter of obtaining suitable sequences, typically from cDNA or genomic DNA libraries. However, once the DNA sequence is found, it is possible to synthesize complete gene sequences in vitro, starting with the individual nucleotide derivatives. The complete genome sequence for a fairly large length (eg 500-1000 bp) genome synthesizes individual overlapping complementary oligonucleotides and uses single stranded with DNA polymerase in the presence of deoxyribonucleotide triphosphate. (single stranded) will be prepared by accumulating in the overlapping part. This approach has been used successfully for the generation of several genes of known sequence. See, eg, Edge, MD, Nature (1981) 292: 756; Nambair, KP, et al., Science (1984) 223: 1299; and Jay, E., J Biol Chem (1984) 259: 6311. .
[210] Synthetic oligonucleotides can be prepared by the phosphotriester method described in the references mentioned above or by Beaucage, SL, and Caruthers, MH, Tet Lett (1981) 22: 1859; and Matteucci, MD, and Caruthers, MH, J Am Chem Soc (1981) 103: 3185, prepared by the phosphoramidite method, and using commercially available automated oligonucleotide synthesizers Can be prepared. Kinase treatment of single strands prior to heat treatment or for labeling is obtained using excess (eg 50 mM Tris, pH7.6, 10 mM MgCl ₂ , 5 mM dithiothreitol, 1-). About 10 units of polynucleotide kinase for 1 nmole substrate in the presence of 2 mM ATP, 1.7 pmole γ- ³² P-ATP (2.9 mCi / mmole), 0.1 mM spermidine, 0.1 mM EDTA)
[211] Once the components of the desired vector are so available, they can be cut out and ligated using standard restriction and ligation procedures. Site-specipic DNA cleavage is a suitable restriction enzyme (or enzymes) under the conditions known in the art, generally known in the art and described by the manufacturer of these commercially available restriction enzymes. It is performed by treatment with (see, eg, New England Biolabs, Product Catalog.). Typically about 1 mg of plasmid or DNA sequence is separated into one unit enzyme in about 20 ml of buffer solution. And in the examples herein, an excess of restriction enzyme is typically used to ensure complete digestion of the DNA substrate. Although changes can be tolerated, incubation times of about one to two hours at about 37 ° C. are feasible. After every incubation, the protein is removed by extraction with phenol / chloroform, followed by ether extraction, and the nucleic acid is recovered from the aqueous fraction by precipitation with ethanol. Size separation of the separated fragments, if desired, will be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. A general explanation of size separation is found in Methods in enzymology (1980) 65: 499-560.
[212] Restricted isolated fragments were prepared using four dishes using an incubation time of about 15 to 25 minutes at 20 ° C. to 25 ° C. in 50 mM Tris pH7.6, 50 mM NaCl, 6 mM MgCl ₂ , 6 mM DTT and 0.1-1.0 mM DTT dNTP. The end will be blunted by treatment with large fragments of E.coil DNA polymerase I (Klenow) in the presence of oxynucleotide triphosphates (dNTPs). The Klenow fragment fills in the 5 'single-stranded overhang, but chews back the protruding 3' single strand despite the presence of four dNTPs. If desired, the selected repair can be performed by supplying only one or selected dNTPs within the limits dictated by the nature of the overhang. After treatment with Klenow, the mixture is extracted with phenol / chloroform and precipitated ethanol. Treatment under appropriate conditions with Si nuclease or BAL-31 results in hydrolysis of any single-stranded moiety.
[213] Ligation is typically performed in 15-50 ml volumes under the following standard conditions and temperatures: for example 20 mM Tris-HCl pH7.5, 10 mM MgCl 2, 10 mM DTT, 33 μg / ml BSA, 10-15 mM NaCl, and 40 μM ATP, 0.01-0.02 (Weiss) unit T4 DNA ligase at 0 ° C. (for 'viscous end' ligation). Intermolecular 'viscous end' nodules are typically performed at 33-100 μg / ml total DNA concentration (5-100 nM total end concentration). Intermolecular blunt end nodules are performed at 1 mM total end concentration.
[214] In vector synthesis using vector fragments, fragments are typically treated with bacterial alkaline phosphate (BAT) or calf intestinal alkali phosphate (CIAP) to remove 5 'phosphate and prevent self-ligation. Digestion is performed approximately at 1 mM EDTA, pH 8 with BAT or CIAP for one hour at 60 ° C. in 10 mM Tris-HCI, 1 unit / mg vector. The preparation is extracted from phenol / chloroform and ethanol precipitates. Alternatively, re-ligation can be prevented in the vector digested twice through the isolation of additional restriction enzymes and unwanted fragments.
[215] Some of the many methods are used to introduce mutations into coding sequences to generate variants of this invention. These mutations include simple removal or insertion, systematic removal, insertion or replacement of a cluster of bases or replacement of a single base.
[216] For example, modifications of the B7-DC DNA sequence (cDNA or genetic DNA) are generated by region-limited mutations. Well known protocols and reactants are commercially available (Zoller, MJ et al., Nucleic Acids Res (1982) 10: 6487-6500 and Adelman, JP et al., DNA (1983) 2: 183-193). . Calibration ligation for plasmid formation can be accomplished by, for example, the first modified E. coli chain MC1061 (Casadaban, M., et al., J Mol Biol (1980) 138: 179-207) or other suitable ligation mixture to the host. Is confirmed. Using conventional methods, the transformants are selected based on the presence of ampicillin-tetracycline or other antibiotic resistance genes (or other selectable markers) depending on the plasmid synthesis mode. The plasmid is then prepared from the transformants with chloramphenic amplification following optical chloramphenicol amplification (Clewll, DB et at., Proc Natl Acad Sci USA (1969)). 62: 1159; Clewell, DB, J Bacteriol (1972) 110: 667). Some mini DNA preparations are used in the usual way. See, e.g., Holmes, Ds, et al., Anal Biochem (1981) 114: 193-197; Birnboim, HC et al., Nucleic Acids Res (1979) 7: 1513-1523. The isolated DNA is further sequenced and / or analyzed by Sanger's didioxy nucleotide method described by Messing.
[217] The above objects, features and advantages will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.
[218] DNA vectors can be inserted into mammalian cells by inventive techniques such as calcium phosphate, calcium chloride co-precipitates, DEAE-dextran-delivery transfection, lipofection or electropolation. Suitable methods for the conversion of host cells can be found in "Sambrook et al. Supra" and other standard texts.
[219] Often, in lysis expression vectors, the hydrolytic cleavage site is employed in the binding of the reporter group and the target protein to allow separation for the target protein from the reporter group that involves purification of the lysed protein. Hydrolytic enzymes for such cleavage and binding include factors Xa, thrombin and enterokinase.
[220] Typical lysis expression vectors include pGEX, pMAL and pRIT5 in proteins of the desired preparation, which dissolve glutathione S-transferase, maltose E binding protein or protein A, respectively.
[221] Derived insoluble expression vectors include pTrc and pET 11d. Gene expression of interest depends on host RNA polymerase transcription from the hybrid trp-lac lysis promoter in pTrc, whereas expression of genes of interest inserted into pET 11d is coexpressed viral RNA polymerase (T7gn1). Depends on the T7 gn10-lacO lysis promoter mediated by).
[222] This viral polymerase contains an intrinsic T7gn1 under the transcriptional control of the lacUV 5 promoter. Provided by host strain BL21 (DE3) or HMS174 (DE3) from the phage.
[223] Promoter and Enhancer
[224] The promoter region of the DNA or RNA molecule binds the RNA polymerase and promotes the transcription of a "workable linked" nucleic acid sequence. As used herein, a "promotor sequence" is a nucleotide sequence of a promoter found on a strand of DNA or RNA transcribed by an RNA polymerase. Two arrays of nucleic acid molecules, having the same promoter and coding sequence, can each be linked to each other in such a way that they can be transcribed into the same RNA transcript or that the RNA transcript originating from one array can be extended to the second array. When, "working associated" is.
[225] As such, two sequences, such as a promoter sequence and a coding sequence of DNA or RNA, are linked to be feasible if they generate an RNA transcript of the coding sequence to which transcription initiated from the promoter sequence is feasible. In order to be "workable linked", the two arrays need not be immediately adjacent to different arrays in the linear array.
[226] Promoter arrangements preferred in the present invention must be feasible in the cells of a mammal and may be feasible in eukaryotic or viral promoters. Appropriate promoters can be induced, restrained, or structural.
[227] An example of a constitutive promoter is the viral promoter MSV-LTR. It is efficient and active for various cell types and, unlike most other promoters, has the same enhancing activity in the arrested or growing cells. Other preferred viral promoters include those present in CMV-LTR (from cytomegalovirus) or RSV-LTR (from Rous sarcoma virus). Also useful are a promoter of a murine metallothionein 1 molecule, a TK promoter of herpes virus, an SV 40 early promoter and a yeast gal4 molecule promoter.
[228] Another example of activity and DNA binding of transcription elements separated from transcriptional elements associated with promoter regions is described in "Keegan et al., Nature 231: 699; Fields et al., Nature (1989) 340: 245; Jones, Cell (1990) 61: 9; Lewin, Cell (1990) 61: 1161; Ptashne et al., Nature (1990) 346: 329; Adams et al., Cell (1990) 72: 306. " It includes. All appropriate published contents of such above listed references are hereby incorporated by reference.
[229] The promoter region may also include an octamer region that can act as a specific enhancer of the tissue through interaction with any protein found in that tissue. The enhancer domain of the DNA structure of the present invention is specialized for transfected target cells or is highly activated by the cellular elements of such target cells.
[230] Examples of vectors (plasmid or retrovirus) are described in U.S. Patent 5,112,767. A general description of enhancers and their behavior in transcription can be found in Lewin, B.M., Genes IV, Oxford University Press, Oxford, (1990), pp. 555-576. Particularly useful in retrovirus enhancers (e.g., LTR of viruses).
[231] Enhancers prefer to settle back from interacting promoters to focus the expression of cells. For the use of retroviral vectors, endogenous viral LTRs are enhancer-less and give other desirable properties, such as tissue specificity or transcriptional efficiency on B7-DC encoded DNA molecules. You can also use other desired enhancer arrays instead.
[232] Nucleic acid sequences of the invention can be synthesized chemically through standard techniques. Various methods are known for chemical synthesis of polydeoxynucleotides, including solid phase synthesis, which is fully automated in commercially available DNA synthesizers, such as peptide synthesis.
[233] Proteins and Polypeptides
[234] The present invention includes an "isolated" B7-DC protein having the arrangement of SEQ ID NO: 2 or SEQ ID NO: 4. While the content presented exemplifies the full-length human and murine B7-DC protein (and DNA), this is a murine of B7-DC from other mammals and modifications including the features so far presented, which are intended in the context of the present invention. Will be understood.
[235] Also included are "functional derivatives" of B7-DC, meaning amino acid substitution modifications. And, it includes the "flagment" or "chemical derivative" of the B7-DC, which will be described later. Functional derivatives retain the activity of important B7-DCs (preferably, binding to T cell receptors and costimulating T cell activity). "Functional derivative" includes here "variants" and "fragments" regardless of whether the term is used in combination or optionally.
[236] Functional mocha includes the biochemical and biological activity of the above. In view of this functional characterization the use of homologous protein B7-DCs from other species (including proteins not yet discovered) is an aspect of the present invention if these proteins have sequence similarity and encouraged biochemical and biological activity. Becomes
[237] To define the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal similar use (eg, the gap is the first and second amino acid sequences for optimal alignment, or May be in one or both of the nucleic acid sequences, for purposes of comparison non-homologous sequences are not considered)
[238] In a first sorting method, Cys residues are arranged.
[239] In a preferred embodiment, the length of the arrays to be compared is at least 30% of the length of the reference array, preferably at least 40%, more preferably 50%, more preferably 60%, 70%, 80% and 90 %to be.
[240] For example, the amino acid sequence having residues of 276 amino acids (SEQ ID NO: 2) when arranging the second sequence to human B7-DC protein, at least 83, preferably at least 110, more preferably at least 138, more preferably Is at least 166, and even more preferably at least 193, 221 or 248 amino acid residues arranged. This is then compared to the position of amino acid residues or nucleotides at the corresponding amino acid position. In the first sequence, when a position is occupied by the same amino acid residues or nucleotides, as the corresponding position in the second sequence, the molecules occupy the same position (where the amino acid or nucleic acid "identity" As used equivalently to the "homology" of this amino acid or nucleic acid). Percent identity between two sequences is a function of the same number of positions divided by the sequence of gaps, taking into account the number of gaps needed to guide the optimal arrangement of the two sequences, the length of each gap, and so on.
[241] The comparison of sequences between two sequences and the determination of percent identity are made using a mathematical algorithm. In a preferred embodiment, the percent identity between the two amino acid sequences is achieved by the Needleman and Wunsch algorithms (J. Mol. Biol. 48: 444-453 (1970)). The algorithm uses the NWSgapdna CMP matrix and 40, 50, 60, 70 Or incorporated into the GAP program in the GCG software package (available at http://www.gcg.com) using a gap weight of 80 and a length weight of 1, 2, 3, 4, 5, 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using an algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989)), which has a gap length penalty of 12. The ALIGN (Version 2.0) program was incorporated using the PAM120 residual weighting table, which applies a gap length penalty of 4 and 4.
[242] Nucleic acid and protein sequences of the present invention are further used as "query sequences" to perform searches against public databases, eg, to identify other family members or related sequences. These searches are described in NBLAST and Altschul et al. (1990) J. Mol. Biol. 215: 403-10 via the XBLAST program (version 2.0). BLAST nucleotide searches are performed with the NBLAST program with nucleotide sequence score = 100, word length = 12 to obtain nucleotide sequence cognates for humans or for murine B7-DC nucleic acid molecules.
[243] BLAST protein irradiation is performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences consistent with human or murine B7-DC protein molecules. To obtain gapped alignments for comparison purposes, Gapped BLASTrk Altschul et al. (1997) Nucleic Acids Res. It can be used as shown in 25: 3389-3402. When using the BLAST and Gapped BLAST programs, the default values of each program (eg XBLAST and NBLAST) can be used. See http: ///: www.ncbi.nlm.nib.gov.
[244] Therefore, the consensus with the B7-DC protein described above is (a) the functional activity of the native B7-DC, and (b) at least 30% (at the amino acid level), as determined above, preferably at least about 50%, more preferably at least about 70%, even more preferably at least about 90% of natural B7-DC (such as SEQ ID NO: 4).
[245] This is in the description of the area for expressing and obtaining proteins using DNA probes based on the closed sequences of B7-DC. Therefore, protein biochemistry and biological activity can be tested using known techniques such as those described herein, eg, basic T cell proliferation or cytokine secretion assays. T cell co-activity biological assays indicate whether an entity has the activity necessary to authorize an activity entity.
[246] The previous assay measures functional properties, such as stimulation of cytostatic synthetic T cells that rely on co-stimulatory signals as well as fixation or cross-linking of TCR (major activation signal). Fixing B7-DC to the natural ligands of its T cells carries a signal that causes increased cytoplasmic production, such as IL-2, which in turn stimulates measurable mesenchymal proliferation.
[247] Modifications of B6-DC refer to molecules of fragments in which the entire protein or substantially one or more amino acids are substantially substituted, subtracted or added to the residue. B6-DC fragments indicate a subset of molecules, preferably one of the polypeptides shorter than the total polypeptide length including the ECD.
[248] The number of processes can be used to generate fragments, mutations and transforms of an isolated DNA sequence. Small subregions and fragments (eg, 1-30 bases in length) of the nucleic acid encoding the B7-DC protein can be prepared by standard chemical synthesis.
[249] Oligonucleotides and primers derived from RNA or complementary DNA are used to generate larger synthetic fragments.
[250] The derivative of the selected function is a mixed protein, ie a polypeptide, which has a functional fragment of B7-DC.
[251] A useful derivative of B7, for example, is the B7-DC-Ig mixed protein, which forms a polypeptide corresponding to the ECD and Ig C regions of B7-DC.
[252] The presence of a mixed partner can alter the solubility, affinity and / or balance of the B7-DC protein (defined here as the number of possible linkage sites per molecule). have.
[253] Soluble B7-DC mixed protein, although still bound to the receptor on T cells, inhibits T cell stimulation by APC, i.e., antagonistic binding rather than costimulation, phase It may have a distinct biological effect compared to the original protein expressed in.
[254] Since the excess cell domain (ECD) of B7-DC is described herein as the entire excess cell portion of the protein or any fragment thereof, it may be necessary to replace other receptors on T cells that are not PD-1 or CD28 or CTLA-4. Perceived or tied up.
[255] Preferably, the ECD of B7-DC is such that the moiety encoded by the amino acid has approximately residues from position 26 to position 221 of SEQ ID NO: 2 or SEQ ID NO: 4.
[256] "Soluble B7-DC" is intended as a cell-free form of B7-DC which is diverted, hidden or otherwise extracted from producing cells. Therefore, soluble B7-DCs are soluble mixed proteins such as BC-DC-Ig, B7-DC free ECD or B7-DC ECD mixed (both biologically or chemically) up to the biologically active molecule. Include, but are not limited to this way.
[257] As noted above, the present invention also includes modified mixed proteins between the B7-DC domain and other B7 family proteins that appear on the cell surface in costimulatory form.
[258] The group selected among the B7-DC variants has been replaced by at least one amino acid residue or only one residue with other residues. For a detailed description of protein chemistry and structure, see Schultz, Ge et al., Principles of Protein Structure, pringer Verlag, New York, 1978, "Creighton, TE, Proteins L Stru8cture and Molecular Properties" and " WH Freeman & Co. San Francisco, 1983 ".
[259] The forms of substitutions made in protein molecules are described in Schultz et al. (supra) 'table 1-2 and' Creighton (supra) 'based on the analysis of the frequency of imino acid modification between homologous proteins of different species as shown in Figures 3-9. Based on this analysis, conservative substitutions were defined as corresponding to exchanges belonging to one of the five groups below.
[260] OneLow fat, nonpolar or low polar contentAla, Ser, Thr (Pro, Gly) 2Extremely negatively charged ingredients and their amidesAsp, Asn, Glu, Gln 3Extremely positively charged ingredientHis, Arg, Lys 4Large fat, nonpolar ingredientsMet, Leu, Ile, Val (Cys) 5Large fatPhe, Tyr, Trp
[261] Incidentally, the three amino acid components play a role in protein structure. Sugars are the only constituents that do not have a chain surface, thus giving the chain flexibility.
[262] Because of its unusual structure, pro tightly constrains the chain.
[263] cys may be involved in the disulfide band structure important in the protein layer.
[264] More substantial changes in biochemistry are made by less conservative substitutions in which functional values are chosen in five groups.
[265] Such changes will be more clearly different in the effect of maintaining a), b), c).
[266] a) the structure of the protein backbone in the substitution region, for example as a lamina or helical structure
[267] b) hydrophobicity of the charge or molecule at the target site
[268] c) or chain size such substitutions
[269] i) sugar (and / or) pro or deletion or insertion of sugar or pro by another amino acid
[270] ii) substitution of the hydrophilic component, for example ser or thr, hydrophobic component, for example by Leu, Lie, Phe, Val, Ala
[271] iii) substitution of other components of the cys component for
[272] iv) Substitution of the component has a positive chain surface. For example, Lya, Arg, His, components (for, by) have a negative electric charge. For example Glu or Asp or
[273] v) The substitution of components has a bulky chain surface. For example Phe
[274] The component has such a chain surface. For example
[275] The most acceptable deletions, insertions and substitutions according to the present invention, do not produce a fundamental change in the nature of the B7-DC protein due to T cell costimulatory behavior.
[276] However, when it is difficult to anticipate the exact effects of substitution, thanks to the deletion, insertion, and skill in art, one of the advances in doing so can be assessed by ordinary screening analysis without the inconsistent experimental requirements described here. something to do.
[277] Short chain variants, on the other hand, can be made by chemical synthesis. For the present invention, preferred long chain variants are typically made by site-specific mutation of nucleic acids. Input B7-DC polypeptides, expression of variant nucleic acids in cell culture and additionally washing of polypeptides from cell culture, e.g. immunoaffinity chromatography, utilize a fixed, specific antibody on the column (at least one epitope that is bound). To absorb variants by)
[278] Chemical Derivatives of B7-DC
[279] Chemical derivatives of B7-DCs include extraneous additional chemical moieties of the protein moiety. Covalent modifications of polypeptides are included within this invention.
[280] Such induced fractions may have resulted in solubility, absorption, biological half-life, and such improvements.
[281] A portion of the dose that mediates such effects is revealed. E.g
[282] Remington's Science of Pharmaceutical Sciences 16th Edition Mark Publishers Eastern Pimay (1980)
[283] Such transient variation can be introduced into the molecule by re-reaction of the desired amino acid component of the tissue derivative polypeptide with a selected chain face or terminal component, which is a re-reaction dose. Another transient variant is the circulation of the protein.
[284] Below is a chemically derived example of a polypeptide.
[285] Lysinyl and amino-terminal components have been derived from succinate and other carboxylic acid accompany.
[286] Inducers with cyclic carboxylic accompaniments have the effect of reversing the charge of lysine components. Other suitable reactants to induce a component comprising amino include methyl picolininidate, pyridoxinic phosphate, pyridoxinic colborohydride, trinitrobenzenisulfonic acid, o-methyllysorrea, 2,4 penta Includes imidoesters such as transsaminas-valatylated reactions with nidion and glyoxylate.
[287] The carboxyl side groups are asphaltyl or glutamiyl, 1-cyclonucleus-3- (2-morpholinyl- (4-ethyl) carbondiimide or 1-ethyl-3- (4-magonia-4, 4 With carbodiimides (RN = C = NR), such as -dimethylpentyl) carbodiimide, and optionally modified by re-reaction.
[288] Furthermore, the aspartyl and glutamic components have ammonia and can be reversed into asparaginyl and glutaminyl components by re-reaction.
[289] Other transient variations include the phosphorylation or threonyl component of the hydrosyl group of the hydroxylation of proline and lysine, the methylation of the lysine amino group (Creighton, supra, pp. 79-86), the acetylation of N-terminal amines, And the migration of the C-terminal carboxyl group.
[290] Also included in one or more D-amino acids is substituted for one or more L-amino acids.
[291] Multiple Binding Peptides
[292] Recent inventions also include longer polypeptides, wherein the basic polypeptide sequence obtained from the sequence of B7-DC is repeated about 2 to 100 times with or without voids or linkers. Such multiple bonds are believed to be made from any peptide variant defined herein. Moreover, peptide multiple bonds can consist of the binding of different peptide monomers and the substitution modifications revealed therefrom. Such oligomers or multiple binding peptides can be made by the chemical synthesis or DNA recombination techniques mentioned herein. When produced recombinantly, multiplexes can have as many repetitions as the expression system allows, for example about 2 to 100 times.
[293] In a serially arranged multimer, preferably a duplex or trimers, of a peptide or polypeptide of B7-DC, bound by an intersulfide disulfide bond or "artificial" covalent bonds between other chains. The chains in are bound side-by-side rather than end-to-end.
[294] Preferred duplexes or triplets are fusion proteins between B7-DCs, such as B7-DC-Ig, as described herein.
[295] Antibody Specification for epitope of B7-DC
[296] In the following description, references will be made to the various systematic classifications known by those techniques in immunology, cell biology, and molecular biology techniques. The reference was made in systematic taxonomy, where the publications and other materials that published such known systematic taxonomy were incorporated here as a whole, even if they were published as a whole. Standard reference materials that publish the laws of general immunology are described in A.K. Abbas et al., Cellular, Molecular Immunity (Fourth Edition), W. B. Saunder Co., Philadelphia, 2000; C. A. Janeway Publishing Co., New York, 1999; Roitt, I. et al., Immunology, (current version) C. V. Mosby Co., St. Louis, MO (1999); Klein, J., Immunology, Blackwell Sciectific Publications, Inc., Cambridge, MA, (1990)
[297] Monoclonal antibodies (mAbs) and methods for producing them are described below. Kohler and Milstein, Nature 256: 495-497 (1975); U.S.Patent No. 4,376,110; Hartlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988); Monoclonal Antibodies and Hybridomas; A New Dimension in Biological Analyses, Plenum Press, New York, NY (1980); H. Zola et al., In Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, 1982).
[298] Immunological validation methods are also described below. Coligan, J.E. et al., eds., Current Protocols in immunology, Wiley-Interscience, New York 1991 (or current edition); Butt, W. R. (ed) Practical Immunoassay: The State of the Art, Dekker, New york, 1984; Bizollon, Ch. A., ed., Monoclonal Antibodies and New York, 1984; Butler, J.E., ElISA (Chapter 29), IN: van Oss, C.J. etal., (eds),
[299] IMMUNOCHEMISTRY, Marcel Dekker, Inc., New York, 1994, pp. 759-803; Butler, J. E. (ed), Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton, 1991; Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986; Work, T.S. et al., Laboratory Techniques and Biochemistry in Molecular Biology, North Holland Publishing Company, NY, (1978) (Chapter by Chard, T., "An Introduction to Radioimmune Assay and Related Techniques").
[300] Anti-idiotypic antibodies are described, for example, in the following references. Idiotypy in biology and Medicine, Academic Press, New York, 1984; Immunological Reviews Volume 79, 1984; Immunological Reviews Volume 90, 1986; Curr. Top. Microbiol., Immunol. Volume 119, 1985; Bona, C. et al., CRC Crit. Rev. Immunol., Pp. 33-81 (1981); Jerne, NK, Ann. Immunol. 125 C: 379-389 (1974); Jerne, NK, In: Idiotypes-Antigens on the Inside, Westen-Schnurr, I., ed, Editiones Roche, Basel, 1982, Urbain, J et al., Ann. Immunol. 133D: 179- (1982); Rajewsky, K. et al., Ann. Rev. Immunol. 1: 569-607 (1983)
[301] Recent invention provides new epitopes of B7-DCs for polyclonal, monoclonal, and reactive antibodies, which have been removed from known B7 family proteins. Antibodies can be in the form of xenogeneic, allogeneic, syngeneic, or humanized or unmodified antibodies such as. Antiidiotypic antibodies against the idiotype of anti-B7-DC antibody were also included. The term antibody is also used to refer to fragments as well as intact molecules, which contain an antigen binding site and have the ability to bind to the B7-DC epitope. These include Fab and F (ab ') 2, which lack Fc fragments of intact antibodies, clean more quickly from circulation, and bind less non-specific tissue than intact antibodies (Whal et al., J). Nucl.Med 24: 316-325 (1983). It also includes Fv fragments (Hochman, J. et al. (1973) Biochemistry 12: 1130-1135; Sharon, J. et al (1976) Biochemistry 15: 1591-1594). These various pieces are made using traditional techniques such as protease cleavage or chemical cleavage (eg Rousseaux et al., Meth. Enzymol., 121: 663-69 (1986)).
[302] Polynominal antibodies are obtained from the serum of immunized animals, eg rabbits, goats, rodents, etc., and can be used directly without further manipulation, or by simple proliferation or ammonium sulfate precipitation, ion conversion chromatography, affinity chromatography It can be obtained by the same purification method (see Zola et al., Supra).
[303] Immune cells can be obtained from complete B7_D6 proteins or their tissues or derivatives. Previous immune cells are part or all of the ECD of human B7-DC (amino acid residues 26-221), an amino acid residue containing post-translational modifications such as glycosylation found in natural B7-DCs. I could get it from Immune cells synthesized in the residual cell region are produced by a variety of methods known as expression of cloned cells using convenient recombinant methods, isolation from myoblasts, and cell populations expressing high levels of B & -DC.
[304] mAbs can be made using simple complex techniques such as the method devised by Kohler and Milstein (Nature, 256: 495-97 (1975)) (see above references). Preferably, an animal such as a mouse obtains an antibody obtained preferably in a purified animal and is purified by immunization with immune cells as above.
[305] B lymphocytes from the peripheral blood of lymph glands, splins or clarified animals generally fuse with bone marrow cells along with promoters such as polyethylene glycol (PEG). Some of the numerous mouse bone marrow cell lines have been used for: P3-NS1 / 1-Ag4-1, P3-x63-k0Ag8.653, Sp2 / 0-Ag14, or HL1-653 bone marrow (available by ATTC, Rockville, MD). The next process involves proliferation in a selective medium to keep only complex cells alive and completely kill unfused bone marrow cells and donor lymph cells. They can be cloned and multiplied to the desired properties on their surface, such as the ability to replicate positive clones using B & -DC-IG synthetic proteins by immunoassay techniques or to isolate mAbs by limited dilution. Allow antibodies to develop.
[306] Complexes produced from this method were grown in vitro or in vivo (in multiple flows) using known techniques (see generally Fink et al., Prog. Clin. Pathol., 9: 121-33 (1984)). Can be done. In general, individual cell lines are grown in culture, and cultures containing high concentrations of single mAbs can be obtained by decantation, filtration, or centrifugation.
[307] Antibodies can be obtained with a single chain antibody or scFv instead of a round multimeric structure. Single chain antibodies regenerate antigen by binding to the native Ig site, which contains a hypervariable region from the interest of Ig and remains intact, in the form of an unprocessed Ig size fragment (Skerra, a. Et al. (1988) Science, 240: Pluckthun, A. et al. (1989) Methods Enzymol. 178: 497-515; Winter, G. et al. (1991) Nature, 349: 293-299); Bird et al., (1988) Science 242: 423; Huston et al. (1988) Proc. Natl. Acad. Sct. USA 85: 5879; Jost CT et al. J Biol Chem. 1994 269: 26267-26273; U.S.Patents No. It is associated with solutions involving unknown amounts of 4,704,692, 4,853,871, 4,946,778, 5,260,203, 5,455,0Kn dms labeled antibodies (this is called reporter molecule). After the second proliferation period for the labeled antibody to synthesize with the antigen bound to the solid support via the unlabeled antibody, the solid support is washed twice to release the unlabeled labeled antibody. This type of forward sandwich assay is simply used to determine the amount that may be made by comparing the amount of labeled antibody obtained by a standard sample, including whether there is an antigen or the amount of known antigen. yes / no ".
[308] Another type of sandwich assay, called "simultaneous" or "inverse", is used. Simultaneous assays include a single culture step in which both the antibody bound to the solid support and the labeled antibody are added to the sample to be tested at the same time. After incubation is complete, the solid support is washed to remove the labeled antibody that is not synthesized with the rest of the liquid sample. Current labeled antibodies associated with a solid support are determined as in a convenient prospective sandwich assay. In a "reverse" assay, one is used, step by step, at the beginning of the solution of the labeled antibody to the liquid sample following the addition of the unlabeled antibody bound to the solid support after a suitable incubation period. After the secondary incubation, the solid phase is washed in a convenient manner and placed in the solution of the labeled antibody which does not react with the sample even after it is being tested. The determination of labeled antibodies associated with a solid support is determined by "simultaneous" and "forward" assays.
[309] The foregoing antibodies are useful for inhibiting T cell responses and for treating diseases associated with undesirable T cell activation such as transplant rejection or free immunity. Such methods include the management of subjects such as the need for the management of efficient amounts of human or humanized mAb traits for the simultaneous reaction epitope of antibodies, preferably mAbs, more preferably B7-DC. Management of antibodies should prevent T cell responses or abolish antigen T cell responses and therefore be effective in inhibiting target T cell responses. Appropriate dosages are described below.
[310] Use of Phosphoric Acid Encoded with B7-DC Protein
[311] In the present invention, phosphoric acid is useful diagnostically in monitoring disease progression by measuring the expression of B7-DC in cells in assaying biological samples or B7-DC expression drug effects. Rather, this is accomplished by measuring the mRNA level of the cell. Phosphoric acid in this diagnostic method
[312] Therapeutic composition and management
[313] Polypeptide cells such as B6-DC polypeptide or DC, tumor cells were administered to mammals such as humans. Aggregations or immobilized forms of polypeptides are used to enhance the T lymphocyte response and the resulting immunity. The B6-DC-Ig lysing protein is combined in two molecules as shown in the example and co-stimulates T cells. The soluble monomeric form of the B6-DC polypeptide can be immobilized to the T cells of the receptor without stimulation, thereby being regarded as a T cell co-stimulatory response inhibitor or antagonist by the stimulatory form of the molecule. Fixation of antagonists such as B6-DC suppresses the progression of T cell responses or exhibits joint-stimulatory signals of their endogenous B6-DC or other B7 family (eg CD28 or CTLA-4) that act through receptors It may interfere with the effect.
[314] The composition with the activity of B7-DC described herein is administered on a pharmaceutically acceptable basis in the amount of biological or therapeutic effect. The B7-CD polypeptide (or cells synthesized from the polypeptide) may be given alone or in a form contained in a peptide compound having the activity of the B7 family and other immunosuppressive molecules. The therapeutic agent may also include administration of an adjuvant that is used in a broad sense, including an unspecified immunostimulatory mixture such as interferon. Supplements carefully include nonionic surfactants such as resorcinol, polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.
[315] The following dosages are relevant when administering an antibody of the invention to a subject. A therapeutically effective amount is a single dose given at effective time intervals that can achieve the expected immunological or clinical effect.
[316] The amount of polypeptide having a therapeutically effective B7-DC effect (or anti-B7-DC antibody) depends on factors such as the disease's condition, age, sex, and the body's weight and the peptide's ability to elicit the desired response. Can vary. Dosage doses can be adjusted to provide the optimum therapeutic response. For example, a single dose may be divided into three or four doses throughout the day, and may be instructed to decrease proportionally depending on the urgent condition of the treatment. The amount of therapeutically effective protein can be presented in terms of protein or cell equivalent conditions in the form of cell combination.
[317] Thus, the effective amount is about 1 ng to 1 gram per kg, more preferably about 1 μg to 100 mg, more preferably about 100 μg to 100 mg, depending on the weight of the doser. Single dosage forms suitable for administration in the body preferably comprise from about 0.1 mg to 500 mg of active ingredient per unit. The active ingredient may vary within 0.5-95% of the weight based on the total weight of the compound. Alternatively, the effective dose of the cell showing a B7-DC converting the cells such as DC's or activity of the tumor cells in approximately 10 ⁴ to 10 ⁹ cells per subject, more preferably from about 10 ⁶ to 10 ⁸ cells, preferably If divided, take it. Techniques gained from such immunotherapy will allow for adjustment of these doses without undue experimentation.
[318] Immediately acting compounds (eg, cells converted to B6-DC polypeptide or B6-DC DNA) may be administered by any convenient method (eg, by a convenient and effective route by injection). Such routes include subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection and the like. Other possible routes are oral administration, inhalation, transdermal ointment, or suppositories. Direct injection can also be used to treat tumors that have not been completely cut off.
[319] Depending on the route of administration, the immediate-acting compound may be coated with a substance to protect it from enzymes, acids, and other natural conditions that can inactivate it. Thus, when a polypeptide or peptide with activated B7-DC is administered by the route through the intestine, it may be necessary to be coated with or administered with a compound of a substance that prevents its inactivation. In one example, the peptide may be administered individually or in combination with an enzyme inhibitor (e.g., pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEP), trasylol) in a suitable medium such as diluent or adjuvant. Alternatively, liposomes (including liposomes such as conventional water-in-oil-in-water) may be suitable mediators. (Strejan et al., (1984) J. Neuroimmunol 7:27).
[320] As used above, "pharmaceutically acceptable carrier" includes any / all solvents, dispersion media, coatings, antibiotics, antifungal agents, isotonic absorption delaying agents, and the like. The use of media and agents for pharmacologically activating a substance is well known in the art. Existing media and agents can be used in therapeutic mixtures, except that they are incompatible with the active compound. Supplementary active compounds can also be mixed in a mixture.
[321] Preferred pharmacologically acceptable diluents include saline and aqueous buffer solutions. Pharmacological mixtures suitable for infusion include sterile aqueous solutions (herein water soluble) or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. Isotonic agents, such as sugar, polyalcohol (eg, mannitol, sorbitol), sodium chloride, and the like, can be included in the pharmacological mixture. In all cases, the mixture should be sterile and fluid. It must be stable under the conditions of manufacture and storage and must contain a preservative to prevent contamination by microorganisms such as bacteria and fungi. In addition, the dispersion should be prepared in glycerol, liquid polyethylene glycols, and mixtures or oils thereof. Under ordinary conditions of storage and use, such preparations include preservatives to prevent the growth of microorganisms. can do.
[322] The carrier may be a solvent or dispersion medium comprising water, ethanol, polyols (eg glycerol, propylene glycol, liquid polyethylene glycols, etc.), and appropriate mixtures thereof. Proper fluidity can be maintained by the use of a coating such as lecithin, by the maintenance of the required particle size in the dispersion, and by the use of surfactants.
[323] Prevention of the activity of microorganisms can be achieved by a variety of antimicrobial and antifungal agents (eg, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.).
[324] The delay in absorption of the injectable mixture is achieved by the inclusion of agents (eg, aluminum monostearate and gelatin) which delay absorption in the mixing.
[325] Parenteral compositions are formulated completely in dosage units for uniformity of dosage and convenience of administration. Dosage unit form refers to physical separation, such as a single dose for a mammalian subject. Each dosage unit comprises a predetermined amount of active compound calculated to exert the desired therapeutic effect associated with the required pharmacological carrier. Detailed descriptions of the dosage units of the present invention are directed to, for example, (1) the unique properties of the active compounds, the specific therapeutic effects that can be exerted, and (2) the therapeutic sensitivity for each individual, such as active compounds. It is described here, while directly depending on the inherent constraints in the mixing scheme.
[326] In lung instillation, an aerosolized solution is used. In sprayed aerosol preparations, the active protein may be combined with a solid or liquid inert carrier material. It can also be packaged in a squeeze bottle or a mixture with a compressed and volatile gas propellant. Aerosol preparations include solvents, buffers, surfactants, antioxidants in addition to the proteins of the invention.
[327] In topical applications, the protein of the present invention is implemented by topical mixing in a topical medium such as an ointment, which has the effect of softening the skin and means of directly administering the active ingredient to the affected area. Has
[328] Carriers for the active ingredient may be in the form of Frerays or Bissprays. The Bisprays may be in the form of semisolids or solids, which include carriers inherent in topical treatments, and which have a much higher dynamic viscosity than water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, slaves, and the like. If desired, such agents may be sterilized or mixed (eg, preservative, stabilizers, wetting agents, buffers, or salts affecting osmotic pressure, etc.). Preferred as non-spray topical therapeutic vehicles are ointment bases (e.g. polyethylene glycol-1000 (PEG-1000)), conventional creams such as HEB cream, gels, petroleum jelly ( petroleum jelly).
[329] Other pharmacologically acceptable carriers for the B7-DC polypeptides according to the invention are liposomes, pharmacological preparations scattered or present in a variety of corpus consisting of aqueous concentric layers attached to the lipid layer (their Contains the active protein in the stomach). The active protein is completely present in the water-soluble and lipid layers, in / out of the layer, in a heterogeneous system known as liposomes at any event. The hydrophobic layer, or lipid layer, is generally phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, and some ionic surface actives, such as dicetylphosphate, stearylamine ) Or phosphatidic acid, and / or other hydrophobic substances, but is not necessarily limited thereto.
[330] Alterations of boil cells expressing B7-DCs and complex costimulatory molecules
[331] Another aspect of the invention is the cell, rather the boil cell changed to express a complex costimulatory molecule. There is an expression of the time of costimulatory molecules in other activated B cells for B7, B7-2 and B7-3. For example, because B7-3 is later expressed, B7-2 is early expressed as the next B cell activation. During the course of the immune response, other costimulatory molecules may benefit this distinctive function. Lymphocyte responses differentiated in T cells that are effective from complex costimulatory molecules may require lymphocytes differentiated in T cells to receive costimulatory codes.
[332] Therefore, the present invention surrounds boil cells. It is genetically altered or represents one or more costimulatory molecules. For example, the boil cell can be altered to express B7-DC and one or more of B7, B7-2, B7-3.
[333] Prior to the change, cells such as tumor cells cannot express some costimulatory molecules, or precise costimulatory molecules cannot express others as well. As described herein, boil cells can be altered by transfection with the nucleic acid alone recoding B7-DC, or with another costimulatory molecule (s). . For example, in boil cells, nucleic acids that encode B7-DC can be transfected with more and more nucleic acids encoding B7. The sequence of cDNA molecules encoded by human or mouse B7-DC proteins is SEQ ID NO: 1, respectively, and is encoded by the coding of SEQ ID NO: 3. Alternatively, more than one type of modification may be used. For example, a boil cell is transfected with the nucleic acid encoded. The B7-DC may be stimulated with zeros with the agent who persuaded the representation of B7-1 (B7-2 ID B7-3).
[334] Antigens bound to pathogens
[335] The present invention is mainly used as a component of a vaccine for treating fatal cancer and chronic viral infection. Such vaccinias are designed to remove infected cells. This requires lymphocyte answers differentiated in T cells as antigen epitopes themselves.
[336] (a) Vectors such as exposed DNA, exposed RNA include RNA replicons, viruses including vaccinia, adenoviruses, adeno-associated virus (AAV) Copying, lentiviruses and RNA alphaviruses self-pollinates.
[337] (b) Antigens attack or process signals such as HSP70, caleticurin, flt-3 combiners in extracellular regions, pefedomnas exotoxin ETA in regions, single VP22 herpes to attack proteins, and the like. (US Patent Application 09 / 421,608; 09 / 501,097; 09 / 693,450; 60 / 222,9002; 60 / 222,985; 60 / 268,575 and chang, WF et al., J. Virol. 75: 2368-2376 (2001) See)
[338] (c) costimulatory signals, B7-DC proteins, derivative or functional derivatives of currently invented or soluble proteins (alone or other known costimulatory proteins such as B7.1, B7.2 that can dissolve CD40, etc.)
[339] Tumor cells or other forms of host cells containing APCs are modified, nuclear clones are modified or antigens that require complete response replication or otherwise require a response to immunity. These antigens are rather antigens that produce microorganisms.
[340] The host is defended by T cell responses, cytotoxic T lymphocytes (CCLs) and effectors that are postponed in hypersensitive form. These typically included viruses such as maleia and bacteria and intracellular parasites grow intracellularly, such as mycobacteria and listeria. Therefore, antibiotics in the vaccine component of the present invention include such pathogens (addition or direction of boil-specific antibiotics).
[341] Particular viral antigens are boil antigens, especially in the case of viruses that cause cancer.
[342] In fact, the world's most common hepatoma and servical cancer are caused by viral infections. Hepatinis B virus (Beasley, RP et al., Lancet 2, 1129-1133 (1981)) has been affected as a cause of cancer-causing agents: 80-90% of uterine cancers are HPV, a very dangerous papillomavirus in humans -16, HPV-18, HPV-31 and HPV-45 (Gissmann, L. et al., Ciba Found Symp. 120, 190-207 (1986); Beaudenon, S. et al.nature 321, 246-249 ( It is expressed as E6 and E7 antigens from one of 1986. HPV E6 and E7 antigens are the most obvious targets for cancer-associated viruses in personal immunity because they are ubiquitous in Svecal cancer. The most important goal of viruses associated with boil and boil antigens is an ideal volunteer for disease prevention vaccines, in fact the introduction of HBV vaccines for disease prevention in Asia has led to the development of hepatoma (Chang, MH, et al. New. Engl j. Med. 336, 1855-1859 (1997), the incidence of cancer is decreasing, and It represents the biggest impact.
[343] Among the most important viruses in chronic human virus hepatitis are: "Human papilloma virus (HPV), pathologic hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), Epsteinbar virus (EBV). And a simple herpes virus.
[344] In addition to the adaptability of the virus to human cancer and hepatitis diseases, the present invention is also designed for the treatment of animal diseases in the field of veterinary medicine. Thus, the approach described here is probably veterinary herpes, including horse herpesviruses, bovine herpesviruses, Marek's disease virus found in chickens and other birds, animal tumor diseases, rabies and rabies, etc. It will be applied by one learned in the field of virus therapy.
[345] The following references are based on four principles and information commonly available in the field of basic, medical and veterinary viruses and are incorporated by this reference (Fields Virology, Fields, BN et al., Eds., Lippincott). Williams & Wilkins, NY, 1996)
[346]
[347] <Drug Targeting Molecules>
[348] Many proteins with varying modes of action bind or bind to antigens, preferably soluble poly, to target antigens in cells and subcellular compartments that promote the preparation of antigens in T cells in a more efficient and effective manner. It is affected by "drug targeting" molecules that bind to the peptide.
[349] Binding of antigens in heat shock proteins (HSPs) suggests a potential approach for increasing the potential of nucleic acid based (and other) vaccines. Heat shock proteins (HSPs) appear to act as supplements in the natural biology of cancer and viral vaccines. Both gp96 heat shock protein (HSPs) and cytosol heat shock protein 70 resident in the ER (S) act as an adjuvant (Srivastava, PK et al., Semin Immunol. 3, 57-64 (1991); Udono , Het al., Proc. Natl. Acad. Sci. USA 91, 3077-3081 (1994)). These heat shock proteins (HSPs) or chapononins are bound by a broad peptide (Lammert, E., et al. Eur. J. Immunol. 27, 923-927 (1997)). Heat shock protein (HSPs) 70 is one keperonin capable of drug targeting proteins bound to proteasomes (protease complexes of major cells that produce peptides for binding MHC class I molecules). Therefore, antigens directly linked to heat shock proteins (HSPs) 70 are more efficiently represented by MHC class I molecules (leading, inter alia, to CTL response). Both aspects are caused by heat shock (HSP) aids. First, in vitro, gp96-loaded peptides meet antigen and MHC class I progression pathways. Second, the bundle of gp96 in the micropages induces secretion of the pro-inflammatory cytokine, thus debating the function of cells in peptide antigens. Immune bodies, along with heat shock protein (HSP) complexes isolated from pathological tumors or isolated from virus infected cells, induce potential anti-tumor immune or anti-viral immune bodies (Srivastava, PK et al., Int J Cancer. 33: 417-22, 1984; Srivastava, PK et al., Proc Natl Acad Sci USA.83: 3407-11, 1986; Udono, Het al., J Immunol.152: Tamura, Y et al., Science.278: 117 -20, 1997; Janetzki, S et al., J Immunother. 21: 269-76, 1998). In vitro mixing of heat shock protein (HSP) and peptides produces an immunogenic heat shock protein peptide complex (Ciupitu, AM et al., J Exp Med. 187: 685-91, 1998; Blachere, NE et al., J Exp Med. 186: 1315-22, 1997). Most recently, the present inventors and their colleagues (eg, Chen, CH et al., Canc. Res. 60: 1035-1042 (2000)) form heat shock proteins in the form of DNA or RNA replicon vaccines that can be processed. HSP). They used HPV-16 E7 as Mycobacterium tuberculosis HSP 70 antigen. Increased expansion and activity of E7-specific CD 8 + T cells due to potential anti-tumor immunity against tumor establishment (Lin, KY. Et al., Cancer Res. 56: 21-26., 1996) .
[350] Another useful drug targeting molecule is the metastasis of the domain of Pseudomonas exotoxin A-ETA, ie the domain II (dII) domain of ETA (rotational residual 253-364). Transition domains are one polypeptide that induces the transfer of a protein or polypeptide that is linked to the cytosol-anneal of a cell. For example, similarly applied polypeptides are derived from Diphtheria, Clostridia (botulinum, tetani), Anthrax, Yersinia, Vibriocholerae, or Bordetella pertusis toxin. The toxin domain of the toxin-encoded DNA is preferably aged or deleted in preparation of such constructs.
[351] The CRT is a rich 46 kDa protein whose location is in the endoplasmic reticulum (ER) cell gap, which shows lectin activity and is known to be involved in the folding and assembly of early glycoproteins (CRTs are TAP-A and TAP-). It is associated with a peptide delivered to the ER by a carrier associated with an antigenic process such as 2. CRT forms a complex with the peptide in vitro. This complex elicits peptide directed CD8 + T cell responses when applied to mice. Purified CRTs from mouse tumors elicited specific immunity to tumors used as the source of CRTs, not antigenically distinct tumors. By sending DCs to the peptide with CRT bound in vitro, the peptide was reproduced within the DC Class I molecular structure and stimulated peptide directed CTL.
[352] Flt-3 ligands stimulate the growth of DC precursors and can promote the generation of multiple DCs in vivo. Flt-3 is a murine tyrosine kinase receptor and is one of a family of III receptor kinases. Expression of Flt-3 in hematopoietic tissue is limited to CD34-positive species. Flt-3 is used to determine the properties of the corresponding ligand, ie Flt3-ligand, and then replicate. A distinct form of Flt3-ligand is synthesized as a membrane protein, in which functionally similar soluble ECDs are produced from the membrane protein by proteolytic digestion. These proteins are bound to and activate unique tyrosine kinase receptors. The expression of the Flt3 receptor among hematopotic cells is mainly limited to the most primitive progenitor cells, including DC precursors. The ECD of Flt3-ligand produced a strong anti-tumor effect against several murine model tumors, including fibrosacoma, breast cancer, liver cancer, lung cancer, melanoma and lymphoma. The present inventors group linked the DNA encoding HPV Ek7 protein to the DNA encoding Flt3-ligand ECD. Immunity to this results in rapidly increased swelling and activation of E7 antigen-directed CD8 + T cells, resulting in potential anti-tumor immunity against preformed E7-expressing metastatic tumors.
[353] HSV-1 protein VP22 is a primitive protein that contributes to the propagation of antigen because of the pronounced nature of intracellular transport. For example, VP22 or thymidine kinase linked to p53 promoted the propagation of proteins linked to surrounding cells in vitro and treatment of model tumors. VP22 linked to the HPV-16 E7 antigen in the DNA vaccine construct led to a sharp increase in the number of E-7 oriented CD8 + T cell precursors in vaccinated mice (approximately 50-fold) and less effective DNA The vaccine was converted to one with great potency against E7-expressing tumors. VP22 variants that did not spread did not extend the vaccine's power. VP22 and proteins, which have similar types of action, contribute to expanded vaccine potency in several ways: (1) Promotes the propagation of antigens to the surrounding APCs from the cells containing the nucleic acid, thereby enhancing the MHC class I pathway. Increase the number of APCs that give antigen; (2) confer antigen more effectively in cells loaded with nucleic acid; (3) Perform crosspriming, whereby the release of VP22 / antigen fusion protein leads to lifting and processing for presentation by DCs (or other APCs) via the MHC-I restriction pathway.
[354] Those skilled in the art will know how to find the appropriate antigenic element (eg CTL antigenic element) of the relevant protein from a pathogen of use according to the invention.
[355] Delivery of B7-DC DNA to Cells and Animals
[356] For example, DNA delivery to perform what is commonly known as gene therapy involves introducing foreign DNA into cells and ultimately living animals. Some general strategies for gene therapy have been studied and extensively reviewed.
[357] One approach is to systematically autograft mutated cells ex vivo to a host or to a particular organ or tissue, or to transfer nucleic acids from the culture to early cells.
[358] Nucleic acid therapy for achieving the object of the present invention is carried out by directly moving functionally activated DNA into somatic cells, or tissues in vivo in mammals. DNA transfer can be done in several ways as described below. Whether these systems have been successfully expressed in vivo is detected by the appropriate immunoassay in the presence of B7-DC expression agents (after treatment with attractants in the case of an attractant) using antibodies against the substance. Selectable markers such as G418 Resistance can be used to select infected clones expressing the DNA. The efficiency of procedures such as DNA uptake, plasmid fusion, and stability of fused plasmids can be improved by unifying plasmid DNA with existing methods and using high molecular weight mammalian DNA as a "carrier."
[359] Conventional examples of successful "gene transfer" include (a) direct injection of plasmid DNA into rat muscle tissue resulting in expression of marker genes for an indefinite period of time (Wolff, JA, et al., Science 247: 1465 (1900); Acsadi, G. et al., The New Biologist 3:71 (1991)), (b) the effect of a vector of retroviruses on in vivo and in situ infection of vascular tissues; (c) Cases of retroviral preparations in which the portal vein injection and direct injection resulted in gene transfer and expression in vivo (Horzaglou, M. et al., J. Boil. Chem. 265: 17285 (1990); Koleko , M. et al., Human Gene Therapy 2:27 (1991); Ferry, N. et al., Proc. Natl. Acad. Sci. USA 88: 8387 (1991)); (d) Cases showing that intratracheal injection of transgenic adenovirus into lung tissue was effective for in vivo transfer of foreign genes to long-term expression of external genes in lung respiratory epithelial tissues (Rosenfeld, MA, et al. Science 252: 431 (1991); (e) Examples of simple herpes virus vectors translocated to brain tissue in vivo (Ahmad, F. et al., eds, Miami Short Reports Advances in Gene Technology: The Molecular Biology of Human Genetic Disease, Vol. 1, Boerringer Manneheim Biochemicals, USA, 1991).
[360] Retroviral-mediated human theraphy uses a retroviral system that is amphotrophic and has an antibiotic nature (Temin, HM, Human Gene Therapy 1: 111 (1990); Temin et al., US Pat. No. 4,980,289; Temin et al., US Pat. No. 4,650,764; Temin et al., US Pat. No. 5,124,263; Wills, JW US Pat. No. 5,175,099; Miller, AD, US Pat.No. 4,861,719). Such vectors have been used to inject functional DNA into human cells or tissues. For example, adenosine deaminase genes are injected into lymphocytes, or NPT-II genes and tumor necrosis factor are injected into tumor penetrating lymphocytes. Retrovirus-mediated gene transfer usually requires cell proliferation to move genes (Miller, DG et al., Mol. Cell. Biol. 10: 4239 (1990). Gene therapy of bladder fibrosis with petroleum vectors and migration by plasmids using several methods is described in US Pat. No. 5,240,846 (Collins). et al.,).
[361] DNA molecules encoding B7-DC sequences are known from the prior art (Cone, RD et al., Proc. Natl. Acad. Sci. USA 81: 6349-6353 (1984); Mann, RF et al., Cell 33: 153-). 159 (1993); Miller, AD, et al., Molec. Cell. Biol. 5: 431437 (1985); Sorge, J. et al., Molec. Cell. Biol. 4: 1730-1737 (1984); Hock Packaged cells that produce aberrant retroviruses, as described in RA et al., Nature 320: 257 (1986); Miller, AD et al., Molec. Cell. Biol. 6: 2895-2902 (1986). Columns can be used to batch insert retroviral vectors. New phage cell rows that are effective and safe for gene transfer are described in U.S. Pat. 5,278,056 (Bank et al.).
[362] This approach can be used for specific methods of delivering retroviral vectors to selected tissues or organs. As an example a catheter delivery system (Nabel, E.G. et al., Science 244: 1342 (1989)) can be used. Such methods using retroviral vectors or liposome vectors are particularly useful for delivering the nucleic acid to be expressed to the blood vessel wall or into the blood circulation of the tumor.
[363] Recombinant adenovirus (Horowitz, MS, In: Virology, Fields, BN et al., Eds, Raven Press, New York, 1990, p.1679; Berkner, KL, Biotechniques 6: 6169191988, Strauss, SE, In: The Other viral vectors are also available, such as Adenoviruses, Ginsberg, HS, ed., Plenum Press, New York, 1984, chapter 11), and simple herpes virus (HSV) for delivery and persistence to specific neurons. The advantage of using adenovirus vectors for human gene therapy is that recombination is rare, and humans do not develop malignancies due to the use of such viruses, and the genome of the adenovirus consists of two strands of DNA, up to 7.5 kb in size. It can accept and replicate foreign genes, and live adenovirus is a safe human vaccine construct. In addition, according to the present invention adeno-associated viruses are useful for the treatment of humans (smulski, R. J. et al., EMBO J. 10: 3941 (1991)).
[364] Another vector that can express the DNA molecule of the invention and is useful in setting up this therapy (especially how to treat humans) is the Bexonia virus. Bexonia virus is a non-replicating virus (US Patent No. 5,225,336; 5,204,243; 5,155,020; 4,769,330; Sutter, G et al., Proc. Natl. Acad. Sci. USA (1992) 89: 10847-10851; Fuerst, TR et al., Proc. Natl. Acad. Sci. USA (1989); 86: 2549-2553; Falkner, FG et al.,; Nucl.Acids Res (1987) 15: 7192; Chakrabarti, S et al., Molec Cell Biol. (1985) 5: 3403-3409). Recommends regarding recombinant Bexonia virus and other viruses, including xenograft DNA, and their use in immune DNADY therapy are reviewed in the following documents: Moss, B., Curr. Opin. Genet. Dev. (1993) 3: 86-90; Moss, B. Biotechnology (1992) 20: 345-362; Moss, B., Curr Top Microbiol Immunol (1992) 158: 25-38; Moss, B., Science (1991) 252: 1662-1667; Piccini, A et al., Adv. Virus Res. (1988) 34: 43-64; Moss, B. et al., Gene Amplif Anal (1983) 3: 201-213.
[365] In addition to naked DNA and RNA, ie viral vectors, engineered bacteria can be used as vectors. Salmonella, BCG and Listeria monocytogenes (LM) (Hoiseth & Stocker, Nature 291, 238-239 (1981); Poirier, TP et al. J. Exp. Med. 168, 25-32 (1988); (Sadoff, JC, et al., Science 240, 336-338 (1988); Stover, CK, et at., Nature 351, 456-460 (1991); Aldovini, A. et al., Nature 351, 479-482 (1991 Schafer, R., et al., J. Immunol. 149, 53-59 (1992); Ikonomidis, G. et al., J. Exp. Med. 180, 2209-2218 (1994)). They have two promising properties that are good for use as vaccine vectors: (1) one is for enteric infections that provide oral vaccine deliverability, and (2) one is monosite against the antigen against professional APC. Infection of macrophages.
[366] In addition to in vivo migration of genes mediated by viruses, plasmid DNA management (Wolff et al., 1990, supra) and gene transfer via particle shock (Yang, NS et al., Proc. Natl. Acad. Sci. USA 87: 9568 (1990); Williams, RS et al., Proc. Natl. Acad. Sci. USA 88: 2726 (1991); Zelenin, AV et al., FEBS Lett. 280: 94 (1991); Zelenin, Conventional physical means such as AV et al., FEBS Lett. 244: 65 (1989); Johnston, SA et al., In Vitro Cell. Dev. Biol. 27:11 (1991)) Can be used. In addition, according to the present invention, DNA molecules may be transferred to tissues in vivo using electroporation, a well-known means for transferring genes to cells in vivo (Titomirov, AV et al., Biochim. Biophys. Acta 1088: 131 ( 1991).
[367] "Transport mediated gene transfer" is also described in the following literature: Wu, C.H. et al., J Biol. Chem. 264: 16985 (1989); Wu, G.Y. et al., J. Biol. Chem. 263: 14621 (1988); Soriano, P. et al., Proc. Natl. Acad. Sci. USA 80: 7128 (1983); Wang, C-Y. et al., Proc. Natl. Acad. Sci. USA 84: 7851 (1982); Wilson, J.M. et al., J. M. et al., J. boil. Chem. 267: 963 (1992). Preferred carriers include targeted liposomes (Nicolau, C. et al., Proc. Natl. Acad. Sci. USA 80: 1068 (1983); Soriano et al., Supra).
[368] Polyconjugates such as asialoglycoprotein or polylysine are used in conjugates containing particulates that are recognized as target tissues (asialoorosomucoid of soy). (Wu et al. , 1989, supra) and DNA binding synthesize DNA to infect. Polylysine is an example of the combination of microparticles and DNA so that DNA is not damaged. This conjugate is synthesized for transfer with prisma DNA and in accordance with the present invention.
[369] Prisma DNA used for infection or microinjection is purified by conventional methods and then prepared by conventional methods known in the art, such as Quiagen treatment.
[370] In other words, as described above, the practical use of the converted B7-DC fine particles according to the present invention does not require a detailed description. A brief description of the polytide is sufficient to describe the modified cells for performing their immunogenic and costimulatory activities.
[371] In order to help the understanding of the present invention, the present invention has been described in more detail through the following examples. The following examples do not limit the invention.
[372] Example 1
[373] Materials and methods
[374] Cell preparation and culture
[375] A 6-12 week old female rat BALB / c was purchased from NCI and used for DC and macrophage preparation.
[376] DCs from bone marrow were fed with 5% fetal calf serum (FCS) (Hyclone), penicillin / Streptomycin (JRH Biosciences), Gentamycin (Sigma), Nonessential amino acids (JRH Biosciences) supplied with RPMI1640 (Gibco BRL) medium. ), L-Glutanmate (JRH Biosciences), Sodium Pyruvate (Sigma), 2 mercaptoethanol (Sigma) and (26) 1000 units / ml recombinant murine GM-CSF (Immunex) described above. DCs obtained from DAY-8 bone marrow are stained with monoclonal antibodies using conventional methods. Monoclonal antibodies, in contrast to MHC class II and 14-4-4, are clarified from the hyplicoma supernatant. Dr. William Baldwin (University of John Hopkins) supplied CTLA4-Ig cease-fire particles. MHC class I (28-14-8), F4 / 80 (C1.A3-1), B7.1 (1G10), B7.2 (GL1), Fc gamma RII / III (2.4G2) and Mac-1 ( Antibodies of M1 / 70) were purchased from PharMingen. For the preparation of the experimental cDNA, DAY-8 cells were clarified by a cell sorter using 14-4-4s and CTLA4-Ig at the Johns Hopkins University Oncology Center. After alignment, the purity of MHC class Ii ^hi and B7 ^hi is 93-98%.
[377] Macrophages from the bone marrow were described above with 10% FCS, Penicillin / Streptomycin, non-essential amino acids, sodium pyruvate, L glutamine, 2-mercaptoethanol, 250 units / ml recombinant murine M-CSFs (27). Cultured in medium of RPMI-1640 supplied with treatment at 500 units / ml gamma -IFN (Pharmingen) and 5 mu g / ml LPS (sigma). After stimulation, cell surface expression of MHC class II and B7 cells was established by routine flow cytometric analysis at 10 days of culture.
[378] Macropage cell lines, WEHI-3, RAQ264.7, J774A.1, and PUS-1.8 were provided by Joshua Pavelel of NIAID, National Institutes of Health. These were incubated with ATCC medium.
[379] Allogeneic Mixed Lymphocyte Reaction
[380] DCs extracted from DAY 8 BM, characterized by MHC class Iihi and B7hi, were tested for their ability to stimulate allogeneic T cells in MLC. MLC reactions were performed on 96 well flat bottom microplates by increasing the number of BALB / C stimulating cells to 3 × 10 ⁵ allogeneic C57BL / 6 lmphocytes. After 3 days of culture, T cell division is assigned by adding 1 mu Ci of [ ³ H] -methyl-thymidine to each well for the last 18th culture. The cells are then harvested and the incorporation of radioactivity is determined using a beta count.
[381] CDNA subtractive hybridization
[382] Aligned DCs and total macrophages from total RNA are extracted with TRIZOL (Gibco BRL). Messenger RNA (messenger RNA) is purified by Oligotex mRNA Purifier (Qiagen). To amplify the CDNA we used a PCR based SMART cDNA synthesis system (Clonetech) according to the PCR based subtraction system PCR select (Clonetech). Subtraction was performed according to the provider's machine language. After the last PCR subtractive, the DNA fragments are ligated with the plasma vector pCR2.1 (Invitrogen) or pCR Blunt (Invitrogent). After modification, each clone is grown for plasma amplification and miniprop DNA, and then digested with EcoRI to confirm the actual presence of inserts. Plasmid dot blots are performed to confirm that the cloned cDNA is dendritic cell specific. Alkaline denatured miniprep DNA is hybridized with SMART cDNA probes derived from DC or activated macrophages marked and spotted on the Hybond N + membrane. The above cDNA probes are labeled with ³² P levels using the random primer labeling method (Stratagene Prime-It II). Hybridization and washing were done as described above (28). Membranes were developed by exposure to a film (Amersham) for 1-2 days.
[383] Plasmid Dot Blot Analysis
[384] Alkaline denatured miniprep DNA samples are crossed on probe-derived SMART DNA or activated macrophages from DCs placed and sorted on a Hybond N + membrane (Amersham). These cDNA probes were ³² P named using the Landon Primer Labeling Method (Stratagene Prime-It II). Hybridization and washing were done as described above. In autoradiography, membranes were developed after exposure to film (Amersham) for 1-2 days.
[385] cDNA library construction and screening-Cloning of B7-DC
[386] Bone marrow-drived DCs were harvested on day 8 without sorting. About 20% -40% of these cells showed MHC class II and B7. Total RNA extraction after poly A RNA washing was done as described above. For oligo dT primed DC library construction, a Lambda ZAP Express cDNA synthesis system (Stratagene) was used. PCR DNA fragments of B7-DC were detected and used for screening. Membrane transfer, denaturation and renaturation were performed using the Stratagene protocol. Radiolabling, hybriddiazation, washing and automated roentgen photography of the probes were performed as described above. Positive clones were isolated and secondary screening was performed. After the second screening the plasmid was excised by in vivo excision and tested by dot blotting and screening. Sequencing was done by the Core Facility at John Hopkins University Pharmacy. The BLAST program was used to perform homology studies of nucleotide sequences for Genbank (NCBI) for similarity to previously reported genes. Full length B7-DC cDNA clones were extracted from DC cDNA libraries. 5'RACE was performed using SMART RACE cDNA amplification kit (Clontech). The 5'RACE product was asexually reproduced and sequenced with the pCR2.1 vector. More than two full length B7-DC clones were obtained by RT-PCR and the sequences were compared to prevent sequence errors.
[387] Human B7-DCs were asexually reproduced as follows. Human DCs are obtained from normal peripheral blood mononuclear cells by culturing in GM-DCF + IL-4 or GM-CSF + Flt-3L above (29). RNA is extracted as described above. The A BLAST investigation identified the overlapping EST clone, GenBank Accession Number AK001879, as homologous to rat B7-DC. 5'RACE is performed as described above. The 5'RACE fragment was sequenced and the primers corresponding to the 5'-UTR of human B7DC were designed. In 5'-URT and 3'URT the following primers were used to amplify full length human B7-DC.
[388] 5'-GGAGCTACTGCATGTTGATTGTTTTG-3 '[SEQ ID No: 6]; And
[389] 5'-TGCAAACTGAGGCACTGAAAAGTC-3 '[SEQ ID No: 7]
[390] Human full length cDNA sequences and murine B7-DC cDNAs have been deposited as EMBL / GenBank / DDBS under access numbers AF329193 and AF142780.
[391] BAC (129SVJ) library screening / Genomic cloning and mapping
[392] BAC library screening used the following producer protocol (Genome systems, Inc.) primers.
[393] 5'-TTGTTGTCTCCTTCTGTCTCCCAAC-3 '[SEQ ID No: 8]; And
[394] 5'-ACAGTTGCTCCTTGTATCAGGTTC-3 '[SEQ ID No: 9]
[395] BAC library screening obtained tripositive clones. Chromosomal location mapping is performed by fluorescence in situ hybridization (Genome Systems Inc.). A total of 80 metaphase cells were analyzed by 79 display specific labling. Human B7-Dc mapping was performed using the bioinformatic tool, NCBI's BLAST program, and the International RH Mapping Consortium. The hB7-DC sequence was examined in htsg and was found to map to two DBc clones RP11-574F11 (AL162253) and Rp11-635N21 (AL354744) localized to chromosome 9.
[396] Virtual Northern Blotting
[397] 4-6 week old female Balb / c mice were purchased from NCI and used for tissue RNA preparation. RNA extraction and SMART cDNA tissue synthesis, sorted DC and activated macropages were performed as described above. SMART PCR cDNAs were cleaned by PCR Cleaning Kit (Qiagen). 0.5 μg / lane wash DNA was performed on 1% agarose gel and transferred to Nytran nylon membrane Schleier and Schuell. DNA was amplified by PCR using a set of primers immediately adjacent to the cloning site of the plasmid DNA, and washed PCR DNA of each clone for the cross probe was used. The nucleotide sequence of these primers is as follows.
[398] 5'-GTAACGGCCGCCAGTGTGCTG-3 '[SEQ ID NO: 10] and
[399] 5'-CGCCAGTGTGATGGATATCTGCA-3 '[SEQ ID NO: 11]
[400] Virtual northern analysis of total RNA and control placenta of human DCs was also performed. Probes and RNA preparation used were as described above. Radiolabling, mating, washing and automatic roentgen photography of the probe are performed as described above.
[401] Hamster Anti-mB7-DC Antibody Production
[402] Stable transfectants of B7-DC of DC2.4, RAW246.7 and RENCA cell lines were used to immunize Armenian hamsters. The B7-DC is cloned into a modified pCAGGS vector 30. The hamster is injected three times with a plasmid containing B7-DC (Rockland). The anti-B7-DC antibody used in this study is one of three sera of three hamsters immunized.
[403] DC28-Ig, CTLA4-Ig and PD-1-Ig Binding Assays
[404] 293T cells are transfected using only vectors using B7.1-pCAGGS, B7-DC-pCAGG, PD-1-pCAGGS or lipofectamine 2000. After 24 hours, the cells are resuspended in FACS buffer (consisting of 1 × HBSS, 2% calf serum, 10mMHEPES and 0.1% NaN ₃ ) and spun at 1000 rpm for 5 minutes at 4 c. The buffer is poured out separately, the antibody is poured into a tube, incubated for 20 minutes at about 4 C and washed twice with the FACS buffer for the secondary antibody. Pass the sample through FACScan. B7.1 antibody is used at a dilution of 1: 5 and 10 g / sample (Cal-Tag), recombinant CD28-Ig, CTLA-4-Ig and PD-1-Ig chimera are 2 g / ml, 10 l / sample (R & D system, Inc). Goat F (ab ') ₂ anti-human IgG-PE is used at a dilution ratio of 1:20 (Southern Biotechnology Associates, Inc.).
[405] B7-DC-Ig dimer synthesis
[406] The B7-DC-Ig construct is a sequence encoding the N7-terminal amino acid of B7-DC without the transmembrane domain in the frame of human IgG ₁ F _{c in} the Pig-Tail Plus vector (R & D system). By fusion to a sequence coding C-terminal amino acid. COS-7 cells are transiently infected with pIg / B7-DC using LipofectAMINE 2000 (GIBCO BRL) or GINE JAMMER (Stratagene). The B7-DC-Ig fusion protein is obtained by clarifying the serum-free supernatant using saturated ammonium sulfate precipitate. The purity of SDS-PAGE and silver staining was over 90%.
[407] T-cell proliferation and cytoplasmic assay
[408] For interstimulatory assays involving anti-CD3, 96 well flat bottom plates (immulon 4 supplied from Dynex, emulon) used anti-CD3 antibodies (2C11, palmigen: pharmigen) and B7.1- Pre-applied with Ig (R & D system), B7-DC-Ig or isotope control (Sigma) at 100 ng / ml is diluted in conditions of 37 C for two hours in PBS (Gibco) PH 7.4 once. Subsequently, the bottom plate was washed three times with PBS once to remove T cells with RPMI1640 medium supplied with 10% FCS, penicillin / streptomycin, non-essential amino acid, sodium pyruvate, L glutamine, 2-Muractetoethanol. Block for an hour and a half before adding. Spleens and lymph nodes are obtained from BALB / c mice 6 to 10 weeks old. RBCs are cleaved (lyse) using ACK buffer and T cells are clarified indirectly using dynabeads M-280 (Dynex). Beads are washed twice with PBS PH 7.4 + 1% FCS before adding the cells and an antibody cocktail consisting of anti-IE ^d and B220 / CD45RO or CD8 (palmigen: pharmigen) is added to the cells Incubate for 30 minutes at 4 C in both directions. The cells are separated by placing the tube in Dynal MPC for 5 minutes and centrifuged at 1500 rpm for 5 minutes to wash twice with PBS pH 7,4 + 1% FCS to remove unbound antibodies. The procedure is repeated for 15 minutes of incubation and cells consisting of 2 10 ⁵ cells / well. After incubation for 72 hours, 10 l of ³ H-thymidine (1 Ci / well) is added to each well and incubated for 18 hours. Cells are harvested using the Packard Micromate Cell Harvester, and the Packard Matrix 96 direct counter reads Peter.
[409] For interstimulatory assays using the RENCA system for current HA antigens, RENCA cells are incubated with RPMI-1640 medium containing 10% FCS, penicillin / streptomycin, non-essential amino acids, sodium pyrubin. Induction with IFN- (75U / ml) for 72 hours for MHC class II expression. Cultured cells are lighted for 13,200 rad and made into 2 10 ⁴ cells / well (96 well plate bottom plate). The HA110-120 peptide was added at 2.5 g / well and Ig-fusion molecules at various concentrations were added. Transgenic IE ^d + HA specific T cells (kind gift of H. von Boehmer, Harvard University) were isolated and made 4 10 ⁵ cells / well as described above. After 48 hours of incubation, 10 l of ³ H-thymidine (1 Ci / well) is added to each well and incubated for 18 hours. Cells are harvested using the Packard Micromate Cell Harvester, and the Packard Matrix 96 direct counter reads Peter.
[410] For cell division production analysis using ELISA, the culture is set up as described above and the supernatant is harvested at the specified time. IL-2, IL-10 and IFN- concentrations are determined using commonly available ELISA meters (endogens) and IL-4 and IL-6 (R & D system).
[411] In Vivo Costimulation
[412] The deceased mesenchymal, groin, cervical and mesenteric LNs from TCR transgenic mice showing TCR recognizing I-Ed-restricted HA epitope (110SFERFEIFPKE120 [SEQ ID NO: 12]) in the genetic background were identified as RPMI medium (GIBCO BRL). It is separated in, passed through a 100um nylon cell strainer and washed in sterile Hank's buffer (GIBCO BRL). After FACS staining to determine the proportion of clonotipid CD4 cells, cell preparations containing 2.5 × 10 6 clonotipic cells in 0.2 ml sterile Hank's buffer are injected intravenously into the tail vein of recipient B10.D2 mice. Three days after this adopted transfer, the animal develops subventricular injection by subcutaneous injection in the hind paw. Each rat received two-way injections of one of three preparations.
[413] (A) 10 g synthetic HA (footpad: per footpad) (HA peptide (110-120) bound with a 1: 1 v / v ratio with incomplete Freund's Adjuvant (IFA)) (Sigma);
[414] (B) HA-IFA mixture mixed with 25 g B7-DC-Ig; or
[415] (C) HA-IFA mixture mixed with 25 g isotope control antibody. After 7 days drained LN nodes are taken; 1.5 10 ⁵ LN cells are cultured in circular lower 96 well tissue plates at the specified synthetic HA peptide concentration. Proliferation assays are performed by pulsing the culture with 1Ci [ ³ H] thymidine for 48 hours and additionally incubating for 12 hours before obtaining and determining the amount of radiation involved.
[416] Example 2
[417] Identification and Characterization of B7-DC
[418] The B7-DC is separated from the (partly) removed library between the DC and the active macropage. The two populations used for cDNA reduction are bone marrow-derived GM-CSF cultured DCs as tester populations and (gamma) as interoperation (interferon + LPS activated adhesive bone marrow) as driver populations. Bone marrow-derived M-CSF macropage. Day 8 MHC Class IihhiB7hi Aged DCs are classified as 93% pure as a source of tester cDNA. DCs are specified at the MHC II class level, almost 50 times higher than macropages, by flow cytometry. Although the B7.2 level is significantly higher in DCs, both populations represented B7.1 and B7.2. F4 / 80 and cd16 were represented at a higher level on the macropage population. The functional comparison of the two populations shows that DC population affects approximately 100 times more than the macropage population in stimulation of allogeneic mixed lymphcyte responses.
[419] After RNA extraction from both populations, PCR selection, ie PCR based on cDNA synthesis system followed by PCR based on subtractive procedures, is used. One of the differentially expressed clones symbolized a portion of the new immunoglobulin supergene system named B7-DC. The murine B7-DC cDNA, which encodes a 247 amino acid precursor protein with a 23 aa N-terminal signal peptide and a predicted molecular weight of 25 kd (Table 1), has a length of ˜1.7 kb. The leader sequence of the estimation and the domain of the transmembrane (which occurs through the membrane) are identified using the SOSUI program 31. Two charged aa are found in the 23 aa transmembrane region of mB7-DC proposed as a junctionable partner. At aa level, murine B7-DC is equivalent to 70% of human B7-DCs representing orthologue (see table below). The hB7-DC is somewhat different from the murine B7-DC due to the longer cytoplasmic tail.
[420] Through kinship studies, B7-DC was found to be important for B7-H1 (34% identity, 48% similarity) and smaller size butyrophylline (30% identity, 45% similarity) (Table 1) and B7.1. And B7.2 have less than 20% identity. Phylogenetic studies may be related to butyrophylline B7 through exon suffling. Each of them has a normative IgV-IgC structure and transmembrane region. In contrast to other B7 family members, murine B7-DCs have an extremely short cytoplasmic tail (4 aa).
[421]
[422]
[423]
[424] Comparison of amino acid sequences of murine B7-DC and human B7-DC. The leader and transmembrane regions of the mB7-DC estimation are shown in the upper row. Arrays were made using the Clustalw-Gonnet Pam25 matrix. [*] Denotes the same amino acid and [:] denotes a traditional substitution. Cysteine surpluses important for the formation of adjuvant bonds in immunoglobulin V or C are shown in italics.
[425] Table 2
[426] Comparison of amino acid sequence of mB7-DC and mB7-H11.
[427] Table 3
[428] Amino Acid Sequence Comparison of B7-DC in B7 Family Members
[429] In order to determine the genomic structure of MB7-DC, the present invention screened bacteria artificial chromosomes (BAC) using probes from 5 'and 3' UTRs and isolated them into genomic clones. Chromosomal location mapping is performed using the BAC clone. Chromosomal localization of B7-DC was achieved using fluorescence in situ crosses. The measurement of ten specifically classified chromosomes 19 is located at the position corresponding to the interface between 19C2 and 19C3 and 47% of the distance from the heterochromatin-true chromosome border to the distal particles of chromosome 19. Certain cross signs are detected by culturing slides mated to anti-digotigenin antibodies fluoresced in opposite staining with DAPI. This position corresponds to the region of human chromosome 9 to which hB7-H1 is mapped.
[430] hB7-DC was found by positioning on two chromosome 9 BAC clones. In addition, hB7-DC and hB7-H1 were found located on a single chromosome 9 BAC clone with an insertion size of approximately 164 kb (FIG. 1). The genomic proximity of B7-DC and B7-H1 recalls the B7.1 / B7.2 pairs mapped in megabases of each other.
[431] Example III
[432] B7-DC is selectively expressed in dendritic cells.
[433] To determine the expression pattern of B7-DC, a virtual Northern analysis was performed using RNA extracted from multiple tissues, macrophage cell lines, macrophage cultures and dendritic cells derived from bone marrow and spleen. 4 macrophage cell lines, activated BMmachrophage or peritoneum, while strong crosses were detected using B7-DC probes on immature (4, 6 days) and mature (8 to sorted MHC IihiB7hi) bone marrow-induced DCs and DCs of the spleen. No signs were found anywhere in machrophage (Figure 2).
[434] Strong expression of hB7-DC is detected in human DCs raised from surrounding blood molecular cells with GM-CSF plus of IL-4 or Flt-3L. To confirm the cell surface representation of B7-DC, anti-mB7-DC antibody was used to color DC. Blocking staining with solvent B7-DC-Ig is observed in DC (FIG. 4).
[435] B7-DC is not connected to CD28 or CTLA-4 but to PD-1.
[436] Although the B7-DC has structural and thermal cognates in the B7 system, the putative CD28 / CTLA-4 binding column, SQDXXXELY (SEQ ID NO: 13) or XXXYXXRT [SEQ ID NO: 14] (34) (X = any Amino acids). In order to directly access binding, the ability of aneuploid CD28-Ig and CTLA-4-Ig to stain 293T cells infected with B7-DC or B7.1 was studied. Strong binding is observed in the B7.1 transfectant, whereas there is no binding in the B7-DC transfectant (FIG. 5). Based on homology and genomic proximity between the B7-DC and B7-H1 / PD-L1s, the experiment was performed by testing PD-1 as a candidate binding partner for B7-DC. In fact, PD-100IG binds to the B7-DC transfectant but not to the B7.1 transfectant. Although specific, the binding of BPD-1-Ig to B7-DC transfectant is smaller than the binding of CTL-4-It and CD28-Ig to B7.1. Confirmation of the binding of PD-1 to B7-DC is obtained from positive staining of PD-1-Ig and stable B7-DC-GFP transfectants. As B7-H1 and B7h / B7Rb-1, B7-DC does not use CD28 or CILA-4 as the acceptor. PD-1 appears as a receptor for B7-DC.
[437] Example 4
[438] B7-DC functions as a costimulatory molecule for T cells.
[439] Soluble B7-DC-Ig fusion molecules that can be added to T cell stimulation analytes are produced for use in testing the activity of co-stimulators containing B7-DC. The proliferative response of T cells is stimulated by an increase in the amount of immobilized anti-CD3 when either B7-DC-Ig, B7.1-Ig or isotype is present. Figure 6 (left) shows that at suboptimal anti-CD3 concentrations, B7-DC costimulated a greater T cell proliferative response than did B7.1. Moreover, the B7-DC co-stimulated proliferative response is higher in cd4 than in CD8 cells (FIG. 6). B7-DC cannot stimulate T cells when there is no TCR-focused stimulus indicating that B7-DC was supplying a true co-stimulation signal.
[440] B7-DC also co-stimulated the proliferative response when “signal 1” was fed by the MHC-peptide combination. RENCA cells (expressing no endogenous B7.1, B7.2 or B7-DC by RT-PCR analysis) are treated with (gamma) -IFN to induce MHC class-II expression. These cells are fed with IE ^d restricted HA 110-120 peptide (FERFEIFPKE) 35 [SEQ ID NO: 15]. IE ^d + HA 110-120 Purified spleen T cells from mouse mating with specific TCR transgene genes were added and proliferative response was present when either B7-DC-Ig, B7.1-Ig or isotypes were present. Was measured. 7 shows that B7-DC has greater costimulatory activity than did B7.1.
[441] Types of Lymphokine Products Co-Stimulated by B7-DC
[442] The most characteristic T cell response to costimulation by B7-based molecules is the lymphokine reactant. These lymphokines are important mediators of T cell effects. Analyzes the product of a number of different lymphokines by T cells stimulated with anti-CD-3 or B7-DC-Ig, B7.1-Ig or HA antigen co-stimulated with isotype (FIG. 8) Has been studied. The type of lymphokine co-stimulation is highly related to whether an anti-CD3 or MHC-peptide complex was used as “signal 1.” Remarkably, B7-DC is less than B7.1 (gamma) -IFN In addition, B7-DC costimulates a significant amount of IL-6 product, while B7.1 virtually nothing costimulates, while both molecules costimulate IL-2, B7 .1 co-stimulated more efficiently than B7-DC, therefore, the type of co-stimulation between B7-DC and B7.1 is distinct and B7-DC is used to stimulate important proinflammotory lymphokines. This is more efficient.
[443] Example 5
[444] B7-DC enhances the Vivo immune response.
[445] To determine whether B7-DC dominates in vivo biologic activity, the inventors asked whether B7-DC-Ig increased the immune response to the peptide vaccine. B7-DC-Ig or isotype control antibody was added to an immunogenic mixture of HA110-120 peptide and IFA. Mice were transferred with 2.5 × 10 ⁶ anti-HA 6.5 T cells 3 days prior to immunization so that HA-specific CD4 cells in Vivo were counted. After 7 days of immunization, water-flowing LN cells were obtained and cells in Vivo were stimulated for two days with changes in the HA110-120 peptide amount. 9 shows that the addition of B7-DC-Ig indeed increases the proliferative response to HA. The total number of HA-specific T cells in water-flowing LNs is approximately doubled in groups receiving B7-DC-Ig relative to isotype antibody control. Thus, it can be concluded that B7-DC has the ability to increase antigen-specific responses even at the cell basis.
[446] Example 6
[447] Discussion and conclusion
[448] The inventors have discovered and characterized new B7-based components that have a strongly limited representation in DCs and have unique costimulatory properties for T cells. The humane orthologue of B7-DCs is also expressed in DCs.
[449] This restricted expression type contrasts with previously expressed B7 family components, which, unlike the known B7.1 / 2 pathway, suggest that B7-DC is involved in other immune responses. While weak B7-DC signals are detected by RT-PCR in activated macropages, preliminary real-time RT-PCR analysis indicates that B7-DC mRNA expression in DSs is 15 times more than in activated macropages. see. Something like antibody contamination senses very low levels of B7-DC on the surface of activated macroorganisms. It is unclear whether this is sufficient for important T cell activation.
[450] The abnormal type of lymphokine product that B7-DC costimulates involves a unique biological role compared to other B7 groups. Cytokines are generally classified as follows. Th1 cytokines include IL-2, -IFN, and lymphotoxin, and Th2 cytokines include IL-4, IL-5, IL-6, and IL-13. B7-DC does not contain typical Th1 or Th2 lymphokines. B7-DC contains trace amounts of IL-4 but no IL-10. However, IL-6 is considered a Th2 cytokine. Low IL-2 and high -IFN mutually stimulated by B7-DC proportional to B7.1 do not follow the typical Th1 type. Nevertheless, the high -IFN product stimulates B7-DCs to produce important T cell effector functions.
[451] B7-DC is excellent for stimulating IL-6. The strong proliferative response of T cells induced by B7-DC is partially explained by the strong stimulation of B7.1 and unobserved IL-6 product. IL-6 strongly expands T cell proliferation in cooperation with other proliferative stimuli. IL-6 is a multifunctional cytokine that regulates T cell function as well as inflammatory responses, monocyte differentiation, B cell differentiation, thrombopoiesis, bone resorption and the growth of certain hematopoietic tumors. When inducing chemokines and leukocyte supplementation, IL-6 can work with the water soluble IL6 receptor (sIL-6R). IL-6 can mediate strong antiapoptotic effects through Stat-3 activation. Other reports suggest that Stat-3 is left without T cells, but ILt-dependent Stat-3 activation in T cells has been reported to be an important pathway for activating activated T cells.
[452] If B7-DC fails to bind CD28 or CTLA-4, it binds to PD-1, the receptor for B7-H1PC-L1. It is not known whether it binds to ICOS, a receptor for B7h / B7RP-1. The homology between B7-DC and B7-H1 / PDL-1, the tight physical binding of hB7-H1 / PD-L1 and hB7-DC, and their binding to common receptors have been associated with recent gene duplication. Suggest that there is. This is similar to the relationship between B7.1 and B7.2, which places genes in the megabases of rat chromosome 16 and human chromosome 3.
[453] As mediated by PD-1 and other virtual receptors, it will be important to identify the relative biological role of B7-DC vs. B7-H1 / PD-L1. PD-1 appears to follow the activation of T cells. PD-1 induces apoptosis by stimulating high levels of anti-CD3 and T cells. Mice overwhelmed by PD-1 develop into reverse syndrome while being a clinical sign of hypertrophic cardiomyopathy. In contrast, Dong et al reported that B7-H1 / PD-L1 interacts with T cell proliferation and cytokine secretion at low levels of anti-CD3. By analogy with the relationship of CD28 / CTLA-4, PD-L1 is an unknown receptor and will be a semireceptor. Despite sharing binding to PD-1, B7-DC and B7-H1 are distinguished in terms of lymphokine stimulation types. B7-H1 stimulates T cell IL-10 product but not B7-DC. Differential cell expression and stimulation of B7-DCs play a unique role in immune function.
[454] Although the present invention has been described in connection with specific details, it may be modified. Although the technical idea of the present invention has been described in detail according to the above preferred embodiment, it should be noted that the above-described embodiment is for the purpose of description and not of limitation. In addition, those skilled in the art will understand that various embodiments are possible within the scope of the technical idea of the present invention.
[455]
[456]
[457]
[458]

权利要求:
Claims (68)
[1" claim-type="Currently amended] A free nucleic acid molecule encoding a B7-DC terminal mammalian protein that is selectively expressed on dendritic cells as compared to activated macrophages.
[2" claim-type="Currently amended] The method of claim 1,
Comprising a nucleic acid sequence selected from base sequence 1 or base sequence 5
Nucleic Acid Molecules.
[3" claim-type="Currently amended] An liberated nucleic acid molecule that hybridizes with the nucleic acid molecule according to claim 1 under stringent hybridization conditions.
[4" claim-type="Currently amended] The method of claim 2,
Comprising base sequence 1
Nucleic Acid Molecules.
[5" claim-type="Currently amended] The method according to any one of claims 1 to 4,
Encoding a protein having an amino acid sequence selected from SEQ ID NO: 2 and SEQ ID NO: 4, or encoding another functional derivative, homologue, or biologically active fragment of the protein
Nucleic Acid Molecules.
[6" claim-type="Currently amended] The method of claim 6,
Encoding a protein comprising SEQ ID NO: 2 or encoding another functional derivative, homologue or biologically active fragment of SEQ ID NO: 2
Nucleic Acid Molecules.
[7" claim-type="Currently amended] A fragment of a nucleic acid molecule according to any one of claims 5 or 6 which encodes part or all of the extracellular region of said B7-DC protein.
[8" claim-type="Currently amended] The method of claim 5,
In a nucleic acid molecule encoding a B7-DC fusion polypeptide, the molecule is
(a) a first nucleic acid sequence encoding a first polypeptide that is part or all of B7-DC protein nucleotide sequence 2 or nucleotide sequence 4;
(b) optionally, a linker nucleic acid sequence encoding a linker peptide, fused in frame with the first nucleic acid sequence; And
(c) a second nucleic acid sequence linked to said first nucleic acid sequence or said linker nucleic acid sequence in a frame and encoding a second polypeptide
Nucleic acid molecule comprising a.
[9" claim-type="Currently amended] The method of claim 8,
The second polypeptide consists of one or more Ig heavy chain constant region.
Nucleic Acid Molecules.
[10" claim-type="Currently amended] The method of claim 9,
The second polypeptide consists of two regions of the human IgG constant region
Nucleic Acid Molecules.
[11" claim-type="Currently amended] Comprising a nucleic acid according to any one of claims 1 to 10,
(a) promoter and (b) optionally, operatively linked with additional regulatory sequences that regulate expression of said nucleic acid in eukaryotic cells
Expression vector.
[12" claim-type="Currently amended] The method of claim 11,
Plasmid
Expression vector
[13" claim-type="Currently amended] The method of claim 11,
Viral vector
Expression vector.
[14" claim-type="Currently amended] (a) a first recombinant expression vector having the incorporated nucleotide sequence in its nucleic acid, wherein the nucleotide encodes a relevant antigen for the immune response to be elicited; And
(b) a second recombinant expression vector having in its nucleic acid one or more nucleotide sequences encoding the mutually stimulating polypeptides, wherein at least one of said mutually stimulating polypeptides is B7-DC, or another functional derivative, homologue or biological thereof Active fragment;
Wherein the expression vectors can mutually infect or transfect host cells, resulting in co-expression of the antigen and the mutually stimulating polypeptide, fragment homolog or derivative.
Vector composition comprising a.
[15" claim-type="Currently amended] The method of claim 14,
The second vector comprises base sequence 1 or base sequence 5
vector.
[16" claim-type="Currently amended] The method of claim 14 or 15,
The second vector comprises the base sequence 1
vector.
[17" claim-type="Currently amended] A cell transformed or transformed using the nucleic acid molecule according to any one of claims 1 to 10.
[18" claim-type="Currently amended] A cell transformed or transformed using the expression vector according to any one of claims 1 to 16.
[19" claim-type="Currently amended] The method of claim 18,
The cell is a mammalian cell
cell.
[20" claim-type="Currently amended] The method of claim 19,
The cell is a human cell
cell.
[21" claim-type="Currently amended] The method of claim 19,
The cell is dendritic cell or its origin
cell.
[22" claim-type="Currently amended] The method of claim 19,
The cell is a tumor cell
cell.
[23" claim-type="Currently amended] Exogenous nucleic acid encoding a mammalian B7-DC protein or biologically active fragment, homolog or other functional derivative such that when a protein, fragment, homolog or other derivative is expressed by a tumor cell, the tumor cell contacts the T cell In isolated mammalian tumor cells that are transfected with molecules,
(i) the B7-DC protein, fragment, homologue or derivative is joined to the T cell;
(ii) the tumor cells interstimulate the T cells to produce, secrete or propagate cytokines.
Tumor cells.
[24" claim-type="Currently amended] The method of claim 23, wherein
The exogenous nucleic acid molecule comprises SEQ ID NO: 1, SEQ ID NO: 5 or a fragment of the sequence.
Tumor cells.
[25" claim-type="Currently amended] It is selectively expressed on dendritic cells in comparison to activated macrophages, and has the following functional characteristics:
(a) binding a binding partner on T cells;
(b) a polypeptide having the characteristic of stimulating said T cells to produce, secrete or propagate cytokines.
[26" claim-type="Currently amended] The method of claim 25,
The polypeptide, fragment, homologue or other derivative is encoded by a nucleic acid molecule having the sequence SEQ ID NO: 1 or SEQ ID NO: 5, or a fragment, homologue or equivalent of the nucleic acid molecule.
Polypeptide.
[27" claim-type="Currently amended] The method of claim 26,
A polypeptide having a SEQ ID NO: 2 or SEQ ID NO: 4 amino acid sequence, or a fragment, homologue or equivalent thereof
Polypeptide.
[28" claim-type="Currently amended] A polypeptide or biologically active fragment, homologue or other functional derivative thereof produced by recombinant expression of the nucleic acid of claim 1.
[29" claim-type="Currently amended] The method according to any one of claims 25 to 28,
An extracellular region of a B7-DC protein comprising a region comprising amino acid residues 26-221 of SEQ ID NO: 2 or SEQ ID NO: 4, or an inter-stimulatory fragment, homologue or other functional derivative of said region Containing
Polypeptide.
[30" claim-type="Currently amended] The method according to any one of claims 25 to 28,
To a region comprising amino acid residues 26-221 of SEQ ID NO: 2 or SEQ ID NO: 4, or an extracellular region of B7-DC, comprising an inter-stimulatory fragment, homologue or other functional derivative of said region Required
Polypeptide.
[31" claim-type="Currently amended] (i) fused directly to a second polypeptide, or
(ii) optionally, a B7-DC fusion polypeptide having a first fusion partner comprising all or part of a B7-DC protein fused to a linker peptide sequence fused to said second polypeptide.
[32" claim-type="Currently amended] A B7-DC fusion polypeptide comprising the polypeptide of any one of claims 25 to 30 fused to a second polypeptide.
[33" claim-type="Currently amended] 33. The method of claim 32,
In the face of sufficient stimulation of the T cell receptor, it binds to a binding partner molecule to the T cell and cross-stimulates the T cell.
Fusion polypeptides.
[34" claim-type="Currently amended] The method of claim 33, wherein
The binding partner molecule is PD-1
Fusion polypeptides.
[35" claim-type="Currently amended] The method according to any one of claims 31 to 33, wherein
The second polypeptide is one or more regions of Ig heavy chain constant region.
Fusion polypeptides.
[36" claim-type="Currently amended] 36. The method of any of claims 31-35,
The first fusion partner comprises amino acid residues 26-221 of SEQ ID NO: 2 or SEQ ID NO: 4, or comprises a co-stimulatory fragment, homologue or other functional derivative thereof
Fusion polypeptides.
[37" claim-type="Currently amended] The method of claim 36,
The first fusion partner comprises amino acids 26-221 of SEQ ID NO: 2, or comprises a co-stimulatory fragment, homologue or other functional derivative thereof
Fusion polypeptides.
[38" claim-type="Currently amended] The method of claim 35, wherein
The polypeptide has a hinge of a human immunoglobulin C _γ 1 chain, a C _H 2 and an amino acid sequence corresponding to a C _H 3 region.
Fusion polypeptides.
[39" claim-type="Currently amended] The method according to any one of claims 31 to 38,
Dimer or trimer fusion polypeptides are fusion polypeptides of serially linked dimers or trimers
Fusion polypeptides.
[40" claim-type="Currently amended] The method according to any one of claims 31 to 38,
The construction of a linear multimer, which is two or more repeats of the monomer that is the first fusion partner, is linked to the end and the end either directly or by a linker sequence present between the mono repeats.
Fusion polypeptides.
[41" claim-type="Currently amended] The antibody is specific for the epitope of the B7-DC protein and the epitope is not present in the absence of a known B7 family of proteins.
Antibodies.
[42" claim-type="Currently amended] 42. The method of claim 41 wherein
The epitope is a linear or epitope that matches the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4
Antibodies.
[43" claim-type="Currently amended] The method of claim 42,
The epitope is linear or an epitope that matches the polypeptide of SEQ ID NO: 2.
Antibodies.
[44" claim-type="Currently amended] 43. The method of claims 41 to 42 wherein
The antibody is a monoclonal antibody
Antibodies.
[45" claim-type="Currently amended] The antibody of claim 43, wherein the antibody is a human or humanized monoclonal antibody
Antibodies.
[46" claim-type="Currently amended] The method of identifying or quantifying cells expressing B7-DC polypeptide on a surface in a cell population,
(a) contacting an antibody of any of claims 41-45 with a cell of said population such that said antibody binds to a cell expressing said epitope;
(b) confirming the presence or analyzing the number of cells bound to the antibody.
Methods for Identifying and Quantifying B7-DC Polypeptide Expressing Cells.
[47" claim-type="Currently amended] A method of isolating cells expressing B7-DC polypeptide on a surface from a cell population,
(a) contacting the antibody of any one of claims 41-45 with a cell of said population to bind said antibody to a cell expressing said epitope;
(b) taking cells bound to the antibody and not taking cells not bound to the antibody
Isolation of B7-DC Polypeptide Expressing Cells.
[48" claim-type="Currently amended] The method of detecting or analyzing the presence of a B7-DC polypeptide or fragment or homologue in a sample,
(a) contacting a sample with the antibody of any one of claims 41-45, such that the antibody and polypeptide or fragment carrying the epitope bind;
(b) detecting the presence or amount of the polypeptide or fragment to which the antibody is bound
A method for detecting and analyzing the presence of a B7-DC polypeptide or fragment or homologue comprising a.
[49" claim-type="Currently amended] Induce or increase the expression of B7-DC polypeptides in antigen-presenting cells or their progeny to increase the activity of said cells co-stimulating T cells in vitro or in vivo, where appropriate stimulation to the T cell receptor is present. The method comprises transforming or infecting the antigen-expressing cells or their progenitor cells with the expression vector of any one of claims 11 to 13 so that expression of the B7-DC polypeptide is induced or increased in the cells. Involving
A method of inducing or increasing expression of a B7-DC polypeptide.
[50" claim-type="Currently amended] Induce or increase the expression of B7-DC polypeptides in antigen-presenting cells or their progeny to increase the activity of said cells co-stimulating T cells in vitro or in vivo, where appropriate stimulation to the T cell receptor is present. The method comprises transforming or infecting the antigen-expressing cells or their progenitor cells with the expression vector of any one of claims 11 to 13 so that expression of the B7-DC polypeptide is induced or increased in the cells. Involving
A method of inducing or increasing expression of a B7-DC polypeptide.
[51" claim-type="Currently amended] The method of claim 49 or 50,
The antigen-presenting cell is a dendritic cell, and the source is a dendritic cell progeny.
A method of inducing or increasing expression of a B7-DC polypeptide.
[52" claim-type="Currently amended] A method of increasing the T cell response of an antigen-stimulated mammal, the method comprising administering an effective amount of a cell according to any one of claims 17 to 24 associated with an antigen, wherein said cell is characterized in that said antigen is stimulated. Characterized in that the cells are effective in increasing the T cell response to
How to increase T cell response.
[53" claim-type="Currently amended] The method according to any one of claims 22 to 24,
The tumor cells exhibit the antigen, and administration of the tumor cells is effective to increase the T cell response of the patient to the tumor antigen stimulation.
Way.
[54" claim-type="Currently amended] The method according to any one of claims 25 to 30,
Administration of the polypeptide is effective to increase the T cell response of the patient to the antigen stimulation.
Way.
[55" claim-type="Currently amended] The method according to any one of claims 31 to 40,
Administration of the polypeptide is effective to increase the T cell response of the patient to the antigen stimulation.
Way.
[56" claim-type="Currently amended] The method according to any one of claims 41 to 45,
Administration of the antibody is effective in blocking stimulation of T cells or eliminating antigen-responsive T cells, thereby inhibiting T cell responses.
Way.
[57" claim-type="Currently amended] The method of claim 56, wherein
The T cell response is directed to tissue transplantation or autoantigen such that the method inhibits transplant rejection or autoimmune response.
Way.
[58" claim-type="Currently amended] A method for increasing the immune response of a mammalian patient to antigenic stimulation,
(a) obtaining T cells from (i) said patient, (ii) an immunologically easy donor for said patient or (iii) an immunologically easy or acceptable culture cell line;
(b) ex vivo contacting said T cells with an effective amount of cells according to any of claims 17 to 24, wherein said contacting step is effective to increase the response of said T cells to antigenic stimulation. Im-; And
(c) administering to said patient the T cells of step (b) to increase said immune response of said patient
How to include.
[59" claim-type="Currently amended] A method for increasing the immune response of a mammalian patient to antigenic stimulation,
(a) obtaining T cells from (i) said patient, (ii) an immunologically easy donor for said patient or (iii) an immunologically easy or acceptable culture cell line;
(b) ex vivo contacting said T cells with an effective amount of cells according to any one of claims 17 to 24, wherein said contacting step is effective to increase the response of said T cells to antigenic stimulation. Im-; And
(c) administering to said patient the T cells of step (b) to increase said immune response of said patient
How to include.
[60" claim-type="Currently amended] A method for increasing the immune response of a mammalian patient to antigenic stimulation,
(a) obtaining T cells from (i) said patient, (ii) an immunologically easy donor for said patient or (iii) an immunologically easy or acceptable culture cell line;
(b) ex vivo contacting said T cells with an effective amount of the polypeptide, fragment, homologue or functional derivative of any one of claims 25-30, wherein said contacting comprises said T for antigen stimulation. Effective in increasing cellular response; And
(c) administering to said patient the T cells of step (b) to increase said immune response of said patient
How to include.
[61" claim-type="Currently amended] A method for increasing the immune response of a mammalian patient to antigenic stimulation,
(a) obtaining T cells from (i) said patient, (ii) an immunologically easy donor for said patient or (iii) an immunologically easy or acceptable culture cell line;
(b) ex vivo contacting said T cells with an effective amount of said fusion polypeptide of any one of claims 31-40, wherein said contacting step is effective to increase the response of said T cells to antigenic stimulation. -; And
(c) administering to said patient the T cells of step (b) to increase said immune response of said patient
How to include.
[62" claim-type="Currently amended] A vaccine composition useful for promoting a protective immune response against an antigen associated with a pathogenic cell or microorganism,
(a) a modified or transfected cell of any one of claims 17 to 24, which selectively expresses the antigen;
(b) if the cells of (a) do not express the antigen, an additional source of said antigen;
(c) optionally, a universal immunostimulatory drug or adjuvant; And
(d) pharmaceutical and immunologically acceptable excipients or carriers for (a), (b) and (c)
Vaccine composition comprising a.
[63" claim-type="Currently amended] A vaccine composition useful for promoting a protective immune response against an antigen associated with a pathogenic cell or microorganism,
(a) a source of antigen for which an immune response is desired;
(b) the polypeptide, fragment, homolog or functional derivative of any one of claims 25-30;
(c) optionally, a universal immunostimulatory drug or adjuvant; And
(d) pharmaceutical and immunologically acceptable excipients or carriers for (a), (b) and (c)
Vaccine composition comprising a.
[64" claim-type="Currently amended] A vaccine composition useful for promoting a protective immune response against an antigen associated with a pathogenic cell or microorganism,
(a) a source of antigen for which an immune response is desired;
(b) the fusion polypeptide of any one of claims 31-40;
(c) optionally, a universal immunostimulatory drug or adjuvant; And
(d) pharmaceutical and immunologically acceptable excipients or carriers for (a), (b) and (c)
Vaccine composition comprising a.
[65" claim-type="Currently amended] In an inter-stimulatory composition used in conjunction with an antigen or a vaccine to increase the immunogene of an antigen or vaccine,
(a) a polypeptide, fragment, homologue or functional derivative of any one of claims 25-30; And
(b) pharmaceutically and immunologically acceptable excipients or carriers
Inter-stimulating composition comprising a.
[66" claim-type="Currently amended] In an inter-stimulatory composition used in conjunction with an antigen or a vaccine to increase the immunogene of an antigen or vaccine,
(a) the fusion polypeptide of any one of claims 31-40; And
(b) pharmaceutically and immunologically acceptable excipients or carriers
Inter-stimulating composition comprising a.
[67" claim-type="Currently amended] 65. A method for promoting or enhancing an immune response to an antigen in a mammalian patient comprising administering to said patient an effective amount of the vaccine composition of any one of claims 62 to 64.
[68" claim-type="Currently amended] 67. A method for enhancing an immune response to an antigen or vaccine in a mammalian patient comprising administering the inter-stimulatory composition of claim 65 or 66 in combination with the antigen or vaccine to the patient.

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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:
2000-04-28|Priority to US20058000P

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2000-04-28|Priority to US60/200,580

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2000-10-13|Priority to US24016900P

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2000-10-13|Priority to US60/240,169

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2001-04-27|Application filed by 더 존스 홉킨스 유니버시티

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2001-04-27|Priority to PCT/US2001/013430

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2003-04-08|Publication of KR20030028466A

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2009-02-23|Application granted

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2009-02-23|Publication of KR100884766B1

优先权:

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申请号 | 申请日 | 专利标题

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US20058000P| true| 2000-04-28|2000-04-28|

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US60/200,580|2000-04-28|

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US24016900P| true| 2000-10-13|2000-10-13|

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US60/240,169|2000-10-13|

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PCT/US2001/013430|WO2001083750A2|2000-04-28|2001-04-27|Dendritic cell co-stimulator molecules|