HK1199271B - Use of anti-cd83 agonist antibodies for treating autoimmune diseases - Google Patents

Description

Use of anti-CD 83 agonist antibodies for the treatment of autoimmune diseases

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application serial No. 61/504,127 filed on 1/7/2011 and japanese patent application No. 2011-one 285585 filed on 27/12/2011 and 2011-one 285595 filed on 27/12/2011, each of which is incorporated by reference in its entirety.

Technical Field

The present invention relates to anti-CD 83 agonist antibodies and methods for treating autoimmune diseases, such as inflammatory bowel disease, using anti-CD 83 agonist antibodies.

Background

Inflammatory Bowel Diseases (IBDs), such as ulcerative colitis and crohn's disease, are characterized by chronic and recurrent inflammation of the intestinal tract. Recent evidence suggests that IBD is due to a loss of tolerance to commensal gut flora, where the mucosal immune system is unable to distinguish between pathogens and commensal organisms. Thus, IBD patients develop immune recognition of the normal flora, which produces a gastrointestinal inflammatory response. The degree of seroreactivity to normal flora roughly correlates with disease duration and disease severity (Lodes et al, J Clininvest.,113:1296-1306, 2004). More than 25 alleles have been identified that overlap between ulcerative colitis and clonopathy genetics (Umeno et al, inflam Bowel dis. in press, 2011). These IBD genetic alleles converge in several common pathways that result in 1) a defect in epithelial integrity, 2) a defect in the production of myeloid microbial responses and proinflammatory cytokines, and 3) an increase in T helper 17 cells and T helper 1 cell responses. Studies have shown that most IBD patients have spontaneous relapses and remissions cycles, where they achieve clinical remission with reasonable frequency, but do not maintain remission for a long period. This observation indicates that there is an as yet undefined mucosal homeostatic mechanism that can drive the remission phase of IBD (Schirbel et al, Expert RevGastroenterol hepatol, 5(1):33-41,2011). The identification of molecules and mechanisms involved in maintaining mucosal homeostasis is therefore beneficial for driving and maintaining IBD remission.

CD83 is a highly conserved 45 kilodalton transmembrane glycoprotein that is expressed predominantly on the surface of dendritic cells and thymic epithelial cells. In addition, CD83 is transiently expressed on the surface of other activated cells of the immune system and is also found in soluble form. Structural analysis of the predicted amino acid sequence of CD83 shows that it is a member of the immunoglobulin superfamily, indicating a role in the immune system. The literature reports suggest that soluble CD83(sCD83) plays an immunomodulatory role due to the observation that sCD83 released by HCMV-infected mature dendritic cells inhibits T cell proliferation (Senechal et al, blood, 103(11): 4207-. However, no ligand for CD83 was identified and little is still known about the function of CD83. Interestingly, CD83 gene expression is down-regulated on the mucosal surface of human clonopathy (Silva et al, dig. Dis. Sci.,53(7):1917-1928,2008), suggesting that CD83 may be involved in maintaining mucosal homeostasis and thus be a therapeutic target for modulating the immune system in IBD.

All references, including patent applications and publications, cited herein are incorporated by reference in their entirety.

Summary of The Invention

Provided herein are methods for treating or preventing an autoimmune disease in an individual comprising administering to the individual an effective amount of an anti-CD 83 agonist antibody. In some embodiments, the individual is a human.

In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), lupus nephritis, ulcerative colitis, wegener's disease, inflammatory bowel disease, Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, raynaud's syndrome, sjogren's syndrome, and glomerulonephritis. In some embodiments, the autoimmune disease is associated with myeloid cell activation (dendritic cells and macrophages), such as multiple sclerosis and inflammatory bowel disease.

In some embodiments, the individual has or is diagnosed with an autoimmune disease. In some embodiments, the individual has or is diagnosed with inflammatory bowel disease (e.g., crohn's disease, ulcerative colitis, and indeterminate colitis).

In some embodiments, the anti-CD 83 agonist antibody inhibits the release of proinflammatory cytokines from mature dendritic cells (e.g., inhibits the release of proinflammatory cytokines MCP-1 and/or IL-12p 40). In some embodiments, the anti-CD 83 agonist antibody induces the release of an anti-inflammatory cytokine from mature dendritic cells (e.g., induces the release of the anti-inflammatory cytokine IL-1 ra). In some embodiments, the anti-CD 83 agonist antibody induces a decrease in cell surface expression of CD83 and/or HLA-DR on mature dendritic cells. In some embodiments, the anti-CD 83 agonist antibody inhibits activation of MAPK and/or TOR signaling in mature dendritic cells. In some embodiments, inhibition of activation of MAPK signaling is determined by a decrease in phosphorylation of p38 and/or CREB proteins in mature dendritic cells. In some embodiments, inhibition of mTOR signaling activation is determined by a decrease in phosphorylation of the mTOR protein in mature dendritic cells. In some embodiments, the anti-CD 83 agonist antibody upregulates the expression of wound healing genes (e.g., vcan, spock2, and fbn 2) in mature dendritic cells.

In some embodiments, the anti-CD 83 agonist antibody is a monoclonal antibody. In some embodiments, the anti-CD 83 agonist antibody is an antigen binding fragment, e.g., selected from the group consisting of Fab, Fab '-SH, Fv, scFv, and (Fab')₂A fragment of a fragment. In some embodiments, the anti-CD 83 agonist antibody is a humanized antibody. In some embodiments, the anti-CD 83 agonist antibody is a human antibody. In some embodiments, the anti-CD 83 agonist antibody is a chimeric antibody. In some embodiments, the anti-CD 83 agonist antibody comprises a heavy chainA variable domain comprising HVR-H1 comprising the amino acid sequence of SEQ ID NO. 31, HVR-H2 comprising the amino acid sequence of SEQ ID NO. 32, and HVR-H3 comprising the amino acid sequence of SEQ ID NO. 33; and/or a light chain variable domain comprising HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, HVR-L2 comprising the amino acid sequence of SEQ ID NO:38, and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the anti-CD 83 agonist antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO. 30, and/or a light chain variable domain comprising the amino acid sequence of SEQ ID NO. 36.

In some embodiments, the anti-CD 83 agonist antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

Also provided herein are articles of manufacture or kits comprising anti-CD 83 agonist antibodies. In some embodiments, the article of manufacture or kit can further comprise a package insert comprising instructions for using the anti-CD 83 agonist antibody to treat or prevent an autoimmune disease in an individual.

Also provided herein are isolated anti-CD 83 antibodies comprising a variable domain comprising at least one, two, three, four, five, or six hypervariable region (HVR) sequences selected from the group consisting of: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 32; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO 38; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39. Also provided herein are isolated anti-CD 83 antibodies comprising a heavy chain variable domain and a light chain variable domain, the variable domains comprising the following six HVR sequences: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 32; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO 38; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, an antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33. In some embodiments, the antibody further comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 38; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, an antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO 38; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, wherein the antibody is a chimeric antibody or a humanized antibody. In some embodiments, the antibody comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO 30, and/or a light chain variable domain comprising the amino acid sequence of SEQ ID NO 36.

Also provided herein are pharmaceutical compositions comprising an anti-CD 83 antibody described herein and a pharmaceutically acceptable carrier.

Also provided herein are isolated nucleic acids comprising a nucleotide sequence encoding an anti-CD 83 antibody described herein. Provided herein are vectors comprising nucleic acids. In some embodiments, the vector is an expression vector. Also provided herein are host cells comprising the vectors. In some embodiments, the host cell is prokaryotic or eukaryotic.

Also provided herein are methods for making an anti-CD 83 antibody, the methods comprising culturing a host cell described herein under conditions suitable for expression of a nucleic acid encoding an anti-CD 83 antibody described herein. In some embodiments, the method further comprises recovering the anti-CD 83 antibody produced by the host cell.

It will be understood that one, some or all of the properties of the various embodiments described herein may be combined to form further embodiments of the invention. These and other aspects of the invention will become apparent to those skilled in the art.

Brief Description of Drawings

Figure 1 shows CD83 involved in homotypic binding on the surface of cells expressing CD83. (a) CD83 expressed on the surface of stably transfected CHO-hCD83 cells was quantified by flow cytometry. (b) Expression of CD83 expressed on MUTZ-3 derived mature DCs was quantified by flow cytometry. (c and d) CD83.fc binding to CHO-hCD83 and mature DCs.

Figure 2 shows that CD83 is required for binding to cd83. fc. (a) As quantified by flow cytometry, cd83.fc bound CHO-hCD83 cells, but binding was blocked by anti-CD 83 antibody. (b) The efficacy of siRNA specific for CD83 (siCD83) in MUTZ-3 mature DCs, as shown by taqman analysis of total RNA normalized to 18S. (c) CD83 was expressed on the surface of mature DCs treated with non-targeting control (sinct) siRNA, but not on mature DCs treated with siCD 83. (d) Fc binds mDC, but binding is eliminated by the siCD83 treatment. (e) Knockdown of CD83 reduced the surface expression of MHCII in mature DCs. (f) Knockdown of CD83 did not alter surface expression of CD86 in mature DCs.

Fig. 3 provides a microscope image at 10X showing that CD83 expression results in cell aggregation in suspension culture. (a) Control CHO cells did not aggregate. (b) Cells expressing CD83 form aggregates when in suspension culture. (c) Fc protein pretreatment CHO-hCD83 cells blocked aggregation. (d) Pretreatment of CHO-hCD83 cells with Ig control protein did not block aggregation.

FIG. 4 shows that treatment of CD83 in mature DCs with CD83.fc or HB15e antibody reduced the expression of the surface activation markers CD83 and HLA-DR.

Figure 5 ELISA data show altered cytokine release in CD 83-treated human monocyte-derived DCs. (a and c) the pro-inflammatory cytokines IL12-p40 and MCP-1 were reduced following CD83.fc or HB15e antibody treatment. (b) The anti-inflammatory cytokine IL-1ra was significantly increased in CD 83-treated DCs. (d) IL-8 did not differ in CD 83-treated or control-treated DCs. Indicates that the values have significant differences; p <0.01, p < 0.001. The figures represent at least four different donors.

Figure 6 ELISA data show altered cytokine release in CD 83-treated MUTZ-3 derived DCs. (a and c) the pro-inflammatory cytokines IL12-p40 and MCP-1 were reduced following CD83.fc or HB15e antibody treatment. (b) The anti-inflammatory cytokine IL-1ra was significantly increased in CD 83-treated DCs. (d) IL-8 did not differ in CD 83-treated or control-treated DCs. Supernatants from each well were run in triplicate.

Fig. 7 shows that treatment of CD83 with either cd83.fc or HB15e antibodies in DCs resulted in up-regulation of genes involved in wound healing as provided by TaqmanqPCR analysis of vcan, spot 2 and fbn2 normalized to gapdh. Mean relative expression (2. sup. DELTA. CT) + SEM.

FIG. 8 shows that the CD83 isotype interaction can occur in reverse to mediate an anti-inflammatory response in DCs. (a and b) ELISA assays for cytokine production showed that co-culture of CHO cells overexpressing CD83 with mDCs inhibited the release of the pro-inflammatory cytokines IL12-p40 and MCP-1. (c) Mature mouse bone marrow-derived DCs (BMDCs) (gray bars) generated from CD83 knockdown (CD83KO) and Wild Type (WT) littermates produced similar levels of IL-12p40 after 24h LPS stimulation. (d) Immature BMDCs cultured with WT mature BMDCs produced significantly less IL-12p40 than those cultured with CD83 deficient mature BMDCs, p = 0.0372.

FIG. 9 shows that the CD83 isotype interaction mediates anti-inflammatory responses in DCs. (a) As shown by ELISA for MCP-1, siRNA knockdown of CD83 abrogated the response to either CD83.fc or HB15e antibody. (b) Schematic representation of CD83 lentiviral expression constructs of full-length CD83 and cytoplasmic truncated CD83. (c) ELISA generated by MCP-1 showed that expression of cytoplasmic truncated CD83 blocked the inhibitory effect of the CD83.fc protein.

Figure 10 shows that antibody cross-linking is sufficient to drive an anti-inflammatory response through the CD83 cytoplasmic domain in DCs. (a) A schematic of a CD83 chimeric lentiviral expression construct depicting a CD79a extracellular domain and a full-length or truncated cytoplasmic domain fused transmembrane to CD83. (b) ELISA generated by IL12-p40 showed that overexpression of the truncated chimera inhibited the response of DCs against CD79a antibody.

Figure 11 shows that CD83 contains a class of III PDZ ligand motifs at the C-terminus that mediate anti-inflammatory responses. (a) Amino acid alignment schemes of the last 15 amino acids of the CD83 cytoplasmic domain show the conserved type of III PDZ ligand motif. (b) Schematic representation of a CD83 lentiviral expression construct of a C-terminal PDZ ligand motif V205A mutant. (c) ELISA generated by MCP-1 shows that the expression of V205APDZ ligand motif mutant blocks the inhibitory effect of CD83.fc protein.

Figure 12 shows that the immunosuppressive effects of CD83 isotype interactions are mediated by the p38MAPK and mTOR signaling pathways. (a-c) shows that HB15e antibody treatment resulted in a significant reduction in phosphorylation of mTOR, p38, and CREB. (d) HB15e antibody treatment did not inhibit phosphorylation of STAT3, which is activated by the TNF pathway. (e) Western blot analysis confirmed the reduction of p38 phosphorylation after HB15e antibody treatment. (f) No significant difference in phosphorylation of STAT3 was seen after HB15e antibody treatment. Total p38 and STAT3 were used as loading controls.

FIG. 13 shows that overexpression of CD83 in the colon results in the down-regulation of surface activation markers on DCs in the lamina propria. (a) Schematic representation of the transgene construct used to generate CD83 transgenic mice. (b) Immunohistochemical staining revealed expression of the transgene in colonic epithelial cells. (c and e) expression of surface markers on DCs isolated from the colon and spleen of CD83 transgenic mice was quantified by FACS and measured as Mean Fluorescence Intensity (MFI). (d and f) no significant difference in T cell surface activation markers was found in the colon or spleen of CD83 transgenic mice. (g) Taqman qPCR analysis showed increased wound healing gene expression in DCs isolated from CD83 transgenic mice.

Fig. 14 depicts FACS gating strategy for analyzing DC subsets isolated from CD83 transgenic mice, showing that CD83 transgene has no effect on the production of DC subsets. (a and b) MHCII + and CD11c hi express gated DCs. (c) Gating of T cells. (d and e) flow cytometry analysis of subsets of DCs isolated from colon and spleen.

Figure 15 shows that CD83 overexpression protects mice from Dextran Sodium Sulfate (DSS) -induced colitis. (a) Describes that CD83 transgenic mice lost less weight when treated with DSS compared to wild type mice. (b) Hematoxylin-eosin staining of colon sections of mice with DSS-induced colitis. (c) Histological scores of wild type and CD83 transgenic mice. (d) ELISA of serum cytokine levels in CD83 transgenic mice treated with DSS compared to wild-type littermates. (e) qPCR for IL-12p40 gene expression in colon lamina propria DCs from CD83Tg mice treated with DSS, compared to wild type littermates.

Fig. 16 shows CD83 knockdown in DCs-exacerbated colitis. (a) Schematic representation of the CD83 conditional knockout strategy. (b) FACS curves gated on TCRb + lymphocytes in the spleen. In the spleen, CD83^fl/flThe CD11c-Cre mouse has 48.6% CD4 positive T cells, CD83w^t/wtCD11c-Cre mice had 48.1% CD4 positive T cells. (c) Histogram showing CD83^{wt/ wt}CD11c-Cre (black line) and CD83^fl/flRelative expression of CD83 on splenic DCs of CD11c-Cre (gray line) mice. (d) CD83^fl/flCD11c-Cre and CD83^wt/wtDSS colitis survived in CD11c-Cre mice. Conditional knockout animals had significantly less survival (Log rank test, p = 0.0186). (e) And CD83^wt/wtCD11c-Cre comparison with littermates on day 8 CD83^fl/flThe body weight of CD11c-Cre mice was significantly lower. (f) Incidence of bright blood around the anus of animals. 100% CD83^fl/flCD11c-Cre mice had significant occult blood by day 7. In CD83^wt/wtNo bright blood was observed in the CD11c-Cre littermates.

Figure 17 shows anti-human CD83 antibodies that bind to CD 83-expressing cells. Quantification by flow cytometry revealed that anti-CD 83 antibodies (a)35G10, (b)40a11, (C)54AD1, (d)59G10, (e)75a1, and (f)7C7 specifically bound to human CD 83-expressing CHO cells (black line), but not to mouse CD 83-expressing cells (hatched line). (g) anti-CD 83 antibody 60B10 bound CHO cells expressing human CD83, but also showed cross-reactivity with CHO cells expressing mouse CD83. No binding was seen in the parental CHO cell line (solid grey bar).

Figure 18 shows anti-mouse CD83 antibodies that bind CHO cells expressing mouse CD83. Quantification by flow cytometry revealed that the anti-CD 83 antibodies (a)42C6 and (b)39a2 specifically bound CHO cells expressing mouse CD83 (hatched), but not CHO cells expressing human CD83 (black line) or the parental CHO cell line (filled grey bar graph).

Figure 19 shows that anti-CD 83 antibodies significantly reduced pro-inflammatory cytokine production (a) ELISA data analysis showed that anti-CD 83 antibodies 60B10, 35G10, 40a11, or 7C7 significantly reduced MCP-1 production from mDCs compared to the use of isotype control antibodies (ISO): indicating significantly different values from the treated isotype control, p =0.0303, p =0.0309, p =0.0369, p =0.0247, respectively no significant difference was seen in cells given 54D1, 59G10, or 75a1 antibodies (B) quantitative PCR analysis of IL-12p40 expression in mouse bone marrow derived dcs (bmdcs) showed that anti-CD 83 antibodies 39a2 or 42C6 significantly reduced LPS-induced CD83 expression compared to the use of isotype control antibodies 0.0372 6 expression is normalized against each of CD 12p40 representing a sign from actin-2, 0.0372 9;，p=0.0438。

figure 20 shows that anti-CD 83 antibody protected mice from DSS-induced colitis. Mice given anti-CD 83 antibody had significantly reduced histological scores compared to those given anti-gD control antibody or DSS only: 39a2, mean =5.3, indicates p =0.0011, 42C6, mean =5.5,p =0.0015, 60B10, mean =5.4, p = 0.0059.

Detailed Description

I. General technique

The techniques and methods described or referenced herein are generally well understood by those skilled in the art and generally employ conventional methods, such as the widely employed methods described below: sambrook et al, Molecular Cloning: ALaberration Manual version 3 (2001) Cold spring harbor Laboratory Press, Cold spring harbor, N.Y.; Current Protocols in Molecular Biology (F.M.Ausubel., et al, eds. (2003)), the services Methods in enzymology (Academic Press, Inc.; PCR2: A practical applications (M.J.MacPherson, B.D.Hames and G.R.Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory, and Animal Cells (R.I.Freeshney, eds. (1987), Synthesis, filtration, Cell, edit: catalog J.E.S.C., Culture, Cell J.E.S.C.; primer, Cell, and Cell, 7, Cell J.E.S.C.; Culture, Cell, edit: 7, Cell J.E.S.C.; Molecular Culture, Culture J.S.C.; Culture, Culture J.S.S.S.C. and Cell, Culture J.E.S.S.S.S.S.S.C. (1987, Culture, Cell, edit, Cell J.S.S.S.S.S.C.; Culture, Culture J.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.C. 7, Culture, C. 7, Culture, edit, Culture J.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.C. Ed and C.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S.S, (Mullis et al, eds., 1994); Current Protocols in immunology (J.E.Coligan et al, eds., 1991); Short Protocols in molecular biology (Wiley and Sons,1999); immunology (C.A.Janeway and P.travers,1997); antibiotics (P.Finch,1997); Antibodies: A Practical Approach (D.Catty., eds., IRL Press, 1988) 1989); Monoclonal Antibodies: A Practical Approach (P.Shell and C.Denan, eds., Oxford University, 2000); Utility Antibodies: A Laboratory Manual (E.Harlow and D.Laplacian (spring, Inc., Japan, edited., variance, Japan, 1995, Press, J.S.; color et al, Huang.

Definition of

The term "autoimmunity" refers to the process by which immune system components, such as antibodies or lymphocytes, attack or damage the molecules, cells or tissues of the organism from which they are produced. "autoimmune disorder" refers to a disease in which damage, such as tissue damage or pathogenesis, results at least in part from an autoimmune process. For example, "autoimmune diseases" include rheumatoid arthritis, juvenile rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), lupus nephritis, ulcerative colitis, wegener's disease, inflammatory bowel disease, Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, raynaud's syndrome, sjorgen's syndrome, and glomerulonephritis. In some embodiments, the autoimmune disorder is associated with myeloid cell activation (dendritic cells and macrophages), such as multiple sclerosis and inflammatory bowel disease.

"inflammatory bowel disease" or "IBD" refers to a group of disorders that cause inflammation of the intestinal tract, which is generally manifested by symptoms including abdominal cramps and pain, diarrhea, weight loss, and intestinal bleeding. IBD includes Ulcerative Colitis (UC), crohn's disease, and indeterminate colitis.

"ulcerative colitis" or "UC" is an unscheduled chronic inflammatory disease of the large intestine and rectum characterized by bloody diarrhea. Ulcerative colitis is characterized by chronic inflammation in the colonic mucosa and can be classified according to location: "proctitis" occurs only in the rectum; "rectosigmoiditis" affects the rectum and sigmoid colon; "left hemicolitis" includes the entire left half of the large intestine; and "pan colitis" inflames the entire colon.

"Crohn's disease," also known as "Crohn's disease," is a chronic autoimmune disease that can affect any part of the gastrointestinal tract, but most often occurs in the ileum (the region where the small and large intestines meet). In contrast to ulcerative colitis, crohn's disease is characterized by chronic inflammation that extends through all layers of the intestinal wall and involves the mesentery as well as regional lymph nodes. The basic pathological process is the same whether the small intestine or colon is involved.

In more than 90% of cases, ulcerative colitis and crohn's disease are clinically, endoscopically, pathologically, and serologically distinct from each other; the remaining cases are considered to be uncertain IBD (Harrison's Principles of internal medicine, 12 th edition, page 1271 (1991)).

"indeterminate colitis" refers to an inflammatory bowel disease condition with overlapping features of ulcerative colitis and crohn's disease. A diagnosis is given when history shows acute and chronic inflammation and has structural changes confined to the colon, but it is not clearly indicated whether the individual suffers from crohn's disease or ulcerative colitis.

As used herein, the term "treatment" refers to a clinical intervention designed to alter the natural course of the treated individual or cell in the course of clinical pathology. Desirable therapeutic effects include reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with an autoimmune disease (such as inflammatory bowel disease) are reduced or eliminated.

As used herein, the term "preventing" includes providing prevention in an individual with respect to the occurrence or recurrence of a disease. An individual may be susceptible to, or at risk of developing an autoimmune disease, but has not yet been diagnosed with the disease.

As used herein, an individual "at risk of developing an autoimmune disease" may or may not have a detectable disease or disease symptom, and may or may not have a detectable disease or disease symptom displayed prior to the treatment methods described herein. By "at risk" is meant that the individual has one or more risk factors that are measurable parameters associated with the development of an autoimmune disease, as is known in the art. Individuals with one or more of these risk factors are more likely to develop disease than individuals without one or more of these risk factors.

An "effective amount" refers to an amount that is at least effective at the dosages and for the periods of time necessary to achieve the desired therapeutic or prophylactic result. An effective amount may be provided in one or more administrations.

A "therapeutically effective amount" is at least the minimum concentration required to achieve a measurable improvement in a particular condition (e.g., an autoimmune disease). The therapeutically effective amount herein will vary depending on such factors as the disease state, age, sex and weight of the patient, and the ability of the anti-CD 83 agonist antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which the beneficial therapeutic effect exceeds any toxic or detrimental effects of the anti-CD 83 agonist antibody. A "prophylactically effective amount" refers to an effective amount, in dosages and for periods of time necessary to achieve the desired prophylactic result. Typically, but not necessarily, because a prophylactic dose is used in a subject prior to or at an earlier stage of the disease, the prophylactically effective amount can be less than the therapeutically effective amount.

By "chronic" administration is meant administration of the drug in a continuous manner, as opposed to an acute manner, to maintain the initial therapeutic effect (activity) for an extended period of time. By "intermittent" administration is meant a treatment that is not accomplished continuously with no intervals, but rather is naturally periodic.

As used herein, "administering in conjunction with" another compound or composition includes administering simultaneously and/or at different times. Co-administration also includes administration as a co-formulation or as separate compositions, including at different dosing frequencies or intervals, and using the same route of administration or different routes of administration.

For therapeutic or prophylactic purposes, "individual" refers to any animal, which is classified as a mammal, including humans, domestic and farm animals, as well as zoo, sports, or pet animals, such as dogs, horses, rabbits, cows, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. In some embodiments, the individual is a human.

As used herein, the term "cytokine" generically refers to a protein released by a population of cells that act as intercellular mediators on other cells or have autocrine effects on the cells producing the protein, examples of such cytokines include lymphokines, monokines, interleukins ("ILs"), such as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, IL-17A-F, IL-18 to IL-29 (such as IL-23), IL-31, includingrIL-2, tumor necrosis factors such as TNF- α or TNF- β, TGF- β 1-3, and other polypeptide factors including leukemia inhibitory factor ("LIF"), ciliary neurotrophic factor ("CNTF"), CNTF-like cytokine ("CLC"), cardiotrophin ("CT"), and kit ligand ("KL").

As used herein, the term "CD 83" includes naturally occurring variants of the native sequences CD83 and CD83. CD83 can be isolated from a variety of sources, such as mammalian (including human) tissue types or another source, or CD83 can be prepared by recombinant and/or synthetic methods.

As used herein, the term "anti-CD 83 agonist antibody" refers to an antibody that binds to CD83 expressed on the surface of a cell and activates signaling of CD83 upon binding to CD83 expressed on the surface of a cell.

The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The term "antibody" is used herein in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments,

as long as they exhibit the desired biological activity.

The basic 4 chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. V_HAnd V_LTogether form a single antigen binding site. For the structure and properties of different classes of antibodies see, e.g., Basic and Clinical Immunology, 8 th edition, Daniel p.stites, Abba i.terr and Tristram g.parslow (ed.), Appleton&Lange, Norwalk, CT,1994, page 71 and chapter 6.

L chains from any vertebrate species can be assigned to one of two distinctly different types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of their heavy Chain (CH) constant domains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, have heavy chains designated alpha ("α"), delta ("), epsilon ("), gamma ("γ"), and mu ("μ"), respectively. Based on the relatively small differences in CH sequence and function, the γ and α classes are further divided into subclasses (isoforms), e.g., humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA 2. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Abbas et al, Cellular and molecular immunology, 4 th edition (w.b. saunders co., 2000).

"native antibodies" are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V) at one end_H) Followed by a number of constant domains. Each light chain has a variable domain (V) at one end_L) A constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the heavy chain variable domain. It is believed that particular amino acid residues form the interface between the light and heavy chain variable domains.

An "isolated" antibody is one that has been identified, isolated and/or recovered (e.g., naturally or recombinantly) from a component of its production environment. Preferably, the isolated polypeptide does not bind to all other contaminant components of the environment in which it is produced. Contaminant components from their production environment, such as those derived from recombinant transfected cells, are materials that would normally interfere with the research, diagnostic, or therapeutic uses of antibodies, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide will be purified as: (1) greater than 95% by weight of the antibody, and in some embodiments, greater than 99% by weight, as determined, for example, by the Lowry method; (2) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotor-type protein sequencer, or (3) to an extent such that SDS-PAGE is electrophoretically homogeneous under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. Isolated antibodies include antibodies in situ within recombinant T cells, as at least one component of the natural environment of the antibody is not present. Typically, however, the isolated polypeptide or antibody is prepared by at least one purification step.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "V" respectively_H"and" V_L". These domains are generally the most variable part of an antibody (relative to other antibodies of the same class) and contain an antigen binding site.

The term "variable" refers to the fact that certain segments of a variable domain differ widely in antibody sequence. The V domain mediates antigen binding and defines the specificity of a particular antibody for a particular antigen. However, the variability is not evenly distributed over the full length of the variable domain. Indeed, it is enriched in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, mostly in a β -sheet configuration, connected by three HVRs that form a loop junction, and in some cases, form part of a β -sheet structure. The HVRs in each chain are held together in close proximity by the FR region and promote the formation of antigen binding sites for antibodies with HVRs from the other chain (see Kabat et al, Sequences of immunological interest, 5 th edition, national institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to the antigen, but exhibit a variety of effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

As used herein, the term "monoclonal antibody" refers to an antibody that is obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that are present in minor amounts. Monoclonal antibodies are highly specific for a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used according to the invention can be prepared by a variety of techniques,

such techniques include, for example, Hybridoma methods (e.g., Kohler and Milstein., Nature,256:495-97(1975); Hongo et al, Hybridoma,14(3): 253. sub.260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2 nd edition 1988); Hammerling et al, Monoclonal Antibodies and T-cell hybrids 563. sub.563-681 (Elsevier, N.Y.,1981)), recombinant DNA methods (see, for example, U.S. Pat. No.4,816,567), phage display techniques (see, for example, Clackson et al, Nature,352: 624. sub.628 (1991); Marks et al, J.mol. 581: 597(1992), Sildhu et al, J.1242. sub.338. sub.119: 97 (2004), and Lehol et al, Biostrain II.340. sub.32. sub.472: 120. sub.472 (Legend), and L. sub.340. sub.32. sub.472.310. sub.32. sub.472; Legend. sub.310. sub.32. mu. J.12. sub.201; Legend.32. sub.32. sub.472; SEQ ID. sub.32. sub.3. sub.32. SEQ ID. sub.32. sub.3. sub.32 The techniques of the bodies (see, e.g., WO1998/24893; WO1996/34096; WO1996/33735; WO1991/10741; Jakobovits et al, Proc. Nat' l Acad. Sci. USA90:2551(1993); Jakobovits et al, Nature362:255-258(1993); Bruggemann et al, Yeast in Immunol.7:33(1993); U.S. Pat. No. 5,545,807;5,545,806;5,569,825;5,625,126;5,633,425; and 5,661,016; Marks et al, Bio/Technology10:779-783(1992); Lonberg et al, Nature368:856-859(1994); Morrison, Nature368:812-813 (ImhhFishwold et al, NatureBiotechnol.14: 845; Neugenberg et al, 1996, Huuberg 1996: 1996-93; Nature et al, Nature 93: 1995).

The term "naked antibody" refers to an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

The terms "full length antibody," "intact antibody," or "whole antibody" are used interchangeably to refer to a substantially intact form of an antibody, as opposed to an antibody fragment. Specific whole antibodies include those having heavy and light chains, including an Fc region. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and/or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; linear antibodies (see U.S. Pat. No. 5,641,870, example 2; Zapata et al, Protein Eng.8(10):1057-1062 (1995)); single chain antibody molecules and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, and a remaining "Fc" fragment, the designation reflecting the ability to crystallize readily. The Fab fragment consists of the variable region domains (V) of the entire L and H chains_H) And the first constant domain (C) of a heavy chain_H1) Are combined together. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single identityThe antigen binding site of (a). Pepsin treatment of antibodies produced a single large F (ab')₂Fragments which correspond approximately to two disulfide-linked Fab fragments with different antigen binding activity and which are still capable of crosslinking the antigen. Fab' fragments by cleavage at C_HThe carboxy terminus of domain 1 has some additional residues (including one or more cysteines from the antibody hinge region) that are different from Fab fragments. Fab '-SH is herein the designation for Fab', where the cysteine residues of the constant domains carry a free thiol group. F (ab')₂Antibody fragments are initially generated as a Fab' fragment pair with a hinge cysteine between them. Other chemical couplings of antibody fragments are also known.

The Fc fragment contains the carboxy terminal portions of two H chains linked together by disulfide bonds. The effector function of an antibody is determined by sequences in the Fc region, which are also recognized by Fc receptors (fcrs) found on certain types of cells.

"Fv" is the smallest antibody fragment that contains the entire antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight, non-covalent association. From the folding of these two domains, six hypervariable loops (3 loops from each of the H and L chains) are emitted, which contribute amino acid residues for antigen binding and confer antigen-to-antibody binding specificity. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

"Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains joined to a single polypeptide chain. Preferably, the sFv polypeptide further comprises V_HAnd V_LA polypeptide linker between the domains that enables the sFv to form the desired structure for antigen binding. For an overview of sFv, see Pluckthun, The pharmacy of monoclonal antibodies, Vol.114, edited by Rosenburg and Moore, Springer-Verlag, New York, pp.269-315 (1994).

"functional fragments" of an antibody of the invention comprise portions of an intact antibody, typically including the antigen-binding or variable regions of an intact antibody or the F region of an antibody, which retain or have altered FcR binding capacity. Examples of antibody fragments include linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments.

The term "diabodies" refers to small antibody fragments prepared by a process in which at V_HAnd V_LsFv fragments with short linker (about 5-10) residues were constructed between domains (see above paragraphs) in order to achieve inter-chain pairing, rather than intra-chain pairing, of the V domains, thereby generating bivalent fragments, i.e., fragments with two antigen binding sites. Bispecific diabodies are heterodimers of two "cross" sFv fragments, where the V of both antibodies_HAnd V_LThe domains are present on different polypeptide chains. Diabodies are described in more detail, for example, in EP404,097, WO93/11161, Hollinger et al, Proc. nat' l Acad. Sci. USA90:6444-48 (1993).

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which portions of the heavy and/or light chain are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remaining portions of the chain are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al, Proc. nat' l Acad. Sci. USA,81:6851-55 (1984)). Chimeric antibodies of interest herein includeAn antibody, wherein the antigen binding region of the antibody is from an antibody produced by, for example, immunization of cynomolgus monkeys with an antigen of interest. As used herein, "humanized antibodies" are used as a subset of "chimeric antibodies".

A "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequences from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody), such as mouse, rat, rabbit or non-human primate, having the desired specificity, affinity, and/or capacity. In some cases, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues not found in the recipient antibody or in the donor antibody. These modifications can be made to further define antibody properties, such as binding affinity. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin sequence, and all or substantially all of the FR regions are those of a human immunoglobulin sequence, although the FR regions may comprise one or more single FR residue substitutions that improve antibody performance, such as binding affinity, isomerization, immunogenicity, and the like. The number of these amino acid substitutions in the FR usually does not exceed 6 in the H chain and 3 in the L chain. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al, Nature321:522-525(1986), Riechmann et al, Nature332:323-329(1988), and Presta, curr, Op, struct, biol.2:593-596 (1992). See also, for example, Vaswani and Hamilton, Ann. allergy, Asthma & Immunol.1: 105-.

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or that has been made using any of the techniques for making human antibodies disclosed herein. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J.mol.biol.,227:381(1991), Marks et al, J.mol.biol.,222:581(1991). Also useful for preparing human Monoclonal antibodies are the methods described in Cole et al, Monoclonal antibodies and cancer Therapy, Alan R.Liss, p.77(1985); Boerner et al, J.Immunol.147(1):86-95 (1991). See also van Dijk and van de Winkel, curr. opin. pharmacol.5:368-74 (2001). Human antibodies can be made by administering an antigen to transgenic animals that have been modified to produce such antibodies in response to antigen challenge, but for which the endogenous locus has failed, e.g., immunized xenomite (for xenomite)^TMSee, for example, U.S. Pat. nos. 6,075,181 and 6,150,584). For the production of human antibodies by human B-cell hybridoma technology see also, for example, Li et al, Proc. nat' l Acad. Sci. USA,103:3557-3562 (2006).

As used herein, the term "hypervariable region", "HVR" or "HV" refers to a region of an antibody variable domain which is hypervariable in sequence and/or forms structurally defined loops. Generally, an antibody comprises six HVRs; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Among natural antibodies, H3 and L3 showed the greatest diversity of six HVRs, and H3 was particularly thought to have a unique role in conferring fine specificity to antibodies. See, e.g., Xu et al, Immunity13:37-45(2000); Johnson and Wu, Methods in Molecular Biology248:1-25(Lo, eds., Human Press, Totowa, NJ, 2003)). Indeed, naturally occurring camelid antibodies consisting of only heavy chains are functional and stable in the absence of light chains. See, e.g., Hamers-Casterman et al, Nature363: 446. sub.448 (1993) and Sheriff et al, Nature struct. biol.3: 733. sub.736 (1996).

The delineation of many HVRs is used and included herein. HVRs, which are Kabat Complementarity Determining Regions (CDRs), are based on sequence variability and are the most commonly used (Kabat et al, supra). Chothia instead refers to the position of the structural loop (Chothia and LeskJ. mol. biol.196:901-917 (1987)). AbM HVRs represent a compromise between Kabat CDRs and Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. "contacted" HVRs are based on analysis of available complex crystal structures. Residues from each of these HVRs are indicated below.

The HVRs may comprise the following "extended HVRs": 24-36 or 24-34(L1), 46-56 or 50-56(L2) in VL, and 89-97 or 89-96(L3) and 26-35(H1), 50-65 or 49-65 (preferred embodiments) (H2), and 93-102, 94-102 or 95-102(H3) in VH. For each of these extended HVR definitions, the variable domain residues were numbered according to Kabat et al (supra).

"framework" or "FR" residues are variable domain residues other than the HVR residues as defined herein.

The phrase "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refers to the numbering system of the heavy chain variable domain or the light chain variable domain used for antibody compilation in Kabat et al (supra). Using this numbering system, the virtually linear amino acid sequence may contain fewer or additional amino acids corresponding to truncations or insertions to the FRs or HVRs of the variable domains. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and residues inserted (e.g., residues 82a,82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. For a given antibody, the Kabat numbering of residues can be determined by aligning regions of homology of the antibody sequence with a "standard" Kabat numbered sequence.

The Kabat numbering system is generally used when referring to residues in the variable domain (about residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al, Sequences of immunological Interest, fifth edition, public Health Service, national institutes of Health, Bethesda, Md. (1991)). The "EU numbering system" or "EU index" is generally used when referring to residues in an immunoglobulin heavy chain constant region (e.g., Kabat et al, EU index reported above). "EU index as in Kabat" refers to the residue numbering of the human IgG1EU antibody. Unless otherwise indicated herein, reference to residue numbering in antibody variable domains refers to residue numbering by the Kabat numbering system. Unless otherwise indicated herein, reference to residue numbering in the constant domain of an antibody refers to residue numbering by the EU numbering system (see, e.g., U.S. provisional application No. 60/640,323, EU numbering scheme).

As used herein, a "recipient human framework" is a framework comprising the amino acid sequence of a VL or VH framework from a human immunoglobulin framework or a human consensus framework. A recipient human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence thereof, or it may contain pre-existing amino acid sequence changes. In some embodiments, the number of pre-existing amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Where there are pre-existing amino acid changes in the VH, preferably those changes occur only at three, two or one of positions 71H, 73H and 78H; for example, the amino acid residues at those positions may be 71A, 73T and/or 78A. In one embodiment, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or human consensus framework sequence.

A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a set of human immunoglobulin VL or VH framework sequences. Typically, the human immunoglobulin VL or VH sequences of the set are from a subset of variable domain sequences. Typically, the sequence subgroups are subgroups as in Kabat et al, Sequences of proteins of Immunological Interest, published Health Service, National Institutes of Health, Bethesda, Md. (1991). For VL, examples include subgroups may be kappa I, kappa II, kappa III or kappa IV subgroups as in Kabat et al (supra). Furthermore, for the VH, the subgroup can be subgroup I, subgroup II or subgroup III as in Kabat et al (supra).

The "VH subgroup III consensus framework" comprises the consensus sequence obtained from the amino acid sequences in variable heavy chain subgroup III of Kabat et al (above).

"VL subgroup I consensus framework" comprises the consensus sequence obtained from the amino acid sequences in variable light chain subgroup kappa I of Kabat et al (supra).

For example, "amino acid modification" at a specified position in the Fc region refers to substitution or deletion of a specified residue, or insertion of at least one amino acid residue adjacent to the specified residue. An insertion specifying a residue "adjacent" indicates an insertion within one or both of its residues. The insertion may be either N-terminal or C-terminal to the designated residue. Preferred amino acid modifications herein are substitutions.

An "affinity matured" antibody is one that has one or more alterations in one or more HVRs thereof, resulting in an improved affinity of the antibody for an antigen compared to a parent antibody that does not have such alterations. In one embodiment, the affinity matured antibody has nanomolar or even picomolar affinity for the target antigen. Affinity matured antibodies can be generated by methods known in the art. For example, Marks et al, Bio/Technology10:779-783(1992) describe affinity maturation by VH and VL domain shuffling. For example, Barbas et al Proc Nat. Acad. Sci. USA91:3809-3813(1994), Schier et al Gene169: 147-.

As used herein, the term "specific binding" or "specific" refers to a measurable and reproducible interaction, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that specifically binds a target (which may be an epitope) binds that target with higher affinity, avidity, more stability, and/or longer duration than it binds to other targets. In one embodiment, the degree of binding of the antibody to an unrelated target is less than about 10% of the degree of binding of the antibody to the target, as determined by Radioimmunoassay (RIA), for example. In certain embodiments, an antibody that specifically binds a target has a dissociation constant (Kd) of less than or equal to 1 μ M, less than or equal to 100nM, less than or equal to 10nM, less than or equal to 1nM, or less than or equal to 0.1 nM. In certain embodiments, the antibody specifically binds to an epitope on a protein that is conserved between proteins from different species. In another embodiment, specific binding may include, but need not exclude, binding.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as an extension from position Cys226, or from the amino acid residue on Pro230, to its carboxy terminus. The C-terminal lysine of the Fc region (corresponding to residue 447 of the EU numbering system) is removed, for example, during antibody production or purification, or by recombinant engineering of nucleic acids encoding the heavy chain of the antibody. Thus, the composition of a whole antibody may comprise a population of antibodies with all K447 residues removed, a population of antibodies without K447 residues removed, and a population of antibodies that are a mixture of antibodies with and without K447 residues. Suitable native sequence Fc regions for use in the antibodies of the invention include human IgG1, IgG2, IgG3 and IgG 4.

A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of a naturally found Fc region. Native sequence human Fc regions include native sequence human IgG1Fc regions (non-a and a allotypes); a native sequence human IgG2Fc region; a native sequence human IgG3Fc region; and the native sequence human IgG4Fc region and naturally occurring variants thereof.

A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, preferably one or more amino acid substitutions. Preferably, the variant Fc region has at least one amino acid substitution as compared to the native sequence Fc region or the Fc region of the parent polypeptide, for example from about 1 to about 10 amino acid substitutions, and preferably from about 1 to about 5 amino acid substitutions in the native sequence Fc region or the Fc region of the parent polypeptide. The variant Fc region herein preferably has at least about 80% homology with the native sequence Fc region and/or the Fc region of the parent polypeptide, and most preferably at least about 90% homology therewith, more preferably at least about 95% homology therewith.

"binding affinity" generally refers to the overall strength of a non-covalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to an intrinsic binding affinity that reflects a 1:1 interaction between binding pair members (e.g., antibody and antigen). The affinity of a molecule X for its partner Y is generally represented by the dissociation constant ("Kd," see below). Affinity can be determined by common methods known in the art, including those described herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate easily, whereas high affinity antibodies generally bind antigen faster and tend to remain bound longer. Various methods for determining binding affinity are known in the art, and any of the methods can be used for the purposes of the present invention. Specific illustrative and exemplary embodiments for determining binding affinity are described below.

In one embodiment, the "Kd" or "Kd value" of the present invention is determined by a radiolabeled antigen binding assay (RIA) which is performed using the Fab version of the antibody of interest and its antigen as described in the assays below. By using the minimum concentration of (in the presence of a titration series of unlabelled antigen¹²⁵I) The Fab is labeled for antigen equilibration and the bound antigen is then captured using anti-Fab antibody coated plates to determine the solution binding affinity of the Fabs for the antigen (see, e.g., Chen et al, J.mol.biol.293: 865-. To establish the conditions for the assay, microtiter plates (DYNEX Technologies, Inc., Chantilly, Va.) were coated overnight with 5. mu.g/ml capture anti-Fab antibody (Cappel Labs, Cochranville, Pa.) in 50mM sodium carbonate (pH9.6) and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for 2-5 hours at room temperature (about 23 ℃). In a non-adsorption plate (Nunc #269620, NalgeNunc International, Rochester, NY), 100pM or 26pM [ alpha ], [ beta ]¹²⁵I]Antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of anti-VEGF antibodies, Fab-12, Prestaet al, Cancer Res.57: 4593-. The Fab of interest is then incubated overnight; however, the incubation may be continued for a longer period of time (e.g., about 65 hours) to ensureEquilibrium is reached. The mixture is then transferred to a capture plate for incubation at room temperature (e.g., 1 hour). The solution was then removed and 0.1% TWEEN-20 in PBS was used^TMThe plates were washed 8 times with surfactant. When the plates had dried, 150. mu.l/well of scintillator (MICROSCINT-20) was added^TMPackard) and in TOPCOUNT^TMThe plates were counted on a gamma counter (Packard) for 10 minutes. The concentration of each Fab that produced less than or equal to 20% of maximal binding was selected for competition binding assays.

According to another embodiment, the measurement is carried out by using surface plasmon resonance-2000 orThe Kd was determined with a 3000 apparatus (BIAcore, Inc., Piscataway, NJ) at 25 ℃ using a-10 Response Unit (RU) immobilized antigen CM5 chip. Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N '- (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Before injection at a flow rate of 5. mu.l/min, the antigen was diluted to 5. mu.g/ml (. about.0.2. mu.M) with 10mM sodium acetate, pH4.8, to achieve approximately 10 Response Units (RU) of conjugated protein. After injection of the antigen, 1M ethanolamine was injected to block unreacted groups. For kinetic measurements, the samples will be tested in a solution containing 0.05% TWEEN20^TMTwo-fold serial dilutions of Fab in surfactant PBS (PBST) (0.78nM to 500nM) were injected at 25 ℃ at a flow rate of approximately 25. mu.l/min. Using a simple one-to-one Langmuir binding model: (Evaluation software version 3.2) the association rate (k) was calculated by simultaneous fitting of association and dissociation sensorgrams_on) And dissociation rate (k)_off). Calculating the equilibrium dissociation constant (Kd) as k_off/k_onThe ratio of (a) to (b). See, e.g., Chen et al, J.mol.biol.293:865-881 (1999). If you go toThe binding rate as determined by surface plasmon resonance above is more than 10⁶M^-1s^-1The binding rate is then determined by using a fluorescence quenching technique that measures the increase or decrease in PBS in the presence of increasing concentrations of antigen, 20nM of anti-antigen antibody (Fab format) at 25 deg.C fluorescence emission intensity (excitation =295 nM; emission =340nM,16nM bandpass) at pH7.2, in a spectrometer such as the spectrophotometer of a flow-off device (Aviv Instruments) or 8000-series SLM-AMINCO^TMThe increasing concentration of the antigen was determined in a spectrophotometer (ThermoSpectronic, Madison, WI) using a stirred cup.

Can also be used as described above-2000 orThe "rate of binding", "rate of binding" or "k" of the present invention was determined by the-3000 system (BIAcore, Inc., Piscataway, NJ)_on”。

An "isolated" nucleic acid molecule encoding an antibody herein is a nucleic acid molecule that is identified and isolated from at least one contaminating nucleic acid molecule with which it normally binds in the environment in which it is produced. Preferably, the isolated nucleic acid does not bind to all components of the binding production environment. Isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in forms other than those forms or environments in which they are found in nature. An isolated nucleic acid molecule is thus distinguished from a nucleic acid encoding a polypeptide and antibody herein that occurs naturally in a cell.

As used herein, the term "vector" is intended to mean a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors," or simply "expression vectors. In general, expression vectors utilized in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector.

"polynucleotide," or "nucleic acid," as used interchangeably herein, refers to a polymer of nucleotides of any length, and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and analogs thereof. If present, modifications to the nucleotide structure may be given before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may comprise one or more modifications performed post-synthesis, such as a conjugate label. Other types of modifications include: such as "caps," substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications, such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramides, carbamates, etc.) and charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant groups, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with chimericizers (e.g., acridine, psoralen (psoralen), etc.), those containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), and unmodified forms of the polynucleotide. Furthermore, any hydroxyl groups typically present in sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to a solid or semi-solid support. The 5 'and 3' terminal OH groups may be phosphorylated or partially substituted with amines or organic capping groups of 1-20 carbon atoms. Other hydroxyl groups may also be derivatized as standard protecting groups. Polynucleotides may also comprise similar forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl-, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, alpha-anomeric sugars, epimeric sugars, such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs, and basic nucleotide analogs, such as methyl nucleosides. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein the phosphate ester is substituted with P (O) S

("thioester)"), p (S) S ("dithioester)", (O) NR2 ("amidate"), p (O) R, P (O) OR ', CO, OR CH2 ("formacetal"), wherein each R OR R' is independently H OR a substituted OR unsubstituted hydrocarbyl (1-20C), aryl, alkenyl, cycloalkyl, cycloalkenyl, OR aralkyl (araldyl) group optionally containing an ether (-O-) linkage. All linkages in a polynucleotide need not be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

As used herein, "oligonucleotide" generally refers to a short, generally single-stranded, generally synthetic polynucleotide that is generally, but not necessarily, less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides applies equally and fully to oligonucleotides.

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers which are compatible with exposure to the agent at the dosages and concentrations employedThe cell or mammal is non-toxic. Typically the physiologically acceptable carrier is a pH buffered aqueous solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; examples of salt-forming counterions are sodium; and/or nonionic surfactants such as TWEEN^TMPolyethylene glycol (PEG) and PLURONICS^TM。

A "package insert" refers to instructions typically included in commercial packages of pharmaceuticals that contain instructional information regarding the indications, usage, dosage, administration, contraindications, other pharmaceuticals combined with the packaged product, and/or warnings, etc., typically included in commercial packages of pharmaceuticals.

"pharmaceutically acceptable acids" include inorganic and organic acids which are non-toxic in the concentrations and manner in which they are formulated. For example, suitable inorganic acids include hydrochloric acid, perchloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, sulfonic acid, sulfinic acid, sulfanilic acid, phosphoric acid, carbonic acid, and the like. Suitable organic acids include straight and branched chain alkyl, aromatic, cyclic, cycloaliphatic, arylaliphatic, heterocyclic, saturated, unsaturated, mono-, di-and tri-carboxylic acids, including, for example, formic, acetic, 2-hydroxyacetic, trifluoroacetic, phenylacetic, trimethylacetic, tert-butylacetic, anthranilic, propionic, 2-hydroxypropionic, 2-oxopropionic, malonic, cyclopentanepropionic (cyclopropanopinic), cyclopentanepropionic (cyclopropanoic), 3-phenylpropionic, butyric, succinic, benzoic, 3- (4-hydroxybenzoyl), 2-acetoxybenzoic, ascorbic, cinnamic, lauryl sulfuric, stearic, muconic, mandelic, succinic, pamoic, fumaric, malic, maleic, hydroxymaleic, malonic, fumaric, malic, maleic, malonic, succinic, malonic, succinic, maleic, fumaric, maleic, fumaric, malonic, and maleic acids, Lactic acid, citric acid, tartaric acid, glycolic acid, gluconic acid (glyconic acid), gluconic acid (gluconic acid), pyruvic acid, glyoxylic acid, oxalic acid, methanesulfonic acid, succinic acid, salicylic acid, phthalic acid, palmitic acid (palmoic acid), palmitic acid (palmeic acid), thiocyanic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid (4-chlorobenezenesulfonic acid), naphthalene-2-sulfonic acid (napthalene-2-sulfonic acid), p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4' -methylenebis-3- (hydroxy-2-ene-1-carboxylic acid), hydroxynaphthoic acid.

"pharmaceutically acceptable bases" include inorganic and organic bases which are non-toxic in the concentrations and manner in which they are formulated. For example, suitable bases include those formed from metals that form inorganic bases, such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N-methylglucamine, morpholine, piperidine, and organic non-toxic bases including primary, secondary, tertiary, substituted amines, cyclic amines, and basic ion exchange resins, [ e.g., N (R')₄ ⁺(wherein R' is independently H or C_1-4Alkyl radicals, e.g. ammonium, Tris)]For example, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethylamine (trimethamine), dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine (hydrabamine), choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

Additional pharmaceutically acceptable acids and bases useful in the present invention include those from amino acids such as histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine and asparagine.

"pharmaceutically acceptable" buffers and salts include those from the acid and base addition salts of the acids and bases described above. Specific buffers and/or salts include histidine, succinate and acetate.

A "pharmaceutically acceptable sugar" is a molecule that, when combined with a protein of interest, significantly prevents or reduces chemical and/or physical instability of the protein upon storage. When the formulation is intended to be lyophilized and subsequently reconstituted, the "pharmaceutically acceptable sugar" may also be referred to as a "lyoprotectant". Exemplary sugars and their corresponding sugar alcohols include: amino acids such as monosodium glutamate or histidine; methylamine such as betaine; easily soluble salts such as magnesium sulfate; polyols such as trihydric or higher molecular weight sugar alcohols, for example glycerol (glycerol), dextran, erythritol, glycerol (glycerol), arabitol, xylitol, sorbitol, mannitol; propylene glycol; polyethylene glycol;(ii) a And combinations thereof. Additional exemplary lyoprotectants include glycerol and gelatin, as well as molasses disaccharide, melezitose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose, and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyols. Preferred sugar alcohols are monoglycosides, especially those obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The pendant glycoside group can be a glycoside or a galactoside. Additional examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Preferred pharmaceutically acceptable sugars are the non-reducing sugars trehalose or sucrose. Pharmaceutically acceptable sugars are added to the formulation in "protective amounts" (e.g., prior to lyophilization), which means that the protein substantially retains its physical and chemical stability and integrity during storage (e.g., after reconstitution and storage).

For purposes herein, a "diluent" is a diluent that is pharmaceutically acceptable (safe and non-toxic for administration to a human) and is used to prepare a liquid formulation, such as a formulation that is reconstituted after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline, ringer's solution, or dextrose solution. In alternative embodiments, the diluent may comprise an aqueous salt solution and/or a buffer.

A "preservative" is a compound that can be added to the formulations herein to reduce bacterial activity. The addition of preservatives, for example, may facilitate the production of multi-use (multi-dose) formulations. Examples of possible preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides in which the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butanol and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative herein is benzyl alcohol.

The term "pharmaceutical formulation" refers to a formulation in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that would cause unacceptable toxicity to the subject to which the formulation is administered. Such formulations are sterile.

"sterile" preparations are sterile or free of all living microorganisms and spores thereof.

As used herein, the term "about" refers to the usual error range for various values as would be readily known to one skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments that are directed to the value or parameter itself.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, reference to "an antibody" is a reference to from one to many antibodies, such as molar amounts of the antibodies, and includes equivalents thereof known to those skilled in the art, and so forth.

It is to be understood that the methods and embodiments of the invention described herein include aspects and embodiments that "comprise," consist of … …, "and" consist essentially of … ….

Compositions and methods of the invention

The invention provides methods for treating or preventing an autoimmune disease (such as inflammatory bowel disease) in an individual comprising administering to the individual an effective amount of an anti-CD 83 agonist antibody described herein. In some embodiments, an effective amount of an anti-CD 83 agonist antibody is administered to an individual for treating or preventing ulcerative colitis in the individual. In some embodiments, an effective amount of an anti-CD 83 agonist antibody is administered to an individual for treating or preventing crohn's disease in the individual. In some embodiments, an effective amount of an anti-CD 83 agonist antibody is administered to an individual for treating or preventing indeterminate colitis in the individual.

With respect to all methods described herein, reference to an anti-CD 83 agonist antibody also includes compositions comprising one or more of those agents. Such compositions also comprise suitable excipients, such as pharmaceutically acceptable excipients (carriers), including buffers, acids, bases, sugars, diluents, preservatives and the like, which are well known in the art and described herein. The methods of the invention may be used alone or in combination with other conventional therapeutic methods.

A. anti-CD 83 agonist antibodies

The methods of the invention use anti-CD 83 agonist antibodies, which term refers to anti-CD 83 antibodies that bind to CD83 expressed on the surface of a cell and activate CD 83-mediated signal transduction upon binding to CD83 expressed on the surface of a cell (e.g., CD83 expressed on the cell surface of mature dendritic cells). The anti-CD 83 agonist antibodies described herein have one or more of the following properties: (a) inhibiting activation of MAPK signaling in mature dendritic cells (e.g., causing a decrease in phosphorylation of p38 and CREB proteins in mature dendritic cells); (b) inhibiting the activation of mTOR signaling in mature dendritic cells (e.g., causing a decrease in phosphorylation of mTOR protein in mature dendritic cells); (c) inhibiting the release of one or more pro-inflammatory cytokines (e.g., MCP-1, IL-12p40) from mature dendritic cells; (d) inducing the release of one or more anti-inflammatory cytokines (e.g., IL-1ra) from the mature dendritic cells; (e) inducing a decrease in cell surface expression of a mature dendritic cell activation marker (e.g., CD83, HLA-DR); (f) up-regulating the expression of one or more wound healing genes (e.g., vcan, spot 2, and fbn 2) in mature dendritic cells; and (g) treatment and/or prevention of autoimmune diseases (e.g. IBD). The activity of an anti-CD 83 agonist antibody can be determined in vitro and/or in vivo.

anti-CD 83 antibodies can be generated and screened for one or more of the agonist activities described herein using methods known in the art. See, for example, the method described in example 9.

In some embodiments, the anti-CD 83 antibody specifically binds to the extracellular region of human CD83.

In some embodiments, human CD83 comprises a polypeptide from

MSRGLQLLLLSCAYSLAPATPEVKVACSEDVDLPCTAPWDPQVPYTVSWVKLLEGGEERMETPQEDHLRGQHYHQKGQNGSFDAPNERPYSLKIRNTTSCNSGTYRCTLQDPDGQRNLSGKVILRVTGCPAQRKEETFKKYRAEIVLLLALVIFYLTLIIFTCKFARLQSIFPDFSKAGMERAFLPVTSPNKHLGLVTPHKTELV (SEQ ID NO: 1).

In some embodiments, the anti-CD 83 antibody specifically binds to a polypeptide comprising

TPEVKVACSEDVDLPCTAPWDPQVPYTVSWVKLLEGGEERMETPQEDHLRGQHYHQKGQNGSFDAPNERPYSLKIRNTTSCNSGTYRCTLQDPDGQRNLSGKVILRVTGCPAQRKEETFKK (SEQ ID NO: 2).

In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an isolated antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the antibody is an antibody fragment, such as Fab, Fab' -SH, or a fragment thereof,Fv, scFv or (Fab')₂。

In some embodiments, the anti-CD 83 antibody comprises at least one, two, three, four, five, or six HVRs selected from (a) the amino acid sequence comprising SEQ ID NO:31

HVR-H1; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 32; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO 38; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the anti-CD 83 antibody comprises six HVRs comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 32; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO. 38; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the anti-CD 83 antibody comprises at least one, at least two, or all three VH HVR sequences selected from the group consisting of (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 32; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33. In one embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO. 33. In another embodiment, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO. 33 and HVR-L3 comprising the amino acid sequence of SEQ ID NO. 39. In other embodiments, the antibody comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO. 33, HVR-L3 comprising the amino acid sequence of SEQ ID NO. 39, and HVR-H2 comprising the amino acid sequence of SEQ ID NO. 32. In other embodiments, the antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 32; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the anti-CD 83 antibody comprises at least one, at least two, or all three VL HVR sequences selected from (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO 38; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39. In one embodiment, the antibody comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO 38; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the anti-CD 83 antibody comprises (a) a VH domain comprising at least one, at least two, or all three VHHVR sequences selected from (i) HVR-H1 comprising the amino acid sequence of SEQ ID NO:31, (ii) HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (iii) HVR-H3 comprising the amino acid sequence selected from SEQ ID NO:33, and (b) a VL domain comprising at least one, at least two, or all three VL HVR sequences selected from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, an anti-CD 83 antibody comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO. 32; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO. 33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO 37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO 38; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the anti-CD 83 antibody is humanized. In one embodiment, the anti-CD 83 antibody comprises HVRs as in any of the embodiments above, and further comprises a recipient human framework, e.g., a human immunoglobulin framework or a human consensus framework.

In some embodiments, anti-CD 83 antibodies are provided, wherein the antibodies comprise a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ id No. 30. In certain embodiments, VH sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contain substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but an anti-CD 83 antibody comprising the sequence retains the ability to bind CD83. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO 30. In certain embodiments, substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., FRs). Optionally, the anti-CD 83 antibody comprises the VH sequence of SEQ ID NO 30, including post-translational modifications of this sequence. In particular embodiments, the VH comprises one, two, or three HVRs selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO. 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:32, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33.

In some embodiments, anti-CD 83 antibodies are provided, wherein the antibodies comprise a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ id No. 36. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to a reference sequence, but an anti-CD 83 antibody comprising the sequence retains the ability to bind CD83. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 36. In certain embodiments, substitutions, insertions, or deletions occur in regions outside of the HVRs (i.e., FRs). Optionally, the anti-CD 83 antibody comprises the VL sequence of SEQ ID NO:36, including post-translational modifications of the sequence. In particular embodiments, the VL comprises one, two, or three HVRs selected from: (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO 37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:38, and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, an anti-CD 83 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above. In one embodiment, the antibody comprises the VH and VL sequences of SEQ ID NO 30 and SEQ ID NO 36, respectively, including post-translational modifications of those sequences. In one embodiment, the antibody comprises the heavy chain amino acid sequence shown in SEQ ID NO. 29 and the light chain amino acid sequence shown in SEQ ID NO. 35, including post-translational modifications of those sequences.

In one embodiment, provided herein are anti-CD 83 antibodies that compete for binding to human CD83 with any of the antibodies described herein. In certain embodiments, competitive binding may be determined using an ELISA assay. For example, in certain embodiments, antibodies are provided that compete for binding to human CD83 with an anti-CD 83 antibody, the anti-CD 83 antibody comprising the VH sequence of SEQ ID NO:30 and the VL sequence of SEQ ID NO: 36. In certain embodiments, antibodies are provided that compete with anti-CD 83 antibodies for binding to human CD83, said anti-CD 83 antibodies comprising the heavy chain amino acid sequence shown in SEQ ID NO. 29 and the light chain amino acid sequence shown in SEQ ID NO. 35.

The antibody may have nanomolar or even picomolar affinity for the target antigen CD83. In certain embodiments, the Kd of the antibody is from about 0.05 to about 100 nM. For example, the antibody has a Kd of any one of about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, or about 50pM to any one of about 2pM, about 5pM, about 10pM, about 15pM, about 20pM, or about 40 pM.

B. Recombinant production of anti-CD 83 agonist antibodies

The invention also provides methods for producing anti-CD 83 agonist antibodies using recombinant techniques. For example, isolated nucleic acids encoding such antibodies or fragments thereof, vectors and host cells comprising such nucleic acids can be used to produce polypeptides. Although the methods described in section B generally relate to the production of antibodies, these methods can also be used to produce any of the polypeptides described herein.

For recombinant production of the antibody or fragment thereof, the nucleic acid encoding the desired antibody or antibody fragment is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding a polyclonal or monoclonal antibody can be readily isolated (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody) and sequenced using conventional methods. Many cloning and/or expression vectors are commercially available. Carrier components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, a multiple cloning site containing recognition sequences for a number of restriction endonucleases, an enhancer element, a promoter and a transcription termination sequence.

(1) Component of a Signal sequence

Antibodies or fragments thereof may be produced recombinantly not only directly, but also as fusion proteins, wherein the antibody is fused to a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence of choice is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the eukaryotic host cell. For prokaryotic host cells that do not recognize and process native mammalian signal sequences, the eukaryotic (i.e., mammalian) signal sequence is replaced by a prokaryotic signal sequence selected from the leader sequence of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II genes. For yeast secretion, the native signal sequence may be replaced by, for example, a yeast invertase leader, a factor leader, including yeast (Saccharomyces) and Kluyveromyces (Kluyveromyces) factor leaders, or an acid phosphatase leader, a Candida albicans (C.albicans) glucoamylase leader, or a signal sequence as described in WO 90/13646. In mammalian cell expression, mammalian signal sequences are available as well as viral secretory leaders, such as the herpes simplex virus gD signal sequence.

The DNA of the precursor region is linked in frame to DNA encoding an antibody or fragment thereof.

(2) Origin of replication

Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in cloning vectors the sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes an origin of replication or an autonomously replicating sequence. Such sequences are well known for a variety of bacteria, yeasts and viruses. The origin of replication of plasmid pBR322 is suitable for gram-negative bacteria, the 2 μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, vesicular stomatitis virus ("VSV"), or bovine papilloma virus ("BPV")) are used as cloning vectors in mammalian cells. Generally, mammalian expression vectors do not require an origin of replication component (typically only the SV40 origin is used because it contains an early promoter).

(3) Selection of Gene Components

Expression and cloning vectors may also contain a selection gene, referred to as a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply important nutrients not available from complex media, such as the gene encoding D-alanine racemase for Bacillus (Bacilli).

One example of a selection scheme utilizes drugs to prevent growth of the host cell. Those cells successfully transformed with the heterologous gene produce a protein conferring drug resistance and thus survive the selection protocol. Examples of such dominant selection strategies use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selection markers for mammalian cells are those that enable identification of cells that are competent to take up antibody or antibody fragment encoding nucleic acids, such as dihydrofolate reductase ("DHFR"), thymidine kinase, metallothionein I and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. An exemplary host cell strain used for wild-type DHFR is a chinese hamster ovary ("CHO") cell line (e.g., ATCC CRL-9096) that lacks DHFR activity.

Another example of suitable selection markers for mammalian cells are those that enable identification of cells that are competent to take up antibody or antibody fragment encoding nucleic acids, such as dihydrofolate reductase ("DHFR"), Glutamine Synthetase (GS), thymidine kinase, metallothionein I and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

Alternatively, cells transformed with the GS (glutamine synthetase) gene are identified by culturing the transformants in a medium containing L-methionine sulfoximine (Msx) (an inhibitor of GS). Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acids. The GS selection/amplification system can be used in combination with the DHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding an anti-CD 83 agonist antibody or fragment thereof, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3' -phosphotransferase ("APH"), can be selected by cell growth in media containing a selection agent for the appropriate selectable marker, such as an aminoglycoside antibiotic (e.g., kanamycin, neomycin, G418). See U.S. Pat. No.4,965,199.

A suitable selection gene for yeast is the trp1 gene present on the yeast plasmid YRp7 (Stinchcomb et al, Nature,282:39 (1979)). The trp1 gene provides a selectable marker for mutant strains of yeast (e.g., ATCC No.44076 or PEP4-1) that lack the ability to grow in tryptophan-containing media Jones, Genetics,85:12 (1977). The presence of a trp1 lesion in the yeast host cell genome then provides an effective environment for detecting growth transformation in the absence of tryptophan. Similarly, a Leu 2-deleted yeast strain (e.g., ATCC20,622 or 38,626) can be complemented by a known plasmid carrying the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used to transform Kluyveromyces yeast. Alternatively, expression systems for large scale production of recombinant bovine chymosin are reported for k. Van den Berg, Bio/Technology,8:135 (1990). Stable multicopy expression vectors for the secretion of mature recombinant human serum albumin by industrial strains of kluyveromyces have also been disclosed. Fleer et al, Bio/Technology,9: 968-.

(4) Promoter component

Expression and cloning vectors typically contain a promoter that is recognized by the host organism and operably linked to a nucleic acid encoding an anti-CD 83 agonist antibody or fragment thereof. Promoters suitable for use in prokaryotic hosts include the phoA promoter, the lactamase and lactose promoter systems, the alkaline phosphatase promoter, the tryptophan promoter system, and hybrid promoters, such as the tac promoter, although other known bacterial promoters may also be suitable. Promoters for use in bacterial systems will also contain Shine-Dalgarno (S.D.) sequences operably linked to DNA encoding antibodies and antibody fragments.

For eukaryotes, promoter sequences are known. Virtually all eukaryotic genes have an AT rich region located approximately 25-30 bases upstream of the transcription start site. Another sequence found 70-80 bases upstream from the start of transcription of many genes is the CNCAAT region, where N can be any nucleotide. The 3 'end of most eukaryotic genes is an AATAAA sequence, which can be a signal to add a poly a tail to the 3' end of the coding sequence. All of these sequences can be inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Inducible promoters in yeast have the additional advantage of allowing transcription to be controlled by growth conditions. Exemplary inducible promoters include promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for yeast expression are further described in EP73,657. Yeast enhancers are also advantageously used with yeast promoters.

Transcription of a nucleic acid encoding an antibody or fragment thereof from a vector in a mammalian host cell may be under the control of the following promoters: for example, promoters obtained from the genomes of viruses, such as polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus, and most preferably simian virus 40(SV40), heterologous mammalian promoters, such as the actin promoter or an immunoglobulin promoter, and heat shock gene promoters, provided that such promoters are compatible with the intended host cell system.

The early and late promoters of the SV40 virus are conveniently obtained as SV40 restriction fragments, which also contain the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. patent No.4,419,446. A modification of this system is described in U.S. patent No.4,601,978. For methods of expressing human interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus, see also Reyes et al, Nature297: 598-. Alternatively, the rous sarcoma virus long terminal repeat can be used as a promoter.

(5) Enhancer element Components

Transcription of higher eukaryote DNA encoding antibodies or fragments thereof is often enhanced by inserting an enhancer sequence into the vector. Many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, one skilled in the art will generally use enhancers from eukaryotic viruses. Examples include the SV40 enhancer (bp100-270) on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and adenovirus enhancers. For enhanced elements for activation of eukaryotic promoters, see also Yaniv, Nature297:17-18 (1982). The enhancer may be spliced into the vector at a position 5' or 3' to the coding sequence of the antibody or antibody fragment, but is preferably located at a position 5' to the promoter.

(6) Transcription termination component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are typically available from the 5', and occasionally 3', untranslated regions of eukaryotic or viral DNA or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody or fragment thereof. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vectors disclosed herein.

(7) Selection and transformation of host cells

Suitable host cells for cloning or expressing DNA encoding an anti-CD 83 agonist antibody or fragment thereof in the vectors described herein include prokaryotic cells, yeast cells, or higher eukaryotic cells as described above. Suitable prokaryotes for this purpose include fungi, such as gram-negative or gram-positive organisms, for example, Enterobacteriaceae (Enterobacteriaceae), such as Escherichia (Escherichia), e.g. Escherichia coli (e.coli), Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella), e.g. Salmonella typhimurium (Salmonella), Serratia (serrrata), e.g. Serratia marcescens and Shigella (Shigella), and bacillus, such as bacillus subtilis and bacillus licheniformis (b.licheniformis) (e.g. bacillus 41P in DD266,710 published on 4.12.1989), Pseudomonas (Pseudomonas), such as Pseudomonas aeruginosa (Streptomyces). A preferred E.coli cloning host is E.coli 294(ATCC31,446), although other strains such as E.coli B, E.coli X1776(ATCC31,537) and E.coli W3110(ATCC27,325) are also suitable. These examples are illustrative and not limiting.

Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, particularly when glycosylation and Fc effector function are not required, such as when therapeutic antibodies are conjugated to cytotoxic agents (e.g., toxins). Full-length antibodies have a longer half-life in circulation. Production in E.coli is faster and more cost effective. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237(Carter et al), U.S. Pat. No. 5,789,199(Joly et al), and U.S. Pat. No. 5,840,523(Simmons et al), which describe Translation Initiation Regions (TIRs) and signal sequences for optimized expression and secretion. After expression, the soluble fraction of antibodies or antibody fragments is separated from the E.coli cell pellet and purified, for example, by a protein A or protein G column depending on the isotype. The final purification can be performed by the same method used for purification of antibodies or antibody fragments expressed, for example, in CHO cells.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibodies or antibody-encoding vectors. The most commonly used among lower eukaryotic host microorganisms is Saccharomyces cerevisiae or common baker's yeast. However, a variety of other genera, species and strains are also commonly available and useful herein, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces species (Kluyveromyces pp.) such as Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis) (ATCC12,424), Kluyveromyces bulgaricus (K.bulgaricus) (ATCC16,045), Kluyveromyces kluyveri (K.wickramii) (ATCC24,178), K.wallidii (ATCC56,500), Kluyveromyces drosophilus (K.drosophilarum) (ATCC36,906), Kluyveromyces thermotolerans (K.thermotolerans) and Kluyveromyces marxianus (K.primanus); ascomycete saccharomyces (yarrowia) (EP402,226); pichia pastoris (Pichia pastoris) (EP183,070); candida genus (Candida); trichoderma reesei (Trichoderma reesei) (EP244,234); neurospora crassa (Neurosporacrassa); schwanniomyces (Schwanniomyces) such as Schwanniomyces occidentalis (Schwanniomyces occidentalis); and filamentous fungi such as Neurospora (Neurospora), penicillium (penicillium), torticollis (Tolypocladium), and Aspergillus (Aspergillus) such as Aspergillus nidulans (a. nidulans) and Aspergillus niger (a. niger). For a review discussing the use of yeast and filamentous fungi for the production of therapeutic proteins, see, e.g., Gerngross, nat. Biotech.22: 1409-.

Certain fungal and yeast strains can be selected in which the glycosylation pathway has been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See, for example, Li et al, nat. Biotech.24:210-215(2006) (humanization of the glycosylation pathway in Pichia pastoris is described), and Gerngross et al, supra.

Suitable host cells for expression of glycosylated antibodies or antibody fragments are from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and bombyx mori (moth). Various viral strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV. Such viruses may be used herein as viruses according to the present invention, in particular for transfecting Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, morning glory, tomato, and tobacco may also be used as hosts.

However, vertebrate cells are of great interest, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 cells or subclones for 293 cells grown in suspension culture, Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); chinese hamster ovary cells/-DHFR (CHO, Urlaub et al, Proc. nat' l Acad. Sci. USA77:4216 (1980)); mouse Sertoli cells (TM4, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CVIATCC CCL 70); vero-cell (VERO-76, ATCCCRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCCCCL 34); buffalo rat (buffalo rat) hepatocytes (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCCCCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982)); MRC5 cells; FS4 cells; and a human liver cancer cell line (Hep G2). Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA77:4216 (1980)); and myeloma cell lines such as NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in molecular Biology, Vol.248 (B.K.C.Lo, eds., Humana Press, Totowa, NJ,2003), pp.255-268.

Host cells are transformed with the above-described expression or cloning vectors for the production of antibodies or antibody fragments and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants or amplifying genes encoding the desired sequences.

(8) Culturing host cells

Host cells for producing the anti-CD 83 agonist antibodies or antibody fragments described herein can be cultured in a variety of media. Commercially available media such as Ham's F10(Sigma), minimal essential medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's modified Eagle medium ((DMEM), Sigma) are suitable for culturing the host cells. Furthermore, any medium described in, for example, the following documents may also be used as the medium for the host cells: ham et al, meth.Enz.58:44(1979), Barnes et al, anal. biochem.102:255(1980), U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WIPO publication No. WO90/03430, WO87/00195, or U.S. Pat. No. Re.30,985. If necessary, any of these media was supplemented with: hormones and/or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN)^TMDrugs), trace elements (defined as inorganic compounds typically present at final concentrations on the micromolar scale), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used by the host cell selected for expression, as will be apparent to the ordinarily skilled artisan.

(9) Purification of antibodies

When using recombinant technology, the anti-CD 83 agonist antibody or antibody fragment can be produced intracellularly, in the periplasmic space or directly secreted into the culture medium. If the antibody is produced intracellularly, the first step is to remove particulate debris from the host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al, Bio/Technology10:163-167(1992) describe methods for isolating antibodies secreted in the periplasmic space of E.coli. Briefly, the cell pellet was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. When the antibody is secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). Protease inhibitors, such as PMSF, may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

Antibodies or antibody fragment compositions prepared from such cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique. The suitability of protein a for use as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies or antibody fragments based on human 1,2 or 4 heavy chains (Lindmark et al, J.Immunol. meth.62:1-13 (1983)). G-proteins are recommended for all mouse isotypes and for human 3 heavy chain antibodies or antibody fragments (Guss et al, EMBO J.5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices may be used. Mechanically stable matrices such as controlled pore glass or poly (styrene-divinyl) benzene allow faster flow rates and shorter processing times than can be achieved with agarose. The antibody or antibody fragment comprises C_H3 domain, Bakerbond ABX^TMResins (j.t.baker, phillips burg, NJ) can be used for purification. Depending on the antibody or antibody fragment to be recovered, other protein purification techniques may also be employed, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin, Sepharose^TMOr anion or cation exchange resin (such as polyaspartic acid column) chromatography, and chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

After any of the foregoing purification step or steps, the mixture comprising the antibody or antibody fragment of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer having a pH between about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.25M salt).

In general, various methodologies for preparing antibodies for research, testing, and clinical applications are well established in the art, consistent with the above methodologies, and/or as recognized by those of skill in the art as being suitable for a particular purpose.

C. Antibody preparation

Antibodies useful in the invention can include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab '-SH, Fv, scFv, and F (ab')₂) Chimeric antibodies, bispecific antibodies, multivalent antibodies, heteroconjugate antibodies, fusion proteins comprising an antibody portion, humanized antibodies, and any other modified configuration of an immunoglobulin comprising an antigen recognition site with a desired specificity, including glycosylation variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies. The antibody may be murine, rat, human, or any other source (including chimeric or humanized antibodies).

(1) Polyclonal antibodies

Polyclonal antibodies are generally produced by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant in the animal. It can be used to conjugate the relevant antigen (e.g., purified or recombinant CD83) to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using bifunctional or derivatizing reagents such as maleimido benzoyl sulfosuccinimid ester (conjugated through cysteine residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinyl anhydride, SOCl₂Or R¹N = C = NR, wherein R and R¹Independently is a lower alkyl group. Examples of adjuvants that can be used include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate).

Animals are immunized against the desired antigen, immunogenic conjugate or derivative by combining, for example, 100 μ g (for rabbits) or 5 μ g (for mice) of protein or conjugate with 3 volumes of Freund's complete adjuvant and injecting the solution subcutaneously at multiple sites. After 1 month, the animals were boosted by subcutaneous injection of 1/5-1/10 starting amounts of peptide or conjugate in freund's complete adjuvant at multiple sites. After 7-14 days, the animals were bled and the serum antibody titer was determined. Animals were boosted until the titer reached plateau. The conjugates can also be produced as fusion proteins in recombinant cell culture. Also, aggregating agents, such as alum, are suitable for enhancing immune responses.

(2) Monoclonal antibodies

Monoclonal antibodies are obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that are present in minor amounts. Thus, the modifier "monoclonal" indicates the identity of the antibody as such and not as a mixture of different antibodies.

For example, monoclonal antibodies can be prepared using the hybridoma method first described by Kohler et al, Nature,256:495(1975), or by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization (e.g., purified or recombinant CD 83). Alternatively, lymphocytes may be immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp 59-103 (Academic Press, 1986)).

The immunizing agent typically includes an antigenic protein (e.g., purified or recombinant CD83) or a fusion variant thereof. Generally, if cells of human origin are desired, peripheral blood lymphocytes ("PBLs") are used, whereas if non-human mammalian sources are desired, the spleen or lymph nodes are used. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells. Goding, Monoclonal Antibodies: Principles and practice, Academic Press (1986), pages 59-103.

Immortalized cell lines are generally transformed mammalian cells, in particular myeloma cells of rodent, bovine or human origin. Typically, rat or mouse myeloma cell lines are used. The hybridoma cells thus prepared are seeded and cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which are substances that prevent the growth of HGPRT-deficient cells.

Preferred immortal myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by the selected antibody-producing cells, and are sensitive to a medium, such as HAT medium. Of these, preferred are murine myeloma lines such as those derived from MOPC-21 and MPC-11 mouse tumors (available from the Salk Institute Cell Distribution Center, SanDiego, California USA), as well as SP-2 cells and derivatives thereof (e.g., X63-Ag 8-653) (available from American type culture Collection, Manassas, Virginia USA). Human myeloma and mouse-human heterogeneous myeloma cell lines have also been described for the production of human Monoclonal antibodies (Kozbor, J.Immunol.,133:3001(1984); Brodeur et al, Monoclonal antibody production Techniques and applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

The production of monoclonal antibodies against an antigen (e.g., CD83) in the medium in which the hybridoma cells are grown is determined. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay ((ELISA).

The culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies to the desired antigen (e.g., CD 83). Preferably, the binding affinity and specificity of a monoclonal antibody is determined by immunoprecipitation or by an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay ((ELISA). such techniques and assays are known in the art.

After hybridoma cells producing antibodies with the desired specificity, affinity, and/or activity are identified, the clones can be subcloned by limiting dilution methods and cultured by standard methods (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as tumors in mammals.

Monoclonal antibodies secreted by the subclones can be isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, affinity chromatography, and other methods described above.

Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those disclosed in U.S. patent No.4,816,567 and described above. DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional methods (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells are a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into a host cell, such as an escherichia coli cell, a monkey COS cell, a Chinese Hamster Ovary (CHO) cell, or a myeloma cell that does not produce immunoglobulin, to synthesize monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression of DNA encoding an antibody in bacteria include Skerra et al, curr. Opin. Immunol.,5: 256-188 (1993) and Pl ü ckthun, Immunol. Rev.130:151-188 (1992).

In certain embodiments, antibodies can be isolated from the generated antibody phage library using techniques described in McCafferty et al, Nature,348: 552-. Clackson et al, Nature,352: 624-. Subsequent publications describe the generation of high affinity (nanomolar ("nM") grades) human antibodies by chain shuffling (Marks et al, Bio/Technology,10: 779-. Thus, these techniques are viable alternatives to conventional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies having the desired specificity (e.g., those that bind CD 83).

DNA encoding antibodies or fragments thereof may also be modified, for example, by substituting the coding sequences for the constant domains of the human heavy and light chains with homologous murine sequences (U.S. Pat. No.4,816,567; Morrison, et al, Proc. Natl Acad. Sci. USA,81:6851(1984)), or by covalently linking immunoglobulin coding sequences to all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically, such non-immunoglobulin polypeptides replace the constant domains of an antibody, or they replace the variable domains of one antigen combining site of an antibody, to produce a chimeric bivalent antibody comprising one antigen combining site with specificity for an antigen and another antigen combining site with specificity for a different antigen.

The monoclonal antibodies described herein (e.g., anti-CD 83 agonist antibodies or fragments thereof) can be monovalent, and are prepared as is well known in the art. For example, one approach involves recombinant expression of immunoglobulin light chains and modified heavy chains. The heavy chains are generally truncated at any position in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residue may be substituted with another amino acid residue or deleted to prevent cross-linking. In vitro methods are also suitable for making monovalent antibodies. Digestion of the antibody to produce fragments thereof, particularly Fab fragments, can be accomplished using conventional techniques known in the art.

Chimeric or hybrid antibodies can also be prepared in vitro using methods known in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate (iminothiolate) and methyl-4-mercaptobutylimidate (methyl-4-mercaptobutyliminate).

(3) Humanized antibodies

The antibodies (e.g., anti-CD 83 agonist antibodies) or antibody fragments of the invention also comprise humanized or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fab '-SH, Fv, scFv, F (ab')₂Or other antigen binding subsequence of an antibody) that contains minimal sequences from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat, hamster, or rabbit, having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will also optimally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al, Nature321:522-525(1986); Riechmann et al, Nature332:323-329(1988) and Presta, curr. Opin. struct. biol.2:593-596 (1992).

Methods for humanizing non-human antibodies are well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues,it is usually derived from an "imported" variable domain. Basically according to Winter and colleagues Jones et al Nature321:522-525(1986); Riechmann et al Nature332323-327(1988), Verhoeyen et al, Science239:1534-1536(1988), or by humanization by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which substantially less than the entire human variable domain is replaced by the corresponding sequence of a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains (heavy and light chains) used to make humanized antibodies is important to reduce antigenicity. The variable domain sequences of rodent antibodies are screened against an entire library of known human variable domain sequences according to the so-called "best-fit" approach. The human sequence most similar to the rodent sequence is then accepted into the human Framework (FR) of the humanized antibody. Sims et al, J.Immunol.,151:2296(1993), Chothia et al, J.mol.biol.,196:901 (1987). Another approach uses a specific framework from the consensus sequence of all human antibodies with a specific subset of light or heavy chains. The same framework can be used for several different humanized antibodies. Carter et al, Proc.Nat' l Acad. Sci. USA89:4285(1992); Presta et al, J.Immunol.151:2623 (1993).

Furthermore, it is important to humanize antibodies that retain high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a method of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. A computer program is available which elucidates and displays the possible three-dimensional conformational structures of the selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in performing the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. Thus, FR residues from the receptor and import sequences can be selected and combined to achieve desired antibody properties, such as increased affinity for the target antigen or antigens (e.g., CD 83). In general, CDR residues are directly and most practically involved in influencing antigen binding.

Various forms of humanized antibodies are contemplated. For example, the humanized antibody can be an antibody fragment, such as a Fab, that is optionally conjugated to one or more cytotoxic agents to produce an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

(4) Human antibodies

Alternatively, human antibodies can be produced. For example, transgenic animals (e.g., mice) can now be produced that, upon immunization, are capable of producing all kinds of human antibodies without endogenous immunoglobulin production. Antibody heavy chain joining region (J) in chimeric and germline mutant mice_H) Homozygous deletion of the gene results in complete suppression of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc. Nat' l Acad. Sci. USA,90:2551(1993), Jakobovits et al, Nature,362:255-258(1993), Bruggemann et al, Yeast in Immunol, 7:33(1993), U.S. Pat. No. 5,591,669 and WO 97/17852.

Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from all components of the immunoglobulin variable (V) domain genes of unimmunized donors. McCafferty et al, Nature348:552-553(1990); Hoogenboom and Winter, J.mol.biol.227:381 (1991). According to this technique, antibody V domain genes are cloned in-frame within the major or minor capsid protein genes of filamentous phage (e.g., M13 or fd) and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also results in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell.Phage display can be performed in a variety of formats, reviewed, for example, in Johnson, Kevin S. and Chiswell, David J., curr, OpinStrect, biol.3:564-571 (1993). Several sources of V gene segments are available for phage display. Clackson et al, Nature352:624-628(1991) isolated anti-antibody-cells from a small random combinatorial library of V genes from the spleen of immunized miceA diverse array of oxazolone antibodies. The repertoire of V genes from non-immunized human donors can be constructed and antibodies directed against a variety of antigens, including self-antigens, isolated essentially according to the techniques described by Marks et al, J.mol.biol.222:581-597(1991), or Griffith et al, EMBO J.12:725-734 (1993). See also U.S. Pat. nos. 5,565,332 and 5,573,905.

The techniques of Cole et al and Boerner et al are also available for the preparation of human Monoclonal Antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77(1985) and Boerner et al, J.Immunol.147(1):86-95 (1991.) similarly, human Antibodies can be prepared by introducing human immunoglobulin sites into transgenic animals, such as mice, in which endogenous immunoglobulin genes have been partially or completely inactivated, after challenge, the production of human Antibodies is observed, which closely approximates in all respects to that seen in humans, including gene rearrangement, assembly and antibody repertoires.e.g., in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,9,825, 5,625,126, 5,633,425, 5,661,016 and the scientific publications,

this method is described in Bio/Technology10: 779-.

Finally, human antibodies can also be produced in vitro by activated B cells (see U.S. Pat. nos. 5,567,610 and 5,229,275).

(5) Antibody fragments

In certain circumstances, the use of antibody fragments may be advantageous over the use of whole antibodies. Smaller fragment sizes allow rapid clearance and may lead to improved access to solid tumors.

Various techniques for generating antibody fragments have been developed. Traditionally, these fragments have been obtained by proteolytic digestion of whole antibodies (see, e.g., Morimoto et al, J.biochem.Biophys.method.24:107-117(1992); and Brennan et al, Science229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells, for example using nucleic acids encoding the antibodies to CD83 discussed above. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E.coli, thus allowing direct production of large quantities of these fragments. Antibody fragments can also be isolated from antibody phage libraries as discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab')₂Fragments (Carter et al, Bio/Technology10:163-167 (1992)). According to another method, F (ab') can be isolated directly from recombinant host cell culture₂And (3) fragment. Fab and F (ab')₂Production of antibody fragments. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO93/16185, U.S. Pat. No. 5,571,894 and U.S. Pat. No. 5,587,458. Antibody fragments may also be "linear antibodies," such as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

(6) Bispecific and multispecific antibodies

Bispecific antibodies (BsAbs) are antibodies that have binding specificity for at least two different epitopes, including those on the same or another protein (e.g., CD 83). Such antibodies can be from full-length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies).

Methods for making bispecific antibodies are known in the art. The traditional method of making full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy/light chain pairs, wherein the two chains have different specificities. Millstein et al, Nature,305:537-539 (1983). Due to the random assignment of immunoglobulin heavy and light chains, these hybridomas (cell hybridomas) produce a possible mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, which is generally performed by an affinity chromatography step, is very cumbersome and the product yield is low. Similar methods are disclosed in WO93/08829 and Traunecker et al, EMBO J.,10:3655-3659 (1991).

According to different methods, antibody variable domains (antibody-antigen binding sites) with the desired binding specificity can be fused to immunoglobulin constant domain sequences. The fusion is preferably with a C-linker comprising at least a part of the hinge region_H2 and C_HRegion 3 immunoglobulin heavy chain constant domain fusion. Preferably, the first heavy chain constant region (C) containing the site required for light chain binding is used_H1) Present in at least one fusion. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, may be inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments, as the unequal ratios of the three polypeptide chains used in the construction provide the best yield. However, when expression of an equal ratio of at least two polypeptide chains results in high yield or when the ratio is not of particular significance, the coding sequences for two or all three polypeptide chains can be inserted into one expression vector.

In a preferred embodiment of the method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from the undesired immunoglobulin chain combinations, since the presence of immunoglobulin light chains in only half of the bispecific molecule provides a convenient means of separation. This method is disclosed in WO 94/04690. For more details on the generation of bispecific antibodies, see, e.g., Suresh et al, Methods in Enzymology121:210 (1986).

According to another method described in WO96/27011 or U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. Preferred interfaces comprise an antibody constant domain C_HAt least a portion of zone 3. In this method, one or more small side chains of amino acids from the interface of the first antibody molecule are replaced by a larger side chain (e.g., tyrosine or tryptophan). Complementary "cavities" of the same or similar size to the large side chains can be formed at the interface of the second antibody molecule by replacing the large amino acid side chains with smaller side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over the undesired end products like homodimers.

Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science229:81(1985) describe a method in which intact antibodies are proteolytically cleaved to yield F (ab')₂A method for fragmenting. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize adjacent dithiols and prevent intermolecular disulfide bond formation. The resulting Fab' fragments are then converted to thionitrobenzoic acid (TNB) derivatives. One of the Fab '-TNB derivatives is then reconverted to the Fab' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as reagents in the selective immobilization of enzymes.

Fab' fragments can be directly recovered from E.coli and chemically coupled to form bispecific antibodies. Shalaby et al, J.Exp.Med.175:217-225(1992) describe fully humanized bispecific antibodies F (ab')₂The generation of molecules. Each Fab' fragment was separately secreted from E.coli and directly chemically coupled in vitro to form bispecific antibodies. The bispecific antibody thus formed was able to interact with cells overexpressing the ErbB2 receptor and normal human T cellsThe cell combination can also trigger the lysis activity of human cytotoxic lymphocytes on human breast tumor cells.

Various techniques for the preparation and isolation of bivalent antibody fragments directly from recombinant cell cultures have also been described. For example, a leucine zipper can be used to produce a bivalent heterodimer. Kostelny et al, J.Immunol.,148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. The "diabody" technology described by Hollinger et al, Proc. Nat' l Acad. Sci. USA,90: 6444-. The fragment comprises a light chain variable domain (V) linked to a linker_L) Linked heavy chain variable domains (V)_H) The linker is too short to allow pairing between the two domains of the same strand. Thus, V on a segment_HAnd V_LThe domains are forced to complement V on the other fragment_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific/bivalent antibodies by using single chain fv (sfv) dimers has also been reported. See Gruber et al, J.Immunol.,152:5368 (1994).

Antibodies of more than bivalent are also contemplated. For example, trispecific antibodies can be prepared. Tutt et al, J.Immunol.147:60 (1991). Exemplary bispecific antibodies can bind to two different epitopes on a given molecule (e.g., CD 83).

(7) Multivalent antibodies

Multivalent antibodies internalize (and/or metabolize) faster than bivalent antibodies by cells expressing the antigen to which the antibody binds. The anti-CD 83 antibody or antibody fragment of the invention can be a multivalent antibody (e.g., tetravalent antibody) with three or more antigen binding sites (which is a species other than an IgM species) that can be readily produced by recombinantly expressing nucleic acids encoding the polypeptide chains of the antibody. Multivalent antibodies may comprise dimerizationA formatting domain and three or more antigen binding sites. Preferred dimerization domains comprise an Fc region or a hinge region. In this embodiment, the antibody will comprise an Fc region and three or more antigen binding sites at the amino acid termini of the Fc region. Preferred multivalent antibodies herein comprise three to about 8, but preferably four antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain or chains comprise two or more variable domains. For example, one or more polypeptide chains can comprise VD1- (X1)_n-VD2-(X2)_n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. Similarly, one or more polypeptide chains can comprise V_H-C_H1-Flexible Joint-V_H-C_H1-Fc region chain; or V_H-C_H1-V_H-C_H1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. Multivalent antibodies herein may comprise, for example, about two to about 8 light chain variable domain polypeptides. Light chain variable domain polypeptides contemplated herein comprise a light chain variable domain and optionally further comprise a CL domain.

(8) Heteroconjugate antibodies

Heteroconjugate antibodies (Heteroconjugate antibodies) are also within the scope of the invention. The heteroconjugate antibody is composed of two covalently linked antibodies. For example, one antibody in the heteroconjugate can be conjugated to avidin and the other to biotin. For example, such antibodies have been proposed to target immune system cells to unwanted cells, U.S. Pat. No.4,676,980, and have been used to treat HIV infection. International publication Nos. WO91/00360, WO92/200373, and EP 0308936. It is contemplated that antibodies can be prepared in vitro using methods known in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Suitable examples for this purpose include iminothiolate (iminothiolate) and methyl-4-mercaptobutylimidate (methyl-4-mercaptoimidate), as well as agents such as those disclosed in U.S. Pat. No.4,676,980. The heteroconjugate antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No.4,676,980, along with a number of crosslinking techniques.

(9) Effect function modification

It may be desirable to modify the antibodies of the invention to modify effector function and/or increase the serum half-life of the antibody. For example, Fc receptor binding sites on the constant region can be modified or mutated to remove or reduce binding affinity for certain Fc receptors, such as Fc γ RI, Fc γ RII, and/or Fc γ RIII. In some embodiments, effector function is impaired by removing N-glycosylation of the antibody Fc region (e.g., in the CH2 domain of IgG). In some embodiments, effector function is impaired by modification of regions, such as 233-.

To increase the serum half-life of the antibody, a salvage receptor binding epitope can be incorporated into the antibody (particularly an antibody fragment) as described, for example, in U.S. patent 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an IgG molecule (e.g., an IgG₁、IgG₂、IgG₃Or IgG₄) Is responsible for increasing the in vivo serum half-life of the IgG molecule.

(10) Other amino acid sequence modifications

Amino acid sequence modifications of the antibodies described herein are contemplated. For example, it is desirable to improve the binding affinity and/or other biological properties of an antibody or antibody fragment. Amino acid sequence variants of an antibody or antibody fragment are prepared by introducing appropriate nucleotide changes into a nucleic acid encoding the antibody or antibody fragment, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions are made to arrive at the final construct, provided that the final construct possesses the desired characteristics (e.g., the ability to bind or physically interact with CD 83). Amino acid changes can also alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites.

A useful method for identifying certain residues or regions of an antibody, which are preferred positions for mutagenesis, is referred to as "alanine scanning mutagenesis", e.g., Cunningham and Wells in Science,

244: 1081-. Herein, a residue or group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the target antigen. Those amino acid positions demonstrating functional sensitivity to substitution are then improved by introducing further or other variants at or for the substitution site. Thus, although the site of introduction of the amino acid sequence variation is predetermined, the nature of the mutation itself need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed in the target codon or region and the expressed antibody variants are screened for the desired activity.

Amino acid sequence insertions include amino- ("N") and/or carboxy- ("C") terminal fusions that range in length from one residue to polypeptides containing hundreds or more residues, as well as intrasequence insertions of single or multiple amino residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertional variants of the antibody molecule include fusions of the N-or C-terminus of the antibody with enzymes or polypeptides that increase the serum half-life of the antibody.

Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced by a different residue. The most interesting sites for substitution mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown below in table a under the heading "preferred substitutions". If such substitutions result in an alteration in biological activity, more substantial changes (designated as "exemplary substitutions" in Table A or described further below with reference to amino acid species) can be introduced and the products screened.

TABLE A

Amino acid substitutions

Original residues	Exemplary substitutions	Preferred substitutions
			Ala(A)	val;leu;ile	Val
Arg(R)	lys;gln;asn	Lys
			Asn(N)	gln;his;asp,lys;arg	Gln
Asp(D)	glu;asn	Glu
			Cys(C)	ser;ala	Ser
Gln(Q)	asn;glu	Asn
			Glu(E)	asp;gln	Asp
Gly(G)	ala	Ala
			His(H)	asn;gln;lys;arg	Arg
Ile(I)	leu, val, met, ala, phe, norleucine	Leu
			Leu(L)	Norleucine, ile, val, met, ala, phe	Ile
Lys(K)	arg;gln;asn	Arg
			Met(M)	leu;phe;ile	Leu
Phe(F)	leu;val;ile;ala;tyr	Tyr
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr

Original residues	Exemplary substitutions	Preferred substitutions
			Thr(T)	Ser	Ser
Trp(W)	tyr;phe	Tyr
			Tyr(Y)	trp;phe;thr;ser	Phe
Val(V)	ile, leu, met, phe, ala, norleucine	Leu

Substantial modification of the biological properties of an antibody can be achieved by selecting substitutions that differ significantly in their effect (a) the structure of the polypeptide backbone in the region of the substitution, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chains. Naturally occurring residues are divided into the following groups based on common side chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gln, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

Non-conservative substitutions entail replacing a member of one of these classes with a member of the other class.

Any cysteine residue not involved in maintaining the proper conformation of the antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment, such as an Fv fragment).

A particularly preferred type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody). Generally, the resulting variants selected for further development have improved biological properties compared to the parent antibody from which they were produced. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus produced are displayed in monovalent form on filamentous phage particles as fusions to the gene III product of M13 packaged in each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be advantageous to analyze the crystal structure of the antigen-antibody complex to identify the point of contact between the antibody and the antigen CD83. These contact residues and adjacent residues are candidate residues for substitution according to the techniques detailed herein. Once such variants are generated, a panel of variants can be screened as described herein and antibodies with superior performance in one or more relevant assays selected for further development.

Another type of amino acid variant of an antibody alters the initial glycosylation pattern of the antibody. An alteration refers to the deletion of one or more carbohydrate moieties found in an antibody, and/or the addition of one or more glycosylation sites not present in an antibody.

Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatic attachment of carbohydrates to asparagine side chains. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites in antibodies can generally be achieved by altering the amino acid sequence so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Changes (for O-linked glycosylation sites) can also be made by adding or substituting one or more serine or threonine residues in the original antibody sequence.

Nucleic acid molecules encoding amino acid sequence variants of anti-IgE antibodies can be prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants), or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of a previously prepared variant or non-variant form of the antibody or antibody fragment.

(10) Other antibody modifications

The antibodies or antibody fragments of the invention may be further modified to contain additional non-protein moieties known in the art and readily available. Preferably, the moiety suitable for derivatising the antibody is a water soluble polymer. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in the manufacturing process due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the amount and/or type of polymer used for derivatization may be determined based on considerations including, but not limited to, the particular properties or function of the antibody to be improved, whether or not the antibody derivative is used for therapy under defined conditions, and the like. Such techniques and other suitable formulations are disclosed in Remington, The Science and practice of Pharmacy, 20 th edition, Alfonso Gennaro, ed., Philadelphia College of Pharmacy and Science (2000).

D. Pharmaceutical compositions and formulations

Therapeutic compositions and formulations of anti-CD 83 agonist antibodies described herein can be prepared by mixing the active ingredient with the desired degree of purity, optionally with pharmaceutically acceptable carriers, excipients, or stabilizers (Remington: the science and Practice of Pharmacy, 20 th edition, (Gennaro, a.r., editions, lippincott williams & Wilkins, Publishers, philidelphia, PA 2000.) acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations used, and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite, preservatives, isotonicity agents, stabilizers, metal complexes (e.g., Zn-protein complex), chelating agents such as EDTA and/or nonionic surfactants, and the like.

Buffering agents are used to control the pH within the range that optimizes the therapeutic effect, especially if stability is pH dependent. The buffer is preferably present in a concentration range of about 50mM to about 250 mM. Suitable buffering agents for use in the present invention include inorganic and organic acids and salts thereof, such as sodium citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. In addition, the buffer may comprise histidine and trimethylamine salts, such as Tris.

Preservatives are added to retard microbial growth and are typically present in the range of 0.2% to 1.0% (w/v). Suitable preservatives for use in the present invention include octadecyl dimethyl benzyl ammonium chloride; (ii) chlorhexidine; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

Tonicity agents (sometimes referred to as "stabilizers") are present to adjust or maintain the tonicity of the liquid in the composition. When large charged biomolecules, such as proteins and antibiotics, are used, they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the possibility of intermolecular and intramolecular interactions. Tonicity agents may be present in any amount between 0.1% to 25% by weight, or more preferably between 1% to 5% by weight, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

Additional excipients include agents that may function as one or more of the following: (1) a filler, (2) a solubility enhancer, (3) a stabilizer, and (4) an agent that prevents denaturation or adhesion to the walls of the container. Such excipients include: polyhydric sugar alcohols (listed above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoionitose, inositol (myoionitol), galactose, galactitol, glycerol, cyclitols (e.g., inositol), polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, alpha-monothioglycerol and sodium thiosulfate; low molecular weight proteins such as human serum albumin, bovine serum albumin, gelatin or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose)); trisaccharides, such as raffinose; and polysaccharides such as dextrin or dextran.

Non-ionic surfactants or detergents (also referred to as "wetting agents") are present to help stabilize the therapeutic agent and protect the therapeutic protein from agitation-induced polymerization, which also allows the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant is present in the range of about 0.05mg/ml to about 1.0mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

Suitable non-ionic surface active agentsSex agents include polysorbates (20, 40, 60,65, 80, etc.), polyoxamers (184, 188, etc.),A polyhydric alcohol,Polyoxyethylene sorbitan monoether (A)-20、-80, etc.), lauromacrogol 400, polyoxyethylene (40) monostearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. Anionic detergents that may be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate (dioctyl sodium sulfosuccinate), and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

In order for pharmaceutical formulations comprising anti-CD 83 agonist antibodies to be used for in vivo administration, they must be sterile. The formulation may be rendered sterile by filtration through sterile filtration membranes. The therapeutic compositions herein are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is according to known and recognized methods, such as by single bolus injection or multiple bolus injections or infusions over a prolonged period in a suitable manner, for example by injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained or extended release means.

The anti-CD 83 agonist antibody compositions and formulations herein also contain more than one active compound, as necessary for the particular indication being treated, preferably those active compounds that have complementary activities that do not adversely affect each other. Alternatively, or additionally, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose.

The active ingredients are also encapsulated in the prepared microcapsules or in macroemulsions in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), for example by the coascervation technique or by interfacial polymerization methods, such as hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively. Such techniques are disclosed in Remington's pharmaceutical Sciences, 20 th edition, supra.

The stability of the proteins and antibodies described herein can be enhanced by the use of non-toxic "water-soluble multivalent metal salts". Examples include Ca²⁺、Mg²⁺、Zn²⁺、Fe²⁺、Fe³⁺、Cu²⁺、Sn²⁺、Sn⁴⁺、Al²⁺And Al³⁺. Exemplary anions that can form water soluble salts (with polyvalent metal cations as above) include those formed from inorganic and/or organic acids. Such water soluble salts are soluble in water (20 ℃) to at least about 20mg/ml, alternatively at least about 100mg/ml, alternatively at least about 200 mg/ml.

Suitable inorganic acids that may be used to form the "water-soluble polyvalent metal salt" include hydrochloric acid, acetic acid, sulfuric acid, nitric acid, thiocyanic acid, and phosphoric acid. Suitable organic acids that may be used include aliphatic carboxylic acids and aromatic acids. The aliphatic acid in this definition may be defined as saturated or unsaturated C_2-9Carboxylic acids (e.g., aliphatic mono-, di-, and tricarboxylic acids). For example, exemplary monocarboxylic acids within this definition include saturated C_2-9Monocarboxylic acids acetic, propionic, butyric, valeric, caproic, enanthic, caprylic, pelargonic and capryonic acids, and unsaturated C_2-9Monocarboxylic acids acrylic acid, preprolic acid, methacrylic acid, crotonic acid and isocrotonic acid. Exemplary dicarboxylic acids include saturated C_2-9Dicarboxylic acids malonic, succinic, glutaric, adipic and pimelic acidDiacid, unsaturated C_2-9Dicarboxylic acids include maleic acid, fumaric acid, citraconic acid, and mesaconic acid. Exemplary tricarboxylic acids include saturated C_2-9Tricarballylic acid and 1,2, 3-butanetricarboxylic acid. Additionally, the defined carboxylic acids may also contain one or more hydroxyl groups to form hydroxycarboxylic acids. Exemplary hydroxycarboxylic acids include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, and citric acid. Aromatic acids in this definition include benzoic acid and salicylic acid.

Common water-soluble multivalent metal salts that may be used to help stabilize the encapsulated polypeptides of the present invention include, for example: (1) inorganic acid metal salts of halides (e.g., zinc chloride, calcium chloride), sulfates, nitrates, phosphates, and thiocyanates; (2) metal salts of aliphatic carboxylic acids (e.g., calcium acetate, zinc acetate, calcium propionate, zinc glycolate, calcium lactate, zinc lactate, and zinc tartrate); and (3) metal salts of aromatic carboxylic acids of benzoic acid (e.g., zinc benzoate) and salicylates.

Pharmaceutical formulations of anti-CD 83 agonist antibodies may be designed to release the anti-CD 83 agonist antibody immediately ("immediate release" formulations), to release the antibody gradually over an extended period of time ("sustained release", "controlled release" or "extended release" formulations), or to have alternative release profiles. The additional materials used to prepare the pharmaceutical formulation may vary depending on the therapeutic form of the formulation (e.g., the system is designed for immediate or sustained release, controlled release, or extended release). In certain variations, the sustained release formulation may further comprise an immediate release component to rapidly deliver a priming dose upon drug delivery and a sustained release component. Thus, a sustained release formulation can be combined with an immediate release formulation to provide a rapid "burst" of drug into the system and a longer gradual release. For example, a core sustained release formulation may be coated with a highly soluble layer incorporating the drug. Alternatively, the sustained release formulation and the immediate release formulation may be included as alternating layers in a tablet or as separate granule types in a capsule. Other combinations of different types of pharmaceutical agents may be used to achieve the desired therapeutic plasma profile.

Exemplary sustained release dosage formulations (discussed above in Remington's Pharmaceutical Sciences, 20 th edition) may include a variety of drug delivery systems, including those using the following: (a) a container system in which the drug is coated in a polymeric film, allowing moisture to diffuse through the film to dissolve the drug, which then diffuses out of the device; (b) matrix systems (gradient or monolithic) in which the drug is suspended in a polymeric matrix and gradually diffuses out as the matrix dissolves or disintegrates; (c) microencapsulated and coated particle systems with diameters as small as 1 micron (` mum ` 10^-6m) are coated in a polymeric film, including embodiments in which coated particles of polymers having different release characteristics (e.g., pH-dependent or pH-independent polymers, compounds having different degrees of water solubility, etc.) are delivered together in a single capsule; (d) a solvent activation system comprising (i) an osmotic control device (e.g.,alza corp., Mountain View, CA), in which an osmotic agent and a drug are coated in a semipermeable membrane, whereby an osmotic gradient draws water into the device and increased pressure drives the drug out of the device through pores in the membrane; (ii) a hydrogel swelling system in which the drug is dispersed in a polymer and/or the polymer is coated onto drug particles, wherein the polymer swells upon contact with water (in certain embodiments, swelling may be pH-dependent, pH-independent, or dependent on other physical or chemical properties) allowing the drug to diffuse out of the device; (iii) microporous membrane systems in which a drug is coated in a membrane having a component that dissolves upon contact with water (in certain embodiments, swelling may be pH-dependent, pH-independent, or dependent on other physical or chemical properties), creating pores in the membrane through which the drug diffuses; and (iv) a wax pattern system in which the drug and additional soluble components are dispersed in wax, thereby allowing the drug to dissolve the soluble components when water is dissolvedDiffusion from the system; and (e) a polymeric degradation system comprising (i) bulk degradation wherein the drug is dispersed in the polymeric matrix and degradation occurs in a random manner through the polymeric structure, allowing for drug release; and (ii) surface erosion, wherein the drug is dispersed in the polymeric matrix and delivered as the polymer surface erodes.

E. Method of treatment

The present invention provides methods for treating or preventing an autoimmune disease, such as Inflammatory Bowel Disease (IBD), in an individual comprising administering to the individual an effective amount of an anti-CD 83 agonist antibody described herein. In some embodiments, the individual is a human. In some embodiments, the autoimmune disease is selected from rheumatoid arthritis, juvenile rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), lupus nephritis, ulcerative colitis, wegener's disease, inflammatory bowel disease, Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, raynaud's syndrome, sjogren's syndrome, and glomerulonephritis. In some embodiments, the individual is at risk of developing an autoimmune disease that is associated with myeloid cell activation (dendritic cells and macrophages) in the pathogenesis of the disease. In some embodiments, the subject has or is at risk of developing IBD.

IBD may be Ulcerative Colitis (UC), crohn's disease, or indeterminate colitis. In some embodiments, an individual having IBD is an individual who is experiencing or has experienced one or more signs, symptoms, or other indications of IBD or has been diagnosed with IBD. Individuals with IBD have steroid refractory and/or steroid dependent IBD, steroid refractory and/or steroid dependent UC or steroid refractory and/or steroid dependent crohn's disease. A "steroid-refractory" IBD is an IBD that develops or worsens, even if steroids are administered to a subject suffering from IBD. Individuals with "steroid-dependent IBD" are dependent on the use of steroids and are unable to gradually reduce or withdraw steroid administration without acute exacerbations of clinical symptoms.

Administration of anti-CD 83 antibodies can result in clinical response and/or disease remission. As used herein, "clinical response" refers to an improvement in the symptoms of a disease. By "remission of the disease" is meant essentially no evidence of disease symptoms. Clinical response or disease remission may be achieved within a certain time frame, e.g., within about 8 weeks or about 8 weeks from treatment with the antagonist or from initial dose of the antagonist. Clinical response may also be sustained for a period of time; such as 24 weeks or more, or 48 weeks or more. In some embodiments, the weight loss associated with IBD may be reduced and/or eliminated by treatment with an anti-CD 83 agonist antibody. In some embodiments, treatment with an anti-CD 83 agonist antibody as described herein prevents mucosal damage and/or aids in epithelial repair of gastrointestinal tissue in a patient with IBD.

Symptoms associated with IBD include abdominal pain, vomiting, diarrhea, blood in the stool (bright red blood in the stool), and weight loss. To diagnose IBD, further testing may be performed. For example, total blood counts, electrolyte panels, Liver Function Tests (LFTs), fecal occult blood tests, X-rays (including barium meal enemas and upper gastrointestinal lines), sigmoidoscopy, colonoscopy, and gastrointestinal endoscopy may be used. A variety of scoring systems known in the art can be used to quantitatively assess the severity of the disease.

For the prevention or treatment of disease, the appropriate dosage of the active agent (i.e., anti-CD 83 agonist antibody) will depend on the type of disease to be treated, the severity and course of the disease, whether the agent is administered for prophylactic or therapeutic purposes, previous therapy, the clinical history of the patient and response to the therapeutic agent, and the discretion of the attending physician. The particular dosage regimen, i.e., dosage, timing and repetition, will depend on the particular individual and the medical history of that individual as assessed by the physician. Typically, the clinician will administer an anti-CD 83 agonist antibody until a dosage is reached that achieves the desired result.

The methods of the invention are useful for treating, ameliorating or reducing the symptoms of an autoimmune disease (such as IBD) in an individual, or for improving the prognosis that an individual suffers from an autoimmune disease. Can improve the quality of life of individuals suffering from the disease and can reduce or eliminate symptoms after treatment with anti-CD 83 agonist antibodies. The methods of the invention may also be used to delay the development of or prevent an autoimmune disease (such as IBD) in an individual who is at risk of developing the disease. Any of the anti-CD 83 agonist antibodies described herein can be administered to an individual.

F. Combination therapy

The methods of the invention may be combined with known treatments for autoimmune diseases (e.g., IBD), as a combined or additional treatment step, or as an additional component of a therapeutic formulation. Alternatively, different anti-CD 83 agonist antibodies may be administered in combination. The type of combination therapy chosen depends on the clinical manifestations of the disease.

For example, IBD (such as ulcerative colitis, crohn's disease, or indeterminate colitis) may be treated by a combination therapy comprising administration of an anti-CD 83 agonist antibody in combination with a second drug for IBD. The type of such second drug depends on a variety of factors, including the type of IBD, the severity of the IBD, the condition and age of the subject, the type and dose of the first drug used, and the like. In some embodiments, the second medicament comprises one or more of an aminosalicylic acid, a corticosteroid, and an immunosuppressive agent. In some embodiments, the aminosalicylic acid is one of sulfasalazine, olsalazine, aminosalicylic acid, balsalazide, and mesalamine. In some embodiments, a combination of a plurality of aminosalicylic acids, such as sulfasalazine and olsalazine, is co-administered. In some embodiments, the corticosteroid is budesonide, prednisone, prednisolone, methylprednisolone, 6-mercaptopurine (6-MP), azathioprine, methotrexate, or cyclosporine. In some embodiments, the second drug is an antibiotic, such as ciprofloxacin and/or metronidazole; or antibody-based agents, such as infliximab。

All these second drugs may be used in combination with each other or with the first drug itself, whereby the expression "second drug" as used herein does not mean that it is the only drug, respectively, other than the first drug. Thus, the second drug need not be one drug, but may consist of or comprise more than one such drug.

These second agents as set forth herein are generally used at the same dosages and routes of administration as used above or at about 1-99% of the dosages previously used. Such second agent, if used at all, is optionally used in a lower amount than if the first agent was not present, particularly in a subsequent dose that exceeds the initial dose of the first agent, to eliminate or reduce side effects caused by the treatment.

Combined administration herein includes co-administration using different formulations or a single pharmaceutical formulation, and sequential administration in either order, wherein there is preferably a period during which both (or all) active agents exert their biological activity simultaneously.

G. Dosage of medicament

The dosage and desired drug concentration of the pharmaceutical composition of the present invention may vary depending on the particular use contemplated. Determination of the appropriate dosage or route of administration is well known to those of ordinary skill in the art. Animal experiments provide reliable guidance for determining effective dosages for human therapy. Interspecies Scaling of effective doses can be performed according to The principles set forth in Mordenti, J. and Chappell, W. "The Use of interactions Scaling in The kinetics of drugs," in The chemistry and New drug development, Yacobi et al, eds., Pergamon Press, New York1989, pages 42-46.

For in vivo administration of the polypeptides or antibodies described herein, the normal dosage may vary from about 10ng/kg up to about 100mg/kg of the individual's body weight per day or higher, preferably from about 1 mg/kg/day to 10 mg/kg/day, depending on the route of administration. For repeated administration over several days or longer, depending on the severity of the disease or condition to be treated, the treatment is maintained until the desired suppression of symptoms is achieved.

An exemplary dosage regimen comprises administration of an initial dose of about 2mg/kg of anti-CD 83 agonist antibody followed by a weekly maintenance dose of about 1mg/kg every other week. Other dosage regimens may be useful depending on the pharmacokinetic decay pattern that the physician wishes to achieve. For example, it is contemplated herein to administer to an individual from once to 21 times a week. In certain embodiments, a dosage of about 3 μ g/kg to about 2mg/kg (such as about 3 μ g/kg, about 10 μ g/kg, about 30 μ g/kg, about 100 μ g/kg, about 300 μ g/kg, about 1mg/kg and about 2/mg/kg) may be used. In certain embodiments, the frequency of administration is three times daily, twice daily, once every other day, once weekly, once every two weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, or once monthly, once every two months, once every three months, or longer. The progress of the treatment is monitored by conventional techniques and assays. The dosage regimen, including the anti-CD 83 agonist antibody administered, may vary over time independently of the dosage used.

The dosage of a particular anti-CD 83 agonist antibody can be determined empirically in an individual to whom one or more administrations of the anti-CD 83 agonist antibody have been administered. Administering to the subject an increased dose of an anti-CD 83 agonist antibody. To assess the efficacy of anti-CD 83 agonist antibodies, the clinical symptoms of autoimmune diseases (e.g., IBD) can be monitored.

Administration of an anti-CD 83 agonist antibody according to the methods of the invention may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to the skilled artisan. Administration of the anti-CD 83 agonist antibody is substantially continuous over a preselected period or can be a series of spaced doses, for example, during or after the development of autoimmune diseases such as ulcerative colitis and Crohn's disease.

Guidance regarding specific dosages and methods of delivery is provided in the literature; see, e.g., U.S. patent nos. 4,657,760; 5,206,344, respectively; or 5,225,212. It is within the scope of the invention that different formulations may be effective for different treatments and different conditions, and administration intended to treat a particular organ or tissue may need to be delivered in a different manner than administration to another organ or tissue. In addition, the dose may be administered by one or more separate administrations, or by continuous infusion. For repeated administrations over several days or longer, depending on the condition, the treatment is maintained until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The progress of the treatment is readily monitored by conventional techniques and assays.

H. Administration of the formulations

The formulations of the present invention, including, but not limited to, reconstituted formulations (e.g., formulations of anti-CD 83 agonist antibodies) are administered to an individual, preferably a human, in need of treatment with an anti-CD 83 agonist antibody according to known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, buccal, topical, or inhalation routes.

In a preferred embodiment, the formulation is administered to the individual by subcutaneous (i.e., sub-dermal) administration. For this purpose, the formulation can be injected using a syringe. However, other devices for administering the formulation are available as injection devices (e.g., injection-ase)^TMAnd GENJECT^TMA device); injection pen (e.g. GENPEN)^TM) (ii) a Automatic injector device, needleless device (e.g. MEDIJECTOR)^TMAnd BIOJECTOR^TM) (ii) a And a subcutaneous patch delivery system.

The appropriate dosage ("effective amount") of the anti-CD 83 agonist antibody depends, for example, on the condition to be treated, the severity and course of the condition, whether the anti-CD 83 agonist antibody is administered for prophylactic or therapeutic purposes, previous therapy, the clinical history of the patient and the response to the anti-CD 83 agonist antibody, the type of anti-CD 83 agonist antibody used, and the judgment of the attending physician. The anti-CD 83 agonist antibody may be suitably administered to the patient in one or a series of treatments, and the anti-CD 83 agonist antibody may be administered to the patient at any time prior to diagnosis. anti-CD 83 agonist antibodies can be administered as the sole therapy or as part of a combination therapy in combination with other drugs or treatments useful in the treatment of autoimmune diseases, such as IBD.

For an anti-CD 83 agonist antibody, about 0.1mg/kg to about 20mg/kg may be an initial candidate dose for administration to an individual, e.g., whether by one or more separate administrations. However, other dosage regimens are also useful. The progress of the treatment is readily monitored by conventional techniques.

Uses of anti-CD 83 agonist antibody formulations include the treatment or prevention of autoimmune diseases (e.g., IBD). A therapeutically effective amount (e.g., from about 1mg/kg to about 15 mg/kg) of an anti-CD 83 agonist antibody is administered to the individual depending on the severity of the disease to be treated.

I. Article of manufacture and kit

In another aspect, an article of manufacture or kit is provided containing an anti-CD 83 agonist antibody preparation, and preferably instructions for use thereof in the methods of the invention. Thus, in certain embodiments, an article of manufacture or kit comprises instructions for use of an anti-CD 83 agonist antibody in a method for treating or preventing an autoimmune disease, such as IBD (including ulcerative colitis and crohn's disease), in an individual, the method comprising administering to the individual an effective amount of an anti-CD 83 agonist antibody. In certain embodiments, the individual is a human.

The article of manufacture or kit can also comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber bottles), syringes (e.g., single chamber or dual chamber syringes), and test tubes. The container may be formed from a variety of materials such as glass or plastic. The container contains a formulation. The article of manufacture or kit may further comprise a label or package insert on or associated with the container that indicates the reconstitution and/or use of the formulation. The label or package insert may also indicate that the formulation is for or intended for subcutaneous or other administration for treating or preventing an autoimmune disease (such as IBD) in an individual. The container containing the formulation may be a single use vial or a multiple use vial, which allows for repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. The article of manufacture or kit may also comprise a second container comprising a suitable diluent (e.g., BWFI). When the diluent is mixed with the lyophilized formulation, the final protein, polypeptide, or small molecule concentration in the reconstituted formulation is typically at least 50 mg/ml. The article of manufacture or kit may also include other materials desirable from a commercial, therapeutic, and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The article of manufacture or kit herein optionally further comprises a container comprising a second medicament, wherein the anti-CD 83 agonist antibody is the first medicament, and the article of manufacture further comprises instructions on the package insert for treating the subject with an effective amount of the second medicament. The second drug may be any of those set forth above, with exemplary second drugs being aminosalicylic acid, oral corticosteroids, 6-mercaptopurine (6-MP), and azathioprine (if an anti-CD 83 antibody is used to treat IBD).

In another embodiment, provided herein is an article of manufacture or kit comprising a formulation described herein for administration in an autoinjector device. An auto-injector may be described as an injection device that, upon activation, will deliver its contents without additional necessary activity by the patient or administrator. They are particularly suitable for self-administration of therapeutic agents when the delivery rate must be constant and the delivery time longer than a moment.

The invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. All citations of the disclosure are hereby expressly incorporated by reference.

Examples

CD83 is a very conserved type 1 membrane protein of the Ig superfamily found primarily on the surface of mature Dendritic Cells (DCs). Soluble CD83 has immunosuppressive activity, however, the function of CD83 on DCs and its putative ligand are still unknown. We have identified a CD83 isotype interaction that elicits anti-inflammatory effects on DCs. Treatment with soluble CD83 or anti-CD 83 antibodies during DC maturation results in reduced expression of surface activation markers and secretion of pro-inflammatory cytokines such as IL-12p 40. Knockdown of surface CD83 expression, or truncation of the cytoplasmic region abolished response to CD83 therapy, indicating that CD83 homotypic interactions mediate inhibition of inflammation. Since this inhibition of MAPK and mTOR signaling functions downstream of CD83 treatment inhibits phosphorylation of mTOR and p38 α, mTOR and p38 α are essential for surface activation marker expression and IL-12p40 production. CD83 immunosuppression is critical in maintaining a balance between tolerance and immunity, as mice that overexpress CD83 at mucosal surfaces are more resistant to colitis, resulting in weight retention and reduced serum cytokine levels. Thus, the CD83 isotype interaction modulates DC immune responses, prevents inappropriate inflammation and promotes tolerance.

Example 1 involvement of CD83 in Homotypic binding on cell surface

To determine whether soluble CD83 bound to CD83 expressed on the cell surface, a cell containing amino acid sequence was generated

TPEVKVACSEDVDLPCTAPWDPQVPYTVSWVKLLEGGEERMETPQEDHLRGQHYHQKGQNGSFDAPNERPYSLKIRNTTSCNSGTYRCTLQDPDGQRNLSGKVILRVTGCPAQRKEETFKKYGRAQVTDKAAHYTLCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:3)

CD83.fc and determination of its binding to human stably expressed on CHO cells

CD83(CHO-hCD83) or endogenous CD83 expressed on mature dendritic cells (mDCs) from MUTZ-3. To generate an expression vector for the generation of a stable CHO-hCD83 cell line, a DNA fragment encoding the N-terminal HIS-tagged human CD83(hCD83) was cloned into the neomycin-resistant plasmid pRKneo (Crowley et al, ProcNatlAcad Sci USA.,90(11):5021-5025,1993) at the XbaI and XhoI sites to generate hCD83. pRKneo. CHO cells were transfected with hCD83.pRKneo using Fugene (Roche) and 10% of the CD83 positive cells prior to sorting by FACS, followed by selection with G418(400ug/ml; GIBCO) to generate a stable CHO-hCD83 cell line. To obtain Immature DCs (iDCs) from MUTZ-3 cells, the cells were cultured for 6 days in MEM α + glutamax/20% heat-inactivated FBS containing 150ng/ml rhGM-CSF and 50ng/ml rhIL-4. DCs were matured with a cytokine cocktail containing 25ng/ml rhIL-1 β, 100ng/ml rhIL-6, 50ng/ml rhTNF α, and 1 μ g/ml PGE-2 to surface express CD83.

Expression of CD83 on CHO-hCD83 cells and on mDCs from MUTZ-3 was confirmed by staining the cells with fluorescent dye-conjugated antibodies against CD83 and analyzing the cells by flow cytometry on an LSR II flow cytometer using FACSDiva software (Becton Dickinson). The level of non-specific staining was determined using labeled isotype-matched antibodies. Data analysis and construction of histograms showing total binding using FlowJo v.8.4.5 demonstrated that CD83 was expressed on the surface of stably transfected CHO-hCD83 cells (black line), but not on control CHO cells (dashed line) (fig. 1A). CD83 was expressed on mdcs from MUTZ-3 (black line), but at very low levels on iDC (grey line) (fig. 1B). The filled histogram represents isotype control. With confirmation of cell surface CD83 expression, CHO cells and CHO-hCD83 cells were fixed in 4% PFA for 5 minutes, followed by washing with cold 1 XPBS. Cells were resuspended in FACS buffer containing PBS/2% BSA/2mM EDTA and incubated with 1 μ g PE-labeled cd83.fc or labeled igg. fc control protein on ice for 30 minutes in the dark. Cells were washed with FACS buffer and analyzed by flow cytometry. For iDCs and mDCs, cells were labeled with 10 μ g/ml PE-labeled cd83.fc or labeled igg. fc control protein prior to fixation and flow cytometry. Data analysis demonstrated that cd83.fc bound CHO-hCD83 (black line), but not control CHO cells (dashed line) (fig. 1C). Furthermore, cd83.fc binds to mature DCs (black lines) but not immature DCs (grey lines) (fig. 1D). These results demonstrate that soluble cd83.fc specifically binds to cells expressing CD83 on the cell surface.

To confirm that CD83 on the cell surface is necessary for CD83.fc binding, CHO-hCD83 cells were incubated with 1. mu.g/ml anti-CD 83 antibody (HB15E; Santa Cruz Biotechnology) to determine whether it blocked CD83.fc binding data analysis demonstrated that CD83.fc binding to CHO-hCD83 cells (black line), but binding was blocked by HB15E (dotted line) rather than isotype control (grey line) (FIG. 2A.) the need for CD83 expression for Cd83.fc binding was further determined in MUTZ-3-derived DCs that do not express CD83. after 4 days of incubation in MEM α + glutamax/20% heat-inactivated FBS containing 150/ml rhHarGM-CSF and 50ng/ml rhIL-4. siRNA (catalog No. E-012680; Dharcong 23; or non-targeting catalog (catalog # Dcong 23) was cultured in DCS containing heat-inactivated DCS 3. mu.3. mu.g/ml and DCs containing Dcong-23% heat-5. C3. C-3. mu.g-3. C3. and DCs containing Dcong-7. mu.g-3. C3. and expressing DCs₂The mixture was incubated for 72 hours. On day 7, DCs were treated with maturation stimuli to produce mDCs as described above. The knockdown efficacy of siRNA mediated CD83RNA and protein expression was evaluated by taqman qPCR and western blot of total cell lysates, respectively. Analysis of total RNA normalized to 18S demonstrated that DCs transfected with CD83siRNA exhibited significant down-regulation of CD83RNA levels (fig. 2B), from MUTZ-3mDCs treated with non-targeting control (sinc) siRNA or siRNA specific for CD83 (siCD 83). Knockdown of CD83 in MUTZ-3mDCs was confirmed by staining cells with fluorescent dye-conjugated antibodies against CD83 and analyzing the cells by flow cytometry. Data analysis and construction of histograms showing total binding demonstrated that CD83 was expressed on the surface of mdcs treated with sintcs (black lines), but not on iDC treated with siDC83 (grey lines) or mature DCs (dashed lines) (fig. 2C). The filled histogram represents isotype control. After confirming knock-down of CD83 expression, iDC, siNTC mDC and siCD83 mdcs were incubated with PE-labeled cd83.fc or labeled igg. fc control protein as described above and subjected to flow cytometry. Data analysis demonstrated that cd83.fc binds to siNTC mDC instead of iDC or siCD83mDC, indicating that CD83 expression is required for cd83.fc binding to mature DCs (fig. 2D). Knock-down of CD83 also resulted in decreased expression of MHCII, but not in other activation markers such as CD86 (fig. 2E and F).

To determine whether CD83 mediated cell-to-cell adhesion via homotypic binding, cell aggregation was determined in hCD 83-expressing CHO cells. CHO cells and CHO-hCD83 cells were detached from the flask with 2mM EDTA, washed and washed in 2% FBS/10mM EDTA but lacking Ca²⁺Or Mg²⁺Resuspended in HBSS medium. The cells were then washed with 10⁶The/ml was resuspended and passed through a 70 μm filter to obtain a single cell suspension for plating on a low adhesion 10cm petri dish. After incubation for 90 minutes at 37 ℃ in an orbital platform shaker, cells were fixed with 4% PFA to assess cell aggregation. Microscopic imaging of the cells demonstrated that control CHO cells lacking CD83 expression did not aggregate, but CHO-hCD83 cells expressing hCD83 formed clusters in suspension culture (fig. 3A and B), with pretreatment of CHO-hCD83 cells with 1 μ g of cd83.fc protein blocking aggregation, while with Ig control treatment did not (fig. 3C and D). These results demonstrate that surface expression of CD83 is sufficient for cells to form cell adhesion, and that this interaction can be blocked by the addition of soluble CD83 due to competition for homotypic binding.

Example 2 soluble CD83 treatment of DCs by inhibiting DC maturation and proinflammatory cytokine release produces anti-inflammatory Phenotype

To characterize the immune response induced in DCs by soluble CD83 treatment, the effect of CD83 treatment on mDC surface activation markers was evaluated. To derive iDCs from MUTZ-3 cells, cells were cultured for 6 days in MEM α + glutamax/20% heat-inactivated FBS containing 150ng/ml rhGM-CSF and 50ng/ml rhIL-4. iDCs were treated with a maturation stimulating cytokine cocktail containing 25ng/ml rhIL-1 β, 100ng/ml rhIL-6, 50ng/ml rhTNF α, and 1 μ g/ml PGE-2. mDCs were treated with 10. mu.g/ml CD83.fc, 1. mu.g/ml HB15e or control IgG. fc concurrently with maturation stimulation. Expression of cell surface activation markers CD83 and HLA-dr (MHCII) on MUTZ-3 derived mDCs was examined by staining cells with fluorescent dye-conjugated antibodies against CD83 or MHCII and analyzing the cells by flow cytometry. The level of non-specific staining was determined using labeled isotype-matched antibodies. Data analysis and construction of histograms showing total binding demonstrated that CD83 and MHCII are expressed on MUTZ-3 derived mdcs (black line), but at low levels on iDC (solid line histogram) (fig. 4). Solid wireless histograms represent isotype controls. Treatment with either CD83.fc (grey line) or HB15e (dashed line), both CD83 and MHCII expression were reduced, indicating that CD83 treatment reduced the expression of surface activation markers in mDCs.

Since DCs are known to modulate immune responses by producing cytokines, the effect of soluble CD83 treatment on DCs was evaluated by measuring cytokine secretion from treated human monocyte-derived DCs (mddcs). MDDCs were isolated from whole blood of multiple donors and stimulated with cytokines in the presence or absence of cd83.fc or HB15e to drive maturation. For separation and processing, human whole blood was diluted with PBS, layered on Ficoll histopaque (GE healthcare), and centrifuged at 1500rpm for 30 minutes. The buffy coat was removed and washed with PBS. Monocytes were isolated using human monocyte isolation kit II (Miltenyi) and cultured for 6 days in RPMI/10% FBS/1 XPicillin/streptomycin (R & D systems) containing 125ng/ml rhIL-4 and 50ng/ml rhGM-CSF. The medium was changed every other day to obtain immature DCs. All treatments with 10. mu.g/ml CD83.Fc or 1. mu.g/ml anti-CD 83 antibody (HB15e; Santa Cruz) were given with simultaneous maturation stimulation. Cell culture supernatants were collected 48 hours after maturation of DCs and analyzed for secreted cytokines by ELISA using kits for MCP-1, IL-12p40 and IL-8(Invitrogen) and IL-1ra (cell sciences) detection according to standard manufacturer instructions. Analysis of cytokines secreted by DCs demonstrated that treatment with cd83.fc in conjunction with maturation stimulators altered DC cytokine secretion, with an increase in interleukin-1 receptor antagonist (IL-1Ra) (fig. 5B), which binds to the IL-1 receptor and blocks downstream inflammatory signaling, and a decrease in the production of the proinflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and subunit beta of interleukin-12 (IL-12p40) (fig. 5A and C). Treatment with CD83.Fc or HB15e had no effect on the production of the inflammatory cytokine IL-8 (FIG. 5D).

To further characterize the immune response induced in DCs by soluble CD83 treatment, the effect of CD83 treatment on pro-inflammatory cytokine secretion by mDCs was evaluated. To obtain immature DCs from MUTZ-3 cells, cells were cultured for 6 days in MEM α + glutamax/20% heat-inactivated FBS containing 150ng/ml rhGM-CSF and 50ng/ml rhIL-4. DCs were matured with a cytokine cocktail containing 25ng/ml rhIL-1 β, 100ng/ml rhIL-6, 50ng/ml rhTNF α, and 1 μ g/ml PGE-2 to surface express CD83. mDCs were treated with 10. mu.g/ml CD83.fc, 1. mu.g/ml HB15e or control IgG. fc with maturation stimulation. Cell culture supernatants were collected 48 hours after maturation of DCs and analyzed by ELISA for secreted cytokines using MCP-1, IL-12p40 and IL-8 kit (Invitrogen) and IL-1ra (cell sciences) according to standard manufacturer's instructions. Analysis of cytokines secreted by DCs demonstrated a reduction in the pro-inflammatory cytokines MCP-1 (FIG. 6A) and IL-12p40 (FIG. 6C) in mDCs upon treatment with CD83.fc or HB15 e. In contrast, the anti-inflammatory cytokine IL-1ra was significantly increased in CD 83-treated mDCs (FIG. 6B). The level of released IL-8 demonstrated no difference in CD 83-treated or control-treated mDCs (fig. 6D). Supernatants from each well were run in triplicate and represent significantly different values, # p <0.01, # p < 0.001. Each point represents the average of an individual well. The figures are representative of at least three independent experiments.

To further characterize the anti-inflammatory phenotype of expressed, i.e., DCs treated with CD83.fc or HB15e, microarray analysis was performed on RNA isolated from DCs following treatment. Statistical analysis of microarrays was done using software from the R program (http:// R-project. org) and the Bioconductor program (http:// Bioconductor. org). Background subtracted microarray data were normalized for LOESS within the array and quantile across the array. The normalized data was then log2 transformed and the probes were filtered using the Bioconductor 'gene filter' package, so that only probes mapped to the Entrez gene were retained. A non-specific filtration procedure was then applied which removed 50% of the least variable probes (Bourgon et al, Proc Natl Acad Sci USA, 107(21): 9546-. To identify differentially expressed genes, the limma software package (Smyth., Stat Appl GenetMol biol.,3: Article3,2004) was used to calculate attenuated t statistics. The linear model measures differential expression between immature and mature DCs, as well as differences between CD 83-linked samples (CD83 fc-and HB15 e-treated mature DCs) and control samples (IgG-and untreated mature DCs). The False Discovery Rate (FDR) was calculated using the Benjamini-Hochberg method. Genes are considered to be differentially expressed if they have an FDR of less than 0.01. Analysis of all gene expression changes in DCs from 5 different donors indicated that cd83.fc or HB15e treatment resulted in an anti-inflammatory phenotype. Taken together, measurements of DC cytokine release and gene expression following cd83.fc or HB15e treatment demonstrate that soluble CD83 treatment inhibits the secretion of pro-inflammatory cytokines and induces an anti-inflammatory response.

Example 3 soluble CD83 treatment results in the upregulation of genes involved in wound healing

To further characterize the changes in gene expression induced in DCs treated with cd83.fc or HB15e, RNA isolated from DCs was subjected to microarray analysis after treatment. Statistical analysis of microarrays was done using software from the R program (http:// R-project. org) and the Bioconductor program (http:// Bioconductor. org). Background subtracted microarray data were normalized by LOESS within the array and by fractional bit between arrays. The normalized data was then log2 transformed and the probes were filtered using the Bioconductor 'gene filtration' package, so that only probes mapped to the Entrez gene were retained. A non-specific filtration procedure was then applied which removed 50% of the least variable probes (Bourgon et al, Proc Natl Acad Sci USA, 107(21): 9546-. To identify differentially expressed genes, the limma software package (Smyth., Stat Appl GenetMol biol.,3: Article3,2004) was used to calculate attenuated t statistics. The linear model measures differential expression between immature and mature DCs, as well as differences between CD 83-linked samples (CD83 fc-and HB15 e-treated mature DCs) and control samples (IgG-and untreated mature DCs). The False Discovery Rate (FDR) was calculated using the Benjamini-Hochberg method. Genes are considered to be differentially expressed if they have an FDR of less than 0.01. Analysis of DCs from 5 different donors showed that cd83.fc and HB15e treated cells were isolated and clustered independently from untreated mDCs and iDCs, and that treatment resulted in up-regulation of genes involved in wound healing. Microarray data was confirmed by taqman qPCR analysis of total RNA isolated from mDCs treated with cd83.fc or HB15e (fig. 7). Analysis of gene expression after gapdh normalization demonstrated that the genes involved in wound healing, vacan, spock2 and fbn2, were up-regulated. The mean correlation indicated is expressed as 2^ Δ CT + standard deviation of the mean (SEM). These in vitro results indicate that soluble CD83 treatment can promote the proliferation of healthy cells and migration in vivo for tissue damage healing resulting from inflammatory diseases.

Example 4 interaction of the CD83 isotype mediated anti-inflammatory response

To further characterize the CD83 interaction that modulates anti-inflammatory responses, the anti-inflammatory response of DCs was monitored when co-cultured with CHO cells overexpressing hCD83. To derive iDCs from MUTZ-3 cells, cells were cultured for 6 days in MEM α + glutamax/20% heat-inactivated FBS containing 150ng/ml rhGM-CSF and 50ng/ml rhIL-4. The iDCs produced were co-cultured with either a control CHO cell line or with CHO cells stably expressing human CD83. The mixed cell cultures were then left untreated or treated with a cytokine mixture containing 25ng/ml rhIL-1 β, 100ng/ml rhIL-6, 50ng/ml rhTNF α and 1 μ g/ml PGE-2 to produce mDCs. Cell culture supernatants were collected 48 hours after maturation of DCs and analyzed by ELISA (Invitrogen) for secreted cytokines IL12-p40 and MCP-1 according to standard manufacturer's instructions. Analysis of secreted IL12-p40 and MCP-1 levels showed a significant reduction in the release of pro-inflammatory cytokines in mDCs co-cultured with hCD 83-expressing CHO cells, compared to CHO cells lacking CD83 expression (fig. 8A and B). This data demonstrates that CD83 can participate in trans-isotype interactions to mediate anti-inflammatory responses. These results were confirmed using human monocyte-derived dcs (mddcs) isolated from whole blood of various donors. DCs cultured with CHO-hCD83 cells produced significantly lower IL-12p40 after stimulation than those cultured with CHO cells lacking CD83 expression.

Co-culture of immature BMDCs with mature BMDCs from wild-type or CD 83-deficient animals was studied and IL-12p40 production was determined. A CD83 knockout mouse (CD 83) was generated using a homologous recombination strategy similar to that used in Fujimoto, Y, et al, Cell108,755-767,2002, which is incorporated herein by reference^-/-) This results in a loss of half of the immunoglobulin domain and transmembrane and cytoplasmic domains of CD83. CD83^-/-Mice lack CD4T cells, but are otherwise normal, are produced at the expected Mendelian frequencies (Mendelian frequencies) and reproduce as well as their wild-type littermates. After stimulation with LPS, it was produced from CD83^-/-BMDCs in mice were able to up-regulate the surface maturation marker CD86 and produce cytokines at levels similar to those produced from wild-type littermates (fig. 8C). Upregulation of MHCII was also seen following stimulation with LPS, however BMDCs from CD 83-/-mice expressed lower MHCII than those produced from wild type mice. Fresh immature BMDCs were then co-cultured with mature cells from wild-type or CD 83-/-animals while stimulated with LPS. After 24 hours, culture supernatants were collected and evaluated for IL-12p40 production by ELISA. Immature DCs co-cultured with mature DCs expressing high levels of CD83 produced significantly lower IL-12p40 than those cultured with CD83 deficient mature DCs (fig. 8D).

To verify that the anti-inflammatory response mediated by CD83 treatment requires interaction with cell surface CD83, treatment was assayed in DCs that do not express CD83 4 days after incubation in MEM α + glutamax/20% heat-inactivated FBS containing 150ng/ml rhGM-CSF and 50ng/ml rhIL-4, M was transfected with Accell siRNA targeting CD83 (catalog number E-012680; Dharmacon) or non-targeting control (catalog number D-001910; Dharmacon)UTZ-3 iDC. MUTZ-3iDCs were cultured in Accell delivery medium (Cat. No. B-005000; Dharmacon) containing 10uMsiRNAs supplemented with 3% heat-inactivated FBS, 150ng/ml GM-CSF and 50ng/ml IL-4 at 37 ℃ and 5% CO₂Following incubation for 72 hours at day 7, iDCs were treated with a cytokine mixture containing 25ng/ml rhIL-1 β, 100ng/ml rhIL-6, 50ng/ml rhTNF α and 1 μ g/ml PGE-2 to generate mDCs, while maturation was stimulated, mDCs were treated with 10 μ g/ml CD83.fc, 1 μ g/ml HB15e or control IgG. fc. cell culture supernatants were collected 48 hours after maturation of DCs and analyzed by ELISA using MCP-1 kit (Invitrogen) according to standard manufacturer's instructions analysis of secreted cytokines by siRNA knockdown of CD83 (siCD83) demonstrated that siRNA knockdown of CD83 (FIG. 9A) abolished responses to 83.fc and HB15 antibody (FIG. 9A.) thus, this data shows that CD83 is required to mediate anti-inflammatory responses treated by CD83 on cell surface-the knockdown of secreted IL-12. the results demonstrated by detection of these secreted IL-12. knockdown of CDHB 83.fc-15 antibodies (FIG. 9A) demonstrated that CD 3635 mediated the knockdown of siRNA of CD 736 by CD 387 7. siRNA release of siRNA and CD 3612. siRNA of CD e.

To determine whether anti-inflammatory responses during CD83 treatment require homotypic binding and downstream signaling via cell surface CD83, treatment was assayed in DCs expressing cytoplasmic truncated CD83 constructs. The CD83 lentiviral expression construct was from pgcmv. ires. egfp (pGIPZ derivatives; Openbiosystems) and was prepared as follows: the full-length hCD83 gene (CD83FL) or hCD83 gene segments of the truncated cytoplasmic region (. DELTA.172-205) were amplified by PCR and inserted into the XhoI/EcoRI cloning site (FIG. 9B). To generate lentiviruses for infection of DCs, 293T cells were plated at 1X10⁷Seeded on gelled 10cm petri dishes and allowed to grow for-20 hours to reach 80-90% confluence. The medium was supplemented with 5ml DMEM, 10% FBS, 2 mML-glutamine and cells were transfected with a DNA mixture containing 5. mu.g of expression plasmid, 8.9 and VSVG at a molar ratio of 1:2.3:0.2 using Lipofectamine2000(Invitrogen) for 6 hours at 37 ℃. The transfection medium was supplemented with 6ml of normal growth medium and the cells were incubated at 37 ℃ for an additional 40 hours. The supernatant was harvested, clarified by filtration through a 0.45 μm tube tip filter (Corning), and used according to the manufacturer's instructionsConcentrated in a Lenti-X-concentrator (Clonetech). MUTZ-3 cells at 0.5X10⁶Perml maintenance Medium containing MEM α + Glutamax/20% Heat-inactivated FBS/15% HTB-9 conditioned Medium inoculated in 24-well plates 1, 5-dimethyl-1, 5-diaza-undecamethylene polymethlromide (Polybrene) and concentrated lentivirus supernatant were separately washed with

A final concentration of 4. mu.g/ml and an MOI of 10 were added to the cells. Cells were centrifuged at 1800rpm for 30 minutes at room temperature in an Allegra X-12R benchtop centrifuge followed by incubation at 37 ℃ overnight.

After 2-3 days of culture, the first 10% GFP positive cells were sorted and cultured in MEM α + Glutamax/20% heat-inactivated FBS supplemented with 150ng/ml rhGM-CSF and 50ng/ml rhIL-4 for 6 days and used as iDC_c、HB15_eOr isotype control for 48 hours.

The supernatants were then collected and analyzed for MCP-1 release by ELISA. Analysis of secreted MCP-1 demonstrated that lentiviral overexpression of full-length CD83 did not inhibit the mDC response to either CD83.fc or HB15e, whereas expression of cytoplasmic truncated CD83 blocked the anti-inflammatory effect of CD83 treatment (fig. 9C). These results were confirmed by detection of secreted IL-12p 40. Analysis of secreted Il-12p40 demonstrated that lentiviral overexpression of full-length CD83 did not inhibit the mDC response to CD83.fc or HB15e, whereas expression of cytoplasmic truncated CD83 blocked the anti-inflammatory effects of CD83 treatment.

To determine whether cross-linking alone was sufficient to drive an anti-inflammatory response via the CD83 cytoplasmic domain,

a CD83 chimeric lentiviral expression construct was generated containing the extracellular region of CD79a fused to the CD83 transmembrane and full-length or truncated cytoplasmic domain (fig. 10A). Infection of iDCs with the indicated lentiviral constructs as described above, and subsequent use with maturation stimuli and with 1_μg CD79a antibody (santa cruz) or isotype control for 48 hours. Supernatants were collected and analyzed by ELISA

IL12-p 40. Analysis of secreted IL12-p40 demonstrated that lentiviral overexpression of the full-length CD83 chimera did not inhibit the mDC response to anti-CD 79a antibody, whereas expression of the cytoplasmic truncated CD83 chimera blocked the anti-inflammatory effect of treatment with anti-CD 79a (fig. 10B). Taken together, these results demonstrate the novel finding that the CD83 isotype interaction mediates anti-inflammatory effects as a result of CD83 treatment.

Example 5 CD83 homotypic interactions inhibition of inflammation by inhibition of MAPK and mTOR signaling pathways

Due to the new finding that CD83 homotypic interactions elicit anti-inflammatory effects in DCs, downstream signaling pathways regulated by the CD83 cytoplasmic domain were investigated. Alignment of the last 15 amino acids of the CD83 cytoplasmic domain indicated that the C-terminal class III PDZ ligand motif was conserved in CD83 (fig. 11A). To determine whether this motif mediates an anti-inflammatory response in CD83 treatment, a CD83 lentiviral expression construct was generated that contained full-length CD83 with a valine to alanine mutation at position 205 (V205A) to disrupt the class III PDZ ligand motif (fig. 11B). iDCs were infected with the indicated lentiviral constructs as described above. Cells were treated with maturation stimuli as well as cd83.fc, HB15e or isotype controls for 48 hours. Supernatants were then collected and analyzed for IL12-p40 release by ELISA. Analysis of secreted IL12-p40 demonstrated that lentiviral overexpression of full-length CD83 did not inhibit the mDC response to either CD83.fc or HB15e, whereas expression of the CD83V205A mutant blocked the anti-inflammatory effects of CD83 treatment (fig. 11C). These results suggest that the class III PDZ ligand motif on the CD83 cytoplasmic domain interacts with proteins that mediate anti-inflammatory responses.

To determine whether immunosuppression of the CD83 isotype interaction was mediated by additional signaling pathways, cell lysates from 5 min mDCs treated with cd83.fc or HB15e were subjected to phosphokinase arrays. Human phosphokinase arrays (R & D systems) were performed according to the manufacturer's instructions. Briefly, MUTZ-3DCs were washed with cold PBS, dissolved in lysis buffer 6, and shaken at 4 ℃ for 30 minutes. The lysates were centrifuged at 14,000X g for 5 minutes and the supernatants were transferred to new tubes for analysis of total protein by Bradford (Bio-Rad). Duplicate spotted array membranes containing 46 antibodies were blocked for 1 hour at room temperature and then incubated with diluted cell lysates overnight at 4 ℃. The membrane was washed and then incubated with detection antibody for 2 hours at room temperature. After washing, the membrane was incubated in streptavidin-HRP for 30 minutes at room temperature and washed again before detection with ECL Plus reagent (Amersham). The FILM was exposed on a FUJI FILMLImage Reader LAS-3000 and the average intensity was analyzed by Multi Gauge v3.1(FUJI FILM). HB15e treatment resulted in a significant decrease in phosphorylation of mTOR, p =0.046 (fig. 12A), p38, p =0.008 (fig. 12B) and CREB, p =0.0104 (fig. 12C). In contrast, HB15e antibody treatment did not inhibit phosphorylation of STAT3, which was activated by TNF receptor binding of component TNF α in the maturation stimulus (fig. 12D). Western blot analysis of whole cell lysates from human monocyte-derived dcs (mddcs) treated with cd83.fc or α CD83(HB15E) demonstrated a reduction in phospho-p 38MAPK (fig. 12E) with no significant effect on STAT3 phosphorylation (fig. 12F). These new findings suggest that the anti-inflammatory effects of the CD83 isotype interaction are mediated by p38MAPK as well as mTOR protein signaling.

Example 6 overexpression of CD83 results in reduced expression of surface activation markers on colonic lamina propria DCs

The new finding that the CD83 isotype interaction elicits anti-inflammatory effects in DCs in vitro suggests that it may elicit similar effects in vivo. To investigate the effect of CD 83-mediated immunosuppression in vivo, a transgenic mouse strain was generated that overexpresses CD83 at the mucosal surface (CD83 Tg). To generate fabp.cd83 targeting vectors, the Picomax PCR system was used to amplify full-length mouse CD83(mCD83) from colon tissue for cloning into fabp.sup.lacz vectors using the SpeI/SacII site (fig. 13A). The primers used for PCR were CD83 SPE-forward primers:

5'-GATCAAACTAGTCCACCATGTCGCAAGGCCTCCAGCTCCT-3' and CD83 SACII-reverse primer 5'-CATCATCCGCGGTCATACCGTTTCTGTCTTAGGAAG-3'. After microinjection, 72 founder mice were screened for high expression in the colon and low expression in the kidney. 1 mouse meets these criteria and is used to generate transgenic lines by backcrossing with FVB mice (Jackson labs). Mice were housed in a specific pathogen free barrier facility. All programs were approved by Genentech Animal Care and Use Committee.

Colonic immunohistochemical staining for CD83 showed that expression of CD83 was restricted to gut-associated lymphoid tissues in wild-type animals, but CD83Tg overexpressed CD83 in the colonic epithelium (fig. 13B). To determine whether CD83 overexpression had an effect on DC subsets, T cell populations, and surface markers, the colon was harvested and rinsed with cold HBSS/2% FBS/10mM HEPES. Fat and other tissue accompanying the colon were removed and the colon was flushed with HBSS/2% FBS. The colon was cut longitudinally with scissors and transferred to a 50-ml conical tube with 30-40ml HBSS/2% FBS on ice. The colonic fragments were then transferred to a sterile baffled flask (Corning) containing 10-15ml of pre-heated HBSS/2% FBS/10mM HEPES/1mM EDTA. The flask was shaken at 200rpm for 45 minutes at 37 ℃. The medium was decanted and the colon washed in fresh HBSS/2% FBS/10mM HEPES and the remaining epithelium scraped with a razor blade. The colon was cut into 1-2-mM pieces in RPMI containing 10% FCS, 20mM HEPES, 0.5mg/ml collagenase/dispase, penicillin and streptomycin, and then incubated at 37 ℃ for 45-90 minutes with 200rmp shaking. The suspension was pipetted 4-5 times and filtered through a 100 μm filter followed by centrifugation at 1800rpm for 10 minutes at 4 ℃. Cells were washed with RPMI containing 5% FBS, 20mM HEPES and 0.1mg/ml DNase and filtered through a 70 μm filter. The cells were then washed with FACS buffer and stained with antibodies for analysis of immune cells and DC for RNA isolation and qPCR_sSorting.

DCs isolated from the colon lamina propria or spleen of CD83Tg mice were sorted by flow cytometry for MHCII and CD11c hi expression (fig. 14A and B). Analysis of the subset of DCs showed no significant difference in the number or percentage of plasmacytoid (CD11B-/B220+), myeloid (CD11B +) or lymphoid (CD11B-/B220-/CD8a +) DCs isolated from the colon (FIG. 14D) or spleen (FIG. 14E), indicating that CD83 overexpression had no effect on the subset of DCs in CD83Tg mice. Analysis of the surface activation markers CD83, CD86, and MHCII (I-a/E) demonstrated that they were significantly reduced by p <0.05 and p <0.01 on the surface of DCs isolated from the colon (fig. 13C), while those from the spleen showed no significant differences (fig. 13E). Each dot represents cells collected from three animals. Furthermore, analysis of T cells isolated and sorted from the colon lamina propria or spleen of CD83Tg mice (fig. 14C) showed no significant difference in expression of CD44 surface markers indicative of T cell activation, whether isolated from the colon (fig. 13D) or spleen (fig. 13F). Interestingly, taqman qPCR analysis of total RNA isolated from CD83Tg DCs showed increased wound healing gene expression in the colon, but was undetectable (ND) in the colon of wild type mice (fig. 13G). Data are representative of three independent experiments with n =6 per group. Taken together, these in vivo results are supported by the in vitro results observed with DCs treated with cd83.fc or HB15 e.

Example 7 immunosuppression of CD83 protects mice from DSS-induced colitis

To evaluate the role of CD83 overexpression in a mouse model of inflammatory bowel disease, colitis was induced, produced and characterized in the previous examples in the CD83Tg mouse strain by treatment with sodium dextran sulfate (DSS). CD83Tg mice, 8-10 weeks old, were given ad libitum in drinking water for 6% DSS7 days and were switched to normal drinking water on day 7 until the experiment was stopped on day 12. Mice were weighed on day 0 and daily onwards from day 4 and examined for occult blood, diarrhea and any other abnormal signs. Mice were euthanized if weight loss exceeded 20% of initial weight on day 0. Mice were bled orbitally under anesthesia on day 12 to collect 150-. Serum cytokines were evaluated using the Bio-PlexPro Mouse23-Plex assay (catalog No. M60-009RDPD; Bio-Rad) according to the manufacturer's instructions. Mice were subsequently euthanized and small and large intestine sections were sectioned and stained with H & E for histological analysis. The slices were randomized and scored double blindly. Wild type mice treated with 6% DSS lost-20% of body weight, while CD83Tg mice retained-89% of the initial body weight on day 12 (fig. 15A). Wild-type mice had severe colitis marked by loss of colon structure and increased inflammatory infiltration compared to CD83Tg mice (fig. 15B). H & E stained histological scores from colon sections of wild type and CD83Tg mice demonstrated that wild type mice had an average histological score of 8.2, whereas the histological score of CD83Tg mice was significantly lower (x, P =0.0094), 5.3 (fig. 15B and C). Furthermore, serum levels of proinflammatory cytokines were measured by ELISA and were found to be significantly reduced in CD83Tg mice treated with 6% DSS compared to wild-type littermates (, P <0.05) (fig. 15D). These results demonstrate that mice overexpressing CD83 on mucosal surfaces are more resistant to colitis, resulting in weight maintenance and reduced serum cytokine levels. Thus, the CD83 isotype interaction modulates DC-mediated immune responses, prevents inappropriate inflammation and promotes tolerance.

To determine whether the protection seen during DSS colitis in mice overexpressing CD83 was due to the effect of CD83 on potential lamina propria DCs, IL-12p40 expression was determined in lamina propria DCs from CD83Tg and WT mice with DSS colitis. Mice were euthanized at day 9 after 7 days of DSS treatment, when weight loss was significantly different between CD83Tg and WT siblings. DCs were then sorted from isolated lamina propria immune cells to assess the expression of pro-inflammatory cytokines by qPCR. DSS induced the expression of inflammatory cytokines in both CD83Tg and WT mice. However, DCs isolated from CD83Tg mice had significantly reduced IL-12p40 expression (x, p =0.0315) compared to wild-type littermates (fig. 15E), suggesting that increased mucosal CD83 levels modulate DC immune responses to protect during colitis.

Example 8: loss of CD83 in dendritic cells exacerbates colitis

To determine whether loss of CD83 in DCs would exacerbate colitis, a specificity in DCs was developedMice sexually lacking CD83 expression were tested. Production of CD83 by homologous recombination^fl/flA mouse. First, the CD83 genomic fragment was retrieved from BAC (catalogue number rpci23.c, Invitrogen) using a synthetic CD83 retrieval vector with unique restriction sites:

GCGGCCGCGAGCAACTGATATTATATATGCCTTGAACATGAAACCAGGGGCAGGCTGTGGAATATTTCCAGGCACGCTGTCTCGAGGCACAGTAGATCCTCAACCCAAGTGGATAAGAGATGACAATAGCTTTCCAAGAGAGACAGTTATGAGGGACC(Blue Heron) are provided. Contacting the purified retrieved fragment with a first targeting cassette:

ACAGGTCTCCCAGCCAGTGTTTCTCTCACCCCTGCAGGGTGAAGGCTGTGTTGGTTCCTGGTGCTACAATCACAGCATTGCAGTCTTATCTTGTTTCAAAATAACTTCGTATAATGTATGCTATACGAAGTTATCTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATAACTTCGTATAATGTATGCTATACGAAGTTATGCAAACACAGTCTCAAGAGTTTTTATAGATTCTCTTCTTCTCCCCTGGAATCCTCATTTACAGGGATAGGGGGTGGGGGAGCACCCTGTCTTGCTTTAAA(Blue Heron) co-transformation, which inserts a loxP-flanked kanamycin selection cassette into the retrieved genomic fragment. The purified targeting plasmid was then transformed into arabinose-induced and electrocompetent SW106 cells to allow Cre-mediated selection cassette ejection (pop-out) to leave behind a single loxP site. The purified fragment was then ligated with a synthetic second targeting cassette:

CTCAGTGACACATTACACACTTGTGGTGCAATGTATGGATTACCTGAATACCCACCTTCCCCAGGGAGCAAGCATTTCTCCGTTTTGTGCTTTCTTCAGTGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGGTCTGAAGAGGAGTTTACGTCCAGCCAAGCTAGCTTGGCTGCAGGTCGTCGAAATTCTACCGGGTAGGGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCATCAGTCAGGTACATATATAACTTCGTATAATGTATGCTATACGAAGTTATGCACAGTAGATCCTCAACCCAAGTGGATAAGAGATGACAATAGCTTTCCAAGAGAGACAGTTATGAGGGACCACGCAGAAATGAACAAAGCACAGTTGGT(Blue Heron) for insertion of the PGK-em7-Neo-pA resistance gene flanked by two Frt sites and a single loxP site to create a conditionally targeted vector. The selection box is reserved for use in ES cellsPositively selected conditions in the cell are targeted into the vector and subsequently removed by transient transfection of cDNA encoding Flp in ES cells to generate conditions CD83^flAlleles (fig. 16A). Will then carry CD83^flAllelic ES cells are injected into mouse blastocysts to generate chimeric mice, which are then used to generate homozygous CD83^fl/flA mouse. CD83 knockout mice were generated using a similar strategy as used in Fujimoto, y, et al, cell108,755-767,2002 (which is incorporated herein by reference). Will CD83^fl/flMice were bred to mice with transgenic expression of Cre recombinase under the control of the CD11c promoter to generate mice specifically deficient in CD83 in DCs (CD 83)^fl/flCD11c-Cre)。CD83^fl/flCD11c-Cre mice did not show global morphological abnormalities and, unlike global CD83 knockout mice, when compared to CD83^wt/wtCD11c-Cre had a normal number of CD4T cells in the spleen when compared to littermates, since CD83 expression in thymic epithelial cells was presumably not affected in these mice (fig. 16B). The number of DCs in spleen and colon was also similar, but the expression of CD83 was in CD83^fl/flMost of the DCs were deleted in CD11C-Cre mice (FIG. 16C). Loss of CD83 expression in DCs resulted in decreased survival of DSS colitis (fig. 16D). CD83^fl/flThe CD11c-CreDSS treated mice had severe weight loss, by day 8, with CD83^wt/wtCD11c-Cre retained significantly lower of its initial body weight (77.7%) compared to littermates (84.9%) (fig. 16E). In addition, DSS treatment was at 100% CD83 by day 8^fl/flBright blood was produced around the anus and in the feces in CD11c-Cre mice, which was observed in CD83^wt/wtCD11c-Cre was not observed in littermates (fig. 16F), and therefore required euthanasia for humane reasons. These results indicate that DC CD83 expression is essential for tolerating DSS-induced colitis.

Example 9 Generation and characterization of agonist anti-CD 83 antibodies

Agonist antibodies that specifically bind to an epitope within the extracellular region of human CD83 can be generated by screening antibody libraries, such as phage display libraries. The antibodies in the library may be human, humanized or chimeric antibodies. The antibodies in the library may also be single chain antibodies or single domain antibodies. Alternatively, peptides from the extracellular region of human CD83 can be used to immunize mice and the CD83 agonist activity of anti-CD 83 antibodies identified as described below.

Binding of agonist anti-CD 83 antibody to cell surface CD 83:

to identify antibodies that bind to cell surface CD83 on dendritic cells, the antibodies produced were screened for binding capacity by flow cytometry analysis. Briefly, Immature Dendritic Cells (iDCs) were treated with a mixture of cytokines to induce DC maturation and surface expression of CD83. Prior to fixation and flow cytometry, mDCs were treated with the generated antibodies simultaneously with a DC maturation stimulus. Antibodies that specifically bind CD83 expressed on the surface of mDCs were identified by analyzing flow cytometry data. Alternatively, the anti-CD 83 antibodies produced were screened using a cell aggregation assay. Briefly, hCD 83-expressing CHO cells (CHO-hCD83) were detached from flasks with 2mM EDTA, washed and washed in a flask containing 2% FBS/10mM EDTA but lacking Ca²⁺Or Mg²⁺Resuspended in HBSS medium. The cells were then washed with 10⁶The/ml was resuspended and passed through a 70 μm filter to obtain a single cell suspension for plating on a low adhesion 10cm petri dish. CHO-hCD83 cells were subsequently treated with the generated antibodies and incubated in an orbital platform shaker at 37 ℃ for 90 minutes before fixation with 4% PFA. Antibodies that blocked cell aggregation of CHO-hCD83 cells by competing for CD83 homotypic binding were identified by microscopic imaging of the cells. These antibodies can be further characterized for their binding to human CD83 and agonist activity.

Alterations in cytokine release from mDCs treated with agonist anti-CD 83 antibody:

to identify anti-CD 83 antibodies with CD83 agonist activity, the effect of CD83 treatment on pro-and anti-inflammatory cytokine secretion by mDCs was evaluated. Briefly, iDCs were treated with a cytokine cocktail to induce DC maturation and surface expression of CD83. The generated antibodies were used to treat mDCs, together with a DC maturation stimulus. Cell culture supernatants were collected 48 hours after maturation of DCs and analyzed by ELISA for secretion of the pro-inflammatory cytokines MCP-1 and IL-12p40 and the anti-inflammatory cytokine IL-1 ra. An anti-CD 83 antibody that inhibits the release of the pro-inflammatory cytokines MCP-1 and IL-12p40 and/or induces the release of the anti-inflammatory cytokine IL-1ra is identified as an antibody having agonist activity.

Reduced expression of mDC cell surface activation markers treated with agonist anti-CD 83 antibodies:

to identify agonist anti-CD 83 antibodies that inhibit the activation of mDCs, the antibodies produced were screened for their ability to reduce the expression of cell surface activation markers. Briefly, Immature Dendritic Cells (iDCs) are treated with a mixture of cytokines to induce DC maturation. The generated antibodies were used to simultaneously treat mDCs with a DC maturation stimulus. Expression of cell surface activation markers CD83 and HLA-dr (MHCII) on mDCs was examined by staining cells with fluorescent dye-conjugated antibodies against CD83 or MHCII and analyzing the cells by flow cytometry. An anti-CD 83 antibody that reduces cell surface expression of CD83 and/or HLA-DR on mdcs is identified as an antibody having agonist activity.

Treatment with agonist anti-CD 83 antibody inhibited the MAPK and mTOR signaling pathways:

to identify agonist anti-CD 83 antibodies that inhibit activation of MAPK and mTOR (mammalian target of rapamycin) signaling in mDCs, the antibodies generated were screened for their ability to inhibit phosphorylation of downstream signaling proteins. Briefly, Immature Dendritic Cells (iDCs) are treated with a mixture of cytokines to induce DC maturation. The generated antibodies were used to simultaneously treat mDCs with a DC maturation stimulus. Cell lysates from treated mDCs were subjected to SDS-PAGE followed by western blot analysis with specific phosphoantibodies. An anti-CD 83 antibody that inhibits the MAPK signaling pathway by reduced phosphorylation of p38 and CREG proteins and/or inhibits the mTOR signaling pathway by reduced phosphorylation of mTOR protein in mDCs is identified as an antibody with agonist activity.

Candidate agonist anti-CD 83 antibodies identified from the antibody screening methods outlined above are used at various doses for the treatment of autoimmune diseases in animal models such as the IL-10 knockout mouse model of colitis (Scheinin et al, Clin Exp Immunol.,133:38-43,2003) and the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis (Miller et al, Curr Protoc Immunol., Chapter 15: Unit15.1, 2007).

Example 10 Generation of anti-CD 83 antibodies

Materials and methods

Culture medium and antibody

ClonaCell-HY Medium B (Cat #03802), Medium C (Cat #03803), Medium D (Cat #03804) and Medium E (Cat #03805) are from StemCell technologies. CytofusionMedium C (Cat # LCM-C) used for electrofusion was from Cyto Pulse Sciences. Goat anti-hamster IgG (H + L) -HRP-conjugated antibody (Cat # 127-. TMB Single component HRP microwell substrate (Cat # TMBW-1000-01) and TMB stop reagent (Cat # BSTP-1000-01) were from BioFx Laboratories.

In vivo immunization

Each hamster was immunized with 2 μ g of recombinant mouse and human CD83 resuspended in monophosphoryl lipid a/trehalose dicorynomycolate adjuvant by intraperitoneal injection at 3-4 day intervals for a total of 18 boosts. Three days after the last prefusion boost, lymphocytes from the spleens of immunized hamsters were collected.

Hybridoma production and antibody screening

Isolated hamster spleen cells were fused with PU-1 myeloma cells (American Type Culture Collection) by using a cytopulse CEEF-50 device (cytopulse Sciences). Briefly, after washing twice with CytofusiMedium C, the separated dermal and PU-1 cells were mixed at 1:1 and then weighed at ten million cells/mlSuspended in Cytofusion Medium C. Electrofusion was performed according to the manufacturer's instructions. Fusing the cells in ClonaCell-HYMedium C at 37 deg.C in 7% CO₂The culture was carried out overnight in an incubator. The next day, the fused cells were centrifuged and then resuspended in 10ml of ClonaCell-HY Medium C, followed by gentle mixing with 90ml of methylcellulose-based ClonaCell-HY Medium D containing HAT components. Cells were plated on 100mm dishes (Cat #351029, Becton Dickinson) and allowed to incubate at 37 ℃ in 7% CO₂And (5) growing in an incubator. After 10 days incubation, individual hybridoma clones were picked by ClonePix (Genetix, United Kingdom) and transferred into 96-well cell culture plates (#353075, Becton Dickinson) containing 200. mu.L of Clonacell-HY MediumE per well. Hybridoma culture medium was changed prior to ELISA screening. Three days after medium change, hybridoma supernatants were screened by ELISA against human CD83 or mouse CD83 for identification of ELISA positive clones.

Hamster Abs purification

Hybridoma supernatants were purified by protein a affinity chromatography, then sterile filtered (0.2 μm pore size, NalgeNunc International, NY, USA) and stored in PBS at 4 ℃. Purified mAbs were confirmed by ELISA before further testing using functional assays.

ELISA assay

ELISA assays were performed according to standard protocols. ELISA96 well microtiter plates (Greiner, Germany) were coated with human or mouse CD83 at a concentration of 2 μ g per ml in 0.05M carbonate buffer (pH9.6) per well and incubated overnight at 4 ℃. After washing the wells three times with wash buffer (0.05% Tween20 in PBS, Sigma), the plates were blocked with 100 μ L of ELISA assay diluent containing BSA. Approximately 100. mu.L of culture supernatant or diluted purified mAbs were added and incubated for 1 hour at room temperature. Plates were washed three times and incubated with HRP-conjugated goat anti-hamster IgG (H + L) for 1 hour. After washing the wells three times, bound HRP conjugated antibody was detected by adding 100 μ Ι of TMB substrate per well (biopx laboratories, MD, USA) and the plates were incubated for 5 minutes. The reaction was stopped by adding 100. mu.L of stop reagent (BioFX, Laboratories, MD, USA) per well and at A_630nmColor is detected to identify or confirm antibodies that bind to human or mouse CD83.

FACS binding assay

The antibodies produced were assayed for their ability to bind to human CD83 or mouse CD83 stably expressed on CHO cells. To generate expression vectors for the generation of stable cell lines, DNA fragments encoding either N-terminal HIS-tagged human CD83(hCD83) or N-terminal HIS-tagged mouse CD83(mCD83) were cloned into the neomycin-resistant plasmid pRKneo (Crowley et al, Proc Natl Acad Sci USA, 90(11):5021-5025,1993) at the XbaI and XhoI sites to generate hCD83.pRKneo or mCD83.pRKneo, respectively. CHO cells were transfected with hCD83.pRKneo or mCD83.pRKneo using Fugene (Roche) and 10% of CD83 positive cells prior to sorting by FACS, then selected with G418 (400. mu.g/ml; GIBCO) to generate stable CHO-hCD83 or CHO-mCD83 cell lines, respectively. CHO-hCD83 and CHO-mCD83 cells were resuspended in FACS buffer containing PBS/2% BSA/2mM EDTA and incubated with anti-human CD83 antibody or anti-mouse CD83 antibody on ice for 30 minutes. Cells were washed with FACS buffer and then incubated with anti-hamster IgG secondary antibody conjugated to a fluorescent dye for 30 minutes on ice in the dark. Cells were washed with FACS buffer and analyzed by flow cytometry on an LSR II flow cytometer using FACSDiva software (Becton Dickinson). Data analysis and graph construction were performed using FlowJo v.9.4.11.

Subcloning

Hybridoma clones producing antibodies capable of binding to mouse or human CD83 were subjected to at least 1 round of single cell subcloning for the production of chimeric antibodies containing wild-type mouse IgG2a constant regions and hamster variable regions. The antibody was then sequenced.

Results

Data analysis of the FACS binding assay identified 9 hybridoma clones that produced antibodies with the ability to bind CD83. anti-CD 83 antibodies 35G10, 40a11, 54D11, 59G10, 75a1, and 7C7 bound CHO cells expressing human CD83 (black line), but not cells expressing mouse CD83 (dashed line) or parental CHO cells lacking CD83 expression (filled histogram) (fig. 17A-F). anti-CD 83 antibody 60B10 bound CHO cells expressing human CD83, but also showed cross-reactivity with CHO cells expressing mouse CD83 (fig. 17G). anti-CD 83 antibodies 42C6 and 39a2 specifically bound CHO cells expressing mouse CD83 (dashed lines), but did not bind CHO cells expressing human CD83 (black lines) or the parental CHO cell line lacking CD83 expression (fig. 18A and B).

Sequencing of antibodies isolated from hybridoma clones yielded the following sequences (variable regions underlined and HVRs in bold):

39A2 anti-mouse CD83 heavy chain DNA sequence

CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTAGTGAAGCCCTCACAGTCAATGTCCCTCACTTGCTCT GTCAATGGTTTCTCCATCACCAGTCGTTACTGGTGGACCTGGATCAGGCAGTTCCCAGGGAAGAACCTGGAGTGGAT GGGTTACATAAGTTATAGTGGTGGCACCAGCTACAACCCCTCCCTCAAGAGCCGCATCTCCATCACCCGAGACACAT CCAAGAACCAGTTCTTCCTGCACCTGAACTCTGTGACCACTGCTGACACAGCCACATATTACTGTGCAAGAGATCTC TACGGTACCTACTTTGATTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTATCCACTGGCTCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGACCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTACGCGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAA(SEQ ID NO:4)

39A2 anti-mouse CD83 heavy chain amino acid sequence

QVQLKESGPGLVKPSQSMSLTCSVNGFSITSRYWWTWIRQFPGKNLEWMGYISYSGGTSYNPSLKSRIS ITRDTSKNQFFLHLNSVTTADTATYYCARDLYGTYFDYWGQGTLVTVSSASTKGPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQID NO:5)

39A2 anti-mouse CD83 heavy chain variable region amino acid sequence

QVQLKESGPGLVKPSQSMSLTCSVNGFSITSRYWWTWIRQFPGKNLEWMGYISYSGGTSYNPSLKSRIS ITRDTSKNQFFLHLNSVTTADTATYYCARDLYGTYFDYWGQGTLVTVSS(SEQ ID NO:6)

39A2HVR-H1 amino acid sequence

GFSITSRYWWT(SEQ ID NO:7)

39A2HVR-H2 amino acid sequence

GYISYSGGTSYNPSLKS(SEQ ID NO:8)

39A2HVR-H3 amino acid sequence

ARDLYGTYFDY(SEQ ID NO:9)

39A2 anti-mouse CD83 light chain DNA sequence

CAGTATGAGCTAATTCAGCCAAAGTCTGTGTCAGAGTCTCTAGGGAGAACAGTCACCATCTCCTGCAAA CGCAGCAGTGGCAACATTGGAAATAACTATGTACACTGGTACCAACAGCACTTTGGAAGCTCACCCAAAACTGTGAT CTATGATGACAATAAAAGACCATCTGGGGTTCCTCATAGGTTCTCTGGCTCCATTGACAGCTCCTCAAACTCAGCTT CCCTGACTATCACTGATCTGCAGATTGAAGATGAAGCTGAATACTACTGTCAATCTGCTTGGGTGTTCGGTTCAGGC ACCAAAGTGACTGTCCTACGCGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT(SEQ ID NO:10)

39A2 anti-mouse CD83 light chain amino acid sequence

QYELIQPKSVSESLGRTVTISCKRSSGNIGNNYVHWYQQHFGSSPKTVIYDDNKRPSGVPHRFSGSIDS SSNSASLTITDLQIEDEAEYYCQSAWVFGSGTKVTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ IDNO:11)

39A2 anti-mouse CD83 light chain variable region amino acid sequence

QYELIQPKSVSESLGRTVTISCKRSSGNIGNNYVHWYQQHFGSSPKTVIYDDNKRPSGVPHRFSGSIDS SSNSASLTITDLQIEDEAEYYCQSAWVFGSGTKVTVL(SEQ ID NO:12)

39A2HVR-L1 amino acid sequence

KRSSGNIGNNYVH(SEQ ID NO:13)

39A2HVR-L2 amino acid sequence

DDNKRPS(SEQ ID NO:14)

39A2HVR-L3 amino acid sequence

QSAWV(SEQ ID NO:15)

42C6 anti-mouse CD83 heavy chain DNA sequence

CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTAGTGAAGCCCTCACAGTCAATGTCCCTCACTTGCTCT GTCAATGGTTTCTCCATCACCAGTCGTTACTGGTGGACCTGGATCAGGCAGTTCCCAGGGAAGAACCTGGAGTGGAT GGGTTACATAAGTTATAGTGGTGGCACCAGCTACAACCCCTCCCTCAAGAGCCGCATCTCCATCACCCGAGACACAT CCAAGAACCAGTTCTTCCTGCACCTGAACTCTGTGACCACTGCTGACACAGCCACATATTACTGTGCAAGAGATCTC TACGGTACCTACTTTGATTACTGGGGCCAAGGAACCATGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTATCCACTGGCTCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGACCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTACGCGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAA(SEQ ID NO:16)

42C6 anti-mouse CD83 heavy chain amino acid sequence

QVQLKESGPGLVKPSQSMSLTCSVNGFSITSRYWWTWIRQFPGKNLEWMGYISYSGGTSYNPSLKSRIS ITRDTSKNQFFLHLNSVTTADTATYYCARDLYGTYFDYWGQGTMVTVSSASTKGPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQID NO:17)

42C6 anti-mouse CD83 heavy chain variable region amino acid sequence

QVQLKESGPGLVKPSQSMSLTCSVNGFSITSRYWWTWIRQFPGKNLEWMGYISYSGGTSYNPSLKSRIS ITRDTSKNQFFLHLNSVTTADTATYYCARDLYGTYFDYWGQGTMVTVSS(SEQ ID NO:18)

42C6HVR-H1 amino acid sequence

GFSITSRYWWT(SEQ ID NO:19)

42C6HVR-H2 amino acid sequence

GYISYSGGTSYNPSLKS(SEQ ID NO:20)

42C6HVR-H3 amino acid sequence

ARDLYGTYFDY(SEQ ID NO:21)

42C6 anti-mouse CD83 light chain DNA sequence

CAGTATGAGCTAATTCAGCCAAAGTCTGTGTCAGAGTCTCTAGGGAGAACAGTCACCATCTCCTGCAAA CGCAGCAGTGGCAACATTGGAAATAACTATGTACACTGGTACCAACAGCACTTTGGAAGCTCACCCAAAACTGTGAT CTATGATGACAATAAAAGACCATCTGGGGTTCCTCATAGGTTCTCTGGCTCCATTGACAGCTCCTCAAACTCAGCTT CCCTGACTATCACTGATCTGCAGATTGAAGATGAAGCTGAATACTACTGTCAATCTGCTTGGGTGTTCGGTTCAGGC ACCAAAGTGACTGTCCTACGCGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT(SEQ ID NO:22)

42C6 anti-mouse CD83 light chain amino acid sequence

QYELIQPKSVSESLGRTVTISCKRSSGNIGNNYVHWYQQHFGSSPKTVIYDDNKRPSGVPHRFSGSIDS SSNSASLTITDLQIEDEAEYYCQSAWVFGSGTKVTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQ IDNO:23)

42C6 anti-mouse CD83 light chain variable region amino acid sequence

QYELIQPKSVSESLGRTVTISCKRSSGNIGNNYVHWYQQHFGSSPKTVIYDDNKRPSGVPHRFSGSIDSSSNSASLTITDLQIEDEAEYYCQSAWVFGSGTKVTVL(SEQ ID NO:24)

42C6HVR-L1 amino acid sequence

KRSSGNIGNNYVH(SEQ ID NO:25)

42C6HVR-L2 amino acid sequence

DDNKRPS(SEQ ID NO:26)

42C6HVR-L3 amino acid sequence

QSAWV(SEQ ID NO:27)

60B10 antihuman CD83 heavy chain DNA sequence

CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTCGTGAAGCCCTCACAGTCACTGTCCCTCACTTGCTCT GTCACTGGTTTCTCCATCACCACCGGTGGTTACTGGTGGACCTGGATCAGGCAGTTCCCAGGGCAGAAGCTGGAGTG GATGGGGTACATATTTAGTAGTGGTAACACCAACTACAACCCATCCATCAAGAGCCGCATCTCCATAACCAGAGACA CATCCAAGAACCAGTTCTTCCTGCAGCTGAACTCTGTGACTACTGAGGGGGACACAGCCAGATATTATTGTGCAAGG GCCTACGGTAAGCTAGGCTTTGATTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTATCCACTGGCTCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGGTTATTTCCCTGAGCCAGTGACCTTGACCTGGAACTCTGGATCCCTGTCCAGTGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACCCTCAGCAGCTCAGTGACTGTAACCTCGAGCACCTGGCCCAGCCAGTCCATCACCTGCAATGTGGCCCACCCGGCAAGCAGCACCAAGGTGGACAAGAAAATTGAGCCCAGAGGACCCACAATCAAGCCCTGTCCTCCATGCAAATGCCCAGCACCTAACCTCTTGGGTGGACCATCCGTCTTCATCTTCCCTCCAAAGATCAAGGATGTACTCATGATCTCCCTGAGCCCCATAGTCACATGTGTGGTGGTGGATGTGAGCGAGGATGACCCAGATGTCCAGATCAGCTGGTTTGTGAACAACGTGGAAGTACACACAGCTCAGACACAAACCCATAGAGAGGATTACAACAGTACTCTACGCGTGGTCAGTGCCCTCCCCATCCAGCACCAGGACTGGATGAGTGGCAAGGAGTTCAAATGCAAGGTCAACAACAAAGACCTCCCAGCGCCCATCGAGAGAACCATCTCAAAACCCAAAGGGTCAGTAAGAGCTCCACAGGTATATGTCTTGCCTCCACCAGAAGAAGAGATGACTAAGAAACAGGTCACTCTGACCTGCATGGTCACAGACTTCATGCCTGAAGACATTTACGTGGAGTGGACCAACAACGGGAAAACAGAGCTAAACTACAAGAACACTGAACCAGTCCTGGACTCTGATGGTTCTTACTTCATGTACAGCAAGCTGAGAGTGGAAAAGAAGAACTGGGTGGAAAGAAATAGCTACTCCTGTTCAGTGGTCCACGAGGGTCTGCACAATCACCACACGACTAAGAGCTTCTCCCGGACTCCGGGTAAA(SEQ ID NO:28)

60B10 antihuman CD83 heavy chain amino acid sequence

QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRI SITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSSASTKGPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQID NO:29)

60B10 anti-human CD83 heavy chain variable region amino acid sequence

QVQLKESGPGLVKPSQSLSLTCSVTGFSITTGGYWWTWIRQFPGQKLEWMGYIFSSGNTNYNPSIKSRI SITRDTSKNQFFLQLNSVTTEGDTARYYCARAYGKLGFDYWGQGTLVTVSS(SEQ ID NO:30)

60B10HVR-H1 amino acid sequence

GFSITTGGYWWT(SEQ ID NO:31)

60B10HVR-H2 amino acid sequence

GYIFSSGNTNYNPSIKS(SEQ ID NO:32)

60B10HVR-H3 amino acid sequence

CARAYGKLGFDY(SEQ ID NO:33)

60B10 anti-human CD83 light chain DNA sequence

CAACCTGTGCTGACTCAGTCACCCTCTGCCTCTGCCTCCCTGGGAAACTCAGTCAAAATCACCTGTACC CTGAGTAGTCAGCACAGCACCTATACCATTGGTTGGTACCAGCAACATCCAGACAAGGCTCCTAAGTATGTGATGTA TGTTAATAGTGATGGAAGCCACAGCAAGGGGGATGGGATCCCTGATCGCTTCTCTGGCTCCAGCTCTGGGGCTCATC GTTACTTAAGCATCTCCAACATTCAGCCTGAAGATGAAGCTGACTATTTCTGTGGTTCTTCTGATAGCAGTGGGTAT GTTTTTGGCAGCGGAACCCAGCTCACCGTCCTACGCGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGT(SEQ ID NO:34)

60B10 antihuman CD83 light chain amino acid sequence

QPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGS SSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC(SEQID NO:35)

60B10 antihuman CD83 light chain variable region amino acid sequence

QPVLTQSPSASASLGNSVKITCTLSSQHSTYTIGWYQQHPDKAPKYVMYVNSDGSHSKGDGIPDRFSGS SSGAHRYLSISNIQPEDEADYFCGSSDSSGYVFGSGTQLTVL(SEQ ID NO:36)

60B10HVR-L1 amino acid sequence

TLSSQHSTYTIG(SEQ ID NO:37)

60B10HVR-L2 amino acid sequence

VNSDGSHSKGD(SEQ ID NO:38)

60B10HVR-L3 amino acid sequence

GSSDSSGYV(SEQ ID NO:39)

Example 11 anti-CD 83 antibodies decrease the release of proinflammatory cytokines from mDCs

Anti-inflammatory responses of Dendritic Cells (DCs) were monitored when treated with anti-CD 83 antibodies 60B10, 35G10, 40a11, 54D1, 59G10, 75a1, or 7C 7. For the assay, monocyte-derived dendritic cells were either left as Immature DCs (iDCs) or treated with a cytokine mixture containing 25ng/ml rhIL-1 β, 100ng/ml rhIL-6, 50ng/ml rhTNF α and 1 μ g/ml PGE-2 to produce mature DCs (mDCs). Mature DCs were treated with 10. mu.g/ml of the indicated anti-CD 83 antibody and incubated for 48 hours. After incubation, cell culture supernatants were collected and analyzed for secreted pro-inflammatory cytokines by elisa (invitrogen) according to standard manufacturer instructions. Analysis of secreted cytokine levels showed a significant decrease in the release of the pro-inflammatory cytokine MCP-1 in mDCs treated with anti-CD 83 antibodies 60B10, 35G10, 40a11, or 7C7 (fig. 19A). Treatment with anti-CD 83 antibodies 54D1, 59G10, or 75a1 did not significantly reduce MCP-1 production from mDCs (fig. 19A).

DCs were monitored when treated with anti-mouse CD83 antibody 39A2 or 42C6_sThe anti-inflammatory response of (a). For the assay, mouse bone marrow derived DCs (BMDCs) were either left as Immature DCs (iDCs) or matured with LPS. Mature DCs were treated with 39A2, 42C6 or soluble mouse CD83.Fc protein and subsequently harvested for RNA isolation to determine the expression of the pro-inflammatory cytokine IL-12p 40. Quantitative PCR analysis of IL-12p40RNA levels showed that treatment of DCs with anti-mouse CD83 antibody 39a2 or 42C6 along with LPS stimulators significantly reduced IL-12p40 expression to levels similar to those observed in DCs treated with soluble mouse cd83.fc protein (fig. 19B).

Example 12 use of anti-CD 83 antibodies to protect mice from DSS-induced colitis

To evaluate the effect of anti-CD 83 antibody treatment in a mouse model of inflammatory bowel disease, colitis was induced in mice by treatment with Dextran Sodium Sulfate (DSS). On days-1, 3 and 5 of the study, FVB mice aged 8-10 weeks were administered 200 μ g of anti-CD 83 antibody 39a2, 42C6 or 60B10, or control anti-gD antibody, by intraperitoneal injection once a day. Mice were given 6% DSS7 days in drinking water starting on study day 0. On day 7, mice were changed to normal drinking water until the experiment was stopped on day 12. Mice were subsequently euthanized and small and large intestine sections were sectioned and stained with H & E for histological analysis. The slices were randomized and scored double blindly. Colon sections of mice treated with DSS alone had an average histological score of 8.4 (fig. 20). In comparison, mice given anti-CD 83 antibody had significantly reduced histological scores. The use of the 39a2, 42C6, or 60B10 antibodies resulted in an average histological score of 5.3, 5.5, or 5.4, respectively.

Claims (24)

1. An isolated anti-CD 83 antibody comprising six hypervariable regions (HVRs) which are:

(a) HVR-H1 consisting of the amino acid sequence of SEQ ID NO. 31;

(b) HVR-H2 consisting of the amino acid sequence of SEQ ID NO. 32;

(c) HVR-H3 consisting of the amino acid sequence of SEQ ID NO. 33;

(d) HVR-L1 consisting of the amino acid sequence of SEQ ID NO 37;

(e) HVR-L2 consisting of the amino acid sequence of SEQ ID NO 38;

(f) HVR-L3 consisting of the amino acid sequence of SEQ ID NO: 39.

2. The antibody of claim 1, wherein the antibody is a chimeric antibody or a humanized antibody.

3. An isolated anti-CD 83 antibody comprising a heavy chain variable domain consisting of the amino acid sequence of SEQ ID NO. 30 and a light chain variable domain consisting of the amino acid sequence of SEQ ID NO. 36.

4. The antibody of claim 3, wherein the antibody is a chimeric antibody.

5. A pharmaceutical composition comprising the antibody of any one of claims 1-4 and a pharmaceutically acceptable carrier.

6. An isolated nucleic acid comprising a nucleotide sequence encoding the anti-CD 83 antibody of any one of claims 1-4.

7. A vector comprising the nucleic acid of claim 6.

8. The vector of claim 7, wherein said vector is an expression vector.

9. A host cell comprising the vector of claim 7 or 8.

10. The host cell of claim 9, wherein the host cell is prokaryotic or eukaryotic.

11. A method for making an anti-CD 83 antibody, the method comprising culturing the host cell of claim 9 under conditions suitable for expression of a nucleic acid encoding an anti-CD 83 antibody.

12. The method of claim 11, further comprising recovering the anti-CD 83 antibody produced by the host cell.

13. Use of an anti-CD 83 agonist antibody in the manufacture of a medicament for the treatment or prevention of an autoimmune disease, wherein the anti-CD 83 agonist antibody comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising (a) HVR-H1 consisting of the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 consisting of the amino acid sequence of SEQ ID NO. 32; and (c) HVR-H3 consisting of the amino acid sequence of SEQ ID NO. 33; the light chain variable region comprises (a) HVR-L1 consisting of the amino acid sequence of SEQ ID NO: 37; (b) HVR-L2 consisting of the amino acid sequence of SEQ ID NO 38; and (c) HVR-L3 consisting of the amino acid sequence of SEQ ID NO: 39.

14. Use of an anti-CD 83 agonist antibody in the manufacture of a medicament for the treatment or prevention of an autoimmune disease, wherein the antibody comprises a heavy chain variable domain consisting of the amino acid sequence of SEQ ID No. 30 and a light chain variable domain consisting of the amino acid sequence of SEQ ID No. 36.

15. The use of claim 13 or 14, wherein the anti-CD 83 agonist antibody is a monoclonal antibody.

16. The use of claim 13 or 14, wherein the anti-CD 83 agonist antibody is an antibody fragment selected from the group consisting of Fab, Fab '-SH, Fv, scFv, and (Fab') 2 fragments.

17. The use of claim 13 or 14, wherein the anti-CD 83 agonist antibody is a humanized or chimeric antibody.

18. The use of claim 13 or 14, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, juvenile rheumatoid arthritis, Systemic Lupus Erythematosus (SLE), lupus nephritis, ulcerative colitis, wegener's disease, inflammatory bowel disease, Idiopathic Thrombocytopenic Purpura (ITP), Thrombotic Thrombocytopenic Purpura (TTP), autoimmune thrombocytopenia, multiple sclerosis, psoriasis, IgA nephropathy, IgM polyneuropathy, myasthenia gravis, vasculitis, diabetes, raynaud's syndrome, sjogren's syndrome and glomerulonephritis.

19. The use of claim 13 or 14, wherein the autoimmune disease is associated with myeloid cell activation.

20. The use of claim 13 or 14, wherein the autoimmune disease is crohn's disease.

21. The use of claim 13 or 14, wherein the autoimmune disease is ulcerative colitis.

22. The use of claim 13 or 14, wherein the anti-CD 83 agonist antibody is a human antibody.

23. The use of claim 13 or 14, wherein the anti-CD 83 agonist antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

24. An article of manufacture comprising an anti-CD 83 agonist antibody as defined in any one of claims 13 to 22 and a package insert comprising instructions for using the anti-CD 83 agonist antibody to treat or prevent an autoimmune disease in an individual.