JP2013150592A5 - - Google Patents

Description

In some embodiments, the individual has or has been diagnosed with an autoimmune disease . In some embodiments, the individual has inflammatory bowel disease (such as Crohn's disease, ulcerative colitis, and indeterminate colitis), or inflammatory bowel disease (Crohn's disease, ulcerative colitis, and non- The patient is diagnosed as having confirmed colitis .

In some embodiments, the anti-CD83 agonist antibody is a monoclonal antibody. In some embodiments, the anti-CD83 agonist antibody is an antigen-binding fragment, eg, a fragment selected from the group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab ′) ₂ fragments. In some embodiments, the anti-CD83 agonist antibody is a humanized antibody. In some embodiments, the anti-CD83 agonist antibody is a human antibody. In some embodiments, the anti-CD83 agonist antibody is a chimeric antibody. In some embodiments, the anti-CD83 agonist antibody comprises HVR-H 1 comprising the amino acid sequence of SEQ ID NO: 31, HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and SEQ ID NO: 33. HVR-H3 comprising an amino acid sequence, a heavy chain variable domain, and / or HVR-L 1 comprising an amino acid sequence of SEQ ID NO: 37, HVR-L2 comprising an amino acid sequence of SEQ ID NO: 38, and It has a light chain variable domain comprising HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39. In some embodiments, the anti-CD83 agonist antibody has 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 is (a) HVR-H 1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 32; (c) the sequence ID NO: 33 comprises the amino acid sequence of the HVR-H3; (d) SEQ ID NO: 37 HVR-L 1 comprising the amino acid sequence of; (e) SEQ ID NO: 38 comprises the amino acid sequence of HVR-L2 (F) a variable domain comprising at least 1, 2, 3, 4, 5 or 6 hypervariable regions (HVRs) selected from the group consisting of HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39; An isolated anti-CD83 antibody comprising Also provided herein are the following six HVR sequences (a) HVR-H 1 comprising the amino acid sequence of SEQ ID NO: 31; (b) HVR- comprising the amino acid sequence of SEQ ID NO: 32 H2; (c) HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33; (d) HVR-L 1 comprising the amino acid sequence of SEQ ID NO: 37; (e) the amino acid sequence of SEQ ID NO: 38 An isolated anti-CD83 antibody comprising a heavy chain variable domain and a light chain variable domain comprising: HVR-L2 comprising; (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39. is there.

In some embodiments, the antibody comprises (a) HVR-H 1 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 comprises (a) HVR-L 1 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 ) It further has HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the antibody comprises (a) HVR-L 1 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.

What is also provided herein a method for creating anti-CD83 antibody, under conditions suitable for expression of the nucleic acid encoding the anti-CD83 antibodies described herein, include culturing a host cell as described herein Said method comprising: In some embodiments, the method further comprises recovering the anti-CD83 antibody produced by the host cell.

FIG. 1 shows that CD83 was involved in homotypic binding at the surface of cells expressing CD83. (A) CD83 expressed on the surface of stably transfected CHO-hCD83 cells was quantified by flow cytometry. (B) The expression of CD83 expressed on MUTZ-3-derived mature DC was quantified by flow cytometry. (C and d) CD83. fc bound to CHO-hCD83 and mature DC. 2 shows that CD83 is CD83. It was shown that it was necessary for the binding of fc. (A) CD83. fc bound to CHO-hCD83 cells, but binding was blocked by anti-CD83 antibody and quantified by flow cytometry. (B) Effect of siRNA specific for CD83 (siCD83) in MUTZ-3 mature DC as shown by taqman analysis of total RNA normalized to 18S. (C) CD83 was expressed on the surface of mature DC treated with non-targeted (siNTC) siRNA, but not on mature DC treated with siCD83. (D) CD83. fc bound to mDC, but binding was inhibited by siCD83 treatment. (E) Knockdown of CD83 reduced the surface expression of MHCII in mature DC. (F) CD83 knock down did not change a surface expression of CD86 in mature DC. FIG. 3 provides a microscopic image at 10 × and shows that CD83 expression resulted in cell aggregation in suspension culture. (A) Control CHO cells did not aggregate. (B) Cells expressing CD83 formed clusters in suspension culture. (C) CD83. Pretreatment of CHO-hCD83 cells with fc protein prevented aggregation. (D) Pretreatment of CHO-hCD83 cells with Ig control protein did not prevent aggregation. 4 shows CD83. FIG. 5 shows that CD83 treatment in mature DC with fc or HB15e antibody decreased the expression of surface activation markers CD83 and HLA-DR. ELISA data showed altered cytokine release in human monocyte-derived DCs treated with CD83. (A and c) Pro-inflammatory cytokines IL12-p40 and MCP-1 are CD83. Decreased by fc or HB15e antibody treatment. (B) The anti-inflammatory cytokine IL-1ra was significantly increased in CD83 treated DC. (D) IL-8 showed no difference between CD83 treated or control treated DCs. * Indicates significantly different values; * p <0.01, * p <0.001. The graph represents at least 4 different donors. ELISA data showed altered cytokine release in CD83-treated MUTZ-3-derived DCs. (A and c) Pro-inflammatory cytokines IL12-p40 and MCP-1 are CD83. Decreased by fc or HB15e antibody treatment. (B) The anti-inflammatory cytokine IL-1ra was significantly increased in CD83 treated DC. (D) IL-8 showed no difference between CD83 treated or control treated DCs. The supernatant from each well was run in triplicate. FIG. 7 shows CD83. CD83 treatment in DC with fc or HB15e antibody showed up-regulation of genes involved in wound healing and was provided by Taqman qPCR analysis of vcan, spike2 and fbn2 normalized to gapdh. Mean relative expression (2 ^ ΔCT ) + SEM. FIG. 8 shows that CD83 homotype interactions occur in trans and mediate anti-inflammatory responses in DC. (A and b) ELISA measurements of cytokine production showed that co-culture of CHO cells overexpressing CD83 with mDC inhibited the release of pro-inflammatory cytokines IL12-p40 and MCP-1. (C) Mature mouse bone marrow derived DC (BMDC) (gray bar) generated from CD83 knockout (CD83KO) and wild type (WT) littermates produced similar levels of IL-12p40 after 24 hours of LPS stimulation. . (D) Immature BMDC cultured with WT mature BMDC produced significantly less IL-12p40 than that cultured with CD83-deficient mature BMDC. p = 0.0372. FIG. 9 shows that CD83 homotype interaction mediated an anti-inflammatory response in DC. (A) The siRNA knockdown of CD83 is CD83. The response to the fc or HB15e antibody was abrogated and demonstrated by MCP-1 ELISA. (B) CD83 lentiviral expression on full length CD83 and cytoplasmic truncated CD83. (C) In the MCP-1 production ELISA, the expression of cytoplasmic truncated CD83 is CD83. It was shown that the inhibitory action of fc protein was blocked. FIG. 10 shows that antibody cross-linking was sufficient to trigger an anti-inflammatory response via the CD83 cytoplasmic domain in DC. (A) CD83 chimeric lentivirus expression construct, depicting the extracellular domain and full-length or truncated cytoplasmic domain of CD79a fused to the CD83 transmembrane region. (B) IL12-p40 production ELISA showed that overexpression of the truncated chimera inhibited the DC response to anti-CD79a antibody. FIG. 11 shows that CD83 has a class III PDZ ligand motif that mediates an anti-inflammatory response at the C-terminus. (A) The amino acid sequence comparison of the last 15 amino acids of the CD83 cytoplasmic domain showed a conserved class III PDZ ligand motif. (B) CD83 lentiviral expression construct of C-terminal PDZ ligand motif V205A mutant. (C) In the MCP-1 production ELISA, the expression of the V205A PDZ ligand motif mutant is CD83. It was shown that the inhibitory action of fc protein was blocked. FIG. 12 shows that the immunosuppressive effect of CD83 homotype interaction was mediated by p38 MAPK and mTOR signaling pathways. (Ac) showed that HB15e antibody treatment resulted in a significant decrease in phosphorylation of mTOR, p38 and CREB. (D) HB15e antibody treatment did not inhibit STAT3 phosphorylation activated by the TNF pathway. (E) Western blot analysis confirmed a decrease in p38 phosphorylation due to HB15e antibody treatment. (F) No significant difference was observed in the phosphorylation of STAT3 due to treatment with the HB15e antibody. All p38 and STAT3 were used as loading controls. FIG. 13 shows that CD83 overexpression in the colon resulted in downregulation of surface activation markers on DCs in the lamina propria. (A) Diagram of the gene transfer construct used to create CD83 transgenic mice. (B) Immunohistochemical staining showed transgene expression in colonic epithelium. (C and e) The 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 marker was observed in either 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 shows a FACS gate strategy for analyzing DC subsets isolated from CD83 transgenic mice, which showed that the CD83 transgene had no effect on DC subset production. (A and b) DC gating by MHCII + and CD11c hi expression. (C) Gating of T cells. (D and e) Flow cytometric analysis of DC subsets isolated from colon and spleen. CD83 overexpression protected mice from dextran sulfate sodium (DSS) -induced colitis. (A) shows less weight loss in CD83 transgenic mice compared to wild type mice when treated with DSS. (B) Hematoxylin and eosin staining of colon sections of mice with DSS-induced colitis. (C) Histology score of wild type and CD83 transgenic mice. (D) ELISA of serum cytokine levels in CDS transgenic mice treated with DSS compared to wild-type littermates. (E) qPCR of IL-12p40 gene expression in DCs in the lamina propria from CD83Tg mice treated with DSS compared to wild-type littermates. FIG. 16 shows that CD83 knockout in DC exacerbated colitis. (A) Diagram of the CD83 conditional knockout strategy. (B) FACS plot gated on TCRb + lymphocytes in the spleen. In the spleen, CD83 ^{fl / fl} CD11c-Cre mice had 48.6% CD4 positive T cells and CD83 ^{wt / wt} CD11c-Cre mice had 48.1% CD4 positive T cells. (C) Histogram representing relative expression of CD83 in spleen DC of CD83 ^{wt / wt} CD11c-Cre (black line) and CD83 ^{fl / fl} CD11c-Cre (gray line) mice. (D) DSS colitis survival in CD83 ^{fl / fl} CD11c-Cre and CD83 ^{wt / wt} CD11c-Cre mice. Conditional knockout animals had significantly reduced survival (log rank test, p = 0.0186). (E) Day 8 body weight was significantly lower in CD83 ^{fl / fl} CD11c-Cre mice compared to CD83 ^{wt / wt} CD11c-Cre littermates. (F) Development of obvious blood around the anus of the animal. 100% of CD83 ^{fl / fl} CD11c-Cre had overt occult blood by day 7. No obvious blood was observed in CD83 ^{wt / wt} CD11c-Cre littermates. Figure 17 shows that bound to the cells an anti-human CD83 antibody express CD83. Quantification by flow cytometry was performed using CHO cells in which anti-CD83 antibodies (a) 35G10, (b) 40A11, (c) 54AD1, (d) 59G10, (e) 75A1, and (f) 7C7 express human CD83 ( It specifically binds to the black line), but does not bind to cells expressing mouse CD83 (dashed line). (G) Anti-CD83 antibody 60B10 bound to CHO cells expressing human CD83 and also showed cross-reactivity with CHO cells expressing mouse CD83. No binding was seen in the parental CHO cell line (solid gray histogram). FIG. 18 shows that anti-mouse CD83 antibody bound to CHO cells expressing mouse CD83. Quantification by flow cytometry showed that anti-CD83 antibodies (a) 42C6 and (b) 39A2 specifically bound to CHO cells expressing mouse CD83 (dashed line) but not to the parental CHO cell line. (Solid gray histogram). FIG. 19 shows that anti-CD83 antibody significantly reduced the production of pro-inflammatory cytokines. (A) ELISA analysis showed that anti-CD83 antibody 60B10, 35G10, 40A11 or 7C7 significantly reduced production of MCP-1 from mDC compared to the use of isotype control antibody (ISO); * Indicates that the value is significantly different from the isotype control treatment. P = 0.0303, p = 0.0309, p = 0.0369, p = 0.0247, respectively. No significant differences were seen in cells given 54D1, 59G10, or 75A1 antibodies. (B) Quantitative PCR analysis of IL-12p40 expression in mouse bone marrow derived DC (BMDC) showed that anti-CD83 antibody 39A2 or 42C6 significantly reduced LPS-induced CD83 expression compared to the use of isotype control antibody It shows that. IL-12p40 expression was normalized to β-actin. Each symbol represents a cell from an independent mouse. *, P = 0.0372; †, p = 0.0438. FIG. 20 shows that anti-CD83 antibody protected mice from DSS-induced colitis. Mice given anti-CD83 antibody had a significantly reduced histological score compared to mice given anti-gD control antibody or DSS only: 39A2, mean = 5.3, ** P = 0.0011; 42C6, average = 5.5, † indicates p = 0.015; 60B10, average = 5.4, * indicates p = 0.0059.

As used herein, the term “preventing” includes providing prevention against the occurrence or recurrence of a disease in an individual. An individual may be susceptible to, 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 have a detectable disease or disease symptom or display detectable disease prior to the method of treatment described herein. Or it may have symptoms of the disease. “At risk” means that an individual has one or more risk factors that are measurable parameters known in the art that correlate with the occurrence of an autoimmune disease. An individual with one or more of these risk factors has a higher probability of developing the disease than an individual without one or more of these risk factors.

に由来する成熟アミノ酸配列を有する。 In some embodiments, the anti-CD83 antibody specifically binds to the extracellular region of human CD83. In some embodiments, human CD83 is

It has a mature amino acid sequence derived from

In some embodiments, the anti-CD83 antibody comprises (a) HVR-H 1 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-L 1 comprising the amino acid sequence of SEQ ID NO: 37; (e) comprising the amino acid sequence of SEQ ID NO: 38 And (f) at least 1, 2, 3, 4, 5 or 6 HVRs selected from HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the anti-CD83 antibody comprises (a) HVR-H 1 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-L 1 comprising the amino acid sequence of SEQ ID NO: 37; (e) comprising the amino acid sequence of SEQ ID NO: 38 HVR-L2; and (f) 6 HVRs comprising HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the anti-CD83 antibody comprises (a) HVR-H 1 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) having at least 1, at least 2, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33. In one embodiment, the antibody has HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33. In another embodiment, the antibody has 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 a further embodiment, 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 the amino acid sequence of SEQ ID NO: 32. HVR-H2 In a further embodiment, the antibody comprises: (a) HVR-H 1 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) It has HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the anti-CD83 antibody comprises (a) HVR-L 1 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) having at least 1, at least 2, or all three VL HVR sequences selected from HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39. In one embodiment, the antibody comprises (a) HVR-L 1 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) the sequence HVR-L3 comprising the amino acid sequence of number 39.

In some embodiments, the anti-CD83 antibody comprises (a) (i) HVR-H 1 comprising the amino acid sequence of SEQ ID NO: 31, and (ii) HVR- comprising the amino acid sequence of SEQ ID NO: 32. H2, and (iii) a VH domain comprising at least one, at least two, or all three VH HVR sequences selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33; and (b) (i HVR-L 1 comprising the amino acid sequence of SEQ ID NO: 37; (ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO: 38; and (iii) comprising the amino acid sequence of SEQ ID NO: 39. It has a VL domain comprising at least 1, at least 2, or all three VL HVR sequences selected from HVR-L3.

In some embodiments, the anti-CD83 antibody comprises (a) HVR-H 1 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-L 1 comprising the amino acid sequence of SEQ ID NO: 37; (e) comprising the amino acid sequence of SEQ ID NO: 38 HVR-L2; and (f) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

In some embodiments, the anti-CD83 antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence of SEQ ID NO: 30. Or an antibody having a heavy chain variable domain (VH) with 100% sequence identity. In some embodiments, a VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is replaced with a reference sequence ( An anti-CD83 antibody comprising this sequence, for example having conservative substitutions, insertions or deletions, retains the ability to bind CD83. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 30. In certain embodiments, substitutions, insertions, and / or deletions occur in regions outside HVR (ie, FR). In some cases, the anti-CD83 antibody has a VH sequence in SEQ ID NO: 30 and includes post-translational modifications of the sequence. In certain embodiments, VH comprises (a) HVR-H 1 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) It has 1, 2, or 3 HVRs selected from HVR-H3 comprising the amino acid sequence of SEQ ID NO: 33.

In some embodiments, the anti-CD83 antibody comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the amino acid sequence of SEQ ID NO: 36. Or an antibody having a light chain variable domain (VL) with 100% sequence identity. In certain embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is replaced with a reference sequence ( An anti-CD83 antibody comprising this sequence, for example having conservative substitutions, insertions or deletions, retains the ability to bind CD83. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted in SEQ ID NO: 36. In certain embodiments, substitutions, insertions, and / or deletions occur in regions outside HVR (ie, FR). In some cases, the anti-CD83 antibody has a VL sequence in SEQ ID NO: 36 and includes a post-translational modification of the sequence. In certain embodiments, the VL comprises (a) HVR-L 1 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) It has one, two or three HVRs selected from HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39.

Inducible promoters in yeast have the added effect that transcription is controlled by growth conditions. Exemplary inducible promoters include alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes that govern the use of maltose and galactose Of the promoter region. Vector and promoter suitably used for yeast expression are further described in EP 73,657. Yeast enhancers are also preferably used with yeast promoters.

(5) Enhancer element component Transcription of DNA encoding an antibody or fragment thereof by higher eukaryotes is often enhanced by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin). Typically, however, one skilled in the art will use an enhancer from a eukaryotic cell virus. Examples include the late SV40 enhancer (100-270 base pairs) of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer and the adenovirus enhancer late of the origin of replication. See also Yaniv, Nature, 297: 17-18 (1982) on enhancing elements for activation of eukaryotic promoters. Enhancers can be spliced into the vector at the 5 ′ or 3 ′ position of the antibody or antibody fragment coding sequence, but are preferably located at the 5 ′ position of the promoter.

(3) Humanized antibody The antibody (anti-CD83 agonist antibody and the like) or antibody fragment of the present invention further includes a humanized or human antibody. A humanized form of a non-human (eg, mouse) antibody is a chimeric immunoglobulin, immunoglobulin chain or fragment thereof (eg, Fab, Fab′-SH, Fv, scFv, F (ab ′) ₂ or other antigen binding of an antibody) Subsequence), which includes a minimal sequence derived from non-human immunoglobulin. A humanized antibody is a CDR of a non-human species (donor antibody) such as a mouse, rat, hamster, or rabbit in which the recipient complementarity determining region (CDR) residues have the desired specificity, affinity and ability. Human immunoglobulins (recipient antibodies) substituted by these residues. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, a humanized antibody comprises substantially all of at least one and typically two variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin. However, all or substantially all of the FR region is a human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op Struct. Biol., 2: 593-596 (1992).

Various techniques have been developed to produce antibody fragments. Traditionally, these fragments have been derived through proteolytic digestion of intact antibodies (e.g. Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science , 229: 81 (1985)). However, these fragments can now be possible you to produced directly by recombinant host cells, using nucleic acid encoding an antibody against CD83 discussed above, for example. Since Fab, Fv and scFv antibody fragments are all expressed and secreted in E. coli, it is easy to produce these fragments in large quantities. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab′-SH fragments can be recovered directly from E. coli and chemically combined to form F (ab ′) ₂ fragments (Carter et al., Bio / Technology 10: 163-167 (1992). )). In another approach, F (ab ′) ₂ fragments can be isolated directly from recombinant host cell culture. Production of Fab and F (ab ′) ₂ antibody fragments with increased in vivo half-life is described in US Pat. No. 5,869,046. In other embodiments, the selection antibody is a single chain Fv fragment (scFV). See WO 93/16185; US Pat. No. 5,571,894; and US Pat. No. 5,587,458. The antibody fragment may also be a “linear antibody” as described in, for example, US Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

In a different approach, antibody variable domains with the desired binding specificities (antigen-antibody combining sites) are fused to immunoglobulin constant domain sequences. The fusion is preferably a fusion with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. It is desirable to have a first heavy chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. Fusion of an immunoglobulin heavy chain, a DNA if it encodes an immunoglobulin light chain is desired, it is inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides great flexibility in adjusting the mutual proportions of the three polypeptide fragments in an embodiment where unequal proportions of the three polypeptide chains used in the assembly result in optimal yield. However, when expression at an equal ratio of at least two polypeptide chains yields a high yield, or when the ratio is not particularly important, the coding sequence for all two or three polypeptide chains Can be inserted into one expression vector.

Various techniques for making and isolating bivalent antibody fragments directly from recombinant cell culture have also been described. For example, divalent heterodimers have been produced using leucine zippers. Kostelny et al., J. Immunol. 148 (5): 1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab ′ portions of two different antibodies by gene fusion. Antibody homodimers are reduced at the hinge region to form monomers and then reoxidized to form antibody heterodimers. The “diabody” technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) provided an alternative mechanism for generating bispecific / bivalent antibody fragments. A fragment consists of a heavy chain variable domain (VH) joined to a light chain variable domain (VL) by a linker that is short enough to allow pairing between two domains on the same chain. Thus, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of the other fragment to form two antigen binding sites. Other strategies for producing bispecific / bivalent antibody fragments by using single chain Fv (sFv) dimers have also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

(9) Manipulation of effector function It may be desirable to modify the antibody of the present invention to alter the effector function and / or increase the serum half-life of the antibody. For example, Fc receptor binding site of the constant region, certain Fc receptors, Fc gamma RI, may be modified or mutated to remove or reduce binding affinity to Fc gamma RII, and / or Fc gamma RIII. In some embodiments, effector function is impaired by removing N-glycosylation of the Fc region of an antibody (eg, in the CH2 of IgG). In some embodiments, effector function is impaired by modifying regions such as 233-236, 297, and / or 327-331 of human IgG, and PCTWO 99/58572 and Armour et al., Molecular Immunology 40: 585- 593 (2003); Reddy et al., J. Immunology 164: 1925-1933 (2000).

(10) Other antibody modifications The antibodies or antibody fragments of the present invention can be further modified to have additional non-proteinaceous moieties known in the art and readily available. Preferably, the moiety suitable for derivatization of the antibody is a water soluble polymer. Non-limiting examples of water-soluble poly M a include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3 -Dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer), and dextran or poly (n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / Ethylene oxide copolymer, polyoxyethylene polyol (eg glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can be of any molecular weight and can 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 number and / or type of polymers used for derivatization can be determined based on considerations, including, but not limited to, the specific properties or functions of the antibody being improved, Including whether or not to be used in the treatment under defined conditions. Such techniques and many suitable formulations are disclosed in Remington: The Science and Practice of Pharmacy, 20th Ed., Alfonso Gennaro, Ed., Philadelphia College of Pharmacy and Science (2000).

Example 1. CD83 in order to determine whether soluble CD83 binds to CD83 expressed on the cell surface involved in homotypic binding at the cell surface, the amino acid sequence TPEVKVACSEDVDLPCTAPWDPQVPYTVSWVKLLEGGEERMETPQEDHLRGQHYHQKGQNGSFDAPNERPYSLKIRNTTSCNSGTYRCTLQDPDGQRNLSGKVILRVTGCPAQRKEETFKKYGRAQVTDKAAHYTLCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWENGNGPEPENYKTTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 3)
CD83. It produces fc and binds to either human CD83 stably expressed on CHO cells (CHO-hCD83) or endogenous CD83 expressed on MUTZ-3-derived mature dendritic cells (mDC) The ability was evaluated. To generate an expression vector for production of a stable CHO-hCD83 cell line, a DNA fragment encoding an N-terminal HIS-tagged human CD83 (hCD83) was placed at positions XbaI and XhoI at the neomycin resistance plasmid, pRKneo (Crowley et al., Proc Natl Acad Sci USA., 90 (11): 5021-5025, 1993) and cloned into hCD83. pRKneo was generated. CHO cells were washed with hCD83. Using Fugene (Roche). Transfected with pRKneo and top 10% CD83 positive cells were sorted by FACS and then selected using G418 (400 μg / ml; GIBCO) to generate a stable CHO-hCD83 cell line. To obtain immature DC (iDC) from MUTZ-3 cells, the cells were cultured in MEM α + glutamax / 20% heat inactivated FBS containing 150 ng / ml rhGM-CSF and 50 ng / ml rhIL-4. The DC, with a cytokine cocktail containing 25 ng / ml of rhIL-1b, 100ng / ml of rhIL-6,50ng / ml of rhTNF alpha and 1 mu g / ml of PGE-2, to the surface expression of CD83 And matured.

CD83. To confirm whether cell surface CD83 is required for fc binding, CHO-hCD83 cells were incubated with 1 μg / ml of anti-CD83 antibody (HB15e; Santa Cruz Biotechnology), which was CD83. It was determined whether to block fc binding. Data analysis was performed on CD83. Although fc binds to CHO-hCD83 cells (black line), it was shown that the binding is blocked by HB15e (dashed line) rather than isotype control (gray line) (FIG. 2A). CD83. The need for CD83 expression for fc binding was further assayed in MUTZ-3-derived DCs that did not express CD83. MUTZ-3 iDC were treated with Accel αRNA targeting CD83 after 4 days of culture in MEM α + glutamax / 20% heat inactivated FBS containing 150 ng / ml rhGM-CSF and 50 ng / ml rhIL-4. No. E-012680; Dharmacon) or non-targeting control (Cat. No. D-001910; Dharmacon). MUTZ-3 iDC containing 10 uM siRNA, supplemented with 3% heat inactivated FBS, 150 ng / ml GM-CSF and 50 ng / ml IL-4 (Cat. No. B- 005000; Dharmacon) and incubated at 37 ° C. with 5% CO ₂ for 72 hours. On day 7, DCs were treated with maturation stimuli to produce mDCs as described above. The effects of siRNA-mediated CD83 RNA and protein expression knockdown were assessed by taqman qPCR and Western blot (whole cell lysate), respectively. Analysis of total RNA from MUTZ-3 mDCs treated with non-targeting control (siNTC) siRNA or CD83-specific siRNA (siCD83) normalized to 18S shows that DCs transfected with CD83 siRNA showed CD83 RNA levels Was demonstrated to exhibit significant down-regulation (Fig. 2B). Knockdown of CD83 in MUTZ-3 mDC was confirmed by staining the cells with a fluorescent dye-conjugated antibody against CD83 and analyzing the cells by flow cytometry. Data analysis and the construction of a histogram showing total binding show that CD83 is expressed on the surface of mDCs treated with siNTC (black line), whereas iDCs treated with siCD83 (gray line) or mature DC (dashed line). It was shown not to be expressed (FIG. 2C). A solid histogram shows isotype control. After confirmation of knockdown of CD83 expression, iDC, siNTC mDC, and siCD83 mDC were treated with PE-labeled CD83. Fc or labeled IgG. Incubated with Fc control protein and subjected to flow cytometry. Data analysis was performed on CD83. fc demonstrates binding to siNTC mDC but not iDC or siCD83 mDC, CD83. It was shown that fc binding requires CD83 expression (FIG. 2D). CD83 knockdown also resulted in decreased expression of MHCII, but no other activity markers such as CD86 were altered (FIGS. 2E and F).

To determine if CD83 mediates cell-to-cell adhesion through homotypic binding, cell aggregation was assayed in CHO cells expressing hCD83. CHO cells and CHO-hCD83 cells were detached from the flask with 2 mM EDTA, washed and resuspended in HBSS containing 2% FBS / 10 mM EDTA but lacking Ca ²⁺ or Mg ²⁺ . Thereafter, the cells were resuspended at 10 ⁶ / ml and passed through a 70 μm filter to obtain a single cell suspension for plating on low adhesion 10 cm culture dishes. After 90 minutes incubation at 37 ° C. in an orbital platform shaker, cells were fixed with 4% PFA to assess cell aggregation. Microscopic imaging of the cells showed that control CHO cells lacking CD83 expression did not aggregate and CHO-hCD83 cells expressing hCD83 formed clusters in suspension culture (FIGS. 3A and B). 1 μg of CD83. Pretreatment of CHO-hCD83 cells with fc protein prevented aggregation but not with Ig control (FIGS. 3C and D). These results indicate that surface expression of CD83 is sufficient for cell-to-cell adhesion and this interaction can be blocked by addition of soluble CD83 due to competition for homotypic binding.

Example 2 Soluble CD83 treatment of DC results in an anti-inflammatory phenotype due to inhibition of DC maturation and pro-inflammatory cytokine release.
To characterize the immune response induced by DC by soluble CD83 treatment, the effect of CD83 treatment on mDC surface activation markers was evaluated. To obtain iDCs from MUTZ-3 cells, the cells were cultured for 6 days in MEM α + glutamax / 20% heat inactivated FBS containing 150 ng / ml rhGM-CSF and 50 ng / ml rhIL-4. iDC and, rhIL-1 beta in 25 ng / ml, and treated with a maturation stimulus cytokine cocktail containing rhIL-6,50ng / ml of rhTNF alpha and 1 mu g / ml of PGE-2 in 100 ng / ml. 10 μg / ml of CD83. fc, 1 μg / ml HB15e, or control IgG. Treatment of mDC with fc was performed simultaneously with maturation stimulation. The expression of cell surface activation markers, CD83 and HLA-DR (MHCII) in MUTZ-3-derived mDCs is examined by staining the cells with a fluorescent dye-conjugated antibody against CD83 or MHCII and analyzing the cells by flow cytometry did. In order to determine the level of non-specific staining, labeled isotype matched antibodies were used. Data analysis and histogram construction showing total binding showed that CD83 and MHCII were expressed in MUTZ-3-derived mDCs (black line) but at low levels in iDC (solid line histogram) (FIG. 4). The solid non-linear histogram represents the isotype control. The expression of both CD83 and MHCII is CD83. It is reduced with fc (gray line) or HB15e (dashed line) treatment, indicating that CD83 treatment reduces the expression of surface activation markers in mDC.

Since DCs are known to modulate immune responses through cytokine production, the effect of soluble CD83 treatment on DCs was assessed by detection of cytokine secretion from treated human monocyte-derived DCs (MDDCs). . CD83. To isolate and mature MDDC from whole donor whole blood. Stimulation with cytokines in the presence or absence of Fc or HBI5e. For isolation and processing, human whole blood was diluted with PBS, layered on Ficoll Paque (GE healthcare) and spun at 1500 rpm for 30 minutes. The leukocyte layer was removed and washed with PBS. Monocytes are isolated using Human monocyte isolation kit II (Miltenyi) and 6 in RPM / 10% FBS / 1Xpen / strep containing 125 ng / ml rhIL-4 and 50 ng / ml rhGM-CSF (R & D systems). Cultured for days. To obtain immature DC, the medium was changed every other day. 10 μg / ml CD83. All treatments with Fc or 1 μg / ml anti-CD83 antibody (HBI5e; Santa Cruz) were performed simultaneously with maturation stimulation. Cell culture supernatants were collected 48 hours after DC maturation and secreted cytokines were collected according to standard manufacturer's instructions for MCP-1, IL12-p40 and IL-8 (Invitrogen), and IL-1ra (Cell Sciences) was analyzed by ELISA using a kit. Analysis of cytokines secreted by DC was performed using CD83. Treatment with Fc alters secretion of DC cytokines and increases interleukin-1 receptor antagonist (IL-1ra) that binds to IL-1 receptor and blocks downstream inflammatory signaling (FIG. 5B) Reduced the production of subunit β of pro-inflammatory cytokine monocyte chemotactic protein-1 (MCP-1) and interleukin-12 (1L-12p40) (FIGS. 5A and C). CD83. Treatment with Fc or HBI5e had no effect on the production of the inflammatory cytokine IL-8 (FIG. 5D).

To further characterize DC-induced immune responses by soluble CD83 treatment, the effect of CD83 treatment on pro-inflammatory cytokine secretion by mDC 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 150 ng / ml rhGM-CSF and 50 ng / ml rhIL-4. DCs were matured for CD83 surface expression using a cytokine cocktail containing 25 ng / ml rhIL-1β, 100 ng / ml rhIL-6, 50 ng / ml rhTNFa and 1 μg / ml PGE-2. . 10 μg / ml of CD83. fc, 1 μg / ml HB15e, or control IgG. Treatment of mDC with fc was performed simultaneously with maturation stimulation. Cell culture supernatants were collected 48 hours after DC maturation and secreted cytokines were collected according to standard manufacturer's instructions for MCP-1, IL12-p40 and IL-8 kits (Invitrogen), and IL-1ra ( Analysis by ELISA using Cell Sciences). Analysis of cytokines secreted by DCs showed that the pro-inflammatory cytokines MCP-1 (FIG. 6A) and IL-12p40 (FIG. 6C) were CD83. It was shown that it was decreased in mDC by Fc or HBI5e treatment. In contrast, the anti-inflammatory cytokine IL-1ra was significantly increased in CD83 treated mDC (FIG. 6B). The level of released IL-8 showed no difference between CD83 treated or control treated mDCs (FIG. 6D). Supernatant from each well was performed in triplicate, * indicates significantly different values, * p <0.01, ** p <0.001. Each dot represents the average of an individual well. The graph is representative of at least 3 independent experiments.

Example 3 Soluble CD83 treatment results in upregulation of genes involved in wound healing.
CD83. To further characterize changes in gene expression induced in DCs treated with fc or HB15e, microarray analysis was performed on RNA isolated from DCs after treatment. Statistical analysis on microarrays was performed using software from R project (http://r-project.org) and Bioconductor project (http://bioconductor.org). Background subtracted microarray data was LOESS normalized within the array and quantile normalized between arrays. The normalized data was then log2 transformed and the probes were filtered using Bioconductor's “genefilter” package (eg, only probes that map to the Entrez gene are retained). Next, a non-specific filter that removes 50% minimal variable probe was used (Bourgon et al., Proc Natl Acad Sci USA., 107 (21): 9546-51, 2010). In order to identify differentially expressed genes, the lmma package was used (Smyth., Stat Appl Genet Mol Biol., 3: Article 3, 2004) to calculate the decay t-test. Linear models were tested for differential expression between immature and mature DCs and between CD83-linked samples (CD83fc- and HB15e-treated mature DCs) and control samples (IgG- and untreated mature DCs). The false discovery rate (FDR) was calculated using the Benjamini-Hochberg method. A gene was considered to be differentially expressed if it had an FDR of less than 0.01. Analysis of DCs from 5 different donors is CD83. We showed that fc and HB15e treated cells were separated and clustered independently from untreated mDCs and iDCs, indicating that the treatment up-regulated genes involved in wound healing. The microarray data was recorded on CD83. The isolated total RNA from mDC treated with fc or HB15e was confirmed by taqman qPCR analysis (FIG. 7). After normalization to gapdh, gene expression analysis showed that the genes vcan, spike2, and fbn2 involved in wound healing were upregulated. Average relative expression shown is 2 ^ delta CT + standard error of the mean (SEM). These in vitro results indicate that soluble CD83 treatment can promote normal cell proliferation and migration for the healing of tissue damage caused by inflammatory diseases.

Example 4 CD83 homotype interactions mediate anti-inflammatory responses.
To further characterize CD83 interactions that regulate anti-inflammatory responses, DCs were monitored for anti-inflammatory responses when co-cultured with CHO cells overexpressing hCD83. To obtain iDCs from MUTZ-3 cells, the cells were cultured for 6 days in MEM α + glutamax / 20% heat inactivated FBS containing 150 ng / ml rhGM-CSF and 50 ng / ml rhIL-4. The generated iDCs were co-cultured with a control CHO cell line or a CHO cell stably expressing human CD83. Thereafter, either untreated cultures of mixed cell, or 25 ng / ml of rhIL-1 β, 100ng / ml of rhIL-6,50ng / ml of rhTNF alpha and 1 mu g / ml cytokine containing PGE-2 in Treatment with cocktail produced mDC. Cell culture supernatants were collected 48 hours after DC maturation and secreted cytokines IL12-p40 and MCP-1 were analyzed by ELISA (Invitrogen) according to standard manufacturer's instructions. Analysis of secreted IL12-p40 and MCP-1 levels revealed that pro-inflammatory cytokine release was significantly reduced in mDC co-cultured with CHO cells expressing hCD83 compared to CHO cells lacking CD83 expression (FIGS. 8A and 8B). This data demonstrates that CD83 can participate in trans-homotype interactions and mediate anti-inflammatory responses. This data indicates that CD83 can participate in trans-homotypic interactions to mediate anti-inflammatory responses. These results were confirmed using human monocyte-derived DC (MDDC) isolated from whole blood from multiple donors. After stimulation, DCs cultured with CHO-hCD83 cells produced significantly less IL12-p40 than those cultured with CHO cells lacking CD83 expression.

In order to verify the anti-inflammatory response mediated by CD83 treatment requiring interaction with cell surface CD83, treatment was assayed in DCs that did not express CD83. MUTZ-3 iDC were treated with Accel αRNA targeting CD83 after 4 days of culture in MEM α + glutamax / 20% heat inactivated FBS containing 150 ng / ml rhGM-CSF and 50 ng / ml rhIL-4. No. E-012680; Dharmacon) or non-targeting control (Cat. No. D-001910; Dharmacon). MUTZ-3 iDC containing 10 uM siRNA, supplemented with 3% heat inactivated FBS, 150 ng / ml GM-CSF and 50 ng / ml IL-4 (Cat. No. B- 005000; Dharmacon) and incubated at 37 ° C. with 5% CO ₂ for 72 hours. Day 7, the iDC, 25 ng / ml of rhIL-1 beta, and treated with cytokine cocktail containing PGE-2 in 100 ng / ml of rhIL-6,50ng / ml of rhTNF alpha and 1 μ g / ml mDC Was generated. 10 μg / ml of CD83. fc, 1 μg / ml HB15e, or control IgG. Treatment of mDC with fc was performed simultaneously with maturation stimulation. Cell culture supernatants were collected 48 hours after DC maturation and secreted cytokines were analyzed by ELISA using the MCP-1 kit (Invitrogen) according to standard manufacturer's instructions. Analysis of MCP-1 released by DC showed that CD83 siRNA knockdown (siCD83) was CD83. It was shown that the reaction to the fc and HB15e antibodies was suppressed (FIG. 9). Therefore, this data indicates that CD83 is required on the cell surface to mediate the anti-inflammatory response by CD83 treatment. These results were confirmed by detection of secreted IL-12p40. Analysis of IL-12p40 released by mDC showed that CD83 siRNA knockdown (siCD83) was CD83. It was shown that the reaction to fc and HB15e antibodies was suppressed.

To determine if the anti-inflammatory response during CD83 treatment requires homotypic binding and downstream signaling via cell surface CD83, the treatment was evaluated in DCs expressing cytoplasmic truncated CD83 constructs. The CD83 lentiviral expression construct is pGCMV. IRES. By PCR amplification of segments of the full-length hCD83 gene (CD83FL) or cytoplasmic region (Δ172-205) cleaved hCD83 gene obtained from eGFP (pGIPZ derivative; Openbiosystems) and inserting these fragments into the XhoI / EcoRI cloning site Created (FIG. 9B). To generate lentivirus for DC infection, 293T cells were seeded at 1 × 10 ⁷ in gelatinized 10 cm culture dishes and allowed to grow for ˜20 hours until reaching 80-90% culture density. Medium supplemented with 5 ml DMEM, 10% FBS, 2 mM L-glutamine and expressed 5 μg at a molar ratio of 1: 2.3: 0.2 using Lipofectamine 2000 (Invitrogen) for 6 hours at 37 ° C. Cells were transfected with a DNA mix containing the plasmid, Delta 8.9 and VSVG. The transfection medium was supplemented with 6 ml normal growth medium and the cells were further incubated at 37 ° C. for 40 hours. The supernatant was collected and purified by filtration through a 0.45 μm tube top filter (Corning) and concentrated using a Lenti-X-concentrator (Clonetech) according to the manufacturer's instructions. MUTZ-3 cells were seeded at 0.5 × 10 ⁶ / ml in 24-well culture plates in maintenance medium containing MEM α + Glutamax / 20% heat inactivated FBS / 15% HTB-9 conditioned medium. Polybrene and concentrated lentiviral supernatants were added to the cells at a final concentration of 4 μg / ml and an MOI of 10, respectively. The cells were spun at 1800 rpm in an Allegra X-12R tabletop centrifuge for 30 minutes at room temperature and then incubated overnight at 37 ° C. The medium containing lentivirus was removed and replaced with fresh maintenance medium. After 2-3 days of culture, the top 10% GFP positive cells were sorted and in MEM α + Glutamax / 20% heat inactivated FBS supplemented with 150 ng / ml rhGM-CSF and 50 ng / ml rhIL-4. It was cultured for 6 days and used as iDC. Cells were stimulated for 48 hours, maturation stimulation, as well as CD83. Treated with Fc, HB15e or isotype control. The supernatant was then collected and MCP-1 release was analyzed by ELISA. Analysis of secreted MCP-1 shows that lentiviral overexpression of full-length CD83 is CD83. Although it did not inhibit the mDC response to fc or HB15e, the expression of cytoplasmic truncated CD83 showed that the anti-inflammatory effect of CD83 treatment was blocked (FIG. 9C). These results were confirmed by detection of secreted IL-12p40. Analysis of secreted IL-12p40 shows that lentiviral overexpression of full-length CD83 is CD83. Although it did not inhibit the mDC response to fc or HB15e, it was shown that expression of cytoplasmic truncated CD83 blocked the anti-inflammatory effect of CD83 treatment.

To determine if the immunosuppressive effect of the CD83 homotype interaction is mediated by additional signaling pathways, CD83. Phosphokinase assays were performed on cell lysates from mDCs treated with fc or HB15e for 5 minutes. Human phosphokinase assay (R & D systems) was performed according to the manufacturer's instructions. Briefly, MUTZ-3 DC was washed with cold PBS, solubilized in Lysis Buffer 6 and rocked at 4 ° C. for 30 minutes. The lysate was centrifuged at 14,000 × g for 5 minutes and the supernatant was transferred to a new tube for analysis of total protein by Bradford (Bio-Rad). Array membranes with 46 antibodies spotted in duplicate were blocked for 1 hour at room temperature and then incubated with diluted cell lysate overnight at 4 ° C. 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). Membranes were exposed to FUJI FILM Image Reader LAS-3000 and 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 the phosphorylation of STAT3 activated by TNF receptor binding of TNF alpha is a component of maturation stimulus (Figure 12D). CD83. Western blot analysis of whole cell lysates from human monocyte-derived DCs (MDDC) treated with Fc or αCD83 (HB15e) confirmed a decrease in phosphorylated p38 MAPK (FIG. 12E) but had a significant effect on STAT3 phosphorylation Was not shown (FIG. 12F). These novel findings indicate that the anti-inflammatory effect of CD83 homotype interaction was mediated by p38 MAPK as well as mTOR protein signaling.

Binding of cell surface CD83 by agonist anti-CD83 antibody:
In order to identify antibodies that bind to cell surface CD83 on dendritic cells, the binding affinity of the antibodies produced was screened by flow cytometric analysis. Briefly, immature dendritic cells (iDC) were treated with a cytokine cocktail to induce DC maturation and surface expression of CD83. Prior to fixation and flow cytometry, mDCs were treated with antibodies generated simultaneously with DC maturation stimuli. Antibodies that specifically bind to CD83 expressed on the surface of mDCs were identified by analyzing flow cytometry data. Alternatively, the produced anti-CD83 antibody was screened using a cell aggregation assay. Briefly, CHO cells expressing hCD83 (CHO-hCD83) are detached from the flask using 2 mM EDTA, washed, and washed with HBSS medium containing 2% FBS / 10 mM EDTA but lacking Ca ²⁺ or Mg ^2+. Resuspended. The cells were then resuspended at 10 ⁶ / ml and passed through a 70 μm filter to obtain a single cell suspension for plating on low adhesion 10 cm culture dishes. CHO-hCD83 cells were then treated with the generated antibody and incubated on an orbital platform shaker at 37 ° C. for 90 minutes prior to fixation with 4% PFA. Antibodies that block CHO-hCD83 cell aggregation by competition for CD83 homotype binding were identified by microscopic imaging of the cells. These antibodies may be further characterized for their binding to human CD83 and agonist activity.

Example 11 Anti-CD83 antibodies reduce the release of pro-inflammatory cytokines from mDC.
Dendritic cells (DC) were monitored for anti-inflammatory response when treated with anti-CD83 antibodies 60B10, 35G10, 40A11, 54D1, 59G10, 75AI, or 7C7. Assays leave monocyte-derived dendritic cells as immature DC (iDC), or 25 ng / ml rhIL-1β, 100 ng / ml rhIL-6, 50 ng / ml rhTNF α and 1 μg / ml. Treatment with a cytokine cocktail containing PGE-2 produced mature DC (mDC). Mature DCs were treated with the indicated anti-CD83 antibody at 10 μg / ml and incubated for 48 hours. Following incubation, cell culture supernatants were collected and secreted pro-inflammatory cytokines were analyzed by ELISA (Invitrogen) according to standard manufacturer's instructions. Analysis of secreted cytokine levels showed that the release of pro-inflammatory cytokine MCP-1 was significantly reduced in mDCs treated with anti-CD83 antibodies 60B10, 35G10, 40A11, or 7C7 (FIG. 19). Treatment with anti-CD83 antibody 54D1, 59G10, or 75AI did not significantly reduce MCP-1 production from mDC (FIG. 19A).

Claims (38)

A medicament for treating or preventing an autoimmune disease in an individual, medicaments comprising effective amount of an anti-CD83 agonist antibodies. 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 multiple neuropathy, myasthenia gravis, vasculitis, diabetes, Raynaud's syndrome, Sjogren's syndrome and The medicament according to claim 1, selected from the group consisting of glomerulonephritis. The medicament according to claim 1, wherein the individual has an autoimmune disease associated with activation of myeloid cells. The medicament according to claim 3, wherein the individual has Crohn's disease. The medicament according to claim 3, wherein the individual has ulcerative colitis. The medicine according to any one of claims 1 to 5, wherein the individual is a human. The medicament according to any one of claims 1 to 5, wherein the anti-CD83 agonist antibody inhibits the release of pro-inflammatory cytokines from mature dendritic cells. The medicament according to claim 7, wherein the release of pro-inflammatory cytokines MCP-1 and / or IL-12p40 is inhibited. The medicament according to any one of claims 1 to 5, wherein the anti-CD83 agonist antibody induces the release of anti-inflammatory cytokines from mature dendritic cells. 10. The medicament according to claim 9, wherein release of the anti-inflammatory cytokine IL-1ra is induced. The medicament according to any one of claims 1 to 5, wherein the anti-CD83 agonist antibody induces a decrease in cell surface expression of CD83 and / or HLA-DR on mature dendritic cells. The medicament according to any one of claims 1 to 5, wherein the anti-CD83 agonist antibody inhibits activation of MAPK and / or mTOR signaling in mature dendritic cells. 13. The medicament according to claim 12, wherein inhibition of activation of MAPK signaling is measured by a decrease in phosphorylation of p38 and CREB proteins in mature dendritic cells. The medicament according to claim 12, wherein inhibition of activation of mTOR signaling is measured by a decrease in phosphorylation of mTOR protein in mature dendritic cells. The medicament according to any one of claims 1 to 14, wherein the anti-CD83 agonist antibody is a monoclonal antibody. 16. The medicament of claim 15, wherein the anti-CD83 agonist antibody is an antibody fragment selected from the group consisting of Fab, Fab′-SH, Fv, scFv, and (Fab ′) ₂ fragments. The medicament according to any one of claims 1 to 14, wherein the anti-CD83 agonist antibody is a humanized antibody or a chimeric antibody. The anti-CD83 agonist 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) SEQ ID NO: 33. A heavy chain variable domain comprising HVR-H3 comprising the amino acid sequence of: and (a) HVR-L1 comprising the amino acid sequence of SEQ ID NO: 37, (b) comprising the amino acid sequence of SEQ ID NO: 38 18. The medicament according to any one of claims 1 to 17, which comprises a light chain variable domain comprising HVR-L2 comprising: and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO: 39. Antibody, SEQ ID NO: 30 the heavy chain variable domain comprising the amino acid sequence of, and / or SEQ ID NO: 36 any one of claims 1-17 comprising comprising at light chain variable domain amino acid sequence of The pharmaceutical described. The medicament according to any one of claims 1 to 14, wherein the anti-CD83 agonist antibody is a human antibody. The anti-CD83 agonist antibody is intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, brain The medicament according to any one of claims 1 to 20, which is administered indoors or intranasally.   An article of manufacture comprising an anti-CD83 agonist antibody and a package insert comprising instructions regarding the use of the anti-CD83 agonist antibody to treat or prevent an autoimmune disease in an individual. (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
An isolated anti-CD83 antibody comprising a variable domain comprising at least 1, 2, 3, 4, 5 or 6 hypervariable region (HVR) sequences selected from the group consisting of: 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
An isolated anti-CD83 antibody comprising a heavy chain variable domain and a light chain variable domain comprising The antibody is (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) the amino acid sequence of SEQ ID NO: 33. 25. The antibody of claim 23 or 24 comprising HVR-H3 comprising   The antibody is (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) the amino acid sequence of SEQ ID NO: 39. 26. The antibody of claim 25, further comprising HVR-L3 comprising The antibody is (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) the amino acid sequence of SEQ ID NO: 39. 25. The antibody of claim 23 or 24 comprising HVR-L3 comprising   28. The antibody according to any one of claims 23 to 27, wherein the antibody is a chimeric antibody or a humanized antibody.   An isolated anti-CD83 antibody comprising 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. 30. The antibody of claim 29 , wherein the antibody is a chimeric antibody.   A pharmaceutical composition comprising the antibody of any one of claims 23-30 and a pharmaceutically acceptable carrier.   31. An isolated nucleic acid comprising a nucleotide sequence encoding the anti-CD83 antibody of any one of claims 23-30.   A vector comprising the nucleic acid of claim 32.   34. The vector of claim 33, wherein the vector is an expression vector.   A host cell comprising the vector of claim 33 or 34. 36. The host cell according to claim 35 , wherein the host cell is a prokaryotic cell or a eukaryotic cell.   36. A method of making an anti-CD83 antibody, comprising culturing the host cell of claim 35 under conditions suitable for expression of a nucleic acid encoding the anti-CD83 antibody.   38. The method of claim 37, further comprising recovering the anti-CD83 antibody produced by the host cell.