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Introduction: CD83 is an important marker of activated dendritic cells (DC) but it is also expressed on other immune cells. Polyclonal anti-CD83 antibody depletes activated DC and prevents human peripheral blood mononuclear cell (PBMC) induced xenogeneic graft versus host disease (GVHD) in immunosuppressed SCID mice (J Exp Med 2009;206;387). We therefore generated a potential therapeutic human anti-CD83 mAb (3C12C), which had similar efficacy and T cell sparing effects in the same model (Leukemia 2015; in press). To investigate the specific immunosuppressive effect of 3C12C further, we undertook a comprehensive analysis of CD83 expression and its glycosylation pattern on various immune cell populations and tested the effect of 3C12C on T cell function using preclinical models, including a human CD83 (hCD83) knock in (KI) mouse. Methods: A panel of mouse and recombinant mAbs to hCD83 were used to analyse its expression by flow cytometry on resting and activated healthy donor PBMC. The expression of potential CD83 splice variants was examined by PCR. T cell expression was examined by flow cytometry and confocal microscopy after PHA, CD3/CD28 beads and allogeneic mixed leukocyte reaction (alloMLR) culture. Control human IgG1 (trastuzumab) and 3C12C mAbs were tested (0.125mg d-1) in a xenogeneic model of GVHD utilizing human PBMC transplanted into total body irradiation and anti-NK conditioned SCID mice. The genetically engineered hCD83 KI mouse was shown to be immune-competent and used to test the effect of 3C12C on LPS activated DC and T cells. Results: There were distinct CD83 splice variants (full length CD83, splicing variant CD83a, CD83b and CD83c) in different immune cells. CD83 glycosylation status also differed with high glycosylation required for surface expression on activated DC, whereas its expression on activated B cells and monocytes was resistant to de-glycosylation. Increases in CD83 expression on T cells occurred early with different kinetics, underlining the distinct signal pathway involved. The 3C12C mAb reduced T cell proliferation in the alloMLR but did not affect cytomegalovirus (CMV) or influenza (Flu) specific CD8+T cell numbers. Treatment with 3C12C prevented GVHD in human PBMC transplanted SCID mice, which otherwise developed histological GVHD between d8-13. Human DC were activated by d2 and expressed the CMRF-44 activation marker plus CD83, CD80 and CD86. Treatment with 3C12C mAb eliminated CD83+ CMRF44+ DC early post-transplant and reduced T cell activation. Further studies, established CMV and Flu specific T cells were retained and responded to antigen by IFNg production. Furthermore, Treg numbers were preserved. The 3C12C mAb depleted LPS activated DC in hCD83 KI mice in experiments performed prior to commencing transplant studies. Conclusion: These findings suggest that the potential therapeutic human anti-CD83 mAb induced significant immune suppression, by depletion of activated DC and consequential modulation of T cell activation. The reduction in allo/xeno activated T cells may result in part from a direct effect of anti-CD83 on early T cell responses. This apparently selective immunosuppressive effect preserves anti-viral T cell immunity and Treg pathways, suggesting that 3C12C merits further investigation as a novel agent for GVHD prophylaxis. Disclosures Hart: DendroCyte BioTech Pty Ltd: Equity Ownership. Clark:DendroCyte BioTech Pty Ltd: Equity Ownership.

Abstract 3554 Poster Board III-491 Introduction Host and donor dendritic cells (DC) stimulate alloreactive donor T lymphocytes, and initiate GVHD. We have shown that polyclonal antibody to the DC surface activation marker human CD83 (anti hCD83), which depletes activated DC, can prevent human DC and T cell induced lethal xenogeneic GVHD in SCID mice without impairing T cell mediated anti-leukaemic and anti-viral (CMV and influenza) immunity (J Exp Med 2009; 206: 387). Therefore, we made and tested a polyclonal anti mouse CD83 (RAM83) antibody in murine HSCT models and developed a human mAb against hCD83 as a potential new therapeutic immunosuppressive agent. Methods RAM83 specificity and function was tested by flow cytometry and allogeneic MLRs. It was administered as a single dose on day -1 of murine syngeneic and allo HSCT. Human and mouse single chain variable fragment (scFv) phage libraries were panned on recombinant human CD83 extracellular domain. After screening for specificity and affinity, the clones were reformatted to human IgG1 and expressed by transfected CHO cells. The purified mAb were tested for their ability to block a mixed leucocyte reaction (MLR). Results Lin-,Sca1+,Kit+ murine stem or progenitor cells were CD83-ve. RAM83 treated mice receiving syngeneic HSCT survived >35days (normal day 14 WBC). Full MHC mismatched (2× 60mg/kg cyclophosphamide + 2× 500cGy conditioned), B6 [H-2b] into BALB/c [H-2d]) recipient mice treated with a single dose of RAM83 developed GVHD later and survived longer (mean 26.9 days ±3.3) compared to controls (RAneg 12.3±1.3days, p=0.001; Nil antibody 7.7±1.5 days, p=0.0008). Daily high dose (50mg/kg) cyclosporin A delayed GVHD by a mean 18.2±3.3 days. Four phage clones that bind to human CD83 have been reformatted. Of the two tested to date, one mouse/human chimeric mAb has lost binding affinity, probably as a result of immunoglobulin variable region glycosylation. The other reformatted fully human anti-CD83 (3C12) mAb blocked a MLR, by an ADCC mechanism. Conclusions mAb targeting of activated DC is a promising novel approach to immunosuppression. The 3C12 clone and others will be subjected to preclinical testing in the xenogeneic SCID mouse model. The human mAb, which prevents GVHD without impairing the desired graft versus leukaemia effect will be developed for phase 1 clinical trials in clinical alloHSCT. Disclosures: Hart: CRC-BT: Consultancy.