ALBERT

Description: Each person’s genome sequence has thousands of missense variants. Practical interpretation of their functional significance must rely on computational inferences in the absence of exhaustive experimental measurements. Here we analyzed the efficacy of these inferences in 33 de novo missense mutations revealed by sequencing in first-generation progeny of N-ethyl-N-nitrosourea–treated mice, involving 23 essential immune system genes. PolyPhen2, SIFT, MutationAssessor, Panther, CADD, and Condel were used to predict each mutation’s functional importance, whereas the actual effect was measured by breeding and testing homozygotes for the expected in vivo loss-of-function phenotype. Only 20% of mutations predicted to be deleterious by PolyPhen2 (and 15% by CADD) showed a discernible phenotype in individual homozygotes. Half of all possible missense mutations in the same 23 immune genes were predicted to be deleterious, and most of these appear to become subject to purifying selection because few persist between separate mouse substrains, rodents, or primates. Because defects in immune genes could be phenotypically masked in vivo by compensation and environment, we compared inferences by the same tools with the in vitro phenotype of all 2,314 possible missense variants in TP53; 42% of mutations predicted by PolyPhen2 to be deleterious (and 45% by CADD) had little measurable consequence for TP53-promoted transcription. We conclude that for de novo or low-frequency missense mutations found by genome sequencing, half those inferred as deleterious correspond to nearly neutral mutations that have little impact on the clinical phenotype of individual cases but will nevertheless become subject to purifying selection.

Description: 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.