Introduction: Blockade of the CD40/CD154 pathway is highly effective in preventing rejection in pig-to-nonhuman primate (NHP) xenotransplantation models. One of the most widely used immunosuppressants in preclinical xenotransplantation is anti-CD40 mAb (clone 2C10R4), developed using rhesus CD40 as the immunogen. In our experience, treatment with 2C10R4 was not as effective at prolonging kidney xenograft survival compared to a similar anti-CD154 based regimen, in part possibly due to species-specific differences between rhesus and porcine CD40; whereas anti-CD154 would target recipient NHP T cells, anti-CD40 might be important for CD40 expression not only on recipient NHP antigen presenting cells (APCs), but also on porcine donor APCs cells in a more direct manner. Accordingly, anti-CD40 antibodies which have different binding affinity to porcine CD40 and NHP CD40 may have increased effectiveness. The aim of this study was to compare the efficacy of a variant anti-CD40 antibody with different CD40 selectivity (vCD40) to 2C10R4 in preventing rejection in a pig-to-NHP model of renal xenotransplantation.

Methods: Noncompetitive binding assays were performed in which labeled 2C10R4 and vCD40 were incubated with porcine or NHP peripheral blood mononuclear cells (PBMCs). Pig-to-NHP renal xenotransplants were then performed from GGTA1 KO/CD55 transgenic donor pigs to rhesus macaques (n=12) with low pre-transplant xenoreactive antibody titers. Recipients underwent T cell depletion followed by maintenance treatment with either: anti-CD40 (clone 2C10R4) alone or variant anti-CD40 antibody in combination with another complement system inhibitor; plus mycophenolic acid and steroids.

Results: In vitro assays indicated that 2C10R4 demonstrated poor binding efficacy to porcine PBMCs, while vCD40 demonstrated superior binding to both porcine and NHP PBMCs (Figure 1). Recipients treated with vCD40 in combination with the additional complement system inhibitor showed prolonged survival (n=6, MST 243 days with a maximum survival of >430 days) compared with recipients treated with 2C10R4 alone (n=6, MST 6.5 days) (Figure 2). Three animals in the vCD40 group demonstrated prolonged graft survival >300 days, with two surviving >400 days. Recipients in the 2C10R4 group showed a significant number of early graft failures, with 2/3 of recipients failing within 1 week.

Conclusions: Our findings suggest that the reduced xenograft survival seen with the 2C10R4-based regimen may be due to the failure of 2C10R4 to adequately block porcine CD40. In contrast, the superior binding efficacy of vCD40, an anti-CD40 mAb that binds both porcine and NHP CD40, is associated with prolonged xenograft survival. This mechanistic explanation for our findings provides further rationale for continued development of drugs that manipulate this pathway for eventual translation to human xenotransplant clinical trials and the importance of assessing activity to both human/NHP as well as pig proteins.