Simultaneous Targeting of Immunoglobulin G and M Using Endopeptidase for Humoral Rejection in Xenotransplantation

Abstract The xenotransplantation field has achieved commendable success through the utilization of the non-human primate (NHP) model. Studies to date serve as a proof-of-concept, demonstrating that pig-to-NHP or human transplantation can be achieved without hyperacute rejection and providing insights regarding the human immune response to a xenograft. Xenotransplantation survival in nonhuman primate recipients has markedly improved in the past decade due to 1) the use of genetically modified pig donors, and 2) the treatment of recipients with costimulation blockade, in particular targeting CD40-CD154 signaling pathway. Recently, a pig- to-NHP xenokidney transplantation model employing a TKO pig with additional human proteins under anti- CD154mAb immunosuppression showed the longest graft survival close to 2 years. However, it is imperative to not overlook the outcomes of animals that did not attain long-term graft survival. In this study, 40% of NHP recipients (6 out of 15 animals) did not survive beyond 2 months. The consistent occurrence of early xenograft failures in virtually all NHP xenotransplantation studies is a matter of concern. Considering the two cases of clinical cardiac xenotransplantation outcomes (patient survival of 61 and 40 days) are coincide with the early failure shown in NHP model, clear mechanistic explanation and intervention for this phenomenon is urgently required prior to clinical xenokidney transplantation. Interestingly, we and others observed a clear antibody- mediated rejection (AMR) in early xenograft rejection with high levels of preformed and de novo xenoreactive IgG/IgM. Removal of preformed IgG antibodies with cleaving enzyme such as IdeS (Imlifidase) has shown therapeutic promise in organ transplantation for sensitized recipients. However, strategies to selectively remove IgM, which precedes IgG in triggering immune dysfunction are lacking. Especially, preformed and de novo polyreactive IgM antibodies against porcine glycans were considered detrimental in pig-to-human and pig-to-NHP xenotransplantation. Therefore, in the present study, our focus is on targeting both xenoreactive IgG and IgM using novel endopeptidase(s), IceMG, to eliminate early antibody-mediated injuries. We hypothesize that mitigating the early injuries caused by preformed and/or de novo xenoreactive IgG/IgM prolongs xenograft survival by preventing AMR. This study will validate the role of xenoreactive IgG and IgM in early xenograft rejection, identify potential xenograft injuries independent of AMR, and assess the efficacy of the novel endopeptidase as a therapeutic option for xenotransplantation.