Preventing xenograft rejection is one of the greatest challenges of transplantation medicine. Here, we describe a reproducible, long-term survival of cardiac xenografts from alpha 1-3 galactosyltransferase gene knockout pigs, which express human complement regulatory protein CD46 and human thrombomodulin (GTKO.hCD46.hTBM), that were transplanted into baboons. Our immunomodulatory drug regimen includes induction with anti-thymocyte globulin and αCD20 antibody, followed by maintenance with mycophenolate mofetil and an intensively dosed αCD40 (2C10R4) antibody. Median (298 days) and longest (945 days) graft survival in five consecutive recipients using this regimen is significantly prolonged over our recently established survival benchmarks (180 and 500 days, respectively). Remarkably, the reduction of αCD40 antibody dose on day 100 or after 1 year resulted in recrudescence of anti-pig antibody and graft failure. In conclusion, genetic modifications (GTKO.hCD46.hTBM) combined with the treatment regimen tested here consistently prevent humoral rejection and systemic coagulation pathway dysregulation, sustaining long-term cardiac xenograft survival beyond 900 days.

Xenotransplantation of genetically modified pig organs offers great potential to address the shortage of human organs for allotransplantation. Persistence of hyperacute rejection in Gal KO pigs due to elicited non Gal antibody response required further genetic modifications of donor pigs and better control of the B cell response to xeno antigens. We report significant prolongation of graft survival of 8 months when peri-transplant B-cell depletion was added to an established anti CD154 and MMF based immunosuppressive regimen. Specifically Galactosyl transferase “knock-out” and human CD46 transgenic (GTKO.CD46Tg) pig cardiac xenografts were heterotopically transplanted into specific pathogen free (SPF) baboons. The B cell numbers and non Gal antibody production remained suppressed for over 2 months after only 4 doses of induction treatment with an anti CD20 antibody. Continuous evaluation of the transplanted hearts and recipients by telemetry provided accurate assessment of graft function and aided post operative care, allowing prevention of several major complications. The significant difference in graft survival with the addition of anti CD20 antibody identifies a critical role for B cells in the mechanisms of delayed xenograft rejection and represents a significant advance toward clinical application.

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Objective Cardiac transplantation, along with available mechanical alternatives, are the only possible solutions for end stage cardiac disease. Unfortunately, due to the limited supply of human organs, xenotransplantation may be the ideal method to overcome this shortage. Recently we have seen significant prolongation of heterotopic cardiac xenograft survival from three months to twelve months and beyond. Methods Hearts from genetically engineered (GE) piglets that were alpha 1–3 galactosidase transferase knockout (GTKO) and expressed the human complement regulatory gene, CD46 (hCD46)(Group A, B, C), and the human Thrombomodulin (hTBM) gene (Group D), were heterotropically transplanted in baboons treated with anti-thymocyte globulin (ATG), cobra venom factor (CVF), anti CD20 antibody, and costimulation blockade (anti CD154 antibody (clone 5C8), in group A, or anti-CD40 antibody (clone 3A8;20mg/Kg) in group B, clone 2C10R4 (25mg/Kg) in group C, or clone 2C10R4 (50mg/Kg) in group D, along with conventional non-specific immunosuppressive agents. Results Group A grafts (n=8) survived for an average of 70 days with the longest survival of 236 days. Some animals in this group (n=3) developed microvascular thrombosis due to platelet activation and consumption, which resulted in spontaneous hemorrhage. The median survival time in group B (n=3) was 21 days, group C (n=6) was 80 days, and group D (n=5) was >200days. Three grafts in group D are still contracting well, with the longest ongoing graft survival surpassing the one-year mark. Conclusion GE pig hearts (GTKOhTg.hCD46.hTBM) with modified targeted immunosuppression (anti CD40mAb) achieved long term cardiac xenograft survival. This potentially paves the way for clinical xenotransplantation if similar survival can be reproduced in an orthotopic transplantation model.

Background Recently, we have shown that an immunosuppression regimen including costimulation blockade via anti-CD154 antibody significantly prolongs the cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon heterotopic xenotransplantation model. Unfortunately, many coagulation disorders were observed with the use of anti-CD154 antibody, and recipient survival was markedly reduced by these complications. Material and Methods In this experiment, we replaced anti-CD154 antibody with a more clinically acceptable anti-CD40 antibody while keeping the rest of the immunosuppressive regimen and the donor pig genetics the same. This was carried out to evaluate the antibody's role in xenograft survival and prevention of coagulopathies. Two available clones of anti-CD40 antibody were tested. One mouse anti-human CD40 antibody, (clone 3A8), activated B lymphocytes in vitro and only modestly suppressed antibody production in vivo. Whereas a recombinant mouse non-human primate chimeric raised against macaque CD40, (clone 2C10R4), blocked B-cell activation in vitro and completely blocked antibody production in vivo. Results The thrombotic complications seen with anti-CD154 antibody were effectively avoided but the graft survival, although extended, was not as prolonged as observed with anti-CD154 antibody treatment. The longest survival for the 3A8 antibody group was 27 days, and the longest graft survival in the 2C10R4 antibody group was 146 days. All of the grafts except two rejected and were explanted. Only two recipient baboons had to be euthanized due to unrelated complications, and the rest of the baboons remained healthy throughout the graft survival period or after graft explantation. In contrast to our anti-CD 154 antibody-treated baboons, the non-Gal antibody levels started to rise after B cells made their appearance around 8 weeks post-transplantation. Conclusions Anti-CD40 antibody at the current dose does not induce any coagulopathies but while effective, had reduced efficacy to induce similar long-term graft survival as with anti-CD154 antibody perhaps due to ineffective control of B-cell function and antibody production at the present dose. More experiments are required to determine antibody affinity and effective dose for inducing long-term cardiac xenograft survival.