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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