One-Year Heterotopic Cardiac Xenograft Survival in a Pig to Baboon Model

To the Editor: We have now demonstrated that the heterotopic pig cardiac xenograft survival in a baboon can exceed 1 year by utilizing porcine hearts with customized genetics (alpha galactosyl transferase gene knock out [GTKO] to eliminate alpha Gal antibody-mediated rejection, transgenic expression of human complement regulatory protein [hCD46] to inhibit complement activation and human thrombomodulin molecules [hTBM] to prevent coagulation) (Revivicor, Inc., Blacksburg, VA) and an immunomodulatory treatment regimen consisting of co-stimulation blockade by a primatized anti-CD40 antibody (clone 2C10R4; 50 mg/kg/weekly), anti-CD20 antibody (19 mg/kg on days −14, −7, 0 and 7), antithymocyte globulin (5 mg/kg on days −2 and −1), mycophenolate mofetil (20 mg/kg twice a day) and steroids (2 mg/kg tapered off in 4–6 weeks). Graft survival of all five animals in this group is shown in Table 1.Table 1:Graft survivalNo.Baboon IDGraft survival1All grafts and baboons remain viable/surviving except for baboon #15009 where the graft rejected at 146 days. (days)Graft score2Range: Strong contractility (++++) to rejection (0).LVP3Strong contractility LVP > 60 mm Hg and rejection LVP < 10 mm Hg. (mm Hg)ComplicationsGraft/recipient status1150091460<10CMV infectionEXP/euthanized2510380++++>804Telemetry battery ran out of charge at day 300.Klebsiella infection on day 300/treatedContracting/alive391091++++>70NoneContracting/alive411084++++>70NoneContracting/alive521077+++>60NoneContracting/aliveCMV, cytomegalovirus; EXP, explanted; LVP, left ventricular pressure.1 All grafts and baboons remain viable/surviving except for baboon #15009 where the graft rejected at 146 days.2 Range: Strong contractility (++++) to rejection (0).3 Strong contractility LVP > 60 mm Hg and rejection LVP < 10 mm Hg.4 Telemetry battery ran out of charge at day 300. Open table in a new tab CMV, cytomegalovirus; EXP, explanted; LVP, left ventricular pressure. To the best of our knowledge, this is the first demonstration of long-term vascular xenograft survival beyond 1 year in any large animal xenotransplantation model. All previous reported graft survivals were at least 4 months less (1Kuwaki K Tseng YL Dor FJ et al.Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: Initial experience.Nat Med. 2005; 11: 29-31Crossref PubMed Scopus (588) Google Scholar,2Mohiuddin MM Corcoran PC Singh AK et al.B-cell depletion extends the survival of GTKO hCD46Tg pig heart xenografts in baboons for up to 8 months.Am J Transplant. 2012; 12: 763-771Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Antibodies, both preformed and elicited against various xenoantigens, that mediate graft rejection (3Tazelaar HD Byrne GW McGregor CG Comparison of Gal and non-Gal-mediated cardiac xenograft rejection.Transplantation. 2011; 91: 968-975Crossref PubMed Scopus (23) Google Scholar) and thrombotic microangiopathy or consumptive coagulopathy due to platelet activation (4Lin CC Cooper DK Dorling A Coagulation dysregulation as a barrier to xenotransplantation in the primate.Transpl Immunol. 2009; 21: 75-80Crossref PubMed Scopus (63) Google Scholar), have been the main obstacles to successful xenograft survival. In this study, both these mechanisms were efficiently controlled in all five baboons by altering the genes of the donor pig and recipient treatment with a regimen that included anti-CD40 antibody. It is hard to comment definitively on the advantage of the genetic modification of pigs or the use of anti-CD40 antibody but the combination has clearly played a significant role in prolonging graft survival. All hemodynamic and coagulation parameters remained within the normal range in all the animals in this group. This was especially true of the platelet counts, control of which, historically, had been a key issue in this model. Prevention of thrombocytopenia by an initially low and thereafter tapering dose (20 mg/kg) of anti-CD40 antibody (clone 2C10R4) has also been demonstrated in our laboratory, but all GTKO.hCD46 grafts (n = 6) in that study were rejected within 149 days (5Mohiuddin MM Singh AK Corcoran PC et al.Role of anti-CD40 antibody-mediated costimulation blockade on non-Gal antibody production and heterotopic cardiac xenograft survival in a GTKO.hCD46Tg pig-to-baboon model.Xenotransplantation. 2013; (doi: 10.1002/1111/xen.12066 [Epub ahead of print].)PubMed Google Scholar). Thus, it seems the addition of the hTBM transgene had a further beneficial effect. As shown in Table 1, only one graft out of five in this experimental group ceased function and stopped contracting after surviving for 146 days. This baboon suffered from a prolonged period of infection which was resistant to all available antibiotics. On necropsy, cytomegalovirus (CMV) inclusion bodies were discovered indicating a probable CMV infection. The histology of this rejected heart showed mostly necrotic cardiac myocytes with fibrosis. As of the date of publication, all of the remaining four graft recipient baboons are still healthy with strong xenograft contractile function (graft scores are shown in Table 1). Due to the use of anti-CD20 antibody, no B cells were detected in these baboons for the first 60 days. Both non-Gal IgM and IgG antibodies remained at pretransplant levels throughout all experiments, indicating that the antibody response against xenoantigen was adequately controlled. In addition to the above manipulations in genetics and immunosuppression regimens, in our opinion, another key reason for improved graft survival is our ability to identify and intervene at the earliest sign of any complication due to continuous telemetric and video monitoring of the baboon recipient. Survival of a heterotopic cardiac xenograft for more than 1 year is a significant milestone in the field of xenotransplantation. To advance the field further, the next logical step should be to test the pig genetics used in this experiment along with the optimal immunomodulation protocol utilized in an orthotopic cardiac xenograft model, to investigate the life sustaining capability of this pig xenograft. We hope that this result will drive further activity and innovation in the field to make clinical xenotransplantation a reality. We would like to acknowledge DVR, ASR and flow cytometry core staff of NHLBI for their help in surgery, animal care and FACS analyses; Dr. Tannia Clark for performing ultrasound exam; Ms. Carol Phelps, Mr. Todd Vaught and Ms. Suyapa Ball (of Revivicor, Inc.) for transgenic pig production; and Ms. Patricia Jackson for her administrative help. The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. Dr. David Ayares is the Executive Vice President and Chief Scientific Officer of Revivicor, Inc. Dr. Keith Reimann holds equity in Primatope Therapeutics who has licensed the 2C10 antibody.