Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs
Avneesh K. Singh
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Department of Surgery, School of Medicine, University of Maryland, Baltimore, Maryland
Search for more papers by this authorJoshua L. Chan
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorLaura DiChiacchio
Department of Surgery, School of Medicine, University of Maryland, Baltimore, Maryland
Search for more papers by this authorNaomi L. Hardy
Department of Pathology at the University of Maryland Medical Center, University of Maryland, Baltimore, Maryland
Search for more papers by this authorPhilip C. Corcoran
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorBilleta G. T. Lewis
Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorMarvin L. Thomas
Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorAllen P. Burke
Department of Pathology at the University of Maryland Medical Center, University of Maryland, Baltimore, Maryland
Search for more papers by this authorKeith A. Horvath
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorCorresponding Author
Muhammad M. Mohiuddin
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Department of Surgery, School of Medicine, University of Maryland, Baltimore, Maryland
Correspondence
Muhammad M. Mohiuddin, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD.
Email: [email protected]
Search for more papers by this authorAvneesh K. Singh
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Department of Surgery, School of Medicine, University of Maryland, Baltimore, Maryland
Search for more papers by this authorJoshua L. Chan
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorLaura DiChiacchio
Department of Surgery, School of Medicine, University of Maryland, Baltimore, Maryland
Search for more papers by this authorNaomi L. Hardy
Department of Pathology at the University of Maryland Medical Center, University of Maryland, Baltimore, Maryland
Search for more papers by this authorPhilip C. Corcoran
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorBilleta G. T. Lewis
Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorMarvin L. Thomas
Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorAllen P. Burke
Department of Pathology at the University of Maryland Medical Center, University of Maryland, Baltimore, Maryland
Search for more papers by this authorKeith A. Horvath
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Search for more papers by this authorCorresponding Author
Muhammad M. Mohiuddin
Cardiothoracic Surgery Research Program/National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland
Department of Surgery, School of Medicine, University of Maryland, Baltimore, Maryland
Correspondence
Muhammad M. Mohiuddin, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD.
Email: [email protected]
Search for more papers by this authorAbstract
A combination of genetic manipulations of donor organs and target-specific immunosuppression is instrumental in achieving long-term cardiac xenograft survival. Recently, results from our preclinical pig-to-baboon heterotopic cardiac xenotransplantation model suggest that a three-pronged approach is successful in extending xenograft survival: (a) α-1,3-galactosyl transferase (Gal) gene knockout in donor pigs (GTKO) to prevent Gal-specific antibody-mediated rejection; (b) transgenic expression of human complement regulatory proteins (hCRP; hCD46) and human thromboregulatory protein thrombomodulin (hTBM) to avoid complement activation and coagulation dysregulation; and (c) effective induction and maintenance of immunomodulation, particularly through co-stimulation blockade of CD40-CD40L pathways with anti-CD40 (2C10R4) monoclonal antibody (mAb). Using this combination of manipulations, we reported significant improvement in cardiac xenograft survival. In this study, we are reporting the survival of cardiac xenotransplantation recipients (n = 3) receiving xenografts from pigs without the expression of hTBM (GTKO.CD46). We observed that all grafts underwent rejection at an early time point (median 70 days) despite utilization of our previously reported successful immunosuppression regimen and effective control of non-Gal antibody response. These results support our hypothesis that transgenic expression of human thrombomodulin in donor pigs confers an independent protective effect for xenograft survival in the setting of a co-stimulation blockade-based immunomodulatory regimen.
DISCLOSURE
The following authors of this manuscript have conflict of interests to disclose as described by the Xenotransplantation journal. David Ayares is the CEO & president of Revivicor, Inc Muhammad Mohiuddin, Avneesh K. Singh, Billeta Lewis and Laura DiChiacchio are part of Cardiac Xenotransplantation Program at the University of Maryland. This program is funded by United Therapeutics Inc.
REFERENCES
- 1Mohiuddin MM. Clinical xenotransplantation of organs: why aren't we there yet? PLoS Medicine. 2007; 4: e75.
- 2Thompson P, Cardona K, Russell M, et al. CD40-specific costimulation blockade enhances neonatal porcine islet survival in nonhuman primates. Am J Transplant. 2011; 11: 947-957.
- 3Mohiuddin 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: 763-771.
- 4Mohiuddin MM, Singh AK, Corcoran PC, et al. Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft. Nat Commun. 2016; 7: 11138.
- 5Higginbotham L, Mathews D, Breeden, et al. Pre-transplant antibody screening and anti-CD154 costimulation blockade promote long-term xenograft survival in a pig-to-primate kidney transplant model. Xenotransplantation. 2015; 22: 221-230.
- 6Kim J, Kim DH, Choi HJ, et al. Anti-CD40 antibody-mediated costimulation blockade promotes long-term survival of deep-lamellar porcine corneal grafts in non-human primates. Xenotransplantation. 2017; 24.
- 7Iwase H, Ekser B, Satyananda V, et al. Pig-to-baboon heterotopic heart transplantation–exploratory preliminary experience with pigs transgenic for human thrombomodulin and comparison of three costimulation blockade-based regimens. Xenotransplantation. 2015; 22: 211-220.
- 8Iwase H, Liu H, Wijkstrom M, et al. Pig kidney graft survival in a baboon for 136 days: longest life-supporting organ graft survival to date. Xenotransplantation. 2015; 22: 302-309.
- 9Iwase H, Liu H, Schmelzer E, et al. Transplantation of hepatocytes from genetically engineered pigs into baboons. Xenotransplantation. 2017; 24.
- 10Shah JA, Patel MS, Elias N, et al. Prolonged survival following pig-to-primate liver xenotransplantation utilizing exogenous coagulation factors and costimulation blockade. Am J Transplant. 2017; 17: 2178-2185.
- 11Petersen B, Ramackers W, Tiede A, et al. Pigs transgenic for human thrombomodulin have elevated production of activated protein C. Xenotransplantation. 2009; 16: 486-495.
- 12Mohiuddin MM, Singh AK, Corcoran PC, et al. Genetically engineered pigs and target-specific immunomodulation provide significant graft survival and hope for clinical cardiac xenotransplantation. J Thorac Cardiovasc Surg. 2014; 148: 1106-1113; discussion 1113-1104.
- 13Phelps CJ, Koike C, Vaught TD, et al. Production of alpha 1,3-galactosyltransferase-deficient pigs. Science. 2003; 299: 411-414.
- 14Chan JL, Singh AK, Corcoran PC, et al. Encouraging experience using multi-transgenic xenografts in a pig-to-baboon cardiac xenotransplantation model. Xenotransplantation. 2017; 24(6): e12330.
- 15Horvath KA, Corcoran PC, Singh AK, et al. Left ventricular pressure measurement by telemetry is an effective means to evaluate transplanted heart function in experimental heterotopic cardiac xenotransplantation. Transplant Proc. 2010; 42: 2152-2155.
- 16Buhler L, Awwad M, Basker M, et al. High-dose porcine hematopoietic cell transplantation combined with CD40 ligand blockade in baboons prevents an induced anti-pig humoral response. Transplantation. 2000; 69: 2296-2304.
- 17Harris DG, Gao Z, Sievert EP, et al. Transgenic human thrombomodulin expression reduces xenogeneic thrombosis: a promising means of reducing pig lung xenograft thrombotic injury. J Heart Lung Transpl. 2014; 33: S108-S108.
- 18Lin WL, Chen CC, Shi GY, Ma CY, Chang CF, Wu HL. Monocytic thrombomodulin promotes cell adhesion through interacting with its ligand, Lewisy. Immunol Cell Biol. 2017; 95: 372-379.
- 19Chung DR, Chitnis T, Panzo RJ, Kasper DL, Sayegh MH, Tzianabos AO. CD4+ T cells regulate surgical and postinfectious adhesion formation. J Exp Med. 2002; 195: 1471-1478.
- 20Menzies D. Postoperative adhesions: their treatment and relevance in clinical practice. Ann R Coll Surg Engl. 1993; 75: 147-153.
- 21Galili U. Interaction of the natural anti-Gal antibody with alpha-galactosyl epitopes: a major obstacle for xenotransplantation in humans. Immunol Today. 1993; 14: 480-482.
- 22Dalmasso AP, Vercellotti GM, Fischel RJ, Bolman RM, Bach FH, Platt JL. Mechanism of complement activation in the hyperacute rejection of porcine organs transplanted into primate recipients. Am J Pathol. 1992; 140: 1157-1166.
- 23Loveland BE, Milland J, Kyriakou P, et al. Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non-immunosuppressed baboons. Xenotransplantation. 2004; 11: 171-183.
- 24Kim SC, Wakwe W, Higginbotham LB, et al. Fc-Silent Anti-CD154 Domain Antibody Effectively Prevents Nonhuman Primate Renal Allograft Rejection. Am J Transplant. 2017; 17: 1182-1192.
- 25Dons EM, Montoya C, Long CE, et al. T-cell-based immunosuppressive therapy inhibits the development of natural antibodies in infant baboons. Transplantation. 2012; 93: 769-776.
- 26Higginbotham L, Ford ML, Newell KA, Adams AB. Preventing T cell rejection of pig xenografts. Int J Surg. 2015; 23: 285-290.
- 27Robson SC, Cooper DK, D'Apice AJ. Disordered regulation of coagulation and platelet activation in xenotransplantation. Xenotransplantation. 2000; 7: 166-176.
- 28Byrne GW, Davies WR, Oi K, et al. Increased immunosuppression, not anticoagulation, extends cardiac xenograft survival. Transplantation. 2006; 82: 1787-1791.
- 29Cowan PJ, Aminian A, Barlow H, et al. Protective effects of recombinant human antithrombin III in pig-to-primate renal xenotransplantation. Am J Transplant. 2002; 2: 520-525.
- 30Cozzi E, Simioni P, Boldrin M, et al. Effects of long-term administration of high-dose recombinant human antithrombin in immunosuppressed primate recipients of porcine xenografts. Transplantation. 2005; 80: 1501-1510.
- 31Cozzi E, Bhatti F, Schmoeckel M, et al. Long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts. Transplantation. 2000; 70: 15-21.
- 32Mohiuddin 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. 2014; 21: 35-45.
- 33Iwase H, Hara H, Ezzelarab M, et al. Immunological and physiological observations in baboons with life-supporting genetically engineered pig kidney grafts. Xenotransplantation. 2017; 24.
- 34Knosalla C, Yazawa K, Behdad A, et al. Renal and cardiac endothelial heterogeneity impact acute vascular rejection in pig-to-baboon xenotransplantation. Am J Transplant. 2009; 9: 1006-1016.
- 35Healy AM, Rayburn HB, Rosenberg RD, Weiler H. Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system. Proc Natl Acad Sci U S A. 1995; 92: 850-854.
- 36Gomi K, Zushi M, Honda G, et al. Antithrombotic effect of recombinant human thrombomodulin on thrombin-induced thromboembolism in mice. Blood. 1990; 75: 1396-1399.
- 37Kumada T, DittmanWA, MajerusPW. A role for thrombomodulin in the pathogenesis of thrombin-induced thromboembolism in mice. Blood. 1988; 71: 728-733.
- 38Cooper DK, Ezzelarab MB, Hara H, et al. The pathobiology of pig-to-primate xenotransplantation: a historical review. Xenotransplantation. 2016; 23: 83-105.
