Philip C. Corcoran

Chimeric 2C10R4 anti-CD40 antibody therapy is critical for long-term survival of GTKO.hCD46.hTBM pig-to-primate cardiac xenograft

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.

B‐Cell Depletion Extends the Survival of GTKO.hCD46Tg Pig Heart Xenografts in Baboons for up to 8 Months

Xenotransplantation of genetically modified pig organs offers great potential to address the shortage of human organs for allotransplantation. Rejection in Gal knockout (GTKO) pigs due to elicited non‐Gal antibody response required further genetic modifications of donor pigs and better control of the B‐cell response to xenoantigens. We report significant prolongation of heterotopic alpha Galactosyl transferase “knock‐out” and human CD46 transgenic (GTKO.hCD46Tg) pig cardiac xenografts survival in specific pathogen free baboons. Peritransplant B‐cell depletion using 4 weekly doses of anti‐CD20 antibody in the context of an established ATG, anti‐CD154 and MMF‐based immunosuppressive regimen prolonged GTKO.hCD46Tg graft survival for up to 236 days (n = 9, median survival 71 days and mean survival 94 days). B‐cell depletion persisted for over 2 months, and elicited anti‐non‐Gal antibody production remained suppressed for the duration of graft follow‐up. This result identifies a critical role for B cells in the mechanisms of elicited anti‐non‐Gal antibody and delayed xenograft rejection. Model‐related morbidity due to variety of causes was seen in these experiments, suggesting that further therapeutic interventions, including candidate genetic modifications of donor pigs, may be necessary to reduce late morbidity in this model to a clinically manageable level.

Genetically Engineered Pigs And Target Specific Immunomodulation Provide Significant Graft Survival And Hope For Clinical Cardiac Xenotransplantation

OBJECTIVES Cardiac transplantation and available mechanical alternatives are the only possible solutions for end-stage cardiac disease. Unfortunately, because of the limited supply of human organs, xenotransplantation may be the ideal method to overcome this shortage. We have recently seen significant prolongation of heterotopic cardiac xenograft survival from 3 to 12 months and beyond. METHODS Hearts from genetically engineered piglets that were alpha 1-3 galactosidase transferase knockout and expressed the human complement regulatory gene, CD46 (groups A-C), and the human thrombomodulin gene (group D) were heterotropically transplanted in baboons treated with antithymocyte globulin, cobra venom factor, anti-CD20 antibody, and costimulation blockade (anti-CD154 antibody [clone 5C8]) in group A, anti-CD40 antibody (clone 3A8; 20 mg/kg) in group B, clone 2C10R4 (25 mg/kg) in group C, or clone 2C10R4 (50 mg/kg) in group D, along with conventional nonspecific 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 was 21 days in group B (n = 3), 80 days in group C (n = 6), and more than 200 days in group D (n = 5). Three grafts in group D are still contracting well, with the longest ongoing graft survival surpassing the 1-year mark. CONCLUSIONS Genetically engineered pig hearts (GTKOhTg.hCD46.hTBM) with modified targeted immunosuppression (anti-CD40 monoclonal antibody) 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.

One-year heterotopic cardiac xenograft survival in a pig to baboon model.

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; 50mg/kg/weekly), anti-CD20 antibody (19mg/kg on days 14, 7, 0 and 7), antithymocyte globulin (5mg/kg on days 2 and 1), mycophenolate mofetil (20mg/kg twice a day) and steroids (2mg/kg tapered off in 4–6 weeks). Graft survival of all five animals in this group is shown in Table 1.

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

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.

Early graft failure of GalTKO pig organs in baboons is reduced by expression of a human complement pathway-regulatory protein

We describe the incidence of early graft failure (EGF, defined as loss of function from any cause within 3 days after transplant) in a large cohort of GalTKO pig organs transplanted into baboons in three centers, and the effect of additional expression of a human complement pathway‐regulatory protein, CD46 or CD55 (GalTKO.hCPRP). Baboon recipients of life‐supporting GalTKO kidney (n = 7) or heterotopic heart (n = 14) grafts received either no immunosuppression (n = 4), or one of several partial or full immunosuppressive regimens (n = 17). Fourteen additional baboons received a GalTKO.hCPRP kidney (n = 5) or heart (n = 9) and similar treatment regimens. Immunologic, pathologic, and coagulation parameters were measured at frequent intervals. EGF of GalTKO organs occurred in 9/21 baboons (43%). hCPRP expression reduced the GalTKO EGF incidence to 7% (1/14; P < 0.01 vs. GalTKO alone). At 30 mins, complement deposits were more intense in organs in which EGF developed (P < 0.005). The intensity of peri‐transplant platelet activation (as β‐thromboglobulin release) correlated with EGF, as did the cumulative coagulation score (P < 0.01). We conclude that (i) the transgenic expression of a hCPRP on the vascular endothelium of a GalTKO pig reduces the incidence of EGF and reduces complement deposition, (ii) complement deposition and platelet activation correlate with early GalTKO organ failure, and (iii) the expression of a hCPRP reduces EGF but does not prevent systemic coagulation activation. Additional strategies will be required to control coagulation activation.

Regulatory T cells enhance mesenchymal stem cell survival and proliferation following autologous cotransplantation in ischemic myocardium

OBJECTIVES We sought to investigate if autologous freshly isolated regulatory T cells (Tregs) provide a protective and supportive role when cotransplanted with mesenchymal stem cells (MSCs). METHODS In a porcine model of chronic ischemia, autologous MSCs were isolated and expanded ex vivo for 4 weeks. Autologous Treg cells were freshly isolated from 100 mL peripheral blood and purified by fluorescence-activated cell sorting. MSCs and Treg cells were then cotransplanted into the chronic ischemic myocardium of Yorkshire pigs by direct intramyocardial injection (1.2 × 10(8) MSCs plus an average of 1.5 million Treg cells in 25 injection sites). Animals were killed 6 weeks postinjection to study the fate of the cells and compare the effect of combined MSCs + Treg cells transplantation versus MSCs alone. RESULTS The coinjection of MSCs along with Tregs was safe and no deleterious side effects were observed. Six weeks after injection of the cell combination, spherical MSCs clusters with thin layer capsules were found in the injected areas. In animals treated with MSCs only, the MSC clusters were less organized and not encapsulated. Immunofluorescent staining showed CD25+ cells among the CD90+ (MSC marker) cells, suggesting that the injected Treg cells remained present locally, and survived. Factor VIII+ cells were also prevalent suggesting new angiogenesis. We found no evidence that coinjections were associated with the generation of cardiac myocytes. CONCLUSIONS The cotransplantation of Treg cells with MSCs dramatically increased the MSC survival rate, proliferation, and augmented their role in angiogenesis, which suggests a new way for future clinical application of cell-based therapy.

Postoperative acute kidney injury following intraoperative blood product transfusions during cardiac surgery

Introduction: This study explored the nature of the association between intraoperative usage of red blood cell, fresh frozen plasma, cryoprecipitate or platelet transfusions and acute kidney injury. Methods: A total of 1175 patients who underwent cardiac surgery between 2008 and 2013 were retrospectively analyzed. We assessed the association between: (1) preoperative patient characteristics and acute kidney injury, (2) intraoperative blood product usage and acute kidney injury, (3) acute kidney injury and 30-day mortality or re-hospitalization. Results: In our cohort of 1175 patients, 288 patients (24.5%) developed acute kidney injury. This included 162 (13.8%), 69 (5.9%) and 57 (4.9%) developing stage 1, stage 2 or stage 3 acute kidney injury, respectively. Increased red blood cell, fresh frozen plasma or platelet transfusions increased the odds of developing acute kidney injury. Specifically, every unit of red blood cells, fresh frozen plasma or platelets transfused was associated with an increase in the covariate-adjusted odds ratio of developing ⩾ stage 2 kidney injury of 1.18, 1.19 and 1.04, respectively. Conclusions: Intraoperative blood product transfusions were independently associated with an increased odds of developing acute kidney injury following cardiac surgery. Further randomized studies are needed to better define intraoperative transfusion criteria.

Circulating cell-free DNA as a biomarker of tissue injury: Assessment in a cardiac xenotransplantation model

BACKGROUND Observational studies suggest that cell-free DNA (cfDNA) is a biomarker of tissue injury in a range of conditions including organ transplantation. However, the lack of model systems to study cfDNA and its relevance to tissue injury has limited the advancements in this field. We hypothesized that the predictable course of acute humoral xenograft rejection (AHXR) in organ transplants from genetically engineered donors provides an ideal system for assessing circulating cfDNA as a marker of tissue injury. METHODS Genetically modified pig donor hearts were heterotopically transplanted into baboons (n = 7). Cell-free DNA was extracted from pre-transplant and post-transplant baboon plasma samples for shotgun sequencing. After alignment of sequence reads to pig and baboon reference sequences, we computed the percentage of xenograft-derived cfDNA (xdcfDNA) relative to recipient by counting uniquely aligned pig and baboon sequence reads. RESULTS The xdcfDNA percentage was high early post-transplantation and decayed exponentially to low stable levels (baseline); the decay half-life was 3.0 days. Post-transplantation baseline xdcfDNA levels were higher for transplant recipients that subsequently developed graft loss than in the 1 animal that did not reject the graft (3.2% vs 0.5%). Elevations in xdcfDNA percentage coincided with increased troponin and clinical evidence of rejection. Importantly, elevations in xdcfDNA percentage preceded clinical signs of rejection or increases in troponin levels. CONCLUSION Cross-species xdcfDNA kinetics in relation to acute rejection are similar to the patterns in human allografts. These observations in a xenotransplantation model support the body of evidence suggesting that circulating cfDNA is a marker of tissue injury.

Encouraging experience using multi-transgenic xenografts in a pig-to-baboon cardiac xenotransplantation model

Innovations in transgenic technology have facilitated improved xenograft survival. Additional gene expression appears to be necessary to overcome the remaining immune and biologic incompatibilities. We report for the first time the novel use of six‐gene modifications within a pig‐to‐baboon cardiac xenotransplantation model.