B. Lewis

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.

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.

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.

CD4+CD25HiFoxP3+ regulatory T cells in long-term cardiac xenotransplantation

CD4+CD25HiFoxP3+ T (Treg) cells are a small subset of CD4+ T cells that have been shown to exhibit immunoregulatory function. Although the absolute number of Treg cells in peripheral blood lymphocytes (PBL) is very small, they play an important role in suppressing immune reactivity. Several studies have demonstrated that the number of Treg cells, rather than their intrinsic suppressive capacity, may contribute to determining the long‐term fate of transplanted grafts. In this study, we analyzed Treg cells in PBL of long‐term baboon recipients who have received genetically modified cardiac xenografts from pig donors.

Anti CD154 Mediated Co-Stimulation Blockade in a Pig to Baboon Cardiac Xenotransplantation Model: A Possible Role of T Regulatory Cells Induction in Long Term Graft Survival: 862

Blockade of CD40-CD154 co-stimulation pathway potentially attenuates the T cell immune response. It has been reported that CD4+CD25+ are required for the allograft tolerance induced by via CD40-CD154 co-stimulation pathway blockade in graft versus host model. Several group have also demonstrated the treatment combination of anti CD154 antibody and donor specific blood transfusion for immune tolerance in allo transplantation models. In this study, we have analyzed the Treg cells in the PBL of long term survived baboon recipients who have received GTKOhCD46Tg pig cardiac xenografts and immunosuppression regimen that has included co-stimulation blockade by anti CD154 antibody. Methods: Heterotopic cardiac xenotransplantation (n=8) was performed from GTKOhCD46Tg pigs to baboons using immunosupression regimen that includes anti thymocyte globulin (ATG), anti CD20, mycophenolate mofitel (MMF), cobra venom factor (CVF), and co-stimulation blockade (anti CD154). FACS analysis was done on PBLs labeled with anti-human CD4, CD25, and FoxP3 monoclonal antibodies to analyze the percentage of Treg cells in six baboons that survived longer than 2 months (ranging from 42 days to 236 days) after receiving a pig cardiac xenograft. Results: It was found that the percentage of Treg cells among CD4+ cells was increased (> 40-70%) in PBL of recipients with long term functioning heart as compared to naïve baboon (8-10%). Total percentage of Treg cells within the CD4+CD25Hi cells was significantly increased (>80%) in long-term survivor as compared to naïve baboons. The absolute number of Treg cells was also counted based on their white blood cell (WBC) count. Even though the total WBC count was low due to immunosuppression, the number of Treg cells was maintained to the level of naïve baboons. One of the baboons that underwent rejection after 75 days had a lower percentage Treg cells (< 35%). Conclusion: Our results suggest that Treg cells may be contributing in preventing or delaying the rejection process by controlling the activation/ expansion of donor-reactive T cells, thereby masking the anti-donor response leading to long term survival of cardiac xenografts. 1802

Cardiac xenografts show reduced survival in the absence of transgenic human thrombomodulin expression in donor pigs: XXXX

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.

Expression of Human Thrombomodulin on GTKO. CD46 Donor Pigs and Costimulation Blockade by Anti CD40 Antibody is Critical for Extending Cardiac Xenograft Survival in Non-human Primates

Introduction Heterotopic cardiac transplant model is being used in xenotransplantation to investigate xenograft survival of multi-genie modified donor pig organs and optimizing immunosuppression (IS) regimen in non-human primate model. We have shown earlier that significant graft survival can be achieved in alpha 1,3 alpha[MM1] Galactosidase transferase enzyme knockout pigs (GTKO) also expressing human complement regulatory transgene CD46 (CD46Tg) and thrombomodulin (hTBM). In this study, we evaluated the necessity of hTBM expression in donors hearts for the long-term graft survival using our costimulation blockade-based IS regimen. Materials and Methods Heterotopic cardiac xenotransplantation were performed in specific pathogen-free baboons from genetically engineered (GE) two gene pig donors which do not express hTBM (i.e.GTKO.CD46 only) (n=3) and three gene pigs (GTKO.CD46.hTBM; (n=5). Recipient baboons were treated with a short course of anti CD20 antibody, cobra venom factor, anti-thymocyte globulin, and were maintained on anti CD40 antibody (clone 2C10R4), mycophenolate mofetil and tapering doses of steroids. All baboons received continuous intravenous heparin. The dose of heparin was adjusted based on ACT levels designed to maintain the ACT at twice the baseline level. Graft survival was monitored with continuous telemetry, periodic echocardiography, and manual palpation. Blood work (CBC, chemistry, troponin, and ACT) was performed at 1-2 week intervals. Results Cardiac xenograft survival in recipient baboon receiving xenograft from GE donor pig without hTBM expression was significantly less (Median 70 days) as compared to 3 genes expressing pig (Median 298 days). All the three xenografts from non-hTBM donor pigs failed on 9, 98 and 70 days respectively. Increased troponin release and poor contractility of the transplanted heart were noted at the time of rejection. Non-Gal IgG and IgM antibody levels in recipient baboons from non-TBM GE donor pigs were at the baseline level at the time of rejection. The animals receiving the grafts expressing TBM also maintained their coagulation profile (PTT, PT and Fibrinogen levels) better and had fewer episodes of bleeding. Also, there was considerably less inflammation observed in 3 gene grafts. Conclusion Cardiac xenograft rejection is a complex phenomenon and a combination of manipulations are required to overcome this process. The study described above further supports this notion that hTBM gene expression on donors hearts is required along with the anti-CD40 mAb-based IS regimen and this regimen alone is insufficient to achieve long-term graft survival. This study further reveals that each mechanism of xenograft rejection is needed to be addressed separately. These very critical findings will further help us advance our journey towards clinical xenotransplantation.

Insights into the Development of a Life-Sustaining Orthotopic Cardiac Xenotransplant Model

Introduction Advances in clinical immunosuppression and characterization of optimal donor genetics continue to move the field of xenotransplantation toward a clinical reality. Cardiac xenograft survival in a non-life supporting heterotopic model has been extended to greater than 900 days as previously described by our lab. Orthotopic life supporting models for cardiac xenografts are being developed with the major barrier being significant peri-operative mortality. We detail the development of an orthotopic pig-to-baboon cardiac xenotransplant model, offering insights gained over time for replication of the model. Materials and Methods Specific pathogen free baboons were transplanted with GTKO.CD46 donor pig hearts in the orthotopic position (n = 3) by cardiac transplant and congenital cardiac surgeons. The clinical team also included a perfusionist and cardiac anesthesiologist. Detailed reports were generated regarding equipment and medications required for the cases to approximate clinical orthotopic cardiac transplantation. Monitoring capabilities were optimized as well as resuscitative access and strategy. These baboons were recovered from prior experiments and per institutional protocol were approved for non-survival studies; cases were performed with intended short term survival under anesthesia followed by elective euthanasia. Results and Discussion All recipient baboons were transplanted and weaned off of cardiopulmonary bypass with life sustaining cardiac function. Low dose inotropic support was continued in all cases; vasopressor support was continued in one case. Two cases were electively terminated after wean from bypass; one recipient suffered arrest three hours post wean from bypass. Recipients were monitored using femoral arterial lines, transesophageal echocardiogram, external EKG, continuous pulse oximetry, and serial bloodwork including ABG, CBC, BMP, and ACT. Central venous access with multiple lumens was critical to post-operative care. Invasive hemodynamic monitoring, pretreatment with anti-arrhythmics, and availability of cardioversion for the donor pig allowed rapid reversal of fibrillation episodes during procurement. Ideal cold ischemia time was found to be close to one hour; shorter ischemia time resulted in poorer xenograft function (n = 1) as evidenced by difficult wean from bypass and persistent need for vasopressor support. Atrial and ventricular pacing wires proved integral to the cardiopulmonary bypass weaning process. Conclusion Cardiac xenotransplantation is a potential solution to the paucity of human organs. Technical refinement of the life-supporting orthotopic cardiac xenotransplant with expert clinical help and applying human standards of post-operative care are the critical next step toward a clinical reality. Here we detail our experience developing an operative and critical care approach to the orthotopic cardiac xenotransplantation model.