Marvin L. Thomas

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.

Mixed chimeric hematopoietic stem cell transplant reverses the disease phenotype in canine leukocyte adhesion deficiency.

The genetic disease canine leukocyte adhesion deficiency (CLAD) is characterized by recurrent, severe bacterial infections, typically culminating in death by 6 months of age. CLAD is due to a mutation in the leukocyte integrin CD18 subunit, which prevents surface expression of the CD11/CD18 leukocyte integrin complex. We demonstrate that stable mixed donor:host hematopoietic chimerism, achieved by a non-myeloablative bone marrow transplant from a histocompatible littermate, reverses the disease phenotype in CLAD. Donor chimerism following the transplant was demonstrated both by flow cytometric detection of donor-derived CD18-positive leukocytes in the peripheral blood of the recipient, and by the demonstration of donor-derived DNA microsatellite repeats in the peripheral blood leukocytes of the recipient. These results indicate that mixed hematopoietic chimerism reverses the clinical phenotype in CLAD and represents a potential therapeutic approach for the human disease leukocyte adhesion deficiency.

Left Ventricular Pressure Measurement by Telemetry Is an Effective Means to Evaluate Transplanted Heart Function in Experimental Heterotopic Cardiac Xenotransplantation

Evaluation of the function of heterotopic cardiac transplants has traditionally been accomplished by either manual palpation or serial biopsies. Both methods have drawbacks. Palpation can be difficult to differentiate a pulse from the graft versus a transmitted pulse from the native aorta. Serial biopsies, though accurate, require multiple laparotomies, leading to increased morbidity and possibly mortality rates. In this study we used an advanced telemetry system, consisting of an intra-abdominal implant, that was capable of continuously monitoring simultaneously several parameters of the transplanted heart and the status of the recipient. In a large animal model of heterotopic cardiac xenotransplantation (pig donor to baboon recipient), we implanted the device in 12 animals: 8 with and 4 without immunosuppression. We monitored and continuously recorded the left ventricular pressure (both peak-systolic and end-diastolic [LVEDP]), heart rate, and the electrocardiogram pattern of the transplanted heart as well as the temperature of the recipient. The left ventricular pressure proved to be the most valuable parameter to assess graft heart function. In the 4 nonimmunosuppressed cases, grafts were rejected acutely. In these cases, the end-diastolic pressure increased sharply and the heart stopped contracting when the difference between the systolic and the diastolic pressure decreased to <10 mm Hg. The earliest reproducible sign of rejection was an increased LVEDP. Among long-term survivors, the increase in diastolic pressure was gradual, indicating progressive thickening of the myocardium and decreased compliance of the ventricle. Six of 8 immunosuppressed animals died of other complications before rejecting the transplanted heart. The telemetry was also helpful to indicate early onset of fever in the recipients, thus allowing us to intervene early and prevent potentially lethal septic complications. Continuous monitoring of several parameters via telemetry allowed detection of changes associated with rejection as well as other complications at an early stage, allowing prompt intervention, treatment, and possibly reversal of rejection.

Surgical and Nonsurgical Complications of a Pig to Baboon Heterotopic Heart Transplantation Model

A modified immunosuppressive regimen, developed at the National Institutes of Health, has been employed in a large animal model of heterotopic cardiac xenotransplantation. Graft survival has been prolonged, but despite this, our recipients have succumbed to various surgical or nonsurgical complications. Herein, we have described different complications and management strategies. The most common complication was hypercoagulability (HC) after transplantation, causing thrombosis of both small and large vasculature, ultimately leading to graft loss. While managing this complication we discovered that there was a delicate balance between HC and consumptive coagulopathy (CC). CC encountered in some recipient baboons was not able to be reversed by stopping anticoagulation and administering multiple blood transfusions. Some complications had iatrogenic components. To monitor the animals, a solid state left ventricular telemetry probe was placed directly into the transplanted heart via the apex. Induction of hypocoagulable states by continuous heparin infusion led to uncontrollable intra-abdominal bleeding in 1 baboon from this apical site. This occurrence necessitated securing the probe more tightly with multiple purse strings and 4-quadrant pledgeted stay sutures. One instance of cardiac rupture originated from a lateral wall infarction site. Earlier studies have shown infections to be uniformly fatal in this transplant model. However, owing to the telemetry placement, infections were identified early by temperature spikes that were treated promptly with antibiotics. We had several cases of wound dehiscence due to recipients disrupting the suture line. These complications were promptly resolved by either re-approximating the wound or finding distractions for the baboon. A few of the most common problems we faced in our earlier experiments were related to the jacket, tether, and infusion pumps. It was difficult to keep the jackets on some baboons and the tether had to be modified several times before we assured long-term success. Infusion catheter replacement resulted in transplant heart venous obstruction and thrombosis from a right common femoral venous line. Homeostatic perturbations such as HC and CC and baboon-induced wound complications comprised most complications. Major bleeding and death due to telemetry implantation and infarct rupture occurred in 2 baboons. Despite the variety of complications, we achieved significant graft prolongation in this model.

Sympathetic blockade in a canine model of gram-negative bacterial peritonitis.

We investigated, in a well-established canine model of human sepsis, the effects of two different techniques of sympathetic blockade during bacterial peritonitis on pain relief, hemodynamics, and survival rate. Twenty-two purpose-bred beagles (12–28 months old, weighing 10–12 kg) were studied. Fourteen animals received an epidural infusion of bupivicaine and morphine, and the other eight received either a celiac plexus block (n = 4) or a sham block (n = 4). Eighteen of the 22 animals received an intraperitoneal challenge of Escherichia coli (1–10 × 109 CFU kg−1 body weight). At comparable doses of intraperitoneal-implanted E. coli (2.5–5 × 109 CFU kg−1 body weight), the addition of sympathetic blockade produced a synergistic decrease in survival times (P = 0.002) and mean left ventricular ejection fraction (P = 0.008), and increase in creatinine levels (P = 0.02). There was also a significant increase in tumor necrosis factor (TNF) levels (P = 0.004) and decrease in blood endotoxin clearance (P = 0.006) associated with sympathetic blockade during sepsis. The celiac plexus-blocked animals had no improvement in pain scores, and subjectively looked clinically worse than animals with sepsis without a celiac plexus block. In contrast, the epidural block was effective in blocking the pain and discomfort associated with low lethality doses of intraperitoneal bacteria reflected by no increase in pain scores compared with animals not receiving bacterial challenge. This study shows that during severe bacterial peritonitis, maintenance of sympathetic tone irrespective of pain relief provided is necessary for clearance of bacterial toxins, control of proinflammatory mediator release, hemodynamic stability, and survival.

Dual-contrast agent photon-counting computed tomography of the heart: initial experience

To determine the feasibility of dual—contrast agent imaging of the heart using photon-counting detector (PCD) computed tomography (CT) to simultaneously assess both first-pass and late enhancement of the myocardium. An occlusion-reperfusion canine model of myocardial infarction was used. Gadolinium-based contrast was injected 10 min prior to PCD CT. Iodinated contrast was infused immediately prior to PCD CT, thus capturing late gadolinium enhancement as well as first-pass iodine enhancement. Gadolinium and iodine maps were calculated using a linear material decomposition technique and compared to single-energy (conventional) images. PCD images were compared to in vivo and ex vivo magnetic resonance imaging (MRI) and histology. For infarct versus remote myocardium, contrast-to-noise ratio (CNR) was maximal on late enhancement gadolinium maps (CNR 9.0 ± 0.8, 6.6 ± 0.7, and 0.4 ± 0.4, p < 0.001 for gadolinium maps, single-energy images, and iodine maps, respectively). For infarct versus blood pool, CNR was maximum for iodine maps (CNR 11.8 ± 1.3, 3.8 ± 1.0, and 1.3 ± 0.4, p < 0.001 for iodine maps, gadolinium maps, and single-energy images, respectively). Combined first-pass iodine and late gadolinium maps allowed quantitative separation of blood pool, scar, and remote myocardium. MRI and histology analysis confirmed accurate PCD CT delineation of scar. Simultaneous multi-contrast agent cardiac imaging is feasible with photon-counting detector CT. These initial proof-of-concept results may provide incentives to develop new k-edge contrast agents, to investigate possible interactions between multiple simultaneously administered contrast agents, and to ultimately bring them to clinical practice.