David Sachs - US grants

Non-myeloablative hematopoietic cell transplantation (HCT) followed by delayed donor leukocyte infusion (DLI) is a promising immunotherapeutic approach to treat hematologic malignancies including leukemias and lymphomas. Major limitations of this approach, however, include the risks of graft failure, graft-versus-host disease, (GVHD) and infection. MGH MHC-inbred miniature swine provide a pre-clinical model for studies of transplantation biology with responses to HCT resembling those of humans. Preliminary data suggest that a novel, minimally myelosuppressive preparative regimen leads to stable multilineage chimerism following highdose haploidentical HCT, without causing GVHD. In this proposal we aim to 1) determine the immunological mechanisms involved in controlling hbst-versus-graft (HVG) and GVH responses that allow engraftment without GVHD across MHC barriers in this model. These studies will be performed in close collaboration with Projects 1 and 2 to extend the mechanistic studies in rodent models. We will then 2) optimize the swine model to facilitate translation of this protocol to the clinic and analyze the importance of specific genetic disparities (haploidentical class I and II, class I only, class II only) on engraftment, GVHD and the effects of subsequent DLI. In collaboration with Project 4, we will test novel strategies to improve stem cell harvests following cytokine mobilization through parathyroid hormone (PTH) stimulation. Increased numbers of stem cells in the leukapheresis product following PTH stimulation and cytokine mobilization may enable stable engraftment using lower doses of cells more easily attainable in the clinic. It is hoped that results of these studies will permit the development of tailored approaches to the use of DLI in chimeric patients depending on their HLA disparities from the donor. In addition, we plan to 3) further develop the swine model to allow direct assessment of graft-versus-tumor effects of HCT and DLI. For this purpose, we will establish tumor cell lines derived from inbred miniature swine and adapt these tumor lines for in vivo growth in pigs. With the recent availability of histocompatible miniature swine, and tumor lines derived from these highly inbred animals, we have the unique opportunity to develop transplantable tumors in a preclinical large animal model. These studies could provide a foundation for future immunotherapeutic approaches for the treatment of hematological malignancies that may be translated toward treatment of human disease.

Core B SUMMARY: The Large Animal Core will provide the facilities, management and technical expertise needed to supply miniature swine and baboons and tissue samples for all projects of the proposed program as well as expert veterinary care, immunological reagents, and microbiologic diagnostic assays and support. Clinical samples will be distributed to maximize utilization of each animal for studies of clinically relevant regimens for xenotransplantation. The success of each component of this program is dependent on the quality, predictability, and accessibility of the animals, microbiologic support, and antibodies provided by this Core. Animal care is reviewed weekly at meetings of the principal investigators and the veterinary staff. The Aims of the Large Animal Core include: Aim 1: Purchase and maintenance of non-human primates; Aim 2: Production, quality control, tracking, and maintenance in an SPF facility of MGH MHC inbred miniature swine including the MGH GalT-KO swine, with homozygous disruption of the ?-1,3-galactosyltransferase gene (see Figures 1 and 2), and GalT-KO- CD47 transgenic swine. In the case of donors for Projects 1 and 2, these animals will be derived by Caesarian section into Hepa-filtered cages for assurance of pCMV-free maintenance until use; Aim 3: Develop and maintain new genetically modified swine in conjunction with Project 4; Aim 4: Provide diagnostic and management support related to infectious diseases, including prophylaxis and care paradigms for the care of immunosuppressed xenograft recipients and support of on-going studies by routine assessment of swine and baboon recipients for pCMV, bCMV, PERV and other pathogens; Aim 5: Provide diagnostic and management support related to vascular thrombosis, platelet sequestration and consumptive coagulopathy; Aim 6: Production and characterization murine monoclonal antibodies and swine antisera reactive with swine and baboon cell surface antigens. Animal care is a central component of this program and is reviewed weekly at laboratory meetings in which the principal investigators and veterinary staff participate.

Summary This Project is directed toward the induction of xenotransplantation tolerance through mixed hematopoietic chimerism, which is to date the only treatment regimen that has been successful in translating allogeneic tolerance induction from animal models to the clinic. Prior to the current project period, attempts to achieve mixed chimerism across the pig-to-primate barrier had failed due to rapid clearance of infused porcine cells from the circulation of primates. During this period, we have tested the hypothesis that this rapid clearance might be due to the absence of the primate analog of the species-specific cell-membrane protein CD47 on the surface of porcine cells leading to rapid clearance of pig cells (PBSC) by recipient macrophages. We have produced and tested mobilized peripheral blood stem cells from human CD47 (hCD47) transgenic GalT-KO miniature swine for their ability to induce chimerism in baboons. Our results demonstrated markedly increased survival of porcine cells injected into baboons that was dependent on the level of hCD47 expression on donor cells. Furthermore, recipients of hCD47-expressing swine PBSC demonstrated prolonged survival of donor swine skin xenografts in the absence of immunosuppression. In the current proposal, we will attempt to increase the level and duration of porcine mixed chimerism by: 1) Confirming the effect of high expression of the hCD47 transgene in porcine hematopoietic stem cells (HSC) and the use of intra-bone HSC inoculation on the establishment of chimerism and prolonged pig skin graft survival in baboons; 2) Testing the effect of ex-vivo expanded recipient Tregs on enhancement of xenogeneic chimerism and engraftment of hCD47/GalT-KO HSC; and 3) Testing the effect of new transgenes in genetically engineered swine, produced in Project 4, that are designed to increase the level and duration of donor chimerism and induce tolerance to skin grafts. In addition to advancing our goal toward the induction of xenograft tolerance, the experiments planned will provide basic information on xenogeneic stem cell engraftment and on the immunologic pathways responsible for xenogeneic rejection and tolerance induction in primates. These studies should therefore have both theoretical and practical implications for the eventual application of xenotransplantation as a clinical modality.

Project Summary This laboratory has previously developed a technique for transplanting pancreatic islets as part of a composite islet-kidney (I-K), in which autologous islets are placed under the kidney capsule of the donor animal 6 to 8 weeks prior to transplantation. During this time, the islets become vascularized, so that there is no period of ischemic damage to the islets when the I-K is subsequently transplanted to a recipient animal. There is also no damage due to an immune response during this period, since the islets are autologous. We have demonstrated the advantages of this technique over free islet transplantation for successful allogeneic islet transplantation, both in miniature swine and in non-human primates. However, the applicability of this approach to clinical islet transplantation is limited by the requirement for 6 to 8 weeks for vascularization, since this requirement limits the approach to living donors. Extension of the approach to I-K xenotransplants could eliminate this limitation, providing a cure for the currently unmet needs of many patients with end-stage diabetic nephropathy. Until recently, however, the survival of xenograft kidneys was too short and the immunosuppression required too great for the approach to be considered as a viable clinical option. Our recent studies now suggest that long-term survival of porcine kidneys in baboons is possible using miniature swine donors and a tolerance-inducing approach. The goal of this project is therefore to develop a clinically relevant tolerance induction strategy for pig-to-baboon I-K transplantation. Specifically, we will: 1) Combine IK technology with our mixed chimerism tolerance-inducing approach to determine whether tolerance of kidney extends to porcine islets and reverses diabetes; 2) Combine IK plus VTL technologies to determine whether tolerance of kidney extends to porcine islets and reverses diabetes; and 3) Compare and contrast mixed chimerism and VTL approaches with regard to mechanism of tolerance to kidney and islets antigens. If successful, our approach could overcome the current limitations to treatment of this clinically important entity by providing a virtually limitless donor supply of islets and kidneys, a tolerance approach to avoid the need for long-term, chronic immunosuppression and a donor kidney from inbred miniature swine, with intrinsic capacity to attain a size similar to that of human recipients.