Primate Transplantation Research Articles

361.9: Liver transplantation with delayed donor bone marrow transplantation in non-human primates achieves hematopoietic chimerism.

361.9: Liver transplantation with delayed donor bone marrow transplantation in non-human primates achieves hematopoietic chimerism.

Abstract We031: Optimizing Immunosuppressive Strategies for Enhanced Survival of Allogeneic Pluripotent Stem Cell-Derived Cardiomyocytes in Non-Human Primate Transplantation Model

Introduction: Accumulating evidence suggests that the transplantation of pluripotent stem cell-derived cardiomyocytes (PSC-CMs) regenerates injured hearts in animal studies. Currently, several clinical studies are underway, transplanting allogeneic human PSC-CMs. In these clinical studies, patients have been treated with the same combination of immunosuppressants used after heart transplantation, including steroids, calcineurin inhibitors (CNIs), and mycophenolate mofetil (MMF). However, the optimal treatment following PSC-CM transplantation has yet to be established. The purpose of this study is to determine the optimal treatment for grafted PSC-CM survival by regulating the immune response using an allogeneic non-human primate transplantation model. Methods and Results: We prepared 9 male cynomolgus monkeys as recipient animals. Donor cardiomyocytes were generated from induced pluripotent stem (iPS) cells derived from another monkey, with MHC class I and class II types differing from those in the recipients. The recipient animals underwent ischemia/reperfusion injury of the left anterior descending artery two weeks before iPSC-CMs transplantation. Initially, animals were treated with the same combination of immunosuppressants (steroid/CNIs/MMF: N=3), and grafted PSC-CMs survived without apparent immune rejection. Subsequently, we conducted experiments aiming to reduce immunosuppressants. The animals were treated with the same combination, but steroids were terminated at 4 weeks post-transplantation (N=4). Histological analysis at 8 or 12 weeks post-transplantation revealed that grafted cardiomyocytes were rejected, with a significant infiltration of CD3-positive lymphocytes. When recipients were treated with another combination of immunosuppressants (steroid/abatacept/MMF), and steroids were terminated at 4 weeks post-transplantation, grafted cardiomyocytes survived without apparent infiltration of CD3-positive lymphocytes at 8 weeks post-transplantation (N=2). Conclusion: A different combination of immunosuppressants from heart transplantation allows transplanted allogeneic PSC-CMs to survive without the long-term use of steroids.

Clinical Trial Protocol for Porcine Islet Xenotransplantation in South Korea.

Islet transplantation holds promise for treating selected type 1 diabetes mellitus patients, yet the scarcity of human donor organs impedes widespread adoption. Porcine islets, deemed a viable alternative, recently demonstrated successful longterm survival without zoonotic risks in a clinically relevant pig-to-non-human primate islet transplantation model. This success prompted the development of a clinical trial protocol for porcine islet xenotransplantation in humans. A single-center, open-label clinical trial initiated by the sponsor will assess the safety and efficacy of porcine islet transplantation for diabetes patients at Gachon Hospital. The protocol received approval from the Gachon Hospital Institutional Review Board (IRB) and the Korean Ministry of Food and Drug Safety (MFDS) under the Investigational New Drug (IND) process. Two diabetic patients, experiencing inadequate glycemic control despite intensive insulin treatment and frequent hypoglycemic unawareness, will be enrolled. Participants and their family members will engage in deliberation before xenotransplantation during the screening period. Each patient will receive islets isolated from designated pathogen-free pigs. Immunosuppressants and systemic infection prophylaxis will follow the program schedule. The primary endpoint is to confirm the safety of porcine islets in patients, and the secondary endpoint is to assess whether porcine islets can reduce insulin dose and the frequency of hypoglycemic unawareness. A clinical trial protocol adhering to global consensus guidelines for porcine islet xenotransplantation is presented, facilitating streamlined implementation of comparable human trials worldwide.

Animal models for partial heart transplantation.

Partial heart transplantation delivers growing heart valve implants by transplanting the part of the heart containing the necessary heart valve only. In contrast to heart transplantation, partial heart transplantation spares the native ventricles. This has important implications for partial heart transplant biology, including the allowable ischemia time, optimal graft preservation, primary graft dysfunction, immune rejection, and optimal immunosuppression. Exploration of partial heart transplant biology will depend on suitable animal models. Here we review our experience with partial heart transplantation in rodents, piglets, and non-human primates. This review is based on our experience with partial heart transplantation using over 100 rodents, over 50 piglets and one baboon. Suitable animal models for partial heart transplantation include rodent heterotopic partial heart transplantation, piglet orthotopic partial heart transplantation, and non-human primate partial heart xenotransplantation. Rodent models are relatively cheap and offer extensive availability of research tools. However, rodent open-heart surgery is technically not feasible. This limits rodents to heterotopic partial heart transplant models. Piglets are comparable in size to children. This allows for open-heart surgery using clinical grade equipment for orthoptic partial heart transplantation. Piglets also grow rapidly, which is useful for studying partial heart transplant growth. Finally, nonhuman primates are immunologically most closely related to humans. Therefore, nonhuman primates are most suitable for studying partial heart transplant immunobiology and xenotransplantation. Animal research is a privilege that is contingent on utilitarian ethics and the 3R principles of replacement, reduction and refinement. This privilege allows the research community to seek fundamental knowledge about partial heart transplantation, and to apply this knowledge to enhance the health of children who require partial heart transplants.

(494) - Donor Chimerism Duration Correlates with Heart Allograft Tolerance in Combined Heart and Bone Marrow Transplantation in Nonhuman Primates

(494) - Donor Chimerism Duration Correlates with Heart Allograft Tolerance in Combined Heart and Bone Marrow Transplantation in Nonhuman Primates

Differential induction of donor-reactive Foxp3+ regulatory T cell via blockade of CD154 vs CD40

Recently published studies in both murine models and a meta-analysis of non-human primate renal transplant studies showed that anti-CD154 reagents conferred a significant survival advantage over CD40 blockers in both animal models and across multiple organs. Here we sought to compare the induction of donor-reactive forkhead box P3+-induced regulatory T cells (Foxp3+ iTreg) in mice treated with anti-CD154 versus anti-CD40 monoclonal antibodies (mAbs). Results indicated that while treatment with anti-CD154 mAb resulted in a significant increase in the frequency of donor-reactive CD4+ Foxp3+ iTreg following transplantation, treatment with anti-CD40 or Cd40 deficiency failed to recapitulate this result. Because we recently identified CD11b as an alternate receptor for CD154 during alloimmunity, we interrogated the role of CD154:CD11b interactions in the generation of Foxp3+ iTreg and found that blockade of CD11b in Cd40−/− recipients resulted in increased donor-reactive Foxp3+ iTreg as compared with CD40 deficiency alone. Mechanistically, CD154:CD11b inhibition decreased interleukin (IL)-1β from CD11b+ and CD11c+ dendritic cells, and blockade of IL-1β synergized with CD40 deficiency to promote Foxp3+ iTreg induction and prolong allograft survival. Taken together, these data provide a mechanistic basis for the observed inferiority of anti-CD40 blockers as compared with anti-CD154 mAb and illuminate an IL-1β-dependent mechanism by which CD154:CD11b interactions prevent the generation of donor-reactive Foxp3+ iTreg during transplantation.

Novel factors potentially initiating acute antibody-mediated rejection in pig kidney xenografts despite an efficient immunosuppressive regimen.

Antibody-mediated rejection (AMR) is a common cause of graft failure after pig-to-nonhuman primate organ transplantation, even when the graft is from a pig with multiple genetic modifications. The specific factors that initiate AMR are often uncertain. We report two cases of pig kidney transplantation into immunosuppressed baboons in which we identify novel factors associated with the initiation of AMR. In the first, membranous nephropathy was the initiating factor that was then associated with the apparent loss of the therapeutic anti-CD154 monoclonal antibody in the urine when severe proteinuria was present. This observation suggests that proteinuria may be associated with the loss of any therapeutic monoclonal antibody, for example, anti-CD154 or eculizumab, in the urine, resulting in xenograft rejection. In the second case, the sequence of events and histopathology tentatively suggested that pyelonephritis may have initiated acute-onset AMR. The association of a urinary infection with graft rejection has been well-documented in ABO-incompatible kidney allotransplantation based on the expression of an antigen on the invading microorganism shared with the kidney graft, generating an immune response to the graft. To our knowledge, these potential initiating factors of AMR in pig xenografts have not been highlighted previously.

Human PD-L1 overexpression decreases xenogeneic human T-cell immune responses towards porcine kidneys.

Xenotransplantation offers a promising alternative to circumvent the lack of donated human organs available for transplantation. Different attempts to improve the survival of xenografts led to the generation of transgenic pigs expressing various combinations of human protective genes or knocked out for specific antigens. Currently, testing the efficiency of porcine organs carrying different genetic modifications in preventing xenogeneic immune responses completely relies on in vitro assays, humanized mouse models, or non-human primate transplantation models. However, these tests are often associated with major concerns due to reproducibility and generation of insufficient data as well as they raise ethical, logistical, and economic issues. In this study, we investigated the feasibility of specifically assessing the strength of human T-cell responses towards the kidneys of wild-type (WT) or transgenic pigs overexpressing human programmed death-1 ligand 1 (hPD-L1) during ex vivo kidney perfusion (EVKP). Human T cells were shown to adhere to the endothelium and transmigrate into WT and hPD-L1 kidneys. However, transcript levels of TNF-a and IFN-y as well as cytotoxic molecules such as granzyme B and perforin secreted by human T cells were significantly decreased in the tissue of hPD-L1 kidneys in comparison to WT kidneys. These results were confirmed via in vitro assays using renal endothelial cells (ECs) isolated from WT and hPD-L1 transgenic pigs. Both CD4+ and CD8+ T cells showed significantly lower proliferation rates after exposure to hPD-L1 porcine renal ECs in comparison to WT ECs. In addition, the secretion of pro-inflammatory cytokines was significantly reduced in cultures using hPD-L1 ECs in comparison to WT ECs. Remarkably, hPD-L1 EC survival was significantly increased in cytotoxic assays. This study demonstrates the feasibility of evaluating the human response of specific immune subsets such as human T cells towards the whole xenograft during EVKP. This may represent a robust strategy to assess the potency of different genetic modifications to prevent xenogeneic immune responses and thereby predict the risk of immune rejection of new genetically engineered xenografts.

Early allogeneic immune modulation after establishment of donor hematopoietic cell-induced mixed chimerism in a nonhuman primate kidney transplant model.

Mixed lymphohematopoietic chimerism is a proven strategy for achieving operational transplant tolerance, though the underlying immunologic mechanisms are incompletely understood. A post-transplant, non-myeloablative, tomotherapy-based total lymphoid (TLI) irradiation protocol combined with anti-thymocyte globulin and T cell co-stimulatory blockade (belatacept) induction was applied to a 3-5 MHC antigen mismatched rhesus macaque kidney and hematopoietic cell transplant model. Mechanistic investigations of early (60 days post-transplant) allogeneic immune modulation induced by mixed chimerism were conducted. Chimeric animals demonstrated expansion of circulating and graft-infiltrating CD4+CD25+Foxp3+ regulatory T cells (Tregs), as well as increased differentiation of allo-protective CD8+ T cell phenotypes compared to naïve and non-chimeric animals. In vitro mixed lymphocyte reaction (MLR) responses and donor-specific antibody production were suppressed in animals with mixed chimerism. PD-1 upregulation was observed among CD8+ T effector memory (CD28-CD95+) subsets in chimeric hosts only. PD-1 blockade in donor-specific functional assays augmented MLR and cytotoxic responses and was associated with increased intracellular granzyme B and extracellular IFN-γ production. These studies demonstrated that donor immune cell engraftment was associated with early immunomodulation via mechanisms of homeostatic expansion of Tregs and early PD-1 upregulation among CD8+ T effector memory cells. These responses may contribute to TLI-based mixed chimerism-induced allogenic tolerance.

Progress in Orthotopic Pig Heart Transplantation in Nonhuman Primates.

Xenotransplantation of porcine hearts has become a promising alternative to human allotransplantation, where organ demand still greatly surpasses organ availability. Before entering the clinic, however, feasibility of cardiac xenotransplantation needs to be proven, ideally in the life supporting orthotopic pig-to-nonhuman primate xenotransplantation model. In this review, we shortly outline the last three decades of research and then discuss in detail its most recent advances. These include the genetic modifications of donor pigs to overcome hyperacute rejection and coagulation dysregulation, new organ preservation methods to prevent perioperative xenograft dysfunction, experimental immunosuppressive and immunomodulatory therapies to inhibit the adaptive immune system and systemic inflammation in the recipient, growth control concepts to avoid detrimental overgrowth of the porcine hearts in nonhuman primates, and lastly, the avoidance of porcine cytomegalovirus infections in donor pigs. With these strategies, consistent survival of 6-9months was achieved in the orthotopic xenotransplantation model, thereby fulfilling the prerequisites for the initiation of a clinical trial.

A Comparison Between GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;TBM Pig Kidneys Transplanted in Nonhuman Primates.

Because there is a shortage of donor kidneys, researchers are exploring the possibility of using genetically modified pig kidneys for transplantation. Approaches involving knockout of carbohydrate genes or knockin of protective proteins have been attempted to determine the best gene modifications. In this study, we utilized GalT-/-;hCD39;hCD55 and GalT-/-;hCD39;hCD46;hCD55;thrombomodulin (TBM) pigs for transplantation in nonhuman primates (NHPs). The NHPs survived for 4 weeks after kidney transplantation (4 WAT) from the GalT-/-;hCD39;hCD55 pig and for 6 WAT from the GalT-/-;hCD39;hCD46;hCD55;TBM pig. However, messenger RNA (mRNA) sequencing and immunohistochemistry analysis revealed that the 6 WAT kidney exhibited more severe apoptosis, inflammation, loss of renal function, and renal fibrosis than the 4 WAT kidney. These results indicate that additional knockin of complement regulator (hCD46) and coagulation regulator (TBM) is not enough to prevent renal damage, suggesting that improved immune suppression is needed for more prolonged survival.

The T-cell environment: may the regulatory force be with you

In the study by Sasaki etal. in this issue, the authors studied infusions of exvivo-expanded regulatory T cells in a highly clinically relevant nonhuman primate kidney transplant model. This commentary will aim to discuss the use of regulatory T cells in the wider context of transplantation, with particular emphasis on the milieu and various engineering potentials to enhance their function, as well as their relationship to other cell populations with regulatory capacity.

CD90-Targeted Cocal-Pseudotyped Lentivirus As a Robust Platform for Human HSC Gene Therapy

Ex vivo gene delivery and editing in hematopoietic stem and progenitor cells (HSPCs) provide a promising approach for the treatment of many chronic conditions such as hemoglobinopathies, lysosomal storage diseases, and HIV. However, such approaches require highly specialized biomedical infrastructure limiting its accessibility to most patients who could benefit. The development of a targeted in vivo approach to HSPC gene delivery/editing is therefore necessary for the broadened use of gene therapies. The CD34 +CD90 + subset of HSPCs represents a highly enriched population for both short-term and long-term hematopoietic lineage repopulation capacity as seen in the non-human primate (NHP) transplant model. Furthermore, ex vivo gene editing of solely CD34 +CD90 + Human and NHP hematopoietic stem cells (HSCs) was sufficient to produce therapeutic thresholds for sickle cell phenotypic amelioration after transplantation. Thus, the development of novel gene therapy vectors targeting the CD34 +CD90 + HSC population is crucial for more accessible and robust gene therapy platforms. Here we report the development of a novel CD90-targeted Cocal pseudotyped lentivirus system capable of targeted transduction of HSPCs in vitro and in vivo. Additionally, we compare transduction efficiency and specificity of this new technology with the more routinely used VSVG targeting methodology. To produce targeted lentivirus, native targeting of VSVG or Cocal envelope to the LDL-Receptor (LDLR) was knocked out by amino acid substitution. An anti-CD90 single-chain variable fragment (scFv) was introduced onto the mutated capsids using CD8 hinge and ICAM transmembrane domains. Mutagenesis of Cocal envelope LDLR binding motif was performed by Gibson cloning and confirmed via nanopore sequencing. Successful mutated viral production was assessed using hydrodynamic titration methods followed by functional analysis of transduction on CD90 + cell lines. In vitro transduction specificity and efficiency assays were performed on Human GCSF-mobilized CD34 +enriched apheresis samples and analyzed by flow cytometry. Humanized NBSGW mice were treated with targeted-lentiviral particles via intravenous (IV) and intraosseous (IO) injection. Mice were necropsied 4-8 weeks post injection. Transgene expression was analyzed by flow cytometry or luminescence imaging and confirmed by PCR on human-CD34 + FACS-sorted cells from necropsied mice. CD90-Targeted Cocal-pseudotyped lentivirus (Cocal-CD90) transduced CD34 +CD90 + HSCs 2.12-fold (p=.017) more robustly than CD90-targeted VSVG-pseudotyped lentivirus (VSVG-CD90) in vitro. Cocal-CD90 virus also showed significantly greater on-target transduction specificity of CD34 +CD90 + HSCs (p=.0008) compared to untargeted wild-type Cocal-pseudotyped lentivirus (Cocal-WT); but similar specificity to VSVG-CD90 virus (p=.118). Unlike VSVG-CD90 virus, we did not observe decreased transduction efficiency of CD34 +CD90 + HSCs using Cocal-CD90 virus in comparison to Cocal-WT virus. IV and IO injection of both CD90-targeted viruses in humanized mice resulted in transduction of immobilized human HSPCs (CD34 +CD38 -CD133 +) in the bone marrow. Transduced cells maintained multilineage differentiation capacity as seen by transgene expression of T-cell, B-cell, and myeloid compartments in the bone marrow, spleen, and peripheral blood at necropsy. As observed in vitro, Cocal-CD90 treated mice consistently displayed more robust transgene expression than VSVG-CD90 treated mice. Importantly, transduction of off-target organs (e.g., liver and lungs) post-IV injection with targeted virus was not detected by luminescence imaging during 8 weeks of follow-up or at necropsy. We provide the first evidence of targeted engineering of a knockout Cocal envelope using the attenuation of native LDLR tropism coupled with a CD90-scFv fusogen protein. Cocal-CD90 virus has increased specificity for CD90 + transduction compared to Cocal-WT lentivirus, without lowering transduction efficiency of on-target cells. Furthermore, in comparison to targeted VSVG engineering, Cocal engineered virus does not decrease transduction efficiency of HSCs in vitro or in vivo. Coupled with Cocal lentiviruses' resistance to human serum inactivation and low immunogenicity, targeted Cocal viral vectors are more suitable for future in vivo gene delivery platforms than their VSVG counterparts.

Meeting report: Xenotransplantation Development Conference in Neijiang, China.

A conference on progress in the development of xenotransplantation in China was held in Neijiang, Sichuan, in May 2023, and was attended by approximately 100 established researchers and trainees. Progress in xenotransplantation research was reviewed by both Chinese and foreign experts. The topics discussed ranged from genetic engineering of pigs and the results of pig-to-nonhuman primate organ transplantation to the requirements for designated pathogen-free (DPF) pig facilities and regulation of xenotransplantation. This conference served as an opportunity to collectively advance the development of xenotransplantation in China and pave the way for its clinical application.

Belatacept and carfilzomib-based treatment for antibody-mediated rejection in a sensitized nonhuman primate kidney transplantation model.

One-third of HLA-incompatible kidney transplant recipients experience antibody mediated rejection (AMR) with limited treatment options. This study describes a novel treatment strategy for AMR consisting of proteasome inhibition and costimulation blockade with or without complement inhibition in a nonhuman primate model of kidney transplantation. All rhesus macaques in the present study were sensitized to maximally MHC-mismatched donors by two sequential skin transplants prior to kidney transplant from the same donor. All primates received induction therapy with rhesus-specific ATG (rhATG) and were maintained on various immunosuppressive regimens. Primates were monitored postoperatively for signs of acute AMR, which was defined as worsening kidney function resistant to high dose steroid rescue therapy, and a rise in serum donor-specific antibody (DSA) levels. Kidney biopsies were performed to confirm AMR using Banff criteria. AMR treatment consisted of carfilzomib and belatacept for a maximum of four weeks with or without complement inhibitor. Treatment with carfilzomib and belatacept was well tolerated and no treatment-specific side effects were observed. After initiation of treatment, we observed a reduction of class I and class II DSA in all primates. Most importantly, primates had improved kidney function evident by reduced serum creatinine and BUN as well as increased urine output. A four-week treatment was able to extend graft survival by up to two months. In summary, combined carfilzomib and belatacept effectively treated AMR in our highly sensitized nonhuman primate model, resulting in normalization of renal function and prolonged allograft survival. This regimen may translate into clinical practice to improve outcomes of patients experiencing AMR.

Failure of Costimulatory Blockade-induced Regulatory T Cells to Sustain Long-term Survival of High Ischemic Allografts.

Costimulatory blockade-induced allograft tolerance has been achieved in rodent models, but these strategies do not translate well to nonhuman primate and clinical transplants. One confounder that may underlie this discrepancy is the greater ischemic inflammation imposed on the transplants. In mice, cardiac allografts subjected to prolonged cold ischemic storage (CIS) before transplant have increased ischemia-reperfusion injury, which amplifies infiltrating endogenous memory CD8 T-cell activation within hours after transplantation to mediate acute graft inflammation and cytotoxic lymphocyte-associated molecule-4 immunoglobulin-resistant rejection. This study tested strategies inhibiting memory CD8 T-cell activation within such high ischemic allografts to achieve long-term survival. A/J (H-2 a ) hearts subjected to 0.5 or 8 h of CIS were transplanted to C57BL/6 (H-2 b ) recipients and treatment with peritransplant costimulatory blockade. At 60 d posttransplant, regulatory T cells (Treg) were depleted in recipients of high ischemic allografts with anti-CD25 monoclonal antibody (mAb) or diphtheria toxin. Whereas peritransplant (days 0 and +1) anti-lymphocyte function-associated antigen-1 mAb and anti-CD154 mAb prolonged survival of >60% allografts subjected to minimal CIS for >100 d, only 20% of allografts subjected to prolonged CIS survived beyond day 80 posttransplant and rejection was accompanied by high titers of donor-specific antibody. Peritransplant anti-lymphocyte function-associated antigen-1, anti-tumor necrosis factor-α, and anti-CD154 mAb plus additional anti-CD154 mAb on days 14 and 16 obviated this donor-specific antibody and promoted Treg-mediated tolerance and survival of 60% of high ischemic allografts beyond day 100 posttransplant, but all allografts failed by day 120. These studies indicate a strategy inducing prolonged high ischemic allograft survival through Treg-mediated tolerance that is not sustained indefinitely.

Addition of interleukin-6 receptor blockade to carfilzomib-based desensitization in a highly sensitized nonhuman primate model

Sensitized patients, those who had prior exposure to foreign human leukocyte antigens, are transplanted at lower rates due to challenges in finding suitable organs. Desensitization strategies have permitted highly sensitized patients to undergo kidney transplantation, albeit with higher rates of rejection. This study assesses targeting plasma cell and interleukin (IL)-6 receptor for desensitization in a sensitized nonhuman primate kidney transplantation model. All animals were sensitized using two sequential skin transplants from maximally major histocompatibility complex-mismatched donors. Carfilzomib (CFZ)/tocilizumab (TCZ) desensitization (N=6) successfully decreased donor-specific antibody (DSA) titers and prevented the expansion of B cells compared to CFZ monotherapy (N=3). Dual desensitization further delayed, but did not prevent humoral rebound, as evidenced by a delayed increase in post-kidney transplant DSA titers. Accordingly, CFZ/TCZ desensitization conferred a significant survival advantage over CFZ monotherapy. A trend toward increased T follicular helper cells was also observed in the dual therapy group along the same timeline as an increase in DSA and subsequent graft loss. Cytomegalovirus reactivation also occurred in the CFZ/TCZ group but was prevented with ganciclovir prophylaxis. In accordance with prior studies of CFZ-based dual desensitization strategies, the addition of IL-6 receptor blockade resulted in desensitization with further suppression of posttransplant humoral response compared to CFZ monotherapy.

Trial in Progress: A First-in-Human Phase 1/2 Study of Hematopoietic Stem Cell Transplantation (HCT) with VOR33 (CD33-Deleted Allograft) Followed By Treatment with Gemtuzumab Ozogamicin (GO) in Patients with CD33+ Acute Myeloid Leukemia (AML) Who Are at High-Risk of Post-HCT Relapse

Background: Allogeneic HCT is the standard of care for eligible patients with AML harboring high-risk features such as adverse-risk cytogenetics or minimal residual disease (MRD). However, the majority of such patients still relapse post-HCT and have very poor outcomes. The use of maintenance therapies intended to control post-HCT relapse is often limited by hematopoietic toxicity towards normal engrafted cells. Gemtuzumab ozogamicin (GO, tradename: Mylotarg) is an FDA-approved anti-CD33 antibody-drug conjugate targeting CD33+ AML, but its use is limited by on-target hematopoietic toxicity towards CD33+ myeloid and progenitor cells. VOR33 is a CRISPR/Cas9 gene-edited allogeneic hematopoietic stem and progenitor cell (HSPC) product lacking CD33 protein that was developed to protect normal hematopoietic cells while exclusively targeting leukemia with CD33-directed therapies. VOR33 is manufactured from CD34+ cells isolated from healthy human leukocyte antigen (HLA)-matched donors. Pre-clinical murine and primate transplant studies using CD33-deleted HSPCs demonstrated unaltered long-term engraftment, multi-lineage hematopoietic differentiation and function, as well as long-term persistence of CD33 gene-edited hematopoietic cells. Additionally, subsequent treatment with GO eradicated CD33+ leukemia cells while the normal hematopoietic cells that reconstituted from CD33-edited HSCs were preserved. The improved therapeutic index for GO in combination with VOR33 has the potential to allow effective targeting of residual leukemia in AML patients post-HCT without dose-limiting hematopoietic toxicity. Study Design and Methods: VBP101(NCT04849910) is a first-in-human Phase 1/2 open-label multicenter trial to establish the safety of using VOR33 as a donor allograft for CD33+ AML patients who are at high risk of relapse and undergoing myeloablative HCT followed by treatment with low-dose GO. Participants (18-70 y) must have a diagnosis of CD33+ AML, an HLA-matched related or unrelated (10/10) donor and be a candidate for myeloablative HCT. Additional key inclusion criteria include CR1/CRi/CR2 with intermediate/poor cytogenetics and the presence of MRD. Patients with 5-10% bone marrow-only blasts with any cytogenetic risk are also eligible. Key exclusion criteria include prior auto/allo-HCT or prior treatment with GO. Related and unrelated donors undergo mobilization with G-CSF and plerixafor prior to cell collection. Manufacturing of VOR33 and isolation of an unmanipulated backup graft are performed centrally and both are cryopreserved. Subjects undergo either a busulfan- or TBI-based myeloablative conditioning regimen prior to transplantation with VOR33. After engraftment and recovery of a sufficient CD33-negative neutrophil count (~Day 60), patients begin maintenance therapy with GO. Part 1 of the study will enroll 9-18 evaluable patients to be treated in a 3+3 dose-escalation strategy with GO (0.5-2.0 mg/m2) in a 28-day cycle for up to 4 cycles. Part 1 will evaluate the safety of VOR33/GO and will determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of GO. The Part 2 expansion phase will enroll an additional 12 patients to further evaluate the safety of VOR33 and the preliminary efficacy of VOR33 and GO at the RP2D. A separate cohort is included for early relapse with treatment per the investigator's choice and may include use of GO. The primary endpoint is to assess the safety of VOR33 as measured by successful neutrophil engraftment at 28 days post-HCT. Secondary endpoints include time to neutrophil and platelet engraftment, assessment of the overall safety of VOR33 and GO (GvHD, graft failure, transplant-related mortality, RP2D, and MTD), non-hematological GO-related toxicities, GO pharmacokinetics, the efficacy of VOR33 in conferring protection from GO-related hematological toxicities, percent CD33 negative myeloid cells over time and survival rates (RFS and OS). Exploratory endpoints include the persistence of VOR33 gene editing and immune reconstitution. Study follow-up is 2 y with up to 15 y long term follow-up in a rollover safety study, VBP201 (NCT05309733). A Dose Escalation Committee and Data Safety Monitoring Committee will review GO dosing levels and monitor overall trial safety, respectively. VBP101 is currently open for enrollment and recruiting patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Competency of iPSC-derived retinas in MHC-mismatched transplantation in non-human primates.

Transplantation of embryonic/induced pluripotent stem cell-derived retina (ESC/iPSC-retina) restores host retinal ganglion cell light responses in end-stage retinal degeneration models with host-graft synapse formation. We studied the immunological features of iPSC-retina transplantation using major histocompatibility complex (MHC)-homozygote monkey iPSC-retinas in monkeys with laser-induced retinal degeneration in MHC-matched and -mismatched transplantation. MHC-mismatched transplantation without immune suppression showed no evident clinical signs of rejection and histologically showed graft maturation without lymphocytic infiltration, although immunological tests using peripheral blood monocytes suggested subclinical rejection in three of four MHC-mismatched monkeys. Although extensive photoreceptor rosette formation was observed on histology, evaluation of functional integration using mouse models such as mouse ESC-retina (C57BL/6) transplanted into rd1(C3H/HeJ, MHC-mismatched model) elicited light responses in the host retinal ganglion cells after transplantation but with less responsiveness than that in rd1-2J mice (C57BL/6, MHC-matched model). These results suggest the reasonable use of ESC/iPSC-retina in MHC-mismatched transplantation, albeit with caution.

Identification of Nonhuman Primate Hematopoietic Stem and Progenitor Cells.

The preclinical development of hematopoietic stem cell (HSC) gene therapy/editing and transplantation protocols is frequently performed in large animal models such as nonhuman primates (NHPs). Similarity in physiology, size, and life expectation as well as cross-reactivity of most reagents and medications allows for the development of treatment strategies with rapid translation to clinical applications. Especially after the adverse events of HSC gene therapy observed in the late 1990s, the ability to perform autologous transplants and follow the animals long-term make the NHP a very attractive model to test the efficiency, feasibility, and safety of new HSC-mediated gene-transfer/editing and transplantation approaches.This protocol describes a method to phenotypically characterize functionally distinct NHP HSPC subsets within specimens or stem cell products from three different NHP species. Procedures are based on the flow-cytometric assessment of cell surface markers that are cross-reactive in between human and NHP to allow for immediate clinical translation. This protocol has been successfully used for the quality control of enriched, cultured, and gene-modified NHP CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as sort-purified CD34 subsets for transplantation in the pig-tailed, cynomolgus, and rhesus macaque. It further allows the longitudinal assessment of primary specimens taken during the long-term follow-up post-transplantation in order to monitor homing, engraftment, and reconstitution of the bone marrow stem cell compartment.