A Novel and Clinically Feasible Co-therapy of Deceased Donor Bone Marrow Combined With Donor-Matched Mesenchymal Stem Cells to Establish Immune Tolerance

摘要 用实体器官和血管复合移植物诱导免疫耐受是治疗 移植医学。对不匹配的移植物诱导免疫耐受将消除对生命的需求- 长期免疫抑制，与严重的不良后果有关，如肾功能衰竭、癌症 和感染。目前最有希望的耐受诱导方法是通过建立混合的 供体造血干细胞移植的嵌合状态；然而，耐受性除外 对于肾脏等器官，混合嵌合体方法尚未实现持久的免疫耐受 大多数实体器官或血管复合异体移植(VCA)的临床前或临床试验。令人鼓舞的是， 然而，我们已经成功地在VCA的临床试验中实现了减少免疫抑制的目的 接近。 间充质干细胞(MSC)是干细胞移植(SCT)的一种潜在有用的佐剂。 促进混合嵌合体以及促进互补的外周免疫调节功能。 然而，在这些有希望的临床翻译之前，有许多问题需要解决 治疗性细胞。主要的障碍是MSC的来源，MSC在所有组织中都很罕见，需要侵入性 采购程序。低丰度要求在文化上广泛扩张，以产生足够的 人体剂量的数字。在临床环境中已经观察到扩张的程度是 与结果呈负相关。 铯健康公司通过鉴定与以下相关的初级MSC的丰富来源克服了这一障碍 从已故的器官捐赠者那里获得的椎体的髓质骨。这些脊椎骨粘附物 骨髓间充质干细胞(VBA-MSC)是通过蛋白水解骨碎片，洗脱和冷冻保存后分离出来的 骨髓(BM)。初级VBA-MSC的数值比 可回收活体供体髂骨骨髓抽吸物。VBA-MSC的另一个优势是 与供者匹配，而不是第三方MSC，这提高了安全性和潜在的疗效。隔离 而来自30多名捐赠者的VBA-MSC的鉴定表明，这些细胞与 BM-MSC，但由于其数量多，与传统的BM-MSC不同，可扩增至50亿个细胞 在培养上只有两代人。 我们假设供者匹配的VBA-MSC将增强混合嵌合体的耐受机制 并提供外周免疫调节功能，以实现持久的耐受性 主要组织相容性复合体不匹配的实体器官和血管复合组织移植。这 假设将首先在小鼠原位后肢移植VCA模型中进行验证，然后在小鼠身上进行验证 异位心脏模型。后肢模型研究将使我们能够评估MSC的免疫机制。 耐受性源于含有BM的后肢的耐受性。将对耐久移植物进行T细胞评估 BE的动态(尤其是记忆T细胞和调节性T细胞)与供体特异性免疫耐受 与供者和第三方皮肤移植确认。从这项研究中获得的信息将用于执行 在我们的异位心脏移植模型中进行了类似的研究。 如果成功，这项研究的结果，结合了大量的MSC临床试验以及悠久的历史 移植耐受试验和我们未来的第二阶段研究，以进一步确定小剂量给药参数 和大型动物模型，将为FDA进行临床试验提供令人信服的论据。 关键词：同种异体血管复合移植；实体器官移植；免疫耐受； 免疫调节、调节性T淋巴细胞

ABSTRACT Induction of immune tolerance with solid organ and vascular composite allografts is the Holy Grail for transplantation medicine. Induction of immune tolerance to mismatched grafts would obviate the need for life- long immunosuppression which is associated with serious adverse outcomes, such as renal failure, cancers and infections. Currently the most promising means of tolerance induction is through establishing a mixed chimeric state by transplantation of donor hematopoietic stem cells; however, with the exception of tolerogenic organs such as kidneys, the mixed chimerism approach has not achieved durable immune tolerance in preclinical or clinical trials with most solid organs or vascular composite allotransplants (VCA). Encouragingly, though, we have succeeded in achieving reduced immunosuppression in clinical trials of VCA using this approach. Mesenchymal stem (stromal) cells (MSC) are a potentially useful adjuvant to stem cell transplants (SCT) for promoting mixed chimerism as well as promoting complementary peripheral immunomodulatory functions. However, there are many unresolved issues to address before clinical translation of these promising therapeutic cells. A primary impediment is the source of MSC, which are rare in all tissues and require invasive procedures for procurement. Low abundance mandates extensive expansion in culture to generate sufficient numbers for human dosing. It has been observed in the clinical setting that the degree of expansion is negatively correlated with outcomes. Ossium Health has overcome this obstacle by identifying an abundant source of primary MSC associated with medullary bones of vertebral bodies obtained from deceased organ donors. These vertebral bone adherent MSC (vBA-MSC) are isolated by proteolytic digestion of bone fragments, following elution and cryopreservation of bone marrow (BM). Primary vBA-MSC are obtained at numbers that are 3 orders of magnitude higher than can be recovered from living donor iliac crest BM aspirates. A further advantage of vBA-MSC is they are matched to the donor, as opposed to third-party MSC, which enhances safety and potentially efficacy. Isolation and characterization of vBA-MSC from over 30 donors has demonstrated that the cells are no different than BM-MSC, but, because of their high numbers, unlike traditional BM-MSC, can be expanded to >5 billion cells with only 2 passages in culture. We hypothesize that donor-matched vBA-MSC will augment tolerance mechanisms of mixed chimerism with BM transplant as well as provide peripheral immunomodulatory functions to achieve durable tolerance for major histocompatibility complex mismatched solid organ and vascular composite tissue transplants. This hypothesis will be tested first in a murine orthotopic hind limb transplant VCA model and then in a murine heterotopic heart model. The hindlimb model studies will allow us to evaluate mechanisms of MSC immune tolerance due to the tolerogenic nature of BM-containing hind limbs. Durable grafts will be evaluated for T cell dynamics (especially memory T cells and regulatory T cells) and donor-specific immune tolerance with be confirmed with donor and third-party skin grafting. Information gained from this study will be used to perform similar studies in our heterotopic heart transplant model. If successful, the results of this study, combined with a plethora of MSC clinical trials as well as a long history of transplantation tolerance trials and our future Phase II studies to further define dosing parameters in small and large animal models, will provide compelling arguments to FDA for proceeding to clinical trials. Keywords: vascular composite allotransplantation; solid organ transplantation; immune tolerance; immunomodulation, regulatory T lymphocytes