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同种异体移植物诱导免疫耐受是免疫治疗的圣杯。 移植医学诱导对不匹配移植物的免疫耐受将使生命的需要减少- 长期免疫抑制与严重不良结局相关，如肾衰竭、癌症 和感染目前，最有希望的耐受性诱导方法是通过建立混合的 嵌合状态通过移植供体造血干细胞;然而，除了致耐受性 尽管混合嵌合体方法在诸如肾脏的器官中没有实现持久的免疫耐受， 大多数实体器官或血管复合同种异体移植物（VCA）的临床前或临床试验。令人鼓舞的是， 尽管如此，我们已经成功地实现了减少免疫抑制的VCA的临床试验，使用这种 approach. 间充质干（基质）细胞（MSC）是干细胞移植（SCT）的潜在有用的佐剂，用于治疗骨髓增生异常综合征。 促进混合嵌合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转化之前，还有许多未解决的问题需要解决。 治疗细胞一个主要的障碍是MSC的来源，MSC在所有组织中都很罕见，需要侵入性的移植。 采购程序。低丰度要求文化的广泛扩张，以产生足够的 人给药的数量。在临床环境中已经观察到，扩张程度是 与结果呈负相关。 骨健康已经克服了这一障碍，确定了一个丰富的来源，主要的MSC相关， 从死亡的器官捐献者身上获得的脊椎体的髓骨。这些脊椎骨粘连 MSC（vBA-MSC）通过骨碎片的蛋白水解消化分离，随后洗脱和冷冻保存 骨髓（BM）。初级vBA-MSC以比初级vBA-MSC高3个数量级的数量获得。 可以从活体供体髂嵴骨髓穿刺液中回收。vBA-MSC的另一个优势是 与供体匹配，而不是第三方MSC，这提高了安全性和潜在的有效性。隔离 来自30多个供体的vBA-MSC的表征表明， BM-MSC，但是，由于它们的高数量，与传统的BM-MSC不同，可以扩展到> 50亿个细胞 在培养物中仅传2代。 我们假设供体匹配的vBA-MSC将增强混合嵌合体的耐受机制， 骨髓移植以及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 首先在鼠原位后肢移植VCA模型中检验假设，然后在鼠原位后肢移植VCA模型中检验假设。 异位心脏模型后肢模型研究将使我们能够评估MSC免疫的机制， 由于含BM的后肢的致耐受性性质而导致的耐受。将对耐久性移植物进行T细胞 动态（特别是记忆T细胞和调节性T细胞）和供体特异性免疫耐受， 经捐赠者和第三方皮肤移植证实。从本研究中获得的信息将用于 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，结合大量的MSC临床试验以及悠久的历史， 移植耐受性试验和我们未来的II期研究，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