Bcl-2 and Mcl-1 inhibition for induction of hematopoietic chimerism and renal allograft tolerance without myelosuppression in nonhuman primates

摘要 器官移植已经成为许多终末期疾病的标准护理手段，但目前 需要终生服用有效的免疫抑制药物。这导致发病率增加。 以及因感染、恶性肿瘤和其他代谢紊乱而死亡。建立可靠的方法 在没有持续免疫抑制的情况下实现同种异体移植物存活(I.S.)仍然是一个重要的目标。 我们之前报道过人类移植肾长期无缺血缺乏症存活的结果。 仅通过供者骨髓移植诱导一过性造血细胞嵌合体。 为了扩大我们的方法的应用，必须提高水平和一致性 与当前条件调节相关的不增加骨髓抑制的造血细胞嵌合体 养生法。我们已经在非人类灵长类动物中确定了一种新的策略，通过 B细胞淋巴瘤-2抑制剂(Bcl2)促进选择性造血细胞的内源性凋亡 2i)。这种方法显著提高了嵌合体水平和持续时间，并实现了无I.S.肾功能 无中性粒细胞减少和血小板减少的同种异体移植物存活。这些研究确实揭示了 共刺激阻断(CB)仍然是诱导Bcl2I耐受所必需的。因此，我们将 首先定义仅使用FDA批准(或正在FDA批准的过程中)CB的协议，包括 1)抗CD2单抗；2)Fc修饰的抗CD154单抗；最近，在支持下， 在一项探索性的R21赠款中，我们发现造血嵌合体的诱导似乎是 即使没有任何化疗/放射治疗，如果造血干细胞被充分耗尽，也是可能的 BM利基细胞与另一种抑制髓系细胞的促凋亡药物联合使用 白血病1(Mcl-1)。因此，在我们的提案中，我们将进一步追求最终目标，以促使 不含任何放射或化疗药物的造血嵌合体。 对于更广泛的临床适用性也具有重要意义，我们将推广最成功的 基于BCL-2I的协议与我们新的“延迟容忍”方法相结合。这将是第一次 使用冷冻保存的已故供者同种异体移植物的耐受性诱导策略 BM，以及正在进行的活体肾移植受者，他们的肾脏捐赠者可以提供 造血干细胞。 最后，我们将阐明成功的无iS.肾移植的机制。 通过短暂性造血细胞嵌合体和促凋亡剂存活，利用广泛的体外和 用新的免疫学方法进行活体实验。

SUMMARY Organ transplantation has become the standard of care for many end-state diseases, but currently requires life-long administration of potent immunosuppressive drugs. This results in increased morbidity and mortality from infection, malignancy and other metabolic disorders. Establishing a reliable method to achieve allograft survival without ongoing immunosuppression (I.S.) remains an important goal. We previously reported achievement of long-term I.S.-free renal allograft survival in humans after induction of only transient hematopoietic chimerism through donor bone marrow (BM) transplantation. To expand the application of our approach, it is imperative to improve the levels and consistency of hematopoietic chimerism without increasing myelosuppression associated with the current conditioning regimen. We have identified a novel strategy in nonhuman primates that addresses this obstacle by enhancing intrinsic apoptosis of selective hematopoietic cells using a B cell lymphoma-2 inhibitor (Bcl- 2i). This approach significantly improves chimerism levels and duration and achieves I.S.-free renal allograft survival without neutropenia and thrombocytopenia. These studies did reveal that costimulatory blockade (CB) remains essential for tolerance induction with Bcl-2i. Therefore, we will first define a protocol using only FDA approved (or in the process of being FDA approved) CB, including 1) anti-CD2 mAb, 2) Fc-modified anti-CD154 mAb, and 3) Belatacept. More recently, with the support of an exploratory R21 grant, we have found that induction of hematopoietic chimerism appears to be possible even without any chemo/radiation therapy, if hematopoietic stem cells are adequately depleted from BM niches with a Bcl-2i in combination with another proapoptotic agent that inhibits Myeloid cell leukemia 1 (Mcl-1). In our proposal, we will therefore further pursue the ultimate goal to induce hematopoietic chimerism without any radiation or chemotherapeutic drugs. Also of major importance for more widespread clinical applicability, we will extend the most successful Bcl-2i based protocol to our novel “delayed tolerance” approach. This will, for the first time, make tolerance induction strategies available to recipients of deceased donor allografts using cryopreserved BM, as well as to ongoing living donor transplant recipients whose kidney donor is available to provide hematopoietic stem cells. Finally, we will elucidate the mechanistic pathways involved in successful I.S.-free renal allograft survival by transient hematopoietic chimerism and proapoptotic agents, utilizing extensive in vitro and in vivo experiments with novel immunological approaches.