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

总结 器官移植已成为许多终末期疾病的标准治疗方法，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 以及感染、恶性肿瘤和其他代谢紊乱的死亡率。建立可靠的方法 在不进行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。 我们以前报告过长期IS的实现-移植肾存活率 通过供体骨髓（BM）移植仅诱导短暂造血嵌合体。 为了扩大我们的方法的应用，必须提高 造血嵌合体，不增加与当前条件相关的骨髓抑制 方案.我们已经在非人类灵长类动物中发现了一种新的策略，可以通过以下方式解决这一障碍： 使用B细胞淋巴瘤-2抑制剂（Bcl-2）增强选择性造血细胞的内在凋亡， 2i）。这种方法显著改善了嵌合水平和持续时间，并实现了I.S.-游离肾 无中性粒细胞减少症和血小板减少症的同种异体移植物存活。这些研究确实表明， 共刺激阻断（CB）对于Bcl-2 i诱导耐受仍然是必需的。所以我们会 首先使用FDA批准的（或正在获得FDA批准的）CB定义方案，包括 1)抗-CD 2 mAb，2）Fc-修饰的抗-CD 154 mAb，和3）贝拉西普。最近，在支持 在探索性的R21资助中，我们发现造血嵌合体的诱导似乎是 即使没有任何化疗/放疗，如果造血干细胞被充分耗尽， 与Bcl-2 i联合另一种抑制骨髓细胞凋亡的促凋亡剂 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，以诱导 造血嵌合体，无需任何放疗或化疗药物。 对于更广泛的临床适用性也具有重要意义，我们将扩展最成功的 Bcl-2 i为基础的协议，我们的新的“延迟耐受”的方法。这将首次使 采用冷冻保存的死亡供体同种异体移植物的耐受诱导策略 BM，以及正在进行的活体供体移植受者，其肾脏供体可提供 造血干细胞 最后，我们将阐明成功的I.S.-游离移植肾 通过瞬时造血嵌合体和促凋亡剂的存活，利用广泛的体外和 用新的免疫学方法进行体内实验。

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.