Novel CART Cells for Treating AML

项目摘要 化疗耐药性仍然是成功治疗急性髓系白血病患者的主要障碍。 白血病（AML），导致高复发率和死亡率。开发更有效的治疗方法 对于AML来说，这是必要的，特别是具有替代作用机制的疗法， 化学抗性使用靶向CD19的嵌合抗原受体（CAR）的连续性细胞免疫疗法- 表达T细胞的人T淋巴细胞白血病已经显著改善了患有多次复发/难治性B细胞白血病的患者的治疗。 淋巴母细胞性白血病（B-ALL）和淋巴瘤，并被FDA批准。成功的翻译 AML免疫疗法的发展已经落后，并且仍然是一个重大的未满足的医疗需求。迄今为止， 针对AML靶向CD33或CD123的细胞已经显示出有效的临床前抗AML活性，但也诱导了严重的 通过对造血干细胞（HSC）的靶向/非靶向肿瘤损伤的骨髓毒性。我们已经开发了一个 分离优先结合AML细胞的单域纳米抗体（Nb）的创新系统， 同源CAR T细胞来杀死癌细胞。这些纳米抗体之一，Nb 157，特异性结合到细胞表面 蛋白CD13（氨肽酶N），通常在成人AML标本和白血病干细胞中上调 （LSC）。在初步研究中，我们证明了Nb157/CD13 CAR T细胞（CD13CART）有效地 在临床前动物模型中根除AML细胞。TIM-3是某些免疫细胞的抑制性受体， 在AML母细胞和LSC中上调，但在人HSC中不表达。于是，我们创造了第一个 产生双特异性和分裂的CD13/TIM-3 CART（第一代G bCART），并证明bCART有效地 在临床前动物模型中根除AML细胞，对HSC的毒性显著降低。进一步提高 bCART的安全性特征，第二代bCART已经产生，它们没有诱导明显的 在我们的离体分析中对HSC的毒性。我们假设，进一步开发双特异性或诱导性的 双特异性CART可以在患者来源的异种移植物（PDX）模型中根除AML， 毒性提出了三个具体目标来检验这一假设。目的1将评估第二代双特异性抗体。 CD13/TIM-3CART（bCART）在最大化选择性AML杀伤中的作用。目标2将研究有效性和特异性 诱导型CD13/TIM-3CART（ibCART）在杀伤AML细胞中的作用。目的3：开发CLL-1/TIM-3双特异性抗体 bCART选择性杀死AML细胞。从这些研究中获得的结果是迫切需要翻译的 复发/难治性AML患者的临床治疗。

Project Summary Chemotherapy resistance remains a major barrier to successful treatment of patients with acute myeloid leukemia (AML), contributing to high rates of relapse and mortality. Development of more effective treatments for AML is imperative, particularly therapies with alternative mechanisms of action to circumvent chemoresistance. Adoptive cellular immunotherapy using CD19-targeting chimeric antigen receptor (CAR)- expressing T cells has drastically improved the treatment of patients with multiply relapsed/refractory B-cell lymphoblastic leukemia (B-ALL) and lymphoma and was approved by the FDA. However, successful translation of AML immunotherapies has lagged behind and remains a significant unmet medical need. To date, CAR T cells targeting CD33 or CD123 for AML have shown potent preclinical anti-AML activity, but also induced severe myelotoxicity via on target/off tumor damage to hematopoietic stem cells (HSCs). We have developed an innovative system to isolate single-domain nanobodies (Nb) that preferentially bind AML cells and enable cognate CAR T cells to kill the cancer cells. One of these nanobodies, Nb157, specifically binds to the cell surface protein CD13 (aminopeptidase N), which is often upregulated in adult AML specimens and leukemia stem cells (LSCs). In preliminary studies, we demonstrated that Nb157/CD13 CAR T cells (CD13CARTs) potently eradicated AML cells in preclinical animal models. TIM-3, an inhibitory receptor of certain immune cells, is upregulated in AML blast cells and LSCs, but not expressed in human HSCs. Thus, we generated the 1st generation bispecific and split CD13/TIM-3 CARTs (1st G bCARTs) and demonstrated that the bCARTs potently eradicated AML cells in preclinical animal models, with significantly reduced toxicity to HSCs. To further improve the safety profile of the bCARTs, the 2nd generation bCARTs were generated and they did not induce obvious toxicity to HSCs in our ex vivo analysis. We hypothesize that further development of the bispecific or inducible bispecific CARTs can eradicate AML in patient-derived xenograft (PDX) models with little or tolerable off-tumor toxicity. Three specific aims are proposed to test this hypothesis. Aim 1 will evaluate the 2nd generation bispecific CD13/TIM-3CARTs (bCARTs) in maximizing selective AML killing. Aim 2 will investigate efficacy and specificity of inducible CD13/TIM-3CARTs (ibCARTs) in killing AML cells. Aim 3 will develop bispecific CLL-1/TIM-3 bCARTs to selectively killing AML cells. Results obtained from these studies are imminently translatable to the clinic in the near future for patients with relapsed/refractory AML.