Novel CART Cells for Treating AML

项目概要 化疗耐药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 来说势在必行，特别是采用替代作用机制的疗法来规避 化学耐药性。使用 CD19 靶向嵌合抗原受体 (CAR) 的过继细胞免疫疗法 - 表达 T 细胞极大地改善了多重复发/难治性 B 细胞患者的治疗 淋巴细胞白血病（B-ALL）和淋巴瘤，并获得 FDA 批准。不过翻译成功了 AML 免疫疗法的应用已经落后，并且仍然是一个重大的未满足的医疗需求。迄今为止，CAR-T 针对 AML 的 CD33 或 CD123 细胞已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/脱靶肿瘤损伤产生骨髓毒性。我们开发了一个 分离单域纳米抗体 (Nb) 的创新系统，该纳米抗体优先结合 AML 细胞并能够 同源 CAR T 细胞杀死癌细胞。其中一种纳米抗体 Nb157 特异性结合细胞表面 蛋白质 CD13（氨肽酶 N），在成人 AML 样本和白血病干细胞中经常上调 （LSC）。在初步研究中，我们证明 Nb157/CD13 CAR T 细胞 (CD13CART) 有效 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 中表达上调，但在人 HSC 中不表达。因此，我们生成了第一个 生成双特异性和分裂 CD13/TIM-3 CART（1st G bCART），并证明 bCART 有效 在临床前动物模型中根除 AML 细胞，并显着降低对 HSC 的毒性。为进一步提高 bCART 的安全性，第二代 bCART 已生成，并且没有引起明显的副作用 在我们的离体分析中对 HSC 的毒性。我们假设双特异性或诱导型的进一步发展 双特异性 CART 可以根除患者来源的异种移植 (PDX) 模型中的 AML，且肿瘤外很少或可耐受 毒性。提出了三个具体目标来检验这一假设。目标 1 将评估第二代双特异性 CD13/TIM-3CART (bCART) 最大限度地选择性杀死 AML。目标 2 将调查功效和特异性 诱导型 CD13/TIM-3CART (ibCART) 杀死 AML 细胞的作用。目标 3 将开发双特异性 CLL-1/TIM-3 bCART 选择性杀死 AML 细胞。从这些研究中获得的结果可以立即转化为 在不久的将来，该诊所将为复发/难治性 AML 患者提供服务。

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.