Developing bispecific CAR Ts for treating AML

项目摘要 急性髓系白血病(AML)具有高度侵袭性，大多数AML患者最终变得难治性 接受化疗并死于恶性疾病。由于急性髓系白血病的病因学非常不同，因此具有挑战性 开发一种有效的方法来治疗大多数AML患者。使用嵌合抗原的过继细胞治疗 受体(CAR)表达的T细胞在治疗淋巴细胞性白血病和淋巴瘤方面非常成功 靶向CD19，最近已被FDA批准用于临床。然而，类似的方法已经 尚未被广泛探索以取得急性髓系白血病的成功。一个亟待满足的需求是开发新的 针对急性髓细胞白血病的方法，以改善治疗。我们最近开发了一种创新的系统来分离 结合AML细胞并使同源CAR T细胞杀死癌细胞的抗体。使用这个系统， 我们成功地从免疫的骆驼中分离出能与单个结构域结合表位的纳米体。 其中两个纳米小体与细胞表面的一种蛋白水解酶特异结合，该酶在80%的急性髓系白血病细胞中表达。 急性髓系白血病患者。值得注意的是，CAR T细胞有效和特异地靶向细胞表面的蛋白水解酶 在体外和AML细胞系来源的异种移植中根除AML细胞。为了控制潜在的击中目标，离开目标- 为了进一步研究CAR T细胞的组织毒性，我们进一步开发了条件诱导的CAR以及双特异性和 分离CAR T细胞，在体内外杀伤AML细胞。我们假设纳米车身导向的汽车T 系统可以根除AML患者来源的异种移植物(PDX)，这种方法可以进一步发展到 以可耐受的毒性治疗AML。提出了两个具体的目标来检验这一假设：目标1将调查 纳米体157(Nb157)导向的CAR T细胞对人急性髓系白血病原代细胞的杀伤作用 临床前模型。Aim 2将改进和评估Nb157导向的CAR，并结合其他AML- 联合抗原特异性分裂CAR T增加AML患者来源白血病的AML特异性杀伤作用 细胞，对正常细胞的毒性降低。这些研究可能会建立一种新颖和安全的汽车系统，以 可控地根除AML，为显著改善AML治疗铺平道路。

Project Summary Acute myeloid leukemia (AML) is highly aggressive, and majority of AML patients eventually become refractory to chemotherapy and succumb to the malignancy. As etiology for AML is very heterogeneous, it is challenging to develop an effective approach to treat majority of AML patients. Adoptive cell therapy using chimeric antigen receptor (CAR)-expressing T cells is very successful for treating lymphocytic leukemia and lymphoma by targeting CD19, and has recently been approved by FDA for clinical use. However, the similar approach has not yet been extensively explored to achieve success for AML. A sorely unmet need is to develop new approaches to target AML to improve therapy. We have recently developed an innovative system to isolate antibodies that bind AML cells and enable the cognate CAR T cells to kill the cancer cells. Using this system, we successfully isolated nanobodies, which can bind epitopes with a single domain, from immunized llama. Two of the nanobodies specifically bind to a cell surface protease, which is expressed in AML cells from > 80% of AML patients. Notably, the CAR T cells targeting the cell surface protease potently and specifically eradicated AML cells in vitro and in AML cell line-derived xenograft. To control the potential on-target, off- tissue toxicity of the CAR T cells, we further developed a conditionally inducible CAR as well as bispecific and split CAR T cells to kill AML cells in vitro and in vivo. We hypothesize that the nanobody-directed CAR T system can eradicate AML patient derived xenografts (PDX), and this approach can be further developed to treat AML with tolerable toxicity. Two specific aims are proposed to test this hypothesis: Aim 1 will investigate the impact of the nanobody 157 (Nb157)-directed CAR T cells on eradicating primary human AML cells in preclinical models. Aim 2 will improve and evaluate the Nb157-directed CAR, in combination with other AML- associating antigen-specific split CAR T to increase AML-specific killing of the AML patient-derived leukemia cells, with reduced toxicity to normal cells. These studies will likely establish a novel and safe CAR system to controllably eradicate AML, paving the way to significantly improve AML therapy.