Novel non-genotoxic ligand-based CD117-directed CAR T conditioning in the context of hematopoietic stem cell transplantation and leukemia treatment

项目总结 研究治疗晚期急性髓系白血病(AML)的改进疗法至关重要，因为近 30%的儿童将复发或患有顽固性疾病，使存活率降至仅20%。造血术 干细胞移植(HSCT)是治疗复发AML的有效方法，但骨髓的遗传毒性 条件反射在其使用中是一个很大的障碍。目前的BM调理方案包括 丁硫丹等烷化化疗药物与大剂量全身照射的联合应用 (待定)。类似于用作AML一线治疗的非靶向化疗药物，这些条件作用 药物也是高度遗传毒性的，在造血室之外有几种有害的毒性， 比如器官衰竭和继发性恶性肿瘤。相比之下，细胞疗法为 在复发环境中使用靶向治疗，FDA总共批准了五个嵌合抗原受体 (CAR)用于治疗复发癌症的产品，其中三种在过去一年内获得批准。 因此，我们假设，非遗传毒性调节方案的发展特别针对 造血干细胞(HSCs)和急性髓系白血病(AML)将对疾病的治疗产生革命性的影响。C-的用法 试剂盒(CD117)作为非遗传毒性调节的靶点已经被我们的实验室和其他实验室探索，因为它 在造血干细胞上的表达。高达90%的AML患者有c-kit的表达，这种表达与不良相关 预后和对化疗的耐药性。我们假设专门针对c-kit治疗急性髓系白血病 除了作为造血干细胞移植前的一种非遗传毒性调节方案外，还将减轻肿瘤负担。这 建议探索一种新的基于配体的c-kit定向CAR(SCF CAR)来靶向HSC(特定目标1) 和AML(特异性靶2)，利用c-kit受体的天然配体干细胞因子作为重组 我们汽车的抗原结合区。重要的是，配体提供了对受体-配体的更多了解 相互作用，并可能通过增强蛋白质稳定性来减少紧张性CAR信号，导致T细胞减少 细胞耗尽，以及其他关键优势。此外，我们的实验室还优化了γδT细胞作为 ⍺βT细胞的细胞毒性替代品。γδT细胞对这种情况特别有益，因为它们i)是先天免疫的 不形成与⍺βT细胞相同程度的记忆T细胞表型的细胞，II)不会引起移植物抗宿主 疾病(GvHD)当跨越主要组织相容(MHC)障碍移植时，III)对先天 通过移植物与白血病的相互作用杀伤白血病细胞，因为γδT细胞可以识别癌细胞应激 抗原，和iv)已被证明可以提高存活率和减少HSCT后白血病的复发。因此，我们 建议利用γδT细胞的细胞毒能力来利用这些效应，因为我们的实验室已经优化了 通过开发无血清方案体外扩增和修饰γδT细胞，使我们的实验室 非常适合进行这些实验。我们的目标是将CAR T疗法的使用推广到患有 并阐明癌症和骨髓调理联合治疗的更广泛应用。

PROJECT SUMMARY It is essential to study improved therapies for the treatment of advanced acute myeloid leukemia (AML), as nearly 30% of children will relapse or have refractory disease, bringing the survival down to a mere 20%. Hematopoietic stem cell transplantation (HSCT) is curative for relapsed AML; however, the genotoxicity of bone marrow (BM) conditioning represents a substantial barrier in its use. Current BM conditioning regimens consist of a combination of alkylating chemotherapeutic agents such as busulfan and high doses of total body irradiation (TBI). Similar to the non-targeted chemotherapeutics used as first-line treatment for AML, these conditioning agents are also highly genotoxic and have several harmful toxicities outside of the hematopoietic compartment, such as organ failure and secondary malignancies. In contrast, cellular therapies provide strong support for the use of targeted therapies in the relapsed setting, as evident by five total FDA approved chimeric antigen receptor (CAR) products for the treatment of relapsed cancers, three of which have been approved within the last year. Therefore, we hypothesize that the development of non-genotoxic conditioning regimens that specifically target hematopoietic stem cells (HSCs) and AML will be transformative to the treatment of the disease. The use of c- kit (CD117) as a target for non-genotoxic conditioning has been explored by our lab and others due to its expression on HSCs. Up to 90% of AML patients have c-kit expression, and this expression correlates with poor prognosis and resistance to chemotherapy. We hypothesize that specifically targeting c-kit for AML treatment will reduce tumor burden in addition to serving as a non-genotoxic conditioning regimen prior to HSCT. This proposal seeks to explore a novel ligand-based c-kit directed CAR (SCF CAR) to target HSCs (Specific Aim 1) and AML (Specific Aim 2) by utilizing the c-kit receptor’s natural ligand stem cell factor (SCF) as the recombinant antigen binding domain of our CAR. Importantly, ligands offer a greater understanding of receptor-ligand interactions and can potentially reduce tonic CAR signaling through enhanced protein stability, leading to less T cell exhaustion, among other key advantages. Furthermore, our lab has optimized the use of γδ T cells as a cytotoxic alternative to ⍺β T cells. γδ T cells are uniquely beneficial for this setting since they i) are innate immune cells that do not form memory T cell phenotypes to the same extent as ⍺β T cells, ii) do not cause graft-vs-host disease (GvHD) when transplanted across major histocompatibility (MHC) barriers, iii) contribute to the innate killing of leukemia cells via graft-vs-leukemia (GvL) interactions, as γδ T cells can recognize cancer cell stress antigens, and iv) have been shown to enhance survival and decrease leukemia relapse post-HSCT. We therefore propose utilizing the cytotoxic abilities of γδ T cells to capitalize on these effects, as our lab has optimized the ex vivo expansion and modification of γδ T cells via the development of a serum-free protocol, making our lab uniquely suited to perform these experiments. Our goal is to advance the use of CAR T therapy to patients with AML and to elucidate the broader application of a combination cancer and bone marrow conditioning treatment.