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

项目摘要 研究改善治疗晚期急性髓细胞白血病（AML）的疗法至关重要，因为近几年来， 30%的儿童会复发或患有难治性疾病，使生存率下降到仅20%。造血 干细胞移植（HSCT）是治愈复发性AML;然而，骨髓（BM）的遗传毒性 调节在其使用中是一个实质性的障碍。目前的BM预处理方案包括： 烷基化化疗剂如白消安和高剂量全身照射的组合 （TBI）。与用作AML一线治疗的非靶向化疗药物类似，这些条件治疗可用于治疗急性粒细胞白血病。 试剂也是高度遗传毒性的并且在造血区室之外具有几种有害毒性， 例如器官衰竭和继发性恶性肿瘤。相比之下，细胞疗法为治疗癌症提供了强有力的支持。 在复发背景下使用靶向治疗，如FDA批准的总共五种嵌合抗原受体所证明的， (CAR)用于治疗复发性癌症的产品，其中三种在去年获得批准。 因此，我们假设，非遗传毒性的预处理方案，具体目标的发展， 造血干细胞（HSC）和AML的联合应用将对疾病的治疗具有变革性意义。使用C- 试剂盒（CD 117）作为非遗传毒性调节的靶点，由于其 HSC的表达。高达90%的AML患者有c-kit表达，并且这种表达与AML患者的不良预后相关。 预后和对化疗的抵抗。我们假设特异性靶向c-kit治疗AML 除了作为HSCT前的非遗传毒性预处理方案外，还将降低肿瘤负荷。这 一项提案旨在探索一种新的基于配体的c-kit定向CAR（SCF CAR），以靶向HSC（特异性目的1） 和AML（Specific Aim 2），利用c-kit受体的天然配体干细胞因子（SCF）作为重组 我们的CAR的抗原结合结构域。重要的是，配体提供了更好的理解受体-配体 相互作用，并可能通过增强蛋白质稳定性来减少紧张性CAR信号传导，导致T 细胞衰竭，以及其他关键优势。此外，我们的实验室已经优化了γδ T细胞的使用， 细胞毒性替代物，以替代α β T细胞。γδ T细胞对于这种情况是独特有益的，因为它们i）是先天免疫的， 不形成与β-T细胞相同程度的记忆T细胞表型的细胞，ii）不引起移植物抗宿主 iii）当跨越主要组织相容性（MHC）屏障移植时，导致先天性GvHD， 通过移植物抗白血病（GvL）相互作用杀死白血病细胞，因为γδ T细胞可以识别癌细胞应激 抗原，和iv）已经显示出提高HSCT后的存活率和减少白血病复发。因此我们 我建议利用γδ T细胞的细胞毒性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γδ T细胞，使我们的实验室 非常适合做这些实验我们的目标是将CAR T疗法的使用推广到患有 AML和阐明联合癌症和骨髓调理治疗的更广泛应用。

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.