Although chimeric antigen receptor CAR) T cell immunotherapies are an undeniable and unequivocal success, knowledge obtained from the monitoring of the first clinical trials targeting the CD19 antigen in B malignancies, either refractory/relapsed acute lymphoid leukemia (ALL) or lymphomas, contributed to the identification of tumor cell escape in about 30–50% of B-ALL. Resistance occurred due to loss of surface expression of the antigen (rCD19−) or to the early disappearance or inactivation of CAR T cells (rCD19+). In a recently reported clinical case, rCD19− relapse resulted from masking of the antigen by the CAR at the surface of B-ALL leukemia cells following the unexpected viral transduction of a leukemic cell present in the cytapheresis sample. The objective of this work was to reproduce this epitope-masking resistance model, in the context of acute myeloid leukemia (AML), based on our immunotherapeutic CAR T cell model targeting the accessory protein of the interleukin-1 receptor (IL-1RAP) expressed by leukemic stem cells. As AML primary blasts express different levels of IL-1RAP, we modeled transduction of different AML tumor cell lines screened for density of antigenic sites with our lentiviral vectors carrying a third-generation IL-1RAP CAR, an iCASP9 suicide gene, and a truncated CD19 surface gene. We demonstrated that primary AML blasts can be easily transduced (74.55 ± 21.29%, n = 4) and that CAR T cytotoxicity to IL-1RAP is inversely correlated with epitope masking in relation to the number of antigenic sites expressed on the surface of IL-1RAP+ lines. Importantly, we showed that, in vitro, a 24-h exposure of IL-1RAP+/CAR+ leukemia lines to Rimiducid eliminated >85% of the cells. We confirmed that the expression of IL-1RAP CAR by an IL-1RAP+ leukemic cell, by decreasing the membrane availability of the targeted antigen, can induce resistance while a high epitope density maintains sensitivity to CAR T cells. Moreover, the presence of the iCASP9/Rimiducid suicide system safety switch makes this immunotherapy approach safe for application in a future phase 1 clinical trial.

尽管嵌合抗原受体 CAR) T 细胞免疫疗法是不可否认和明确的成功，但从针对 B 恶性肿瘤（难治性/复发性急性淋巴细胞白血病 (ALL) 或淋巴瘤）中针对 CD19 抗原的第一个临床试验的监测中获得的知识有助于在大约 30-50% 的 B-ALL 中发现肿瘤细胞逃逸。由于抗原（rCD19-）的表面表达丧失或 CAR T 细胞（rCD19+）的早期消失或失活而发生抗药性。在最近报道的一个临床病例中，rCD19- 复发是由于细胞单采样本中存在的白血病细胞意外病毒转导后，B-ALL 白血病细胞表面的 CAR 掩盖了抗原所致。这项工作的目的是重现这种表位掩蔽抗性模型，在急性髓性白血病 (AML) 的背景下，基于我们针对白血病干细胞表达的白细胞介素 1 受体 (IL-1RAP) 的辅助蛋白的免疫治疗性 CAR T 细胞模型。由于 AML 原发性原始细胞表达不同水平的 IL-1RAP，我们使用携带第三代 IL-1RAP CAR、iCASP9 自杀基因和截短 CD19 的慢病毒载体模拟不同 AML 肿瘤细胞系的转导，筛选抗原位点的密度表面基因。我们证明了原发性 AML 原始细胞很容易被转导（74.55 ± 21.29%，我们使用携带第三代 IL-1RAP CAR、iCASP9 自杀基因和截短的 CD19 表面基因的慢病毒载体模拟了不同 AML 肿瘤细胞系的转导，筛选了抗原位点的密度。我们证明了原发性 AML 原始细胞很容易被转导（74.55 ± 21.29%，我们用携带第三代 IL-1RAP CAR、iCASP9 自杀基因和截短的 CD19 表面基因的慢病毒载体模拟了不同 AML 肿瘤细胞系的转导，筛选了抗原位点的密度。我们证明了原发性 AML 原始细胞很容易被转导（74.55 ± 21.29%，n  = 4) 并且 CAR T 对 IL-1RAP 的细胞毒性与表位掩蔽呈负相关，表位掩蔽与 IL-1RAP+ 系表面上表达的抗原位点的数量有关。重要的是，我们发现，在体外，IL-1RAP+/CAR+ 白血病细胞系暴露于 Rimiducid 的 24 小时消除了超过 85% 的细胞。我们证实，IL-1RAP+ 白血病细胞表达 IL-1RAP CAR，通过降低靶向抗原的膜可用性，可以诱导耐药性，同时高表位密度保持对 CAR T 细胞的敏感性。此外，iCASP9/Rimiducid 自杀系统安全开关的存在使得这种免疫治疗方法可以安全地应用于未来的 1 期临床试验。