Targeting AML by chimeric antigen receptor T-cells (CAR-T) is challenging due to the promiscuous expression of AML-associated antigens in healthy hematopoiesis and high degree of inter- and intratumoral heterogeneity. Here, we present single-cell expression data of AML-associated antigens in 30 primary pediatric AML samples. We identified CD33, CD38, CD371, IL1RAP and CD123 as the most frequently expressed. Notably, high variability was observed not only across the different patient samples but also among leukemic cells of the same patient suggesting the necessity of multiplexed targeting approaches. To address this need, we utilized our modular Adapter CAR (AdCAR) platform, enabling precise qualitative and quantitative control over CAR-T-cell function. We show highly efficient and target-specific activity for newly generated adapter molecules (AMs) against CD33, CD38, CD123, CD135 and CD371, both in vitro and in vivo. We reveal that inherent intratumoral heterogeneity in antigen expression translates into antigen escape and therapy failure to monotargeted CAR-T therapy. Further, we demonstrate in PDX models that rational combinatorial targeting by AdCAR-T-cells can cure heterogenic disease. In conclusion, we elucidate the clinical relevance of heterogeneity in antigen expression in pediatric AML and present a novel concept for precision immunotherapy by combinatorial targeting utilizing the AdCAR platform.

由于 AML 相关抗原在健康造血系统中的混杂表达以及肿瘤间和肿瘤内的高度异质性，通过嵌合抗原受体 T 细胞 (CAR-T) 靶向 AML 具有挑战性。在这里，我们展示了 30 个原发儿科 AML 样本中 AML 相关抗原的单细胞表达数据。我们确定 CD33、CD38、CD371、IL1RAP 和 CD123 是最常表达的。值得注意的是，不仅在不同患者样本中观察到了高变异性，而且在同一患者的白血病细胞中也观察到了高变异性，这表明多重靶向方法的必要性。为了满足这一需求，我们利用模块化的 Adapter CAR (AdCAR) 平台，实现对 CAR-T 细胞功能的精确定性和定量控制。我们在体外和体内表现出新生成的接头分子 (AM) 针对 CD33、CD38、CD123、CD135 和 CD371 的高效和靶标特异性活性。我们揭示了抗原表达中固有的肿瘤内异质性转化为抗原逃逸和单靶点 CAR-T 疗法的治疗失败。此外，我们在 PDX 模型中证明，AdCAR-T 细胞的合理组合靶向可以治愈异源性疾病。总之，我们阐明了儿科 AML 抗原表达异质性的临床相关性，并提出了利用 AdCAR 平台进行组合靶向的精准免疫治疗的新概念。