目前的 CD33 靶向免疫疗法通常识别 CD33 的膜远端 V-set 结构域。在这里，我们证明，缩短 T 细胞与白血病细胞膜之间的距离可以提高 CD33 嵌合抗原受体 (CAR) T 细胞的功效。因此，我们生成并优化了第二代 CAR 构建体，其中包含来自针对近膜 C2-set 结构域的抗体的单链可变片段，无论 V-set 结构域是否存在（CD33PAN 抗体），该构建体都会结合 CD33。 CD33PAN CAR T 细胞可有效清除肿瘤，并提高携带人类 AML 细胞异种移植物的免疫缺陷小鼠的生存率，并且在 CD33 表达有限的 AML 模型中，迫使其逃避 CD33neg 白血病。与 CD33V-set CAR T 细胞相比，CD33PAN CAR T 细胞对几种具有不同 CD33 水平的人 AML 细胞系表现出更高的体外和体内功效，且耗竭标记物的表达没有增加。 CD33PAN 部分在人白血病干细胞上检测到的频率更高，并且 CD33PAN CAR T 细胞对原代人 AML 细胞具有更大的体外功效。总之，我们的研究证明 CAR T 细胞在细胞膜附近结合 CD33 可以提高疗效，为进一步研究 CD33PAN CAR T 细胞以实现可能的临床应用提供了理论依据。© 2024 作者。

Current CD33-targeted immunotherapies typically recognize the membrane-distal V-set domain of CD33. Here, we show that decreasing the distance between T cell and leukemia cell membrane increases the efficacy of CD33 chimeric antigen receptor (CAR) T cells. We therefore generated and optimized second-generation CAR constructs containing single-chain variable fragments from antibodies raised against the membrane-proximal C2-set domain, which bind CD33 regardless of whether the V-set domain is present (CD33PAN antibodies). CD33PAN CAR T cells resulted in efficient tumor clearance and improved survival of immunodeficient mice bearing human AML cell xenografts and, in an AML model with limited CD33 expression, forced escape of CD33neg leukemia. Compared to CD33V-set CAR T cells, CD33PAN CAR T cells showed greater in vitro and in vivo efficacy against several human AML cell lines with differing levels of CD33 without increased expression of exhaustion markers. CD33PAN moieties were detected at a higher frequency on human leukemic stem cells, and CD33PAN CAR T cells had greater in vitro efficacy against primary human AML cells. Together, our studies demonstrate improved efficacy with CAR T cells binding CD33 close to the cell membrane, providing the rationale to investigate CD33PAN CAR T cells further toward possible clinical application.© 2024 The Author(s).