T cells expressing CD26-specific chimeric antigen receptors exhibit extensive self-antigen-driven fratricide

Background: Immunotherapy utilizing T cells genetically modified to express chimeric antigen receptors (CARs) is rapidly emerging as a promising novel treatment for hematological and nonhematological malignancies. In order to target the TKI-insensitive leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) by CAR T cells, we chose CD26 as a cell surface tumor-associated antigen due to preferentially expression on LSCs. Additionally, CD26 has also been suggested to be a multipurpose therapeutic target for other cancer. Therefore, developing the CD26-targeting CAR T cells may be a promising therapy for not only LSCs but also other CD26+ cancer cells. Methods: We designed the second-generation CD26-targeting CAR utilizing 4-1BB (CD137) as costimulatory domain, and transduced T cells with CD26-CAR containing lentiviral. Then we evaluated the transduction efficiency and expansion ability, and demonstrated the existence of self-antigen-driven fratricide by cytokine assay and cytotoxicity assay. Results: Anti-CD26-4-1BB-CAR T cells exhibited poor viability, multiple cytokine secretion, down-regulation of CD26 and direct cytotoxicity against themselves, indicating self-antigen-driven fratricide. Conclusion: Eradicating CML-LSCs via anti-CD26-4-1BB-CAR T cells is not applicable, and optimized design or alternative target is needed.

背景：经基因修饰以表达嵌合抗原受体（chimeric antigen receptor，CAR）的T细胞介导的免疫疗法，正快速成为治疗血液系统与非血液系统恶性肿瘤的极具前景的新型疗法。为借助CAR-T细胞靶向慢性髓系白血病（chronic myeloid leukemia，CML）中对酪氨酸激酶抑制剂（tyrosine kinase inhibitor，TKI）耐药的白血病干细胞（leukemia stem cells，LSCs），我们选择CD26作为细胞表面肿瘤相关抗原，因其在LSCs上呈优先表达。此外，现有研究提示CD26亦可作为多种其他癌症的多用途治疗靶点。因此，开发靶向CD26的CAR-T细胞，有望同时针对LSCs与其他表达CD26的癌细胞。
方法：我们设计了以4-1BB（CD137）作为共刺激结构域的第二代靶向CD26的CAR，并使用携带CD26-CAR的慢病毒转导T细胞。随后我们评估了转导效率与扩增能力，并通过细胞因子检测实验与细胞毒性实验证实了自身抗原驱动的细胞自相残杀现象的存在。
结果：抗CD26-4-1BB-CAR-T细胞表现出存活能力低下、多细胞因子分泌、CD26表达下调以及对自身的直接细胞毒性，这表明存在自身抗原驱动的细胞自相残杀。
结论：通过抗CD26-4-1BB-CAR-T细胞清除CML-LSCs的策略并不可行，亟需优化设计或更换靶点。