肿瘤细胞治疗研究
英文原题：Preclinical evaluation of CD70-specific CAR T cells targeting acute myeloid leukemia.
我们的研究揭示，抗 CD70 CAR-T 细胞是 AML 的一种新的潜在治疗手段。
背景：嵌合抗原受体（CAR）T细胞疗法治疗血液系统恶性肿瘤取得了前所未有的成功。然而，由于缺乏理想的细胞表面靶点，即靶点仅表达于急性髓系白血病（AML）白血病原始细胞和白血病干细胞（LSC），而不表达于正常造血干细胞（HSC），这种细胞疗法治疗AML受到限制。方法：我们检测AML细胞系、原代AML细胞、HSC和外周血细胞表面的CD70表达，并采用含人源化41D12来源单链可变片段（scFv）以及4-1BB-CD3胞内信号结构域的构建体，制备第二代CD70特异性CAR-T细胞。通过抗原刺激条件下的细胞毒性、细胞因子释放和增殖实验、CD107a实验及CFSE实验，评估其体外抗白血病活性。建立Molm-13异种移植小鼠模型评价CD70 CAR-T细胞的体内抗白血病活性，并采用集落形成单位（CFU）实验评估其对HSC的安全性。结果：CD70在AML原代细胞中呈异质性表达，包括白血病原始细胞、白血病祖细胞及干细胞，但不表达于正常HSC及大多数血细胞。与CD70阳性AML细胞系共培养时，抗CD70 CAR-T细胞表现出强效细胞毒作用、细胞因子生成和增殖能力。在Molm-13异种移植小鼠模型中，该细胞也显示强劲抗白血病活性并延长生存，但未能在体内完全清除白血病。讨论：本研究揭示抗CD70 CAR-T细胞是AML的一种新型潜在治疗方法。然而，该疗法未能在体内完全清除白血病，提示未来需开发创新联合CAR构建体，或提高白血病细胞表面CD70表达密度，以延长CAR-T细胞在循环中的存留时间并优化AML治疗应答。
BACKGROUNDS: Chimeric antigen receptor (CAR)-T cell therapy has achieved unprecedented success in treating hematopoietic malignancies. However, this cell therapy is hampered in treating acute myeloid leukemia (AML) due to lack of ideal cell surface targets that only express on AML blasts and leukemia stem cells (LSCs) but not on normal hematopoietic stem cells (HSCs). METHODS: We detected the CD70 expression on the surfaces of AML cell lines, primary AML cells, HSC, and peripheral blood cells and generated a second-generation CD70-specific CAR-T cells using a construct containing a humanized 41D12-based scFv and a 41BB-CD3 intracellular signaling domain. Cytotoxicity, cytokine release, and proliferation in antigen stimulation, CD107a assay, and CFSE assays were used to demonstrate the potent anti-leukemia activity in vitro. A Molm-13 xenograft mouse model was established to evaluate the anti-leukemic activity of CD70 CAR-T in vivo . CFU assay was explored to assess the safety of CD70 CAR-T on HSC. RESULTS: CD70 heterogeneously expressed on AML primary cells, including leukemia blasts, leukemic progenitor, and stem cells, but not expressed on normal HSCs and majority of blood cells. Anti-CD70 CAR-T cells exhibited potent cytotoxicity, cytokines production, and proliferation when incubated with CD70 + AML cell lines. It also displayed robust anti-leukemia activity and prolonged survival in Molm-13 xenograft mouse model. However, such CAR-T cell therapy did not completely eliminate leukemia in vivo . DISCUSSION: Our study reveals that anti-CD70 CAR-T cells are a new potential treatment for AML. However, such CAR-T cell therapy did not completely eliminate leukemia in vivo , suggesting that future studies aiming to generate innovative combinatorial CAR constructs or to increase CD70 expression density on leukemia cell surface to prolong the life-span of CAR-T cells in the circulation will be needed in order to optimize CAR-T cell responses for AML.
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