Background: Claudin18.2 (CLDN18.2) is highly expressed during the development of various malignant tumors, especially gastric cancer, and CAR-T cells targeting CLDN18.2 have therapeutic potential. However, their dependence on the major histocompatibility complex (MHC) for antigen recognition limits their application. Human Gamma Delta (γδ) T cells, with strong MHC-independent cytotoxicity to most solid tumors both in vivo and in vitro, are emerging as ideal cells for the generation of robust universal CLDN18.2 CAR-T cells to treat solid tumors. Our aim was to construct a universal CAR-γδ T cell targeting CLDN18.2. Methods: We constructed novel CAR-CLDN18.2-γδ T cells by lentiviral infection and compared their superior efficacy in the treatment of CLDN18.2-positive solid tumors in vivo and in vitro. Results: CD3ζ expression was verified in HEK293T cells after lentiviral transfection of CLDN18.2 CAR, and the lentivirus was packaged and concentrated to a titer of 4.90 × 108TU/mL. Primary γδ T cells and αβ T cells were infected with efficiencies of approximately 31.76 ± 4.122% and 44.13 ± 4.436%, respectively. CAR-CLDN18.2-γδ T cells exhibited specific cytotoxicity against CLDN18.2-positive gastric cancer cells and secreted relatively high levels of Granzyme-B, Perforin-1, and IFN-γ. CAR-γδ T cells also showed superior cytotoxicity to target cells compared to classical CAR-αβ T cells in vitro. Finally, the antitumor activity of γδ T-CAR-CLDN18.2 cells was evaluated in tumor-bearing NSG mice, and CAR-CLDN18.2-γδ T cells significantly inhibited tumor growth and prolonged the survival of the mice. Conclusions: Our results demonstrate that universal CAR-CLDN18.2-γδ T cell is promising for the treatment of CLDN18.2-positive solid tumor and provide insights for the development of more universal CAR-γδ T-cell strategies for tumor immunotherapy.
背景：Claudin18.2（CLDN18.2）在多种恶性肿瘤尤其是胃癌的发生发展中高表达，靶向CLDN18.2的CAR-T细胞具有治疗潜力。然而，其抗原识别对主要组织相容性复合体（MHC）的依赖性限制了其应用。人γδ T细胞在体内外对大多数实体瘤具有不依赖MHC的强效细胞毒性，正成为构建强效通用型CLDN18.2 CAR-T细胞治疗实体瘤的理想选择。本研究旨在构建靶向CLDN18.2的通用型CAR-γδ T细胞。方法：通过慢病毒感染构建新型CAR-CLDN18.2-γδ T细胞，并在体内外比较其治疗CLDN18.2阳性实体瘤的优越疗效。结果：慢病毒转染CLDN18.2 CAR后，在HEK293T细胞中验证了CD3ζ的表达，慢病毒包装浓缩后滴度达4.90×10⁸ TU/mL。原代γδ T细胞与αβ T细胞的感染效率分别约为31.76±4.122%和44.13±4.436%。CAR-CLDN18.2-γδ T细胞对CLDN18.2阳性胃癌细胞表现出特异性细胞毒性，并分泌较高水平的颗粒酶B、穿孔素-1和干扰素-γ。体外实验中，CAR-γδ T细胞相较于经典CAR-αβ T细胞对靶细胞展现出更优的细胞毒性。最后，在荷瘤NSG小鼠体内评估γδ T-CAR-CLDN18.2细胞的抗肿瘤活性，结果显示CAR-CLDN18.2-γδ T细胞能显著抑制肿瘤生长并延长小鼠生存期。结论：本研究证实通用型CAR-CLDN18.2-γδ T细胞在治疗CLDN18.2阳性实体瘤方面具有良好前景，并为开发更广泛的通用型CAR-γδ T细胞肿瘤免疫治疗策略提供了新思路。
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