CLDN18.2-靶向嵌合抗原受体（CAR）修饰的T细胞是目前治疗实体肿瘤效果显著的细胞疗法之一，然而，其长期治疗疗效不满意，短暂反应时间。转基因表达白细胞介素-15（IL-15）已被报道能促进T细胞扩增、存活和功能，并增强体外和体内工程T细胞的抗肿瘤活性。因此，本研究旨在探讨IL-15修饰是否能增强免疫能力的小鼠肿瘤模型中CLDN18.2靶向CAR修饰的T细胞的抗肿瘤活性。通过转染小鼠脾脏T细胞，制备了带（H9 CAR-IL15）或不带转基因IL-15表达（H9 CAR）的CLDN18.2特异性CAR-T细胞。体外实验中，与H9 CAR T细胞相比，H9 CAR-IL15 T细胞在无抗原刺激的情况下表现出更好的扩增和存活能力，并呈现较少分化和T细胞疲竭表型；虽然IL-15修饰对短期杀伤实验中的效应细胞因子产生和细胞毒性活性没有影响，但它适度改善了CAR-T细胞对表达CLDN18.2的肿瘤细胞的体外迭代杀伤活性。在体内，H9 CAR T细胞在无淋巴灭活预处理的免疫竞争小鼠模型中对CLDN18.2表达的胰腺肿瘤没有抗肿瘤活性；然而，H9 CAR-IL15 T细胞产生了显著的肿瘤抑制效应。此外，结合淋巴灭活预处理，H9 CAR-IL15 T细胞在体内扩增和肿瘤浸润能力更强，导致在两个小鼠肿瘤模型中表现出更优异的抗肿瘤活性，并在一个肿瘤模型中具有更长的生存优势。我们进一步证明了H9 CAR-IL15 T细胞治疗后复发肿瘤下调了CLDN18.2表达，表明在持续的CAR-T细胞免疫压力下通过选择抗原阴性细胞进行免疫逃逸。总之，我们的研究结果提供了临床评估表达IL-15的CLDN18.2 CAR-T细胞治疗携带CLDN18.2阳性肿瘤患者的临床前证据。版权所有 © 2023 资治、李、戴、薛、赵、田、宋、王、陈、张、周、魏和秦。

Claudin 18.2 (CLDN18.2)-targeting chimeric antigen receptor (CAR)-modified T cells are one of the few cell therapies currently producing an impressive therapeutic effect in treating solid tumors; however, their long-term therapeutic efficacy is not satisfactory with a short duration of response. Transgenic expression of interleukin (IL)-15 has been reported to promote T-cell expansion, survival, and function and enhance the antitumor activity of engineered T cells in vitro and in vivo. Therefore, this study aimed to explore whether IL-15 modification would increase the antitumor activity of CLDN18.2-targeting CAR-modified T (CAR-T) cells in immunocompetent murine tumor models. CLDN18.2-specific CAR-T cells with (H9 CAR-IL15) or without transgenic IL-15 expression (H9 CAR) were generated by retroviral transduction of mouse splenic T cells. In vitro, compared with H9 CAR T cells, H9 CAR-IL15 T cells exhibited better expansion and viability in the absence of antigen stimulation, with a less differentiated and T-cell exhausted phenotype; although IL-15 modification did not affect the production of effector cytokines and cytotoxic activity in the short-term killing assay, it moderately improved the in vitro recursive killing activity of CAR-T cells against CLDN18.2-expressing tumor cells. In vivo, H9 CAR T cells showed no antitumor activity against CLDN18.2-expressing pancreatic tumors in immunocompetent mice without lymphodepleting pretreatment; however, H9 CAR-IL15 T cells produced significant tumor-suppressive effects. Furthermore, H9 CAR-IL15 T cells exhibited greater in vivo expansion and tumor infiltration when combined with lymphodepleting preconditioning, resulting in superior antitumor activity in two murine tumor models and a survival advantage in one tumor model. We further demonstrated that recurrent tumors following H9 CAR-IL15 T-cell therapy downregulated CLDN18.2 expression, suggesting immune escape through the selection of antigen-negative cells under persistent CAR-T-cell immune pressure. In conclusion, our findings provide preclinical evidence supporting the clinical evaluation of IL-15-expressing CLDN18.2 CAR-T cells in patients with CLDN18.2-positive tumors.Copyright © 2023 Shi, Li, Dai, Xue, Zhao, Tian, Song, Wang, Chen, Zhang, Zhou, Wei and Qin.