CAR-T细胞在实体瘤中的应用带来了一些挑战，包括T细胞归巢能力差、T细胞浸润有限以及免疫抑制的肿瘤环境。在本研究中，我们开发了一种解决这些障碍的新方法，通过设计共表达 IL-21 和 CXCL9 的 GPC3 特异性 CAR-T 细胞（21 × 9 GPC3 CAR-T 细胞）并阻断其上的 PD-1 表达。体外评估了所示 CAR-T 细胞的增殖、细胞表型、细胞因子分泌和细胞迁移。在体外和体内评估了基因工程 CAR-T 细胞的细胞毒活性。与传统的GPC3 CAR-T细胞相比，21 × 9 GPC3 CAR-T细胞在体外表现出优异的增殖、细胞因子分泌和趋化能力。此外，当与PD-1阻断相结合时，21 × 9 GPC3 CAR-T细胞表现出增强的增殖、细胞因子分泌以及效应T细胞（例如CTL、NKT和TEM细胞）的富集。在异种移植肿瘤模型中，PD-1阻断21 × 9 GPC3 CAR-T细胞有效抑制HCC异种移植物生长并增加T细胞浸润。总体而言，我们的研究成功生成了同时表达 IL-21 和 CXCL9 的 GPC3 CAR-T 细胞，证明联合 PD-1 阻断可以通过促进增殖、细胞因子分泌、趋化性和抗肿瘤活性来进一步增强 CAR-T 细胞功能。这些发现为 GPC3 阳性 HCC 患者提供了一种充满希望且可能有效的策略。© 2024。作者。

The application of CAR-T cells in solid tumors poses several challenges, including poor T cell homing ability, limited infiltration of T cells and an immunosuppressive tumor environment. In this study, we developed a novel approach to address these obstacles by designing GPC3-specific CAR-T cell that co-express IL-21 and CXCL9 (21 × 9 GPC3 CAR-T cells) and blocking the PD-1 expression on it. The proliferation, cell phenotype, cytokine secretion and cell migration of indicated CAR-T cells were evaluated in vitro. The cytotoxic activities of genetically engineered CAR-T cells were accessed in vitro and in vivo. Compared to conventional GPC3 CAR-T cells, the 21 × 9 GPC3 CAR-T cells demonstrated superior proliferation, cytokine secretion and chemotaxis capabilities in vitro. Furthermore, when combined with PD-1 blockade, the 21 × 9 GPC3 CAR-T cells exhibited enhanced proliferation, cytokine secretion and enrichment of effector T cells such as CTL, NKT and TEM cells. In xenograft tumor models, the PD-1 blocked 21 × 9 GPC3 CAR-T cells effectively suppressed HCC xenograft growth and increased T cell infiltration. Overall, our study successfully generated GPC3 CAR-T cells expressing both IL-21 and CXCL9, demonstrated that combining PD-1 blockade can further enhance CAR-T cell function by promoting proliferation, cytokine secretion, chemotaxis and antitumor activity. These findings present a hopeful and potentially effective strategy for GPC3-positive HCC patients.© 2024. The Author(s).