肿瘤细胞治疗研究
英文原题：Autocrine PD-1-blocking nanobodies enhance the antitumor efficacy of TCR-like CAR-T cells by attenuating t cell exhaustion.
局部自分泌 PD-1 检查点阻断可有效增强 TCRm CAR-T 细胞的效应功能和持久性。这种装甲 TCRm CAR-T 策略是实体瘤免疫治疗的一种有前景的治疗方法，对于靶向不同 peptide-MHC 表位的其他 TCRm CAR 系统具有广泛的转化潜力。
CAR-T（CAR-T）细胞疗法在血液系统恶性肿瘤中取得了显著成功，但其对实体瘤的疗效受到两个核心障碍的严重限制：均匀表达的肿瘤相关抗原（TAA）稀缺，以及免疫抑制性肿瘤微环境（TME），其中 PD-1/PD-L1 信号促进不可逆的 T 细胞耗竭。本研究旨在开发一种新型 T 细胞受体模拟（TCRm）CAR-T 平台，具有自分泌抗 PD-1 纳米抗体分泌功能，以同时克服抗原异质性和 TME 免疫抑制。
构建了编码靶向 HLA-A*02:01 限制性 WT1 的 TCRm CAR 及分泌型抗 PD-1 纳米抗体的慢病毒载体，以生成 WT1-CAR-Nb T 细胞。同时制备了 WT1-CAR T 细胞（仅 CAR 对照）和 Mock T 细胞。体外实验包括抗原特异性细胞毒性、促炎细胞因子（IFN-γ、TNF-α）检测、慢性抗原刺激诱导的 T 细胞耗竭模型以及转录组分析。通过肿瘤生长监测、生存分析、瘤内 T 细胞浸润评估和安全性评价，在人卵巢癌 SKOV3-A2 异种移植小鼠中评估了体内疗效。
靶向HLA-A*02:01限制性WT1并组成性分泌抗PD-1纳米抗体的WT1-CAR-Nb T细胞已成功构建。体外功能实验表明，在特异性抗原刺激下，WT1-CAR-Nb T细胞较传统WT1-CAR T细胞表现出显著增强的抗原特异性细胞毒性，并分泌显著更高水平的促炎细胞因子IFN-γ和TNF-α。关键在于，自分泌PD-1纳米抗体阻断有效缓解了TME免疫抑制，这体现为多种耗竭标志物（TIM-3、CTLA-4、TIGIT）表达下调，以及在慢性抗原刺激下保持增殖潜能。在SKOV3-A2实体瘤异种移植模型中，WT1-CAR-Nb T细胞介导了更优的肿瘤生长控制并显著延长总生存期，这与肿瘤内T细胞浸润增强以及工程化T细胞在体内持续扩增密切相关。
BACKGROUND: Chimeric antigen receptor T (CAR-T) cell therapy has achieved remarkable success in hematologic malignancies, but its efficacy against solid tumors is severely limited by two core barriers: the scarcity of uniformly expressed tumor-associated antigens (TAAs) and the immunosuppressive tumor microenvironment (TME), in which PD-1/PD-L1 signaling promotes irreversible T cell exhaustion. This study aimed to develop a novel T cell receptor-mimic (TCRm) CAR-T platform with autocrine anti-PD-1 nanobody secretion to simultaneously overcome antigen heterogeneity and TME immunosuppression. METHODS: A lentiviral vector encoding a TCRm CAR targeting HLA-A*02:01-restricted WT1 and a secreted anti-PD-1 nanobody was constructed to generate WT1-CAR-Nb T cells. WT1-CAR T cells (CAR-only control) and Mock T cells were also prepared. In vitro assays included antigen-specific cytotoxicity, proinflammatory cytokine (IFN-γ, TNF-α) detection, chronic antigen stimulation-induced T cell exhaustion model, and transcriptomic analysis. In vivo efficacy was evaluated in human ovarian cancer SKOV3-A2 xenograft mice via tumor growth monitoring, survival analysis, intratumoral T cell infiltration assessment, and safety evaluation. RESULTS: WT1-CAR-Nb T cells targeting the HLA-A*02:01-restricted WT1 and constitutively secreting anti-PD-1 nanobody were successfully generated. In vitro functional assays demonstrated that WT1-CAR-Nb T cells exhibited significantly enhanced antigen-specific cytotoxicity and secreted markedly higher levels of proinflammatory cytokines IFN-γ and TNF-α compared with conventional WT1-CAR T cells upon specific antigen stimulation. Critically, autocrine PD-1 nanobody blockade effectively alleviated TME immunosuppression, as reflected by downregulated expression of multiple exhaustion markers (TIM-3, CTLA-4, TIGIT) and preserved proliferative potential under chronic antigen stimulation. In the SKOV3-A2 solid tumor xenograft model, WT1-CAR-Nb T cells mediated superior tumor growth control and significantly prolonged overall survival, which was closely correlated with enhanced intratumoral T cell infiltration and persistent in vivo expansion of engineered T cells. CONCLUSION: Local autocrine PD-1 checkpoint blockade potently enhances the effector function and durability of TCRm CAR-T cells. This armored TCRm CAR-T strategy is a promising therapeutic approach for solid tumor immunotherapy, with broad translational potential for other TCRm CAR systems targeting distinct peptide-MHC epitopes.
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