Sun Minmin, Bian Linke, Wang Hongye, Liu Xin, Li Yantao, Wu Zhaorong, Zhang Shuangshuang, Hao Ruidong, Xin Hong, Zhai Bo, Zhang Xuemei, Cheng Yuanguo
School of Pharmacy, Fudan University, Shanghai, P.R. China.
China State Institute of Pharmaceutical Industry, Shanghai, P.R. China.
Cell Oncol (Dordr). 2025 Jun 19. doi: 10.1007/s13402-025-01066-5.
CAR-T cell therapy has demonstrated remarkable success in hematologic malignancies; however, its effectiveness against solid tumors remains limited due to tumor antigen heterogeneity. NKG2DLs, including MICA/B and the ULBP family, are stress-induced molecules frequently upregulated on the surface of tumor cells and components of the tumor microenvironment, providing attractive targets for immunotherapy. To broaden the targeting capability beyond conventional Claudin18.2-directed CAR-T cells, we engineered a Synthetic NKG2D Receptor (SNR). The SNR comprises the extracellular domain of NKG2D fused with the intracellular signaling domains of DAP10 and DAP12, enabling effective targeting of NKG2D ligands (NKG2DLs).
Expression of NKG2DLs and CLDN18.2 were detected by immunohistochemistry on a gastric cancer tissue microarray. We designed SNR CAR-T cells by linking CLDN18.2 CAR with SNR by a 2A self-cleaving peptide. We assessed their cytotoxicity, tumor infiltration, persistence, and antitumor efficacy using in vitro assays, patient-derived xenograft (PDX) models, and murine syngeneic models. Additionally, transcriptomic analysis and flow cytometry were performed to evaluate exhaustion and memory markers.
SNR CAR-T cells demonstrated enhanced cytotoxicity against tumor cells with heterogeneous CLDN18.2 expression, effectively lysing both CLDN18.2-positive and NKG2DL-positive tumor cells in vitro. In PDX and murine models, SNR CAR-T cells exhibited superior antitumor efficacy, leading to significant tumor regression and CAR-T expansion compared to conventional CAR-T cells. Furthermore, SNR CAR-T cells displayed reduced expression of exhaustion markers and increased expression of memory-associated markers. Enhanced tumor infiltration, proliferation and cytotoxicity within the tumor microenvironment, and a reduced presence of myeloid-derived suppressor cells (MDSCs) and tumor neovasculature were observed. Importantly, SNR CAR-T cell therapy was well-tolerated, with no significant toxicity noted in all the treated animals.
The SNR CAR-T cell approach addresses tumor antigen heterogeneity and suppressive tumor microenvironment, offering a promising therapeutic strategy for solid tumors and paving the way for its future clinical applications.
嵌合抗原受体T细胞（CAR-T）疗法在血液系统恶性肿瘤中已显示出显著成效；然而，由于肿瘤抗原的异质性，其对实体瘤的疗效仍然有限。自然杀伤细胞2D配体（NKG2DLs），包括MICA/B和ULBP家族，是应激诱导分子，常在肿瘤细胞表面和肿瘤微环境成分上上调，为免疫治疗提供了有吸引力的靶点。为了拓宽靶向能力，超越传统的靶向Claudin18.2的CAR-T细胞，我们构建了一种合成NKG2D受体（SNR）。SNR由NKG2D的胞外结构域与DAP10和DAP12的胞内信号结构域融合而成，能够有效靶向NKG2D配体（NKG2DLs）。
通过免疫组化在胃癌组织芯片上检测NKG2DLs和CLDN18.2的表达。我们通过2A自切割肽将CLDN18.2 CAR与SNR连接来设计SNR CAR-T细胞。我们使用体外试验、患者来源异种移植（PDX）模型和小鼠同基因模型评估了它们的细胞毒性、肿瘤浸润、持久性和抗肿瘤疗效。此外，进行了转录组分析和流式细胞术以评估耗竭和记忆标志物。
SNR CAR-T细胞对CLDN18.2表达异质性的肿瘤细胞表现出增强的细胞毒性，在体外能有效裂解CLDN18.2阳性和NKG2DL阳性肿瘤细胞。在PDX和小鼠模型中，SNR CAR-T细胞表现出卓越的抗肿瘤疗效，与传统CAR-T细胞相比，导致显著的肿瘤消退和CAR-T细胞扩增。此外，SNR CAR-T细胞显示出耗竭标志物表达降低，记忆相关标志物表达增加。观察到肿瘤微环境内肿瘤浸润、增殖和细胞毒性增强，以及髓源性抑制细胞（MDSCs）和肿瘤新生血管减少。重要的是，SNR CAR-T细胞疗法耐受性良好，所有治疗动物均未观察到明显毒性。
SNR CAR-T细胞方法解决了肿瘤抗原异质性和抑制性肿瘤微环境问题，为实体瘤提供了一种有前景的治疗策略，并为其未来的临床应用铺平了道路。
