肿瘤细胞治疗研究
英文原题：Improved Antitumor Activity of Interleukin-12-Secreting Chimeric Antigen Receptor T Cells Targeting CD176 across Different Carcinomas.
在识别 CD176⁺ 癌细胞系后，CD176-iIL12-TRUCKs 特异性释放促炎介质（干扰素-、肿瘤坏死因子-），并表现出活化标志物表达增加（CD25、CD69）。
引言：晚期实体瘤的有效治疗方案仍严重不足，尤其在发生转移后死亡率很高。糖类结构CD176已被认为是多种癌症精准免疫治疗的有前景靶点：约90%的癌症表达该结构，而健康组织上不存在可结合的CD176。我们报告了CD176特异性第四代CAR-T 细胞的开发，这类细胞也称为抗原非依赖性细胞因子启动杀伤的T细胞（TRUCK）。为应对限制CAR-T疗效的实体瘤免疫抑制性肿瘤微环境（TME）和抗原表达异质性，我们使其能够释放NFAT诱导型IL-12（iIL12），以增强促炎性自分泌和旁分泌效应。 方法：研究者使用源自JE6-1的报告细胞系，在报告细胞实验中测试CD176-iIL12-TRUCK构建体的靶点特异性。随后利用原代CD8⁺ T细胞制备CD176-iIL12-TRUCK。通过与不同癌种来源的CD176阳性细胞系共培养，评估iIL12对功能的影响，包括T细胞活化水平、细胞毒能力和旁观者免疫细胞募集。 结果：识别CD176阳性癌细胞系后，CD176-iIL12-TRUCK会特异性释放促炎介质（干扰素γ、肿瘤坏死因子α），并提高CD25和CD69等活化标志物表达。通过基于7-AAD的活力检测和基于阻抗的细胞毒性检测，证实CD176-iIL12-TRUCK可清除不同肿瘤类型来源的CD176阳性细胞系。此外，在趋化实验室中，CD176-iIL12-TRUCK释放的iIL12可招募单核细胞和NK细胞系。 讨论/结论：总体而言，释放IL-12显著增强了针对CD176阳性而非CD176阴性细胞的效应功能，提示该策略兼具疗效和特异性。因此，CD176-iIL12-TRUCK凭借强效抗肿瘤能力和调节TME的潜力，有望成为多种晚期实体瘤患者的治疗选择。
INTRODUCTION: Effective therapeutic options for advanced solid tumors remain severely limited, causing high fatality rates especially after metastasis. The carbohydrate structure CD176 has been identified as a promising target for precise immunotherapy in multiple carcinomas, as it is present in about 90% of carcinomas but unavailable for binding on healthy tissue. Here, we report the development of CD176-specific 4th-generation chimeric antigen receptor T cells (CAR-Ts), also known as T cells redirected for antigen-unrestricted cytokine-initiated killing (TRUCKs). To address the immunosuppressive tumor microenvironment (TME) and the heterogeneous antigen expression of solid tumors, which limit the efficacy of CAR-Ts, they were endowed with NFAT-inducible interleukin-12 (iIL12) release to improve pro-inflammatory autocrine and paracrine effects. METHODS: The CD176-iIL12-TRUCK construct was tested for target specificity in a reporter cell assay using a JE6-1-derived reporter cell line. Afterward, CD176-iIL12-TRUCKs were manufactured using primary CD8 + T cells. The influence of iIL12 on functionality of CD176-iIL12-TRUCKs, including T-cell activation levels, cytotoxic capacity, and recruitment of bystander immune cells, was evaluated following cocultures with CD176 + cell lines from different carcinomas. RESULTS: Upon recognition of CD176 + cancer cell lines, CD176-iIL12-TRUCKs specifically released pro-inflammatory mediators (interferon- , tumor necrosis factor- ) and showed an increased activation marker expression (CD25, CD69). Using both a 7-AAD-based viability assay and an impedance-based cytotoxicity assay, elimination of CD176 + cell lines from different tumor entities by CD176-iIL12-TRUCKs was shown. Additionally, iIL12 released by CD176-iIL12-TRUCKs led to recruitment of monocyte and NK cell lines in a chemotaxis chamber assay. DISCUSSION/CONCLUSION: Overall, the IL-12 release substantially improved effector functionality against CD176 + cells but not CD176 - cells, indicating efficacy while maintaining specificity. Thus, CD176-iIL12-TRUCKs, with their potent antitumor efficacy and TME modulation potential, are a promising treatment option for patients with a variety of advanced solid tumors.
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