肿瘤细胞治疗研究
英文原题：Glypican 3-targeted chimeric antigen receptor T cells secreting TROP2-directed bispecific T cell engagers exhibit potent efficacy against lung squamous cell carcinoma.
本研究表明，GPC3 CAR-T。
**背景：**CAR-T 细胞治疗实体瘤面临多重挑战，尤其是肿瘤抗原异质性。磷脂酰肌醇蛋白聚糖3（GPC3）和滋养层细胞表面抗原2（TROP2）在肺鳞状细胞癌（LUSC）中高表达，是开发双靶点疗法的候选抗原。GPC3在成人正常组织中不表达，是理想CAR-T靶点；但TROP2在多种正常组织上皮细胞中表达，直接将其用于CAR-T可能带来较高的“靶向肿瘤外组织”风险。研究者开发了双靶向LUSC疗法：靶向GPC3并分泌靶向TROP2的双特异性T细胞衔接器（BiTE）的CAR-T（GPC3 CAR-T.TROP2 BiTE），并分别在体外和体内验证其抗肿瘤活性。 **方法：**采用免疫组化确认LUSC和正常组织中GPC3及TROP2的表达；通过慢病毒转导CAR基因制备CAR-T细胞；使用实时细胞分析检测其对LUSC细胞的细胞毒作用；以流式细胞术评估CAR-T表型、耗竭和活化，并以ELISA检测细胞因子释放。体内实验中，向荷瘤免疫缺陷小鼠单次静脉注射CAR-T细胞，并定期监测肿瘤负荷和CAR-T扩增。 **结果：**70%的LUSC组织中GPC3过表达，而所有正常组织均为阴性；所有LUSC组织均表达TROP2，许多正常组织也表达。与GPC3 CAR-T或TROP2 CAR-T相比，GPC3 CAR-T.TROP2 BiTE可杀伤GPC3阳性和TROP2阳性LUSC细胞，因此对GPC3和TROP2表达不均一的LUSC细胞杀伤更快、抗肿瘤作用更持久。在荷瘤小鼠中，该疗法表现出强效清除肿瘤的能力。 **结论：**本研究证明GPC3 CAR-T.TROP2 BiTE是治疗LUSC的强效策略，并为克服实体瘤抗原异质性提供了思路。
BACKGROUND: Chimeric antigen receptor T cell (CAR-T) therapy faces multiple challenges in solid tumors, especially the heterogeneity of tumor antigens. Glypican-3 (GPC3) and trophoblast cell-surface antigen 2 (TROP2) are highly expressed antigens in lung squamous cell carcinoma (LUSC) for development of dual-targeted therapy. The absence of GPC3 in any normal tissues of adults makes it an ideal target for CAR-T therapy. However, TROP2 is expressed in the epithelial cells of various normal tissues and thus is not acceptable for direct design of CAR-T therapy due to the high risk of "on-target off-tumor" effects. Here we developed a dual-targeted LUSC therapy featuring a GPC3-targeted CAR-T cell secreting TROP2-directed bispecific T cell engagers (GPC3 CAR-T. TROP2 BiTE), and verified the antitumor activity in vitro and in vivo , respectively. METHODS: Immunohistochemistry (IHC) was used to confirm the expression of GPC3 and TROP2 in LUSC and normal tissues. CAR-T cells were produced through lentiviral transduction of CAR genes. Real-time cytotoxicity assay (RTCA) was used to assess the cytotoxic effect of CAR-T cells on LUSC cells. Flow cytometry was utilized to examine the CAR-T cell phenotype, exhaustion and activation. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the release of cytokines. To evaluate the activity of CAR-T cells in vivo , tumor-bearing immunodeficient mice were given a single intravenous injection of CAR-T cells, and the tumor burden and CAR-T cell expansion were regularly monitored. RESULTS: GPC3 was overexpressed in 70% of LUSC tissues, while negatively expressed in all normal tissues. Positive expression of TROP2 was observed in all LUSC tissues and also in many normal tissues. Compared with GPC3 CAR-T and TROP2 CAR-T, GPC3 CAR-T. TROP2 BiTE exhibited cytotoxicity to both GPC3 + and TROP2 + LUSC cells, and thereby showed faster killing and durable antitumor effect against LUSC cells with heterogenous expression of GPC3 and TROP2. In tumor-bearing mice, GPC3 CAR-T. TROP2 BiTE showed strong ability to eliminate tumors. CONCLUSIONS: This study demonstrated that GPC3 CAR-T. TROP2 BiTE was a potent therapy for LUSC and provided a strategy for overcoming the antigen heterogeneity in solid tumors.
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