新型CAR-T细胞靶向杀伤HPV16阳性宫颈癌细胞的探索研究

Exploration of CAR-T cells targeting HPV16 positive cervical cancer cells

目的:制备特异性杀伤HPV16阳性宫颈癌细胞的CAR-T细胞,初步探索CAR-T细胞治疗宫颈癌的可行性。方法:以HPV16 E711-19:HLA-A*0201复合物为靶点,合成第三代CAR基因序列,并克隆到慢病毒表达质粒,通过293T细胞包装慢病毒,将其转导人外周血T细胞,制备CAR-T细胞,采用流式细胞术检测CAR-T细胞的转导效率。将CAR-T细胞分别与宫颈癌细胞株HPV16+Ca Ski和HPV18+He La共培养,使用CCK-8试剂盒检测CAR-T细胞的杀伤效果,以及利用ELISA测定CAR-T细胞分泌IFN-γ和IL-2的水平。结果:流式细胞术检测显示T细胞的慢病毒转导效率为41.2%,其中CD4+T细胞为85.2%,CD8+T细胞为30.9%。体外共培养实验CAR-T细胞与Ca Ski和He La细胞为10:1时,对应的细胞活力分别为56.8%和98.3%。CART细胞对Ca Ski细胞具有较强的细胞毒性,而对He La细胞的杀伤作用较小。Ca Ski细胞培养基中IFN-γ浓度达到100 pg/mL,IL-2浓度达到24 pg/mL,而He La细胞培养基中IFN-γ为16 pg/mL,IL-2为6 pg/mL。结论:我们成功制备靶向杀伤HPV16阳性宫颈癌细胞的CAR-T细胞,为CAR-T细胞治疗宫颈癌提供了初步的研究支持。

Objective:To prepare CAR-T cells specifically killing HPV16 positive cervical cancer cells,and to preliminary explore the feasibility of CAR-T cells in the treatment of cervical cancer.Methods:The third generation CAR gene sequence was synthesized to target HPV16 E711-19:HLA-A*0201 complex and cloned into lentiviral expression plasmid.The lentiviral vectors were packaged in 293T cells and transduced into human peripheral blood T cells to prepare CAR-T cells.The transduction efficiency of CAR-T cells was detected by flow cytometry.CAR-T cells were co-cultured with cervical cancer cell lines HPV16+Ca Ski and HPV18+HeLa,respectively.The cytotoxicity of CAR-T cells was detected by CCK-8 kit,and the levels of IFN-γand IL-2 secreted by CAR-T cells were determined by ELISA.Results:Lentivirus transduction efficiency of T cells with was 41.2%,as 85.2%of CD4+T cells and 30.9%of CD8+T cells.In vitro co-culture experiment,as the ratios of CAR-T cells to Ca Ski and HeLa cells were 10:1,the cell viability of Ca Ski were 56.8%and 98.3%,respectively,indicating that CAR-T cells were strong cytotoxic to Ca Ski cells and less cytotoxic to HeLa cells.The concentration of IFN-γin Ca Ski cell culture medium reached 100 pg/mL,and that of IL-2 reached 24 pg/mL,while IFN-γand IL-2 of HeLa cell culture medium were 16 pg/mL and 6 pg/mL,respectively.Conclusion:CAR-T cells targeting HPV16 positive cervical cancer cells were successfully prepared,which provided preliminary research data for the treatment of cervical cancer with CAR-T cells.