scFv亲和力对CAR-T细胞抗肿瘤能力的影响

Comparison of antitumor ability of CAR-T cells based on affinity of scFv

目的:比较抗原亲和力不同的scFv片段对CAR-T细胞增殖、分化和抗肿瘤能力的影响。方法:分别构建靶向ErbB2的FRP5(K_D=30 nmol/L)和chA21(K_D=11 nmol/L)CAR慢病毒表达载体,包装病毒后制备CD8^+CAR-T细胞;在CAR-T细胞扩增过程中,通过计数细胞来检测细胞增殖;利用流式细胞术检测CAR-T细胞分化情况;利用荧光素酶活性法检测杀伤活性;用ELISA检测CAR-T细胞在肿瘤识别过程中IL-2和IFN-γ的分泌情况;利用成像流式细胞仪检测scFv亲和力对CAR-T细胞形成免疫突触能力的影响。结果:成功构建了CD8^+ CAR-T细胞,FRP5和chA21 CAR阳性率均达50%左右;CAR-T细胞的增殖和分化状态没有差异;荧光素酶活性法显示chA21-CAR-T细胞裂解肿瘤细胞的能力较强;ELISA检测结果显示chA21-CAR-T细胞分泌细胞因子能力更强;chA21-CAR-T细胞形成免疫突触的能力更强。结论:包含高亲和力scFv的chA21-CAR-T细胞具有较强的抗原依赖性杀伤活性。

Objective:Comparing the effect on the proliferation,differentiation and cytotoxicity of the chimeric antigen receptor-T cells(CAR-T cells) based on the affinity of their single-chain variable fragment(scFv) regions to tumor antigen.Methods:The FPR5-CAR(K_D=30 nmol/L) or chA21-CAR(K_D=11 nmol/L) recognizing ErbB2 were constructed into lentiviral vectors to manufacture CD8^+ T cells.The proliferation of CAR-T cells was detected by cell-counting during their expansion.The differentiation of CAR-T cells was detected by flow cytometry.The cytotoxicity of CAR-T cells was evaluated through luciferase imaging.IL-2 and IFN-γ secretion by CAR-T cells during tumor recognition were detected by ELISA.The ability of CAR-T cells with different affinity to form immune synapses were detected by imaging flow cytometry.Results:CD8^+ CAR-T cells were constructed successfully,and the positive rate of FRP5-CAR-T and chA21-CAR-T was 50%.There was no difference in the ability of cell proliferation or differentiation between the two kinds of CAR-T cells.Compared to FRP5-CAR-T cell,chA21-CAR-T cell could secrete more cytokines,resulting in greater cytotoxicity.The ability of chA21-CAR-T cells to form immune synapses was stronger.Conclusion:The CAR-T cells containing scFv of high affinity have greater antigen-dependent cytotoxicity.