基于BCMA突变体构建BCMA CAR-T细胞体外杀伤功能评价模型

Establishment of an in vitro cytotoxicity evaluation model for BCMA CAR-T cells based on BCMA mutants

目的:为解决野生型B细胞成熟抗原(BCMA)被γ分泌酶切割导致表达不稳定的问题,构建抵抗γ分泌酶切割的BCMA突变体并构建靶细胞,用于评价BCMA CAR-T细胞的杀伤功能。方法:将野生型BCMA的穿膜域替换为人CD8α穿膜域,构建抵抗γ分泌酶切割的BCMA突变体(BCMA-CD8αTM),构建过表达该突变体的U266(U266^(BCMA Mut))、K562(K562^(BCMA Mut))、SKOV3(SKOV3^(BCMA Mut))和CHO(CHO^(BCMA Mut))细胞;构建装载NFAT-EGFP报告基因的BCMA CAR Jurkat细胞(BCMA-CAR-Jurkat-Reporter)与U266^(BCMA Mut)细胞共培养,采用FCM检测该细胞中EGFP表达水平以指示NFAT激活水平,荧光素酶法检测BCMA CAR-T细胞对Luciferase标记的K562^(BCMA Mut)细胞的杀伤作用,实时无标记动态细胞分析技术(RTCA)检测BCMA CAR-T细胞对SKOV3^(BCMA Mut)和CHO^(BCMA Mut)细胞的杀伤作用。结果:应用γ分泌酶抑制剂LY411575抑制γ分泌酶活性,显著增强野生型U266细胞表面BCMA表达水平,平均荧光强度上调10倍以上;但撤除抑制剂后BCMA表达水平逐渐降低(P<0.01);BCMA-CD8αTM突变体可抵抗γ分泌酶的切割作用,在U266细胞表面稳定表达(P>0.05);U266细胞及过表达BCMA-CD8αTM的U266细胞与BCMA-CAR-Jurkat-Reporter细胞共培养后都可激活Reporter系统、增强EGFP表达,但该效应在BCMA-CD8αTM过表达的U266细胞中更显著(P<0.01);BCMA-CD8αTM在BCMA表达阴性的K562、SKOV3和CHO 3种靶细胞中成功过表达,且在LY411575处理下该突变体的表达水平仅有小幅度升高;荧光素酶法检测结果显示,不同效靶比下,BCMA CAR-T细胞均可特异、高效杀伤过表达BCMA-CD8αTM的K562细胞;RTCA结果显示,不同效靶比下,BCMA CAR-T细胞均可有效识别、杀伤过表达BCMACD8αTM的SKOV3和CHO细胞,但同等效靶比下的Mock-T细胞无此效应。结论:本实验构建的BCMA-CD8αTM突变体能够抵抗γ分泌酶的切割,在多种靶细胞表面稳定表达,为评价BCMA CAR-T细胞体外杀伤的有效性和特异性提供多种检测手段。

Objective:To engineer a BCMA mutant resistant toγ-secretase cleavage in order to stabilize wild-type BCMA expression afterγ-secretase cleavage and to generate target cells for measuring BCMA CAR-T cell cytotoxicity.Methods:Our study aimed to engineer a mutant BCMA protein(BCMA-CD8αTM)that could resistγ-secretase cleavage by replacing the transmembrane domain of the wild-type BCMA protein with the human CD8αsequence.Four different types of cells in which this mutant gene was expressed excessively were engineered,including U266(U266^(BCMA Mut)),K562(K562^(BCMA Mut)),SKOV3(SKOV3^(BCMA Mut)),and CHO(CHO^(BCMA Mut))cells.BCMA CAR Jurkat cells,loaded with the NFAT-EGFP reporter gene(BCMA-CAR-Jurkat-Reporter),were engineered and co cultured with U266^(BCMA Mut) cells.The expression level of EGFP was detected by FCM in order to indicate the activation level of NFAT.The cytotoxicity of BCMA CAR-T cells against Luciferase-labeled K562^(BCMA Mut) cells was detected by the luciferase assay.Additionally,real-time cell analysis(RTCA)technique was employed to detect the cytotoxicity of BCMA CAR-T cells against SKOV3^(BCMA Mut) and CHO^(BCMA Mut) cells.Results:Application ofγ-secretase inhibitor LY411575 to inhibitγ-secretase activity significantly enhanced the expression level of BCMA on the surface of wild-type U266 cells,and the average fluorescence intensity was increased by more than 10 times.However,the expression level of BCMA gradually decreased after removal of inhibitors(P<0.01).BCMA-CD8αTM mutant could resist the cleavage ofγ-secretase and expressed stably on the surface of U266 cells(P>0.05).U266 cells and U266 cells overexpressing BCMA-CD8αTM were co-incubated with BCMA-CAR-Jurkat-Reporter cells,both of which could activate the Reporter system and enhance the expression of EGFP,but the effect was more significant in U266 cells overexpressing BCMA-CD8αTM(P<0.01).BCMA-CD8αTM mutants were successfully overexpressed in 3 BCMA-negative target cells,namely K562,SKOV3 and CHO cells,and the expression level of the mutant was only slightly increased under LY411575 treatment.Luciferase assay results showed that under different target-effect ratios BCMA CAR-T cells could all be specific and efficient in killing K562 cells overexpressing BCMA-CD8αTM.RTCA results showed that under different target ratios BCMA CAR-T cells could all effectively recognize and kill SKOV3 and CHO cells overexpressing BCMA-CD8αTM,but Mock-T cells with the same target ratio had no such effect.Conclusion:The BCMA-CD8αTM mutant engineered in this study can resistγ-secretase cleavage and exhibits stable surface expression on various target cells,thus offering various methods for evaluating the efficacy and specificity of BCMA CAR-T cell cytotoxicity in vitro.