目的： 探讨在靶向HER2的CAR的CD3ζ链胞内区引入YRHQ基序对CAR-T细胞的特异性杀伤活性及免疫记忆形成的影响。 方法： 通过DNA合成获得包含靶向HER2的编码抗原受体H28ζ或H28ζ(YRHQ)的DNA片段，通过慢病毒载体将不同CAR的DNA片段分别转导健康人外周血T细胞，制备靶向HER2的H28ζ-CAR-T及H28ζ(YRHQ)-CAR-T细胞。扩增过程中对不同CAR-T细胞进行计数，FCM检测CAR的表达率。将CAR-T细胞分别与HER2阳性的SKOV3、MDA-MB-453或HER2阴性的MCF-7细胞共培养，LDH释放法检测其杀伤活性，ELISA法检测IL-2、IFN-γ和颗粒酶B的水平，WB法检测STAT3磷酸化水平及免疫检查点分子TIM-3和PD-1的表达，通过FCM检测CCR7、CD45RO的表达，分析CAR-T细胞的表型。 结果： H28ζ-CAR-T和H28ζ(YRHQ)-CAR-T细胞扩增能力较好，体外培养7 d时扩增4~5倍。H28ζ-CAR和H28ζ(YRHQ)-CAR表达率分别为（33.3±2.85）%和（28.30±3.2）%。H28ξ(YRHQ)-CAR-T细胞的杀伤活性较H28ζ-CAR-T细胞更高（P<0.05）。经HER2抗原刺激后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞的STAT3磷酸化水平较H28ξ-CAR-T细胞明显升高（P<0.01）；而两者间PD-1和TIM-3的表达无明显差异。未经抗原刺激的CAR-T细胞CCR7和CD45RO表达与正常T细胞比较差异无统计学意义（均P>0.05），与SKOV3细胞共培养后，与T细胞或H28z-CAR-T细胞比较，H28ξ(YRHQ)-CAR-T细胞中 T EM 细胞比例明显增加、T CM 细胞比例明显减少（均P<0.05）。 结论： 在CD3胞内区引入YRHQ基序可在一定程度上提高CAR-T细胞的杀伤潜力。

Objective: To investigate the effect of incorporating YRHQ motif into the intracellular CD3ζ region of chimeric antigen receptor (CAR) targeting HER2 (human epidermal growth factor receptor 2) on the specific killing activity and immune memory formation of CAR-T cells. Methods: DNA fragments encoding the antigen receptor H28ζ or H28ζ(YRHQ) were obtained by DNA synthesis. H28ζ-CAR-T and H28ζ(YRHQ)-CAR-T cells targeting HER2 were developed by transducing different CAR DNA fragments into T cells from healthy human peripheral blood using lentiviral vectors. The number of different CAR-T cells was counted during amplification, and CAR expression rate was detected by FCM. The CAR-T cells were incubated with HER2 positive SKOV3, MDA-MB-453 cells or HER2 negative MCF-7 cells, respectively. Then, the killing activity of CAR-T cells was measured by LDH release assay, the levels of IL-2, IFN-γ and GZMB were measured by ELISA, the phosphorylation level of STAT3 and the expression of immune checkpoint molecules TIM-3 and PD-1 were detected by WB, and the expression of CCR7 and CD45RO was detected by FCM. In addition, the phenotypes of CAR-T cells were analyzed. Results: Both H28ζ-CAR-T and H28ζ(YRHQ)-CAR-T cells had good amplification ability and expanded 4-5 folds at 7th day of culture in vitro. The expression rate of H28ζ-CAR or H28ζ(YRHQ) CAR in T cells were (33.3±2.85)% and (28.30±3.2)%, respectively. A higher cytotoxicity of H28ζ(YRHQ)-CAR-T cells than H28ζ-CAR-T cells was observed (P<0.05). After HER2 antigen stimulation, the STAT3 phosphorylation level of H28ζ(YRHQ)-CAR-T cells was significantly higher than that of H28ζ-CAR-T cells (P<0.01); however, no significant difference in the expressionof PD-1 and TIM-3 was observedbetween two CAR-T cells. The expression of CCR7 and CD45RO in the CAR-T cells without antigen stimulation was not significantly different from that in normal T cells (both P>0.05). After co-culture with SKOV3 tumor cells, compared with T cells or H28ζ(YRHQ) -CAR-T cells, the the proportion of T EM cells increased, while the proportion of the T CM cells decreased significantly in H28ζ(YRHQ)-CAR-T cells (all P<0.05). Conclusion: The incorporation of YRHQ motif in CD3 intracellular region could improve the killing potential of CAR-T cells to some extent.

R730.51

河北省卫生健康委医学科研资助项目（No. 20200037）