CD28突变的CAR T细胞疗法展现出良好的安全性

The mutation of CD28 exhibits improved safety in CAR T immunotherapy

为改善CD19靶向的嵌合抗原受体(chimeric antigen receptor,CAR)修饰的T细胞治疗后潜在的细胞因子风暴问题,提高临床应用的安全性,将CD28共刺激结构域中的YMNM和PYAP基序双突变,并分别构建包含CD28结构域(19-CD28)和CD28突变结构域(19-CD28M)的CD19-CAR,利用慢病毒载体构建CD19-CAR T细胞和CD19-CAR Jurkat细胞。将CD19-CAR Jurkat细胞株与肿瘤细胞共孵育来验证CD19-CAR对Jurkat细胞的特异性活化效果,随后检测CD19-CAR T细胞与靶细胞共孵育后的细胞毒性和细胞因子释放量。结果显示,与对照组比较,19-CD28-CAR Jurkat细胞和19-CD28M-CAR Jurkat细胞与Raji(CD19~+)细胞共孵育后CD69表达水平上调60%以上,CD25表达水平上调10%左右。在效应细胞∶靶细胞分别为0.5∶1、1∶1、5∶1的条件下,19-CD28-CAR T细胞和19-CD28M-CAR T细胞诱导Raji(CD19~+)细胞的凋亡率比对照组分别增加了15%、25%和40%左右,并于效应细胞∶靶细胞为5∶1时达到饱和。与此同时,19-CD28M-CAR T细胞释放细胞因子Granzyme B、IFN-γ、IL-2、TNF-α的量分别下降了14%、28%、34%和33%。该研究提示,CD28共刺激结构域突变的CD19-CAR T细胞毒性并未减弱,同时安全性显著增强。

The goal of this study is to improve the potential cytokine storm problem after CD19-targeted chimeric antigen receptor(CAR) T cell therapy and increase the safety of the clinical application. To this end, the YMNM and PYAP fragments in the CD28 costimulatory domain were double-mutated. The CD19-CAR containing either the CD28 domain(19-CD28) or the CD28 mutation domain(19-CD28 M) were constructed and a lentiviral system was used to generate the CD19-CAR T cells and CD19-CAR Jurkat cells. The CD19-CAR Jurkat cells were then incubated with tumor cells to verify the specific activation of CD19-CAR on Jurkat cells. Then the cytotoxicity and cytokine release of CD19-CAR T cells after incubation with target cells were evaluated. The results showed that compared with control, the 19-CD28-CAR Jurkat cells and 19-CD28 M-CAR Jurkat cells incubated with Raji(CD19) cells showed an increase of CD69 expression by more than 60%, and CD25 by about 10%. The apoptotic rates of Raji(CD19) cells induced by 19-CD28-CAR T cells and 19-CD28 M-CAR T cells were about 15%, 25%, and 40% higher than those of the control group at the ratio of effect to the target of 0.5∶1, 1∶1 and 5∶1, respectively. The killing effect reached saturation at the ratio of effect to the target of 5∶1. Meanwhile, the release of Granzyme B, IFN-γ, IL-2, and TNF-α in the 19-CD28 M-CAR T cells decreased by 14%, 28%, 34%, and 33%, respectively. The results indicate that while the cytotoxicity of CD19-CAR T cells with mutations in the CD28 costimulatory domain does not cause weakened killing activity, the safety is significantly enhanced.