人源化与鼠源CD19嵌合抗原受体-T细胞对急性淋巴细胞白血病细胞及其移植瘤的杀伤活性比较

Activity comparison of humanized CD19 CAR-T cells with murine CD19 CAR-T on Nalm-6 cells and xenograft tumor model

目的比较人源化CD19嵌合抗原受体(CAR)-T细胞与鼠源CD19 CAR-T细胞的功能。方法收集8名健康志愿者的外周静脉血T细胞,行人源化和鼠源CD19 CAR慢病毒感染,制备人源化和鼠源CD19 CAR-T细胞,采用细胞计数8法检测细胞增殖情况。以急性淋巴细胞白血病Nalm-6细胞作为靶细胞,采用乳酸盐脱氢酶法检测对照CD^(3+)T细胞、人源化及鼠源CD19 CAR-T细胞的细胞杀伤活性。将30只Nalm-6细胞荷瘤裸鼠采用随机区组法随机分为3组,每组10只,分别经尾静脉注射人源化CAR-T细胞、鼠源CAR-T细胞和对照CD^(3+)T细胞悬液。采用流式细胞术检测内眦静脉血Nalm-6细胞占外周血细胞比例和CD19 CAR-T细胞占T细胞比例。观察裸鼠的生存时间。结果体外培养24 h,鼠源和人源化CAR-T细胞的增殖率分别为(68.50±0.93)%和(80.63±1.41)%,差异有统计学意义(t=20.353,P<0.001);共培养48 h时,分别为(91.38±1.41)%和(148.13±1.25)%,差异亦有统计学意义(t=85.364,P<0.001)。效靶比为1∶1时,共培养24 h,人源化CD19 CAR-T细胞组与鼠源CD19 CAR-T细胞组对Nalm-6细胞的杀伤活性差异无统计学意义(P=0.169);共培养48 h,人源化CD19 CAR-T细胞组对Nalm-6细胞的杀伤活性高于鼠源CD19 CAR-T细胞组(P<0.01)。效靶比为4∶1时,共培养24和48 h,人源化CD19 CAR-T细胞组对Nalm-6细胞的杀伤活性均高于鼠源CD19 CAR-T细胞组(均P<0.01)。CAR-T细胞治疗第7天,人源化CD19 CAR-T细胞组、鼠源CAR-T细胞组裸鼠外周血中Nalm-6细胞比例下降至最低;21 d后,鼠源CAR-T细胞组裸鼠体内Nalm-6细胞比例高于人源化CAR-T细胞组(P_(21 d)=0.001,P_(28 d)<0.001,P_(35 d)<0.001)。人源化CD19 CAR-T细胞组和鼠源CAR-T细胞组裸鼠外周血中CAR-T细胞比例均于CAR-T细胞治疗第7天达到峰值,人源化CD19 CAR-T细胞组高于鼠源CAR-T细胞组(P_(7 d)=0.002)。鼠源CAR-T细胞组裸鼠外周血中CAR-T细胞于第14天消失,人源化CAR-T细胞组裸鼠外周血中CAR-T细胞至第21天消失。鼠源CAR-T细胞组和人源化CD19 CAR-T细胞组裸鼠的中位生存时间分别为42和63 d,差异有统计学意义(χ^(2)=15.382,P<0.001)。结论人源化CD19 CAR-T细胞比鼠源CD19 CAR-T细胞增殖能力强、对急性淋巴细胞白血病细胞的杀伤活性高、体内存留时间长,用于急性淋巴细胞白血病的临床疗效可能会优于鼠源CD19 CAR-T细胞。

Objective To compare the activity difference of the high affinity humanized CD19 chimeric antigen receptor(CAR)-T cells and murine CD19 CAR-T cells.Methods Peripheral venous blood T cells from 8 healthy volunteers were collected and infected with humanized and murine CD19 CAR lentivirus.Human and murine CD19 CAR-T cells were prepared and cell proliferation was detected by cell counting kit-8(CCK-8)method.The cytotoxicity of CD^(3+)T cells,humanized and murine CD19 CAR-T cells to NALM-6 cells was detected by lactate dehydrogenase assay.Thirty BAL B/c nude mice transplanted with NALM-6 cells were randomly divided into 3 groups with 10 mice in each group and injected humanized CD19 CAR-T cells,mouse CD19 CAR-T cells and control CD^(3+)T cell via tail vein,respectively.The proportion of NALM-6 cells in peripheral blood and the proportion of CD19 CAR-T cells in T cells from the vein of the inner canthus were detected by flow cytometry.The overall survival of BAL B/c nude mice was observed.Results The proliferation of mouse and humanized CD19 CAR-T cells were(68.50±0.93)%and(80.63±1.41)%,respectively(t=20.353,P<0.001)after cultured in vitro for 24 hours,and were(91.38±1.41)%and(148.13±1.25)%,respectively(t=85.364,P<0.001)after cultured for 48 hours.When the effect to target ratio was 1∶1,there was no difference between the humanized and murine CD19 CAR-T cell group after co-culture for 24 hours(P=0.169),while the killing activity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells(P<0.01)after 48 hours of co-culture.When the effect to target ratio was 4∶1,the cytotoxicity of humanized CD19 CAR-T cells against NALM-6 cells was higher than that of murine CD19 CAR-T cells in co-culture for 24 and 48 hours(P<0.01).On the seventh day of CD19 CAR-T cell therapy,the proportion of NALM-6 cells in the peripheral blood of BAL B/c nude mice decreased to the lowest level in the humanized CD19 CAR-T cell group and the murine CD19 CAR-T cell group.After 21 days,the proportion of NALM-6 cells in the murine CD19 CAR-T cell group was higher than that in the humanized CD19 CAR-T cell group(P_(21 d)=0.001,P_(28 d)<0.001,P_(35 d)<0.001).The proportion of humanized and murine CD19 CAR-T cells in the peripheral blood reached the peaks after 7 days of therapy,and the proportion of humanized CD19 CAR-T cells was higher than that of murine CAR-T cells(P_(7 d)=0.002).The CD19 CAR-T cells disappeared in the peripheral blood in the murine CD19 CAR-T cell group after 14 days of therapy,while in the humanized CD19 CAR-T cell group it disappeared after 21 days of therapy.The median survival of BAL B/c nude mice in the murine CD19 CAR-T cell group and the humanized CD19 CAR-T cell group was 42 days and 63 days,respectively(χ^(2)=15.382,P<0.001).Conclusions High affinity humanized CD19 CAR-T cells have stronger proliferation,higher cytotoxicity and longer survival time compared with those of murine CD19 CAR-T cells.The results indicate that the clinical efficacy of humanized CD19 CAR-T cells would be better than that of murine CD19 CAR-T cells.