ABSTRACT
Objective@#To investigate the different functions of humanized and murinized CD19 chimeric antigen receptor (CAR)-T cells against Raji cell line in vitro and in vivo.@*Methods@#Peripheral blood samples were collected from eight patients with lymphoma who were going to receive CD19 CAR-T cell therapy and used for the preparation of peripheral blood mononuclear cells (PBMC) as well as humanized and murinized CAR-T cells. Cell proliferation and cytotoxicity were detected with CCK-8 and LDH assays, respectively. A tumor-bearing mouse model was established by injecting BALB/c female nude mice with fluorescent Raji cells. Changes in tumor volume in these mice were observed by in vivo imaging technology. The transfection efficiency and amount of CAR-T cells in the mice were detected with flow cytometry.@*Results@#No statistical difference in transfection efficiency was found between humanized and murinized CAR-T cells, nor in cell proliferation at 24 h of culture in vitro(P=0.104). The proliferation of humanized CAR-T cells showed a significant increase compared with that of murinized CAR-T cells at 48 h of culture (P=0.009). Similarly, the cytotoxicity of the two types of CAR-T cells against Raji cells showed no significant difference at 24 h at any effector/target (E/T) ratio (1∶1 or 4∶1), and that of humanized CAR-T cells was higher than that of murinized CAR-T cells at both E/T ratios at 48 h (E/T ratio=1∶1, P=0.005; E/T ratio=4∶1, P=0.008). Moreover, the cytotoxicity of CAR-T cells was higher than that of PBMC in any case. Tumor volumes in mice were reduced 14 d after humanized or murinized CAR-T cell therapy, while the mice in the PBMC control group suffered tumor progression. Tumor volume began to increase in mice 21 d after murinized CAR-T cell therapy, while no significant change was observed in the mice treated with humanized CAR-T cells. All of the mice died 25 d after murinized CAR-T cell therapy, while the deaths among those under humanized CAR-T cell therapy occurred on 31 d. The proportion of CAR-T cells in mice reached the peak 7 d after receiving humanized or murinized CAR-T cell therapy, while that in the humanized group was significantly higher than that in the murinized group at any time point (P4 d=0.001, P7 d=0.000, P14 d=0.003). Murinized CAR-T cells became undetectable on 21 d, while humanized CAR-T cells on 35 d. The maximum survival time for mice in the PBMC and murinized and humanized CAR-T cell groups was 20 d, 25 d and 53 d, respectively.@*Conclusions@#Compared with murinized CD19 CAR-T cells, humanized CD19 CAR-T cells showed stronger proliferation potential and cytotoxicity and remained in vivo detectable for a longer period of time. This study indicated that humanized CD19 CAR-T cells were superior to murinized CD19 CAR-T cells for the treatment of B cell lymphoma.
ABSTRACT
Objective To investigate the different functions of humanized and murinized CD19 chimeric antigen receptor ( CAR)-T cells against Raji cell line in vitro and in vivo. Methods Peripheral blood samples were collected from eight patients with lymphoma who were going to receive CD19 CAR-T cell therapy and used for the preparation of peripheral blood mononuclear cells ( PBMC) as well as humanized and murinized CAR-T cells. Cell proliferation and cytotoxicity were detected with CCK-8 and LDH assays, respectively. A tumor-bearing mouse model was established by injecting BALB/c female nude mice with flu-orescent Raji cells. Changes in tumor volume in these mice were observed by in vivo imaging technology. The transfection efficiency and amount of CAR-T cells in the mice were detected with flow cytometry. Re-sults No statistical difference in transfection efficiency was found between humanized and murinized CAR-T cells, nor in cell proliferation at 24 h of culture in vitro(P=0. 104). The proliferation of humanized CAR-T cells showed a significant increase compared with that of murinized CAR-T cells at 48 h of culture ( P=0. 009). Similarly, the cytotoxicity of the two types of CAR-T cells against Raji cells showed no significant difference at 24 h at any effector/target (E/T) ratio (1 : 1 or 4 : 1), and that of humanized CAR-T cells was higher than that of murinized CAR-T cells at both E/T ratios at 48 h (E/T ratio=1 : 1, P=0. 005;E/T ratio=4 : 1, P=0. 008). Moreover, the cytotoxicity of CAR-T cells was higher than that of PBMC in any case. Tumor volumes in mice were reduced 14 d after humanized or murinized CAR-T cell therapy, while the mice in the PBMC control group suffered tumor progression. Tumor volume began to increase in mice 21 d after murinized CAR-T cell therapy, while no significant change was observed in the mice treated with hu-manized CAR-T cells. All of the mice died 25 d after murinized CAR-T cell therapy, while the deaths among those under humanized CAR-T cell therapy occurred on 31 d. The proportion of CAR-T cells in mice reached the peak 7 d after receiving humanized or murinized CAR-T cell therapy, while that in the humanized group was significantly higher than that in the murinized group at any time point (P4 d=0. 001, P7 d=0. 000, P14 d=0. 003). Murinized CAR-T cells became undetectable on 21 d, while humanized CAR-T cells on 35 d. The maximum survival time for mice in the PBMC and murinized and humanized CAR-T cell groups was 20 d, 25 d and 53 d, respectively. Conclusions Compared with murinized CD19 CAR-T cells, humanized CD19 CAR-T cells showed stronger proliferation potential and cytotoxicity and remained in vivo detectable for a longer period of time. This study indicated that humanized CD19 CAR-T cells were superior to murinized CD19 CAR-T cells for the treatment of B cell lymphoma.
ABSTRACT
Objective To study the effect of two-step pretargeting radioimmunotherapy of CD45 McAb and 188Re-Avidin on lymphoma Raji cell line.Methods The CD45 McAb and Avidin were directly labeled with 188Re,and the labeling efficiency and radiochemical purity were measured by the paper chromatography.The specific binding test and competition binding test between 188 Re-CD45 McAb and Raji cells in vitro were also performed.CCK-8 assay was used to determine the inhibition effect on Raji cell proliferation in the pretargeted group,188Re-CD45 McAb,188Re-Avidin and 188ReO4 groups,then the cell survival and proliferation inhibition rate were calculated.Results The specific cell binding rate of 188Re-CD45 McAb with Raji cells was (70.92 ± 1.91) ％,in the competition group,the binging rate of 188Re-CD45 McAb with Raji cells was only (7.96 ± 0.87)％.The Raji cells proliferation was inhibited in all groups with 188Re radiolabel,and the inhibition rate was positively correlated with the radioactivity dose (r=0.907-0.992,P ＜0.05).However,at the same dose,the inhibition effect in the group of two-step pretargeting at each time point were all stronger than those of 188Re-CD45 McAb,188Re-Avidin and 188 ReO4-alone (t =124.76-607.98,P ＜ 0.05).But there was no significantly statistical difference in the inhibitory effect between the groups of 188Re-Avidin and 188ReO4-(P ＞ 0.05).Conclusions It is confirmed that 188Re-CD45 McAb could be specifically bound to Raji cells,and the two-step pretargeting of CD45 McAb and 188Re-Avidin has obvious inhibitory effect on the Raji cell proliferation.