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Archive > Volume 29 Issue 3 > 2022,29(3):181-188. DOI:10.3872/j.issn.1007-385X.2022.03.003 Prev Next
Basic Research
Incorporation of the YRHQ motif into CD3ζ chain enhances the antitumor activity of HER2-targeted CAR-T cells
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Abstract:
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.
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Clc Number:
R730.51
