Decreasing affinity of CAR-T cells targeting HER2 to increase therapeutic outcome against tumors

ZHANG Hao; YE Zhenlong; QIAN Qijun

doi:10.3969/j.issn.1006-0111.2016.03.018

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Objective To investigate whether decreasing affinity of CAR-T cells can increase their therapeutic outcome or not. Methods Moderate affinity La-G3HER2-CAR and high affinity Ha-G3HER2-CAR were constructed, and electroporated to modify T cells. Western blot assay, FCM assay and the RTCA DP cytotoxic equipment were applied to test the CAR expression and cytotoxic function of CAR-T cells. Results 43000 and 58000 exogenous CD3ζ fragments were expressed by both La-G3HER2-CAR-T cells and Ha-G3HER2-CAR-T cells with 58.1% and 69.0% transfection rate respectively. High affinity Ha-G3HER2-CAR-T cells effectively killed all target tumor cells by which HER2 was expressed at variable expression levels, while moderate affinity La-G3HER2-CAR-T cells specifically killed HER2 high-level expressing SK-OV-3 and BT474 cells, and showed weaker cytotoxicity on HER2 moderate-level expressing MDA-MB-231 and HCC-202 cells, and showed no cytotoxicity on HER2 low-level expressing MCF-7 and 293 cells. The underlying mechanic investigation found that La-G3HER2-CAR-T cells and Ha-G3HER2-CAR-T cells were differentially activated by co-culture with MDA-MB-231(CD107a:8.2% vs 71.6%, IFN-γ:66.3% vs 83.4%, TNF-α:73.4% vs 94.1%). Conclusion Moderate affinity La-G3HER2-CAR-T cells have enhanced specific cytotoxicity toward target tumor cells compared to high affinity Ha-G3HER2-CAR-T cells, decreasing affinity of CAR-T cell is a promising strategy to increase the therapeutic outcome of CAR-T cell based immunotherapies.

Decreasing affinity of CAR-T cells targeting HER2 to increase therapeutic outcome against tumors

Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China

Received Date: 2016-04-20

Rev Recd Date: 2016-05-04

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Abstract: Objective To investigate whether decreasing affinity of CAR-T cells can increase their therapeutic outcome or not. Methods Moderate affinity La-G3HER2-CAR and high affinity Ha-G3HER2-CAR were constructed, and electroporated to modify T cells. Western blot assay, FCM assay and the RTCA DP cytotoxic equipment were applied to test the CAR expression and cytotoxic function of CAR-T cells. Results 43000 and 58000 exogenous CD3ζ fragments were expressed by both La-G3HER2-CAR-T cells and Ha-G3HER2-CAR-T cells with 58.1% and 69.0% transfection rate respectively. High affinity Ha-G3HER2-CAR-T cells effectively killed all target tumor cells by which HER2 was expressed at variable expression levels, while moderate affinity La-G3HER2-CAR-T cells specifically killed HER2 high-level expressing SK-OV-3 and BT474 cells, and showed weaker cytotoxicity on HER2 moderate-level expressing MDA-MB-231 and HCC-202 cells, and showed no cytotoxicity on HER2 low-level expressing MCF-7 and 293 cells. The underlying mechanic investigation found that La-G3HER2-CAR-T cells and Ha-G3HER2-CAR-T cells were differentially activated by co-culture with MDA-MB-231(CD107a:8.2% vs 71.6%, IFN-γ:66.3% vs 83.4%, TNF-α:73.4% vs 94.1%). Conclusion Moderate affinity La-G3HER2-CAR-T cells have enhanced specific cytotoxicity toward target tumor cells compared to high affinity Ha-G3HER2-CAR-T cells, decreasing affinity of CAR-T cell is a promising strategy to increase the therapeutic outcome of CAR-T cell based immunotherapies.

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Decreasing affinity of CAR-T cells targeting HER2 to increase therapeutic outcome against tumors

doi: 10.3969/j.issn.1006-0111.2016.03.018

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