Optimization study of CAR-T cell expansion targeting CD99

Abstract

Background and purpose: Chimeric antigen receptor T (CAR-T) cell therapy has shown remarkable efficacy in treating hematological and lymphatic system tumors, but its effectiveness in solid tumors is relatively poor, which is partly attributed to target selection. For Ewing sarcoma (ES), CD99 can be a potential target for CAR-T cells. However, due to T cells’ endogenous expression of CD99 protein, CAR-T cells targeting CD99 face limitations in their expansion capacity in vitro. This study aimed to identify the optimal conditions for preparing CD99 CAR-T cells by incorporating CD99 knockdown short hairpin RNA (shRNA), optimizing the multiplicity of infection (MOI) for lentiviral transduction, and screening for the best culture medium and container for CAR-T cell expansion. Methods: shRNA sequences were screened to enhance the expansion capacity of CD99 CAR-T cells. Different MOI, culture media, and containers were used to assess CAR-T cell transduction efficiency, cell viability, proliferation capacity, specific killing ability, and interferon-γ (IFN-γ) release levels under various conditions, in order to identify the optimal cell preparation conditions. Results: The expansion level of KO-CD99 CAR-T cells obtained through shRNA knockdown was significantly higher than that of CD99 CAR-T cells [(16.40±0.40) vs (6.33±1.53), P<0.01]. The optimal expansion effect was observed when the transduction MOI was between 0.25 and 1.0, and OptiVitro was used as the culture medium. CAR-T cells cultured in ventilated flasks exhibited significantly higher expansion rates compared to cells cultured in bags [MOI=0.25: (50.23±3.32) vs (13.02±4.82); MOI=0.50: (49.96±0.83) vs (18.25±2.88); MOI=1.00: (48.27±5.08) vs (13.16±6.26); P<0.01], with better cell phenotype and higher specific killing ability. Conclusion: KO-CD99 CAR-T cells obtained through shRNA technology can achieve stable expansion. Based on the optimization of expansion conditions, KO-CD99 CAR-T cells exhibit superior expansion capacity and a higher proportion of memory T cells when the MOI is between 0.25 and 1.00, OptiVitro is used as the culture medium, and ventilated flasks are used as the culture container. These findings lay a solid foundation for further clinical trials of CD99 CAR-T cell therapy for ES.

Keywords

• |cd99|chimeric antigen receptor t cell|amplification optimization|ko-cd99