Enhanced anti-ovarian cancer efficacy with MSLN-chimeric antigen receptor T cells secreting anti-CD39 antibody.

Abstract

e17553 Background: The effectiveness of chimeric antigen receptor (CAR) T cell therapy directed at mesothelin (MSLN) in solid tumors, such as ovarian cancer, is hindered by the immunosuppressive tumor microenvironment (TME), where adenosine plays a crucial role in impairing CAR-T cell function. Preclinical and clinical studies have demonstrated that targeting CD39, a pivotal enzyme catalyzing the conversion of extracellular ATP into adenosine, yields significant antitumor effects. Therefore, combining MSLN CAR-T with an anti-CD39 antibody emerges as a potential therapeutic strategy against cancer. Methods: We have previously designed OriA631, an antibody antagonist targeting CD39 in VHH format. Our preclinical investigation has highlighted the outstanding potential of OriA631, showcasing superior performance in binding affinity, effective inhibition of CD39 enzyme activity, and remarkable in vivo high anti-tumor efficacy. Subsequently, we undertook further engineering of MSLN CAR-T cells to secrete the VHH component of OriA631 (MSLN CAR-T-CD39-VHH) and systematically assessed its antitumor effects in NSG mice. Results: Our data reveals that MSLN CAR-T cells exhibit a significant upregulation of CD39 upon activation by ovarian cancer cells in both in vitro and in the tumor microenvironment (TME). To assess the combined effect of MSLN CAR-T with anti-CD39, B-NDG mice were subcutaneously inoculated with 3x10^6 ovarian cancer cells (SKOV3-MSLNhi-CD39hi). Subsequently, different T cells (1x10^7 cells/mouse) were injected when the tumor reached around 80 mm³, and the groups were divided into Mock T, MSLN CAR-T, MSLN CAR-T-CD39-VHH, and MSLN CAR-T in combination with locally injected CD39 Ab (OriA631). The data demonstrated that MSLN CAR-T inhibited tumor growth from approximately 1000mm³ (Mock T group) to 300mm³ at day 30 relative to T cell injection. Tumors in the MSLN CAR-T in combination with OriA631 group were around 100mm³, suggesting a significant combination anti-tumor efficacy of MSLN CAR-T in conjunction with CD39 targeting. Furthermore, MSLN CAR-T-CD39-VHH, where MSLN CAR-T cells secrete the VHH component of OriA631, completely eliminated the tumor at day 18 with no relapse during the experimental period. This result underscores the superior anti-tumor efficacy of MSLN CAR-T-CD39-VHH, with the high level of VHH component of CD39 Ab observed in the TME. Importantly, the low or undetectable level of VHH component of CD39 Ab in the serum also indicates the safety of the product. Conclusions: Our study introduces the innovative therapeutic agent MSLN CAR-T-CD39-VHH, showcasing superior anti-tumor efficacy and the potential for reduced antibody-associated side effects. These findings strongly endorse the application of MSLN CAR-T-CD39-VHH as a promising and impactful therapeutic approach for the treatment of ovarian cancer.