Effect of 4-1BBL-OX40L fusion protein on the anti-tumor activity of CAR-T cells in solid tumors.

Abstract

e14535 Background: Chimeric antigen receptor T (CAR-T) cell therapy has shown clinical success in hematologic malignancies but faces challenges in solid tumors, including limited persistence and tumor infiltration, contributing to relapse and suboptimal efficacy. Both the 4-1BB/4-1BBL and OX40/OX40L pathways are critical for T cell activation, proliferation, differentiation, survival, and antitumor activity. To address these limitations, we engineered CAR-T cells to co-express a membrane-bound fusion protein combining 4-1BBL and OX40L, providing an optimized CAR-independent survival signal for both CD8 + and CD4 + T cells while avoiding 4-1BB-associated liver toxicity. Methods: We developed B7-H3-targeted CAR-T cells co-expressing 4-1BBL or a 4-1BBL-OX40L fusion protein. The expression of 4-1BBL, 4-1BBL-OX40L fusion protein, and CAR was assessed using flow cytometry (FCM). The functionality of 4-1BBL and 4-1BBL-OX40L fusion protein was evaluated through in vitro and in vivo experiments. T cell reactivity was assessed by measuring cytotoxicity, proliferation, IFN-γ secretion, and exhaustion markers (PD1, TIM3, and TIGIT). In vivo anti-tumor activity was assessed in glioblastoma (U87 MG) and colon cancer (LoVo) xenograft mouse models, with CAR-T cell persistence and tumor infiltration quantified via FCM and immunohistochemistry. Results: Both 4-1BBL and the 4-1BBL-OX40L fusion protein significantly enhanced B7-H3 CAR-T cell proliferation and cytotoxicity. However, CAR-T cells co-expressing the 4-1BBL-OX40L fusion protein exhibited superior proliferation compared to those co-expressing 4-1BBL alone. In both the LoVo and U87 MG xenograft mouse models, CAR-T cells co-expressing the 4-1BBL-OX40L fusion protein demonstrated superior anti-tumor activity and T cell infiltration compared to CAR-T cell only and CAR-T co-expressing 4-1BBL. Conclusions: These results suggest that co-expressing 4-1BBL-OX40L fusion protein improves anti-tumor activity of B7-H3-targeted CAR-T cells against solid tumors in vitro and in vivo . B7-H3-targeted CAR-T cells co-expressing 4-1BBL-OX40L fusion protein is a promising treatment for solid tumors.