Abstract 6397: Overexpression of Serpin B9 in anti-tumor T cells restrains granzyme B mediated auto-destruction and improves the therapeutic effect of adoptive cell therapy in ovarian cancer

Abstract Adoptive cell therapy (ACT) has demonstrated success in treating blood cancers. However, it remains less effective against solid tumors, such as ovarian cancers. A major challenge for this therapy is chronic T-cell stimulation within the tumor microenvironment, leading to T-cell dysfunction and activation-induced cell death (AICD). Improving T-cell survival by targeting key T-cell AICD pathways in the tumor microenvironment may augment ACT's effectiveness in solid tumors. We previously reported that overexpression of TCF1 in human T cells significantly inhibits granzyme B (GzmB) expression and improves T-cell survival after TCR stimulations. Based on these findings, we hypothesized that in addition to its critical role in T-cell cytotoxicity, GzmB limits anti-tumor T-cell functions by inducing T-cell autolysis. Therefore, inhibition of GzmB-dependent T-cell autolysis could enhance T-cell survival and anti-tumor function. In this study, we investigated the effect of overexpression of Serpin B9 (Sb9), an endogenous cytosolic inhibitor of GzmB, on anti-tumor functions of human T cells. Using a pH-dependent GFP reporter protein fused to GzmB, we found that GzmB is leaked into the cytosol of effector T cells after TCR stimulations, which is maximized at 2-3 days after stimulation. Silencing GzmB in T cells by shRNA significantly improved their proliferation after repetitive stimulations. Overexpression of Sb9 significantly improved T-cell survival compared to control T cells after repeated TCR stimulations without affecting degranulation and GzmB secretion. We further explored the effect of Sb9 overexpression on anti-tumor responses by HER2-CAR T cells. In vitro, Sb9-CAR T cells showed increased proliferation, IFN-γ production, and cytotoxicity after repeated stimulations with HER2+ ovarian cancer cells, compared to control CAR T cells. In vivo, Sb9-CAR T cells showed significantly improved therapeutic effects over control CAR T cells upon adoptive transfer in A2780 and OV-90 ovarian cancer xenograft models in immunodeficient NSG mice. Additionally, Sb9 overexpression in natural killer (NK) cells, which are also susceptible to GzmB-dependent autolysis, improved their viability, cytotoxicity, and anti-tumor functions, suggesting that Sb9 overexpression can also benefit NK-cell-based ACT. In summary, this study demonstrates that inhibiting GzmB-dependent autolysis without impairing GzmB-dependent cytotoxicity through Sb9 overexpression significantly enhances the survival and anti-tumor function of both CAR T and NK cells, offering a promising approach to improve ACT for solid tumors. Citation Format: Paulina Walczak, Takemasa Tsuji, Junko Matsuzaki, Kunle Odunsi. Overexpression of Serpin B9 in anti-tumor T cells restrains granzyme B mediated auto-destruction and improves the therapeutic effect of adoptive cell therapy in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6397.