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
Authors: Paulina Walczak, Takemasa Tsuji, Junko Matsuzaki, Kunle Odunsi
Journal: Cancer Research (2025)

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.