Abstract B010: A PD-L1 x CD3 bispecific antibody enhances the anti-tumor efficacy of regorafenib in pre-clinical colon cancer models

Abstract Background Colorectal cancer (CRC) remains a leading cause of cancer-related deaths worldwide. The current standard of care for patients may involve surgery, chemotherapy, and immune checkpoint inhibitors (ICIs), but these approaches typically fail to secure durable responses against late-stage disease. Regorafenib (REG) is an FDA approved tyrosine kinase inhibitor with immunomodulating properties (inciting, for example, immunogenic cell death [ICD]) for CRC patients who progress on standard care, but 5-year relative survival rates for individuals dosed with the drug as a monotherapy are poor. We hypothesize that REG may be more appropriately leveraged alongside immunotherapeutic agents that specifically stimulate T cell infiltration and activation within the tumor microenvironment (TME). We, therefore, engineered a PD-L1 x CD3 bispecific antibody (bsAb) that simultaneously binds PD-L1-expressing CRC cells and activates nearby T cells in order to investigate combination strategies with REG in pre-clinical models of CRC. Methods REG’s ability to induce ICD was evaluated by assessing calreticulin expression by flow cytometry and ATP release through luminescence from MC38 and CT26 CRC cells in vitro. A PD-L1 x CD3 bsAb was next engineered, transiently expressed in Expi293 cells, purified through affinity chromatography, and tested for binding cognate ligands and stimulating T cell function through flow cytometry and ELISA. Lastly, the anti-tumor and immune-driven roles of REG + bsAb therapy were explored in MC38 and CT26 in vivo models using a compilation of IHC, IF, qPCR, and ELISA techniques. Results Our in vitro data overall confirm ICD-induction by REG and demonstrate selective binding and T cell stimulation using a newly designed PD-L1 x CD3 bsAb. Combined REG + bsAb therapy also safely initiated and sustained inhibition against MC38 and CT26 progression in vivo, and these effects correlated to improved CD8+ T cell infiltration and activity within a Type-1-prone TME. Additionally, cytotoxic CD8+ T cells from REG + bsAb-sensitized mice more readily engaged tumor cells than animals treated with either agent alone. Conclusions We provide evidence that the immunomodulatory benefits of REG can be effectively paired with a bsAb that works to anchor to CRC cells, diminish immunosuppression (through PD-L1 blockade), and activate/sustain antigen-specific CD8+ T cells within the TME. This newly described REG + bsAb regimen led to improved anti-tumor outcomes pre-clinically, and may represent a promising future approach for CRC patients. Citation Format: Izuchukwu F Okpalanwaka, Elizabeth A Daugherity, Amanda L McCormick, Trevor S Anderson, Savanna L Smith, Caryn Lawrence, Devin B Lowe. A PD-L1 x CD3 bispecific antibody enhances the anti-tumor efficacy of regorafenib in pre-clinical colon cancer models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2024 Oct 18-21; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2024;12(10 Suppl):Abstract nr B010.