Abstract 3597: Multimodal γδ T cell cytotoxicity overcomes cellular therapy reprogramming

Abstract Colorectal cancer (CRC) is a devastating disease that kills ~700, 000 people worldwide annually. Current immunotherapies struggle against both microsatellite stable (MSS) disease and the immunosuppressive CRC tumor microenvironment (TME). Despite these challenges, tumor infiltrating γδ T cells confer a prognostic benefit to CRC patients and can kill cancer via antibody independent cytotoxicity (AIC) and antibody-dependent cellular cytotoxicity (ADCC). We hypothesized that γδ T cells can be exploited as an ‘off-the-shelf’ anti-CRC biotherapeutic but their complex interactions within the CRC TME need to be elucidated. To explore the patient-, donor-, and mechanism-specific interactions of γδ T cells with CRC, we performed single-cell profiling of 1, 000 CRC patient-derived organoid (PDO) and human Vγ9Vδ2 T cell cultures. γδ T cells from multiple donors were used either unmodified or engineered to secrete a modified IL-15 cytokine (stIL15-γδs). The addition of anti-B7-H3 IgG further allowed us to study anti-PDO ADCC, with antigen specificity assessed with B7-H3KO PDO models. Using 126-plex Thiol Organoid Barcoding in situ (TOBis) mass cytometry (MC) (Qin et al. , Nature Methods, 2020) (Sufi and Qin et al. , Nature Protocols, 2021), we measured cell-type specific signaling across multiple γδ donors and CRC PDOs, including post-translational modifications (PTMs), cell-state, and immunological phenotype. We found that stIL15-γδs exhibit superior proliferation, purity and viability both in vitro and in vivo. stIL15-γδs demonstrated significant cytotoxicity against all CRC PDOs tested, commonly outperforming standard-of-care chemotherapies even when challenged against our previously-defined chemorefractory MSS patients (Zapatero et al. , Cell, 2023). The susceptibility of PDOs to anti-B7-H3 ADCC differs substantially and is not associated with B7-H3 antigen density and %positivity or PDO tumor mutational burden. Minimal differences in ADCC capability were seen between stIL15-γδs donors which were not associated with γδ phenotype or pre- and post-experiment FcγR CD16 expression. Crucially, we found that stIL15-γδ signaling is reciprocally regulated in a PDO-specific manner, with significant regulation of stIL15-γδ cell-state, PTM signaling and immunological phenotype observed. Increased dysregulation of stIL15-γδs during AIC negatively correlates with PDO cytotoxicity — indicating PDOs negatively reprogram γδ T cellular therapies. However, when γδ T cells engage in ADCC (via anti-B7-H3 IgG), stIL15-γδ signaling recovers, leading to substantial anti-PDO cytotoxicity. Furthermore, stIL15-γδs could perform significant cytotoxicity against all chemoresistant MSS PDOs tested, representing the patient population with greatest unmet clinical need. These results demonstrate that multimodal γδ T cell cytotoxicity can overcome tumor-specific cellular therapy reprogramming. Citation Format: Callum Baird Nattress, Daniel Fowler, Petra Vlckova, Colin Hutton, Maria Ramos Zapatero, Jahangir Sufi, Ferran Cardoso Rodriguez, Ashley Campbell, Angeliki Kanouta, Magdalena Buschhaus, Kerry Chester, John Anderson, Vivian Li, Marta Barisa, Jonathan Fisher, Christopher Tape. Multimodal γδ T cell cytotoxicity overcomes cellular therapy reprogramming abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3597.