41P Single-cell signalling analysis of engineered γδ T cell biotherapeutics for the treatment of colorectal cancer

Colorectal cancer (CRC) is a devastating disease that kills ∼700,000 people worldwide annually, with immunotherapies struggling against the immunosuppressive CRC tumour microenvironment (TME). Tumour infiltrating γδ T cells confer prognostic benefit to patients and can kill via multiple innate adaptive mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) antigens (e.g., B7-H3). We hypothesise be exploited as an anti-CRC biotherapeutic but their complex interactions within TME need elucidated. performed 3D cultures of patient-derived organoids (PDOs) cancer-associated fibroblasts (CAFs) human Vγ9Vδ2 cells, either unmodified or engineered secrete modified IL15 cytokine. The addition anti-B7-H3 IgG also allows modelling anti-PDO ADCC, CRISPR-Cas9 PDO models generated for demonstration antigen specificity. Using 126-plex Thiol Organoid Barcoding in situ (TOBis) mass cytometry (MC), we measure over 60 parameters per cell across hundreds culture conditions (Qin et al., Nature Methods, 2020) (Sufi Qin Protocols, 2021). Cell-type specific signalling analysis donors PDOs post-translational modifications (PTMs), cell-state immunological phenotype were analysed computationally. Engineered exhibit superior proliferation, purity, cytotoxicity, viability, donor-specific responses cytokine engineering (127IdU+ pSTAT5 [Y694]+) co-culture (GranzymeB+ CD69+). Multiple generate substantial killing various (cCaspase3 [D175]+ cPARP [D214]+). detect transfer Granzyme B from PDOs, executing apoptosis. ADCC varies between associated FcγR CD16 expression. have identified marker signatures selection candidate biotherapeutics. CAFs protect work continuing understand mechanistic basis stromal protection.