41P - Single-Cell Signalling Analysis of Engineered γδ T cell Biotherapeutics for the Treatment of Colorectal Cancer

Background

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 a prognostic benefit to CRC patients and can kill cancer via multiple innate and adaptive mechanisms, including antibody-dependent cellular cytotoxicity (ADCC) against tumour antigens (e.g., B7-H3). We hypothesise that γδ T cells can be exploited as an anti-CRC biotherapeutic but their complex interactions within the CRC TME need to be elucidated.

Methods

We performed 3D TME cultures of CRC patient-derived organoids (PDOs) with CRC cancer-associated fibroblasts (CAFs) and human Vγ9Vδ2 T cells, either unmodified or engineered to secrete a modified IL15 cytokine. The addition of anti-B7-H3 IgG also allows the modelling of anti-PDO ADCC, with 3D CRISPR-Cas9 PDO models generated for demonstration of antigen specificity. Using 126-plex Thiol Organoid Barcoding in situ (TOBis) mass cytometry (MC), we measure over 60 parameters per cell across hundreds of culture conditions (Qin et al., Nature Methods, 2020) (Sufi and Qin et al., Nature Protocols, 2021). Cell-type specific signalling analysis across multiple γδ donors and CRC PDOs including post-translational modifications (PTMs), cell-state and immunological phenotype were analysed computationally.

Results

Engineered γδ T cells exhibit superior proliferation, purity, cytotoxicity, and viability, with γδ donor-specific responses to cytokine engineering (¹²⁷IdU⁺ and pSTAT5 [Y694]⁺) and PDO co-culture (GranzymeB⁺ and CD69⁺). Multiple engineered γδ T cells generate a substantial killing phenotype against various CRC PDOs (cCaspase3 [D175]⁺ and cPARP [D214]⁺). We also detect the transfer of Granzyme B from γδ T cells to PDOs, executing PDO apoptosis. ADCC varies between γδ donors and is associated with FcγR CD16 expression.

Conclusions

We have identified marker signatures for the selection of candidate γδ biotherapeutics. CAFs can protect PDOs from γδ ADCC and work is continuing to understand the mechanistic basis of biotherapeutic stromal protection.

Legal entity responsible for the study

Christopher Tape.

Funding

Cancer Research UK.

Disclosure

All authors have declared no conflicts of interest.