Optimized procurement of cancer-reactive T-cell receptors from clinical samples

Abstract

Abstract T-cell receptors can sense cancer-associated changes in cellular proteins, lipids, metabolites, and stress pathways, enabling immune-mediated tumour elimination in some patients. These receptors underpin T-cell receptor-based immunotherapies, yet their isolation from clinical samples remains challenging due to labour-intensive workflows, dependence on prior antigen knowledge, and loss of cell viability. We developed an optimized flow cytometry-based assay, termed the T107 assay, to isolate viable cancer-reactive T-cells directly from clinical samples. Following short-term exposure to cancer cells, responding T-cells are identified by simultaneous surface detection of tumour necrosis factor and the degranulation marker CD107a, enabling recovery of live antigen-reactive cells for downstream analysis. The T107 assay enabled rapid identification of cancer-reactive T-cells from tumour-infiltrating lymphocyte products derived from melanoma and sarcoma patients. The assay supported phenotypic and functional characterization of αβ and γδ T-cell receptor populations, including CD4+ and CD8+ subsets, responding to both patient-specific and shared cancer antigens. Isolation of viable responding cells enabled high-resolution receptor sequencing, generation of functional T-cell clones, determination of antigen specificity and major histocompatibility complex restriction or independence. Paired receptors were engineered into primary T-cells to generate T-cell receptor-engineered products capable of recognizing multiple cancer types. The T107 assay provides a rapid and robust method for antigen-agnostic procurement of viable cancer-reactive T-cells and their receptors from clinical material. This approach removes key bottlenecks in T-cell receptor discovery and is expected to accelerate development of T-cell receptor-based immunotherapies across a broad range of cancers.