Single cell sequencing analysis of a CD28/CD40-based chimeric costimulatory antigen receptor (CoStAR™) activity reveals multiple functionally validated effector activities in CD4+ and CD8+ T cells.

Tumour infiltrating lymphocyte (TIL) therapy for treatment of metastatic melanoma, has demonstrated clinical efficacy leading to its recent FDA approval. Extending the clinical benefit to patients with other cancers has posed a challenge. Insufficient costimulation in the tumour microenvironment can lead to T cell anergy and exhaustion, in turn leading to reduced anti-tumour activity. Our CoStAR™ platform synergises with TCR signals upon antigen engagement to enhance proliferation and effector function. To explore the underlying biology of CoStAR activity we performed a scRNA-seq analysis. T cells were engineered to express a HLA-A*02/CEA specific TCR and/or FRα-specific CoStAR. Following coculture with tumour cells bearing the cognate pMHC and CoStAR target antigens, samples were processed through a scRNA-seq workflow. Bioinformatic analysis was performed to compare TCR and/or CoStAR+ populations, with functional validation of gene signatures performed using cytokine analysis, flow cytometry and cytotoxicity assays. When compared to TCR alone stimulation, TCR+CoStAR signalling in CD4+ cells resulted in enhanced gene signatures associated with activation and cytotoxicity, and in CD8+ cells, signatures of enhanced cytotoxicity and reduced exhaustion marker expression. Several genes – predominantly costimulatory receptors – were upregulated following CoStAR signalling alone. The cytotoxicity signature in CD4+ T cells was confirmed using xCELLigence cytotoxicity assay, with TCR+CoStAR signalling associated with enhanced cytotoxicity compared to TCR signalling alone. Cytokine and chemokine production was concordant with gene expression data, with flow cytometry confirming upregulation of costimulatory receptors in CD4+ T cells upon CoStAR engagement alone. These data further support the clinical utility of CoStAR engineered TIL.