Comparison of Transcriptional Profiles of T cells after Activation by TCR and Chimeric Antigen Receptors

Chimeric antigen receptor T (CAR-T) cell therapies for B cell malignancies demonstrate high response rate and durable disease control. However, in the case of solid tumors, CAR-T cells have shown dysfunction ascribed to some intrinsic defects in CAR signaling. Here, we construct a multi-chain chimeric receptor, termed as Synthetic T Cell Receptor and Antigen Receptor (STAR), which incorporates antigen-recognition domain of antibody and engages entire CD3 signaling machinery of T cell receptor (TCR). In multiple solid tumor models, STAR-T cells prominently outperform CAR-T cells without notable toxicity. STAR triggers strong and sensitive TCR-like signaling upon antigen stimulation. We compared the transcriptional profiles of STAR/CAR/TCR-T cells after stimulation for different time points (0, 6, 24, 72 hours), in order to figure out whether signaling difference of these receptors led to distinct gene expression. Our results showd that STAR activation phencopied TCR, while CAR drove a different program, displayed as various pathways related to effector function, cytokine response and cell survival were altered. Overall design: For sample 1-36, we compare the cetuximab-derived anti-EGFR STAR-T cells, 28zCAR-T cells with 1G4-TCR which recognized NY-ESO-1 peptide in the context of HLA-A*02:01. EGFR+ A549 cells were over-expressed HLA-A*02:01 and NY-ESO-1 protein, so that it could be used to stimulate STAR, CAR and TCR. Transduced RFP+ T cells from three PBMC donors were sorted by FACS for CD8 positive and conducted coculture with target cells. T cells were retrieved from coculture system using CD3 positive selection kit and subjected to RNA preparation. For sample 37-39, we compared the cetuximab-derived anti-EGFR STAR-T cells, 28zCAR-T cells and BBzCAR-T cells (from a single PBMC donor) after stimulation by immobilized EGFR protein for 72 hours.