Predicting TCR antigen specificity at proteome-scale with synthetic immune cells and machine learning

Abstract TCR specificity to peptide-HLA antigens is central to immunology, impacting responses in infection, autoimmunity and cancer. Achieving precise recognition while avoiding off-target reactivity is critical for effective immunity and safe therapeutic interventions. Comprehensive, proteome-wide specificity profiling of TCRs is challenging with current methods, which notably lack integrated machine learning for large-scale analysis. Here, we report a synthetic immune cell system coupled with machine learning to enable TCR functional and specificity mapping of peptide-HLA antigens at proteome-scale. Multi-step immunogenomic engineering of synthetic antigen-presenting cells (APCs) was performed to enable stable mono-allelic integration and precise display of peptide antigens and HLA class I from a defined genomic locus, ensuring genomically-encoded antigen presentation. Compatible with a synthetic TCR displaying T cell system, this platform incorporates a fluorescent reporter of cytokine-mediated signaling for real-time activation detection in both synthetic APCs and T cells. We combined this screening with diverse peptide antigen libraries and deep sequencing to train supervised machine learning models. These models were applied to predict TCR specificity to peptide-HLA antigens across the entire human proteome. Experimental validation confirmed novel off-targets for therapeutic TCR candidates, including for a clinically-approved TCR therapeutic. This integrated synthetic immune cell and machine learning approach provides unprecedented proteome-wide peptide-HLA specificity mapping to support the development of safer TCR-based therapies. One sentence summary We present a synthetic immune cell platform integrated with machine learning that enables prediction of TCR specificity to peptide-HLA antigens at proteome-scale. Competing Interest Statement ETH Zurich has filed for patent protection on the technology described herein, and K-L.H, J.K., R.V.-L., and S.T.R. are named as co-inventors on this patent (WO2024003416A1). S.T.R. holds shares of Alloy Therapeutics, Engimmune Therapeutics, Encelta and Fy Cappa Biologics. S.T.R. is on the scientific advisory board of Alloy Therapeutics, Encelta, Engimmune Therapeutics and Fy Cappa Biologics. RVL holds shares of Engimmune Therapeutics. B.G., R.A.E., M.H., J.F., S.W., V.J., J.K., S.L., M-C.D., Q.Y., R.V-L are employees of Engimmune Therapeutics.