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

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.