From Epitope to Therapy — Selecting the Right TCR 2259094

Abstract Introduction The effective design of T cell receptor (TCR)-based therapies — whether in a cellular or cell-engager format — is crucially dependent on the identification of TCRs with high specificity for the target MHC:peptide (MHCp) complex. This makes screening candidate TCRs for cross-reactivity towards off-target self-epitopes as well as ranking their affinity for the target (and off-targets) key activities on the path from epitope to therapy. Here, we present a novel, high-throughput assay for characterisation of TCR and TCR mimic molecules in terms of fine specificity (binding motif) and broader cross-reactivity towards unrelated epitopes. The assay can screen hundreds of MHCp complexes in a single run and enables easy access to essential TCR characteristics. In addition, the assay can be extended to include avidity measurements to allow TCR affinity ranking towards multiple targets. Methods TCR-coated beads were stained with pools of DNA barcode-labelled MHC Dextramer reagents carrying positional scanning peptide libraries or selected off-target peptides. Subsequent sequencing of the DNA barcodes associated with TCR binding revealed a detailed picture of the TCR binding preferences. By including MHCp complexes of known affinity towards the TCR, all MHCp could be ranked according to their TCR binding affinity. Results Using our high-throughput assay, we were able to simultaneously characterize five TCR molecules in terms of their fine specificity and cross-reactivity, while also ranking the affinity of each TCR towards the screened MHCp complexes. Conclusion Here, we demonstrate a novel assay for the characterization of TCR fine specificity, cross-reactivity, and affinity in a simple, high-throughput format. The assay enables easy assessment of TCR specificity towards MHCp complexes and is part of a larger pipeline of assays, which combines T cell/TCR discovery with TCR validation and characterisation, an essential part of the development process for TCR-based therapeutics. Funding Source Eurostars Topic Categories Technological Innovations in Immunology (TECH)