A novel method for TCR affinity ranking 9136

Abstract Description Objective The affinity and specificity of a T cell receptor (TCR) towards MHC:peptide (MHCp) complexes plays a central role in the function of T cells as well as in the design of TCR-based therapies. In particular, the strength of a TCR’s interaction with its target (cognate) MHCp complex versus that of unrelated (non-cognate) MHCp complexes determines its therapeutic utility. Here, we present a novel assay that allows ranking of TCR molecules based on their affinity for MHCp complexes by mimicking the binding of a T cell via its TCR to target cells. The assay is both robust and quick, requires only small amounts of TCR and target MHCp complex, and allows for medium to high-throughput screening against cognate and non-cognate MHCp complexes. Methods Binding of fluorescent TCR Dextramer reagents (T cell surrogate) to MHCp-loaded beads (target cell surrogate) was assessed using flow cytometry, and the binding characteristics of each TCR Dextramer reagent was transformed into a binding score. Results Using our bead-based assay, we were able to rank TCR molecules based on their binding score towards MHCp complexes for TCR:MHCp dissociation constants in the high micromolar to the sub-nanomolar range. Summary Here, we demonstrate a novel assay for the ranking of TCR:MHCp affinity in a simple, high-throughput format. The assay enables easy assessment of TCR affinity and specificity towards MHCp complexes with important implications for the development of TCR-based therapeutics. Topic Categories Technological Innovations in Immunology (TECH)