Principal Scientist IMMUDEX ApS Virum, Hovedstaden, Denmark
Disclosure(s):
Thomas H. Blicher, PhD: No relevant disclosure to display
Introduction/Rationale: 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.
