Improving TCR affinity on 293T cells

摘要

This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCR beta, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCR alpha, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers.