Subtle changes at the variable domain interface of the T-cell receptor can strongly increase affinity [Immunology]

December 14th, 2017 by Preeti Sharma, David M Kranz

Most affinity-maturation campaigns for antibodies and T cell receptors (TCRs) operate on the residues at the binding site, located within the loops known as complementarity determining regions (CDRs). Accordingly, mutations in contact residues, or so-called "second shell" residues, that increase affinity are typically identified by group at the α:β interface, at a significant distance from the TCR/pepMHC binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART- 1/HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.directed evolution involving combinatorial libraries. To determine the impact of residues located at a distance from the binding site, here we used single codon libraries of both CDR and non- CDR residues to generate a deep mutational scan of a human TCR against the cancer antigen MART-1/HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (α and β) and surface-exposed framework residues. Mutational analyses showed that both α:β interface and CDR residues were important in maintaining binding to MART- 1/HLA-A2, likely due to either structural requirements for proper α:β association or direct contact with the ligand. More surprisingly, many α:β interface substitutions yielded improved binding to MART-1/HLA-A2. To further explore this finding, we constructed interface libraries and selected them for improved stability or affinity. Among the variants identified, one conservative substitution (β) was most prevalent. Further analysis of β showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the α:β interface, at a significant distance from the TCR/pepMHC binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART- 1/HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.