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

Preeti Sharma, David M. Kranz

Research output: Contribution to journalArticlepeer-review


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 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-1HLA-A2. Non-CDR residues included those at the interface of the TCR variable domains (V and V) and surface-exposed framework residues. Mutational analyses showed that both V/V interface and CDR residues were important in maintaining binding to MART-1HLA-A2, probably due to either structural requirements for proper V/V association or direct contact with the ligand. More surprisingly, many V/V interface substitutions yielded improved binding to MART-1HLA-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 (F45Y) was most prevalent. Further analysis of F45Y showed that it enhanced thermostability and increased affinity by 60-fold. Thus, introducing a single hydroxyl group at the V/V interface, at a significant distance from the TCRpeptideMHC-binding site, remarkably affected ligand binding. The variant retained a high degree of specificity for MART-1HLA-A2, indicating that our approach provides a general strategy for engineering improvements in either soluble or cell-based TCRs for therapeutic purposes.

Original languageEnglish (US)
Pages (from-to)1820-1834
Number of pages15
JournalJournal of Biological Chemistry
Issue number5
StatePublished - Feb 2 2018

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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