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

Area of Science:

• Immunology
• Protein Engineering
• Structural Biology

Background:

• Antibody and T-cell receptor (TCR) affinity maturation typically focuses on binding site residues within complementarity-determining regions (CDRs).
• Mutations in contact or "second shell" residues are often found using directed evolution with combinatorial libraries.
• The impact of residues distant from the binding site on TCR function is less understood.

Purpose of the Study:

• To investigate the effect of non-complementarity-determining region (non-CDR) residues on TCR binding affinity.
• To explore mutations at the TCR variable domain interface (Vα/Vβ) for improved therapeutic potential.

Main Methods:

• Deep mutational scanning of a human TCR against MART-1·HLA-A2 using single-codon libraries for both CDR and non-CDR residues.
• Construction and selection of interface libraries for enhanced stability or affinity.
• Biochemical and biophysical characterization of identified variants, including the F45βY mutation.

Main Results:

• Both CDR and Vα/Vβ interface residues are crucial for maintaining TCR binding to MART-1·HLA-A2.
• Several Vα/Vβ interface substitutions unexpectedly improved binding affinity.
• The F45βY variant demonstrated enhanced thermostability and a 60-fold increase in binding affinity for MART-1·HLA-A2.
• The F45βY variant maintained high specificity for the target antigen.

Conclusions:

• Residues distant from the TCR binding site, particularly at the Vα/Vβ interface, can significantly modulate ligand binding affinity and stability.
• Targeting Vα/Vβ interface residues offers a novel strategy for engineering improved TCRs for therapeutic applications.
• This approach is applicable to both soluble and cell-based TCR engineering.