Abstract
The T-cell receptor (TCR) recognizes peptides bound to major histocompatibility molecules (MHC) and allows T-cells to interrogate the cellular proteome for internal anomalies from the cell surface. The TCR contacts both MHC and peptide in an interaction characterized by weak affinity (KD = 100 nM to 270 μM). We used phage-display to produce a melanoma-specific TCR (α24β17) with a 30,000-fold enhanced binding affinity (KD = 0.6 nM) to aid our exploration of the molecular mechanisms utilized to maintain peptide specificity. Remarkably, although the enhanced affinity was mediated primarily through new TCR-MHC contacts, α24β17 remained acutely sensitive to modifications at every position along the peptide backbone, mimicking the specificity of the wild type TCR. Thermodynamic analyses revealed an important role for solvation in directing peptide specificity. These findings advance our understanding of the molecular mechanisms that can govern the exquisite peptide specificity characteristic of TCR recognition.
Highlights
The molecular principles governing T-cell specificity are poorly understood
The ␣2417 T-cell receptor (TCR) Binds to A2-ELA with High Affinity Due to an Extended Off-rate—To generate a high affinity version of the MEL5 TCR, we implemented phage display as previously described [10]
This process produced a high affinity TCR, ␣2417, that varied from the MEL5 TCR parental sequence at 19 amino acids located within the CDR1␣, CDR2␣, FW␣, CDR3␣, CDR2, and CDR3 loops as well as in the  chain between residues 41 and 45 (Fig. 1A)
Summary
The molecular principles governing T-cell specificity are poorly understood. Results: High affinity binding of a melanoma-specific T-cell receptor (TCR) is mediated through new MHC contacts and distinct thermodynamics. There has been no rigorous testing of the specificity of an enhanced affinity TCR using molecular approaches We explored these issues using phage display to generate a high affinity TCR derived from the MEL5 TCR specific for the heteroclitic version of the HLA-A*0201-restricted primary melanoma antigen recognized by T-cells 1 (MART-126–35) peptide, ELAGIGILTV (A2-ELA) [10, 20]. This TCR, ␣2417, bound to A2-ELA with an affinity 30,000 times stronger than the natural parent MEL5 TCR, resulting in picomolar levels of binding [10]. We provide a new molecular mechanism by which TCRs maintain peptide specificity and show that it is possible to affinity mature TCRs for therapeutic use as soluble molecules without concomitant loss of peptide specificity
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