Directed evolution of human T-cell receptors with picomolar affinities by phage display

Abstract

Peptides derived from almost all proteins, including disease-associated proteins, can be presented on the cell surface as peptide-human leukocyte antigen (pHLA) complexes. T cells specifically recognize pHLA with their clonally rearranged T-cell receptors (TCRs), whose natural affinities are limited to approximately 1-100 muM. Here we describe the display of ten different human TCRs on the surface of bacteriophage, stabilized by a nonnative interchain disulfide bond. We report the directed evolution of high-affinity TCRs specific for two different pHLAs: the human T-cell lymphotropic virus type 1 (HTLV-1) tax(11-19) peptide-HLA-A(*)0201 complex and the NY-ESO-1(157-165) tumor-associated peptide antigen-HLA-A(*)0201 complex, with affinities of up to 2.5 nM and 26 pM, respectively, and we demonstrate their high specificity and sensitivity for targeting of cell-surface pHLAs.

The structure of superantigen complexed with TCR and MHC reveals novel insights into superantigenic T cell activation

“…In addition, we solved a high-resolution structure including only the TCR and SEH at 2.1 Å resolution (Table 1). TCR and MHC were both expressed in E. coli as inclusion bodies and refolded, according to earlier published protocols [12][13][14] . SEH was expressed in the periplasm of E. coli as earlier described by Nilsson et al 15 The proteins were mixed in an equimolar ratio and the crystals were formed in a few weeks.…”

Single and Dual Amino Acid Substitutions in TCR CDRs Can Enhance Antigen-Specific T Cell Functions

“…Previously, a multiple substitution variant of the 1G4 TCR was shown to possess an affinity ϳ1 million-fold higher than the WT 1G4 TCR. As a soluble monomeric protein, this TCR was shown to bind specifically to NY-ESO-1 peptide-pulsed T2 cells but not to a panel of 16 other unrelated control peptide/HLA-A*02 Ags (18). When this TCR and other mutant1G4 TCRs were transfected into CD8 ϩ T cells, Ag cross-reactivity with NY-ESO-1-negative target cell lines was observed with the highest affinity TCR and with those with Ag affinities ranging from 20 to 50 pM to 84 nM.…”

“…Additional studies conducted using bacteriophage display mutation and selection technology have resulted in the identification of high-affinity variants of the 1G4 TCR that recognize the HLA-A*02-restricted peptide 157-165 of the NY-ESO-1 cancer-testis Ag (18). Expression of the NY-ESO-1 molecule has been demonstrated in a wide variety of tumor types that include melanoma, breast, prostate, lung ovarian, thyroid, and bladder cancer, but is limited in normal tissues to the testis (19).…”

“…The current studies were guided by previous bacteriophage display studies that identified high-affinity variants of the 1G4 TCR that contained multiple combined substitutions principally in the CDR2 and CDR3 regions of both TCR chains (18,22). Extending this approach, CDR2␤ chain individual amino acid substitution variants were subsequently screened in two distinct TCRs (DMF4 and DMF5) directed against the immunodominant HLA-A2-restricted peptide epitope corresponding to amino acids 27-35 of the melanoma Ag MART-1 (23), a gene product expressed in 80 -90% of fresh, uncultured melanomas as well as cultured melanoma cell lines (24) but not in additional histologies or normal tissues with the exception of melanocytes.…”

“…The amino acid residues 94 -97 (PTSG) of the 1G4 ␣-chain, which are found at the amino terminus of the CDR3 ␣ loop (36), were then targeted for substitution as mutations in this region had previously been identified by bacteriophage display experiments as important for high-affinity Ag binding (18). Cotransfection of CD8 ϩ PBMC with a WT 1G4 ␤-chain construct and individual ␣-chain 1G4 TCR CDR3 substitution constructs of alanine or leucine for proline at position 94 (designated 1G4 ␣94:A and 1G4 ␣94:L, respectively) resulted in diminished recognition of NY-ESO-1:157-165 peptide-pulsed T2 cells ( Fig.…”