Method for high throughput peptide-mhc affinity screening for tcr ligands

A method for screening for a TCR-binding peptide ligand/MHC molecule complex (pMHC), comprising:

• a) providing a stabilized human HLA-A*0201 molecule capable of binding to a peptide ligand,
• wherein said HLA-A molecule comprises an artificially introduced covalent bridge between a first amino acid within the β1 unit at the amino acid position 22 in the alpha 1 domain and a second amino acid within the α1 unit at the amino acid position 71 in the alpha 1 domain of the HLA-A molecule based on IGMT numbering excluding the first 24 amino acids,
• wherein the first and the second amino acids are each modified to a cysteine forming a first disulfide bridge,
• wherein the first amino acid at position 22 is modified from phenylalanine (F) to cysteine (C) and the second amino acid at position 71 is modified from serine(S) to cysteine (C), and wherein the first disulfide bridge stabilizes the HLA-A molecule in the absence of the peptide ligand,
• b) contacting said suitably stabilized MHC molecule with a multitude of peptide ligands thereof, to form peptide ligand/MHC (pMHC) molecule complexes, and
• c) screening said pMHC molecule complexes for TCR-binding.