Method for high throughput peptide-MHC affinity screening for TCR ligands
The present invention relates to a method for high throughput screening for a TCR-binding peptide ligand/MHC molecule complex, comprising a stabilized peptide-MHC molecule and respective uses of said method. The present invention further relates to polypeptides comprising or consisting of stabilized MHC molecules or peptide binding fragments thereof, pharmaceutical compositions comprising said polypeptides, vaccines comprising said pharmaceutical composition and uses of said vaccine for the manufacturing of a medicament and/or in the prevention of cancer The present invention further relates to nucleic acids encoding said polypeptides and vectors comprising said nucleic acids.
1. A polypeptide comprising 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.
2. A pharmaceutical composition comprising the polypeptide of claim 1
wherein said stabilized HLA-A molecule is bound to a bead, filament, nanoparticle or other suitable carrier, and
wherein the composition is produced by contacting the peptide ligand with the stabilized HLA-A molecule and forming a peptide ligand-loaded HLA-A molecule thereby.
3. The pharmaceutical composition according to claim 2 , further comprising an anti-CD28 antibody or an anti-4-1BB antibody.
4. A vaccine comprising the pharmaceutical composition according to claim 2 .
5. The polypeptide according to claim 1 , wherein the HLA-A molecule further comprises a third amino acid modified in the α1 unit of the alpha 1 domain at the amino acid position 51 and a fourth amino acid modified within the alpha 2 domain at the amino acid position 175 based on IGMT numbering excluding the first 24 amino acids, wherein the third and the fourth amino acids are each modified to a cysteine forming a second disulfide bridge.
6. The pharmaceutical composition according to claim 3 , wherein the composition comprises the anti-CD28 antibody.
7. The pharmaceutical composition according to claim 3 , wherein the composition comprises the anti-4-1BB antibody.
8. The polypeptide of claim 1 , comprising SEQ ID NO: 324.
9. The polypeptide of claim 5 , wherein the polypeptide comprises SEQ ID NO: 325.
10. A pharmaceutical composition comprising the polypeptide of claim 5 ,
wherein the stabilized HLA-A molecule is bound to a bead, filament, nanoparticle or other suitable carrier, and
wherein the composition is produced by contacting the peptide ligand with the stabilized HLA-A molecule and forming a peptide ligand-loaded HLA-A molecule thereby.
11. The pharmaceutical composition of claim 10 , further comprising an anti-CD28 antibody or an anti-4-1BB antibody.