Disulfide trap MHC class I molecules and uses therefor
A disulfide trap, comprising an antigen peptide covalently attached to an MHC class I heavy chain molecule by a disulfide bond extending between two cysteines, is disclosed. In some configurations, a disulfide trap, such as a disulfide trap single chain trimer (dtSCT), can comprise a single contiguous polypeptide chain. Upon synthesis in a cell, a disulfide trap oxidizes properly in the ER, and can be recognized by T cells. In some configurations, a peptide moiety of a disulfide trap is not displaced by high-affinity competitor peptides, even if the peptide binds the heavy chain relatively weakly. In various configurations, a disulfide trap can be used for vaccination, to elicit CD8 T cells, and in multivalent MHC/peptide reagents for the enumeration and tracking of T cells. Also disclosed are nucleic acids comprising a sequence encoding a disulfide trap. Such nucleic acids, which can be DNA vectors, can be used as vaccines.
1. A disulfide trap comprising, in amino terminal-to-carboxy terminal order, an MHC class I ligand peptide, a first linker, said first linker comprising a first cysteine, a β2-microglobulin, a second linker, and an MHC class I heavy chain sequence comprising a second cysteine, wherein said second cysteine is a substitution of an amino acid of the MHC class I heavy chain selected from the group consisting of T80C, Y84C and N86C, and wherein the first cysteine and the second cysteine comprise a disulfide bridge.
2. A disulfide trap in accordance with claim 1 , wherein the MHC class I ligand peptide comprises an MHC class I antigen peptide.
3. A disulfide trap in accordance with claim 2 , wherein the MHC class I antigen peptide is an MHC class I self peptide.
4. A disulfide trap in accordance with claim 2 , wherein the MHC class I antigen peptide is an MHC class I tumor peptide.
5. A disulfide trap in accordance with claim 1 , wherein the MHC class I heavy chain sequence is a human MHC class I heavy chain sequence.
6. A disulfide trap in accordance with claim 1 , wherein the MHC class I heavy chain sequence is a murine MHC class I heavy chain sequence.
7. A disulfide trap in accordance with claim 1 , wherein the MHC class I ligand peptide comprises from about 8 amino acids up to about 13 contiguous amino acid residues.
8. A disulfide trap in accordance with claim 1 , wherein the MHC class I ligand peptide comprises 9 contiguous amino acids.
9. A disulfide trap in accordance with claim 5 , wherein the human MHC class I heavy chain sequence is selected from the group consisting of an HLA-A MHC class I heavy chain sequence and an HLA-B MHC class I heavy chain sequence.
10. A disulfide trap in accordance with claim 5 , wherein the human MHC class I heavy chain sequence is an HLA heavy chain sequence selected from the group consisting of an HLA-A, HLA-B and HLA-C heavy chain sequence.
11. A disulfide trap in accordance with claim 1 , wherein the MHC class I heavy chain sequence is a murine heavy chain sequence selected from the group consisting of an MHC-K, MHC-D and MHC-L murine heavy chain class I sequence.
12. A disulfide trap in accordance with claim 1 , wherein the first cysteine is the first, second or third amino acid of the linker.
13. A disulfide trap in accordance with claim 1 , wherein the second linker comprises at least about 15 amino acids up to 20 amino acids, and wherein at least about 80 percent of the amino acids comprising the second linker are glycines.
14. A disulfide trap in accordance with claim 1 , further comprising a leader peptide.
15. An MHC/peptide multimer comprising a disulfide trap of claim 1 .
16. An MHC/peptide multimer in accordance with claim 15 , further comprising a label.
17. An MHC/peptide multimer in accordance with claim 16 , wherein the label is selected from the group consisting of a hapten, a radioisotope and a fluorophore.
18. An MHC/peptide multimer in accordance with claim 17 , wherein the radioisotope is selected from the group consisting of H 3 , P 32 , P 33 , S 35 , C 14 , I 125 and I 131 .
19. An MHC/peptide multimer in accordance with claim 15 , wherein the multimer is a tetramer.
20. A disulfide trap single chain trimer in accordance with claim 1 , wherein the first linker comprises at least 1 up to 15 amino acids and comprises the first cysteine, and the second linker comprises at least 15 amino acids up to 20 amino acids, and wherein at least 80 percent of the amino acids comprising the second linker are glycines.
21. A disulfide trap in accordance with claim 20 , wherein the first cysteine is the first, second or third amino acid of the linker.
22. A disulfide trap in accordance with claim 1 , wherein the MHC class I heavy chain comprises a non-covalent binding site for the MHC ligand peptide.