Monomeric recombinant MHC molecules useful for manipulation of antigen-specific T cells
The present invention provides, in particular embodiments, for modified recombinant T cell receptor (TCR) ligands (RTLs) comprising a MHC class I or MHC class II component. The modified RTLs have redesigned surface features that preclude or reduce aggregation, wherein the modified molecules retain the ability to bind Ag-peptides, target antigen-specific T cells, inhibit T cell proliferation in an Ag-specific manner and have utility to treat, inter alia, autoimmune disease and other conditions mediated by antigen-specific T cells in vivo.
1. An isolated, modified recombinant T cell ligand (RTL) having a reduced potential for aggregation in solution, comprising:
a major histocompatibility complex-class II (MHC Class II) component in the form of a single chain (sc) polypeptide comprising multiple, covalently-linked MHC domain elements including α1 and β1 domains of an MHC class II polypeptide, wherein the amino terminus of the α1 domain is covalently linked to the carboxy terminus of the β1 domain, wherein the MHC Class II component does not include an α2 or β2 domain, wherein the MHC Class II component comprises the α1 and β1 domains of an HLA-DR protein, and wherein the MHC Class II component is modified by substitution of one or more hydrophobic residues with a polar or charged residue, wherein the one or more hydrophobic residues are selected from residues V6, I8, A10, F12, and L14 of the α1 domain,
whereby the modified RTL exhibits reduced aggregation in solution compared to aggregation exhibited by an unmodified, control RTL comprising α1 and β1 domains of an MHC class II polypeptide, wherein the amino terminus of the α1 domain is covalently linked to the carboxy terminus of the β1 domain, wherein the unmodified, control RTL does not include an α2 or β2 domain.
2. The isolated, modified RTL of claim 1 , wherein the α1 and β1 domains are coupled by a peptide linker.
3. The isolated, modified RTL of claim 1 , further comprising a T cell antigenic determinant bound to the MHC Class II component or covalently linked to the MHC Class II component.
4. The isolated, modified RTL of claim 1 , coupled to a toxin effective to mediate T cell killing.
5. The isolated, modified RTL of claim 1 , wherein the one or more hydrophobic residues are modified by substitution with a serine or aspartate residue.
6. The isolated, modified RTL of claim 1 , wherein each of the residues V6, I8, A10, F12, and L14 of the α1 domain are modified by substitution with a serine or aspartate residue.
7. An assay composition or kit useful to detect, quantify and/or purify antigen-specific T-cells, comprising a modified, recombinant T cell receptor ligand (RTL) according to claim 1 .
8. The isolated, modified RTL of claim 3 , wherein the T cell antigenic determinant comprises a myelin oligodendrocyte glycoprotein (MOG) peptide.
9. The isolated, modified RTL of claim 8 , wherein the MOG peptide is amino acids 35-55 of MOG.
10. The isolated, modified RTL of claim 3 , wherein the T cell antigenic determinant comprises a myelin basic protein (MBP) peptide.
11. The isolated, modified RTL of claim 10 , wherein the MBP peptide is amino acids 85-99 of MBP.
12. An isolated, modified recombinant T cell ligand (RTL) having a reduced potential for aggregation in solution, comprising SEQ ID NO: 9 in which one or more of amino acids V102, I104, A106, F108, and L110 are substituted with a polar or charged residue, whereby the modified RTL exhibits reduced aggregation in solution compared to aggregation exhibited by the unmodified, control RTL (SEQ ID NO: 9).
13. The isolated, modified RTL of claim 12 , further comprising a T cell antigenic determinant.
14. The isolated, modified RTL of claim 12 , wherein each of the residues V102, I104, A106, F108, and L110 are substituted with a serine or aspartate residue.
15. The isolated, modified RTL of claim 13 , wherein the T cell antigenic determinant comprises a myelin oligodendrocyte glycoprotein (MOG) peptide.
16. The isolated, modified RTL of claim 15 , wherein the MOG peptide comprises amino acids 35-55 of MOG.
17. The isolated, modified RTL of claim 13 , wherein the T cell antigenic determinant comprises a myelin basic protein (MBP) peptide.
18. The isolated, modified RTL of claim 17 , wherein the MBP peptide comprises amino acids 85-99 of MBP.
19. The isolated, modified RTL of claim 12 , further comprising substitution of one or more amino acids selected from the group consisting of L9, F19, L28, F32, V45, V51, A133, V138, and L141 with a polar or charged residue.