Disulphide bond-stabilized functional soluble MHC class II heterodimers
The present invention relates to disulphide bond stabilized recombinant MHC class II molecules. In particular, the present invention provides a recombinant MHC class II molecule, which comprises: (i) all or part of the extracellular portion of an MHC class II α chain; (ii) all or part of the extracellular portion of an MHC class II β chain; wherein (i) and (ii) provide a functional peptide binding domain and wherein (i) and (ii) are linked by a disulphide bond between cysteine residues located in the α2 domain of said α chain and the β2 domain of said β chain, wherein said cysteine residues are not present in native MHC class II α2 and β2 domains. Methods of producing these molecules in prokaryotic systems and various uses of these molecules form further aspects.
1. A recombinant MHC class II molecule, which comprises:
(i) all or part of the extracellular portion of an MHC class II α chain;
(ii) all or part of the extracellular portion of an MHC class II β chain;
wherein (i) and (ii) provide a functional peptide binding domain and wherein (i) and (ii) are linked by a disulphide bond between cysteine residues located in the α2 domain of said α chain and the β2 domain of said β chain, wherein said cysteine residues are not present in native MHC class II α2 and β2 domains, and
wherein said recombinant molecule does not comprise a leucine zipper motif.
2. The recombinant MHC class II molecule of claim 1 , wherein said disulphide bond is located between cysteine residues positioned at Pro 96 α 2 -Ser 119 β 2 (rank 1), Ser 95 α 2 -Ser 121 β2 (rank 2) or Arg 94 α2 -Asn 151 β2 (rank 3) of a murine I-E isotype or the equivalent locations in an alternative MHC class II isotype.
3. The recombinant MHC class II molecule of claim 2 , wherein said disulphide bond is located between cysteine residues positioned at 95 α2 -Ser 121 β2 (rank 2) of a murine I-E isotype or the equivalent locations in an alternative MHC class II isotype.
4. The recombinant MHC class II molecule of claim 1 , further wherein one or more of the cysteine residues corresponding to positions 38, 42 or 106 of the reference sequence H-2EB*01 (SEQ ID NO:2) or one or more of the cysteine residues at equivalent locations in an alternative MHC class II isotype are removed.
5. The recombinant MHC class II molecule of claim 1 , wherein said molecule is a soluble molecule.
6. The recombinant MHC class II molecule of claim 5 , wherein (i) or (ii) of said molecule is fused to an Fc portion of an immunoglobulin.
7. The recombinant MHC class II molecule of claim 1 , wherein said molecule is multimeric.
8. The recombinant MHC class II molecule of claim 1 , wherein (i) and (ii) of said molecule are derived from mouse or human MHC class II molecules.
9. The recombinant MHC class II molecule of claim 1 , wherein said molecule is produced by a bacterial host.
10. The recombinant MHC class II molecule of claim 9 , wherein said bacterial host is E. coli .
11. The recombinant MHC class II molecule of claim 1 , wherein said disulphide bond provides the sole means of stabilization of the MHC class II molecule.
12. The recombinant MHC class II molecule of claim 1 , wherein said molecule is capable of staining T cells.
13. The recombinant MHC class II molecule of claim 1 , wherein said molecule further comprises a peptide bound to said peptide binding domain.
14. A method of producing a recombinant MHC class II molecule, which comprises:
(i) all or part of the extracellular portion of an MHC class II α chain;
(ii) all or part of the extracellular portion of an MHC class II β chain;
wherein (i) and (ii) provide a functional peptide binding domain and wherein (i) and (ii) are linked by a disulphide bond between cysteine residues located in the α2 domain of said α chain and the β2 domain of said β chain, wherein said cysteine residues are not present in native MHC class II α2 and β2 domains, and wherein said recombinant molecule does not comprise a leucine zipper motif,
said method comprising expressing said recombinant molecule in a prokaryotic host.
15. A method for identifying an antigenic peptide epitope which can be recognized by a T cell having a T cell receptor specific for said antigenic peptide bound to a MHC class II molecule, wherein said method comprises the steps of:
(i) contacting the recombinant MHC class II molecule of claim 13 with said T cell receptor; and
(ii) detecting binding of said recombinant MHC class II molecule to said T cell receptor.
16. A method of detecting an antigen specific T cell in a sample, wherein said T cell has a T cell receptor specific for an antigenic peptide bound to a MHC class II molecule, and wherein said method comprises the steps of:
(i) contacting a recombinant MHC class II molecule of claim 13 with said sample; and
(ii) detecting binding of said recombinant MHC class II molecule to said T cell.