Biotinylated MHC complexes and their uses

Abstract

The invention demonstrates an improved choice of biotinylation peptide to be used in a combination or fusion with an MHC molecule for immobilizing or multimerising such MHC molecules for a variety of purposes.

Description

EXAMPLE

A chimeric protein (SEQ ID NO: 1) is generated by fusing (in N-terminal to C-terminal direction) (i) amino acids 25-300 of the unprocessed HLA-A*0201 alpha (heavy) chain precursor protein (SEQ ID NO: 2) followed by (ii) one glycine and one serine residue followed by (iii) amino acids 81-156 unprocessed precursor of the BCCP protein of acetyl-CoA carboxylase of E.coli strain K12 (SEQ ID NO: 3). The fusion protein is generated using molecular cloning techniques well known in the art. Purified HLA heavy chain can be obtained by expression in inclusion bodies in E.coli. using a suitable expression system, such as the pET system (Novagen, Milwaukee, Wis., USA). Recombinant biotinylated MHC peptide complexes can be generated according to US2003/0017447A1. Specifically, native HLA-A2 monomeric MHC peptide complexes are refolded from denatured MHC alpha and human beta-2-microglobulin in the presence of the peptide GLCTLVAML (EBV BMLF-1 280-288; SEQ ID NO: 4). This peptide is known to bind strongly to HLA-A2 and is an immunodominant T cell epitope from Epstein Barr Virus (EBV). Refolded complexes are biotinylated overnight with the enzyme BirA as described in US2003/0017447A1. Biotinylated complexes are purified by size exclusion chromatography and the ˜50 kD peak is recovered. The recovered material may be subjected to a second chromatography step, such as ion exchange chromatography, if desired, or may simply be concentrated to a suitable protein concentration appropriate for further use. The protein concentration is determined by the method of Bradford and the level of biotinylation can be determined, e.g. via the EZ™ Biotin Quantitation Kit (Pierce Biotechnology, Inc., Rockford, Ill., USA).

For use in detecting antigen specific T cells, the biotinylated MHC complexes of the invention can be conjugated in a 4:1 molar ration to fluorescent labelled streptavidin, such as streptavidin:PE (Molecular Probes, Eugene, Oreg., USA). The complexes can then be used e.g. in flow cytometry to detect antigen-specific T cells as described in EP812331.

Similarly, for use in detecting anti-HLA antibodies the complexes can be plated into streptavidin-coated ELISA plates using a single-specificity HLA molecule per well (HLA complexes of a single allele in one well, HLA complexes of different alleles in different wells).

A typical ELISA set up would be as follows:

Coat wells with 100 μl Streptavidin (1 ng/μl; 1:1000 dilution of 1 mg/ml stock) in Coating buffer (0.1 M NaHCO₃, pH8.3) and leave at 4° C. overnight. Wash plates 4 times with phosphate buffered saline (PBS)-Tween® (0.1%). Add 200 μl of Blocking buffer (5% bovine serum albumin (BSA)/PBS+5% Glycine) and leave at room temperature for 1 hour. Wash plates 4×PBS-Tween® (0.1%). Add 100 μl MHC monomers (0.5 ng/μl) in coating buffer to each well (single MHC allele per well) and leave at room temperature for 1 hour. Wash plates 4×PBS-Tween® (0.1%). Add 50 μl of serum (1:10 dilution) in PBS to each well and leave at room temperature for 1 hour. Wash plates 4×PBS-Tween® (0.1%). Add 100 μl Rabbit anti Human IgA, G, M, kappa, lambda-horseradish peroxidase (HRP) (1:5000 dilution) in PBS and incubate at room temperature on the shaker at 250 rpm for 1 hour. Wash plates 4×PBS-Tween® (0.1%). Add 50 μl tetramethylbenzidine (TMB) and leave it for 10 minutes. Stop reaction with 50 μl H₂SO₄ and measure OD₄₅₀.

Claims

1. A chimeric peptide comprising an MHC peptide and a biotinylation peptide which either is a natural biotinylation peptide or has a greater than 70% sequence homology to a natural biotinylation peptide.

2. A chimeric peptide comprising an MHC peptide and a biotinylation peptide wherein the biotinylation peptide has a minimal sequence required for being biotinylated that is longer than 50 amino acids in length.

3. A chimeric peptide of claim 1 wherein the MHC peptide is a Class I MHC peptide or a Class II MHC peptide.

4. A chimeric peptide of claim 2 wherein the MHC peptide is a Class I MHC peptide or a Class II MHC peptide.

5. A chimeric peptide of claim 1 wherein the biotinylation peptide is biotinylated.

6. A chimeric peptide of claim 1 wherein the biotinylation peptide is located in the chimeric protein after the C-terminal end of the MHC peptide.

7. A chimeric peptide of claim 1 wherein the MHC peptide and the biotinylation peptide are separated by a linker sequence.

8. A chimeric peptide of claim 1 wherein the biotinylation peptide is selected from the group consisting of the biotinylation domain of BCCP and Proprionibacterifum shermanii 1.3S subunit of transcarboxilase.

9. An MHC peptide complex with the formula (α-β-P), wherein α comprises an α chain of a MHC I or MHC II class molecule, β comprises an β chain of a MHC I or MHC II class molecule, and P is a peptide antigen bound in the binding groove of the MHC molecule, wherein said MHC peptide complex comprises a chimeric protein of claim 1.

10. An MHC peptide complex of claim 9, wherein the peptide antigen P bound in the groove is substantially homogeneous.

11. An Multimeric binding complex having the formula (α-β-P)n, wherein (α-β-P) is an MHC peptide complex of claim 9, and wherein n≧2.

12. A multimeric binding complex of claim 11 wherein the MHC peptide complexes are biotinylated and the multimeric binding complex is formed by binding the biotinylated MHC peptide complexes to a multivalent entity that binds to biotin with high affinity.

13. A multineric binding complex of claim 12 wherein the multivalent entity is an avidin family protein and preferably streptavidin.

14. A multimeric binding complex of claim 11 comprising a label.

15. A method of labelling and or detecting mammalian T cells according to the specificity of their antigen receptor, the method comprising (i) combining a multimeric binding complex according to claim 11 and a suspension or biological sample comprising T cells, and(ii) detecting the presence of specific binding of said complex and at least one of the T cells.

16. A method of separating mammalian T cells according to the specificity of their antigen receptor, the method comprising (i) combining a multimeric binding complex according to claim 11 and a suspension or biological sample comprising T cells, and(ii) separating one or more T cells bound to said complex from unbound cells.

17. A method of detecting the presence of one or more anti-MHC antibodies in a sample comprising contacting said sample with at least one MHC complex according to claim 9 and detecting the binding or absence of binding of the one or-more ainti-MHC antibodies to the MHC complex(es).

18. A method according to claim 17 wherein the antibodies which are detected are IgG, IgM or IgA.

19. A method according to claim 17 wherein the peptide antigen P is derived from an antigen that occurs in less than 5% of a population group.

20. A method according to claim 17 wherein the MHC complex(es) is (are) attached to a solid support.

21. A method according to claim 20 wherein the MHC complex(es) is (are) biotinylated and immobilized to the solid support through binding to an avidin family protein which is itself bound to the solid support.

22. A method of claim 21 wherein the avidin family protein is streptavidin.

23. A method according to claim 20 wherein said solid support is a spherical bead.

24. A method according to claim 23 wherein the bead comprises a detectable label.

25. The method according to claim 20 wherein said solid support is a nitrocellulose strip.

26. The method according to claim 20 wherein said solid support is an ELISA plate.

27. The method according to claim 17 wherein the MHC complex(es) is (are) synthesized in a prokaryotic expression system.

28. The method according to claim 17 wherein the sample is a body fluid sample.

29. The method according to claim 17 wherein the bound antibody or absence thereof is detected via an immunosorbent assay using an antibody conjugated to a signalling means.

30. A method according to claim 17 wherein a single solid support is carrying two or more different ones of the MHC complexes or of the multimeric binding complexes at discrete locations on said solid support.

31. A method according to claim 17 wherein two or more different ones of the MHC complexes or of the multimeric binding complexes are immobilized on a different ones of said solid supports.

32. A method for determining the suitability of an organ to be transplanted for a transplant recipitent, comprising the method of claim 17, wherein the sample is a serum sample of the prospective transplant recipient and the presence of antibodies in the recipient that are reactive to at least one MHC molecule in the organ are detected and at least one MHC allele is determined against which such antibodies are reactive.

33. A method for determining a rejection reaction against a transplanted organ comprising the method of claim 17, wherein the sample is a serum sample of the transplant recipient by detecting the presence of antibodies in the recipient that are reactive to at least one MHC molecule in the organ are detected and at least one MHC allele is determined against which such antibodies are reactive.

34. A method of depleting a sample of anti-MHC molecule antibodies comprising at least the steps of contacting said sample with at least one MHC complex of claim 9, optionally attached to a solid support, and removing at least the MHC complex from the sample to which at least one anti-MHC antibody contained within the sample has bound.

35. A kit comprising at least the following components: a) one or more recombinant MHC complexes according to claim 9; b) optionally a solid support, together with means for attachment of the MHC complex(es); and c) a means for detecting anti-MHC-antibodies.

36. A method for biotinylating a chimeric peptide, the chimeric peptide comprising an MHC peptide and a biotinylation peptide which either is a natural biotinylation peptide or has a greater than 70% sequence homology to a natural biotinylation peptide wherein the chimeric peptide is incubated in a reaction mixture comprising biotin or a biotin analogue and a biotinylating enzyme, resulting in the biotinylation of the chimeric peptide.

37. A method for biotinylating a clhimeric peptide, the chimeric peptide comprising an MHC pepdtide and a biotinylation peptide which is biotinylation peptide is either is a natural biotinylation peptide or has a greater than 70% sequence homology to a natural biotinylation peptide the method comprising (i) constructing a recombinant DNA expression vector that encodes the chimeric peptide, (ii) transforming a recombinant host cell with said vector, and (iii) culturing said host cell in the presence of biotin or a biotin analogue and-under conditions such that said fusion protein and a biotinylating enzyme are expressed, resulting in the biotinylation of said chimeric peptide.

38. A method for biotinylating a chimeric peptide, the chimeric peptide comprising an MHC peptide and a biotinylation peptide which biotinylation peptide has a minimal sequence required for being biotinylated that is longer than 50 amino acids in length wherein the chimeric peptide is incubated in a reaction mixture comprising biotin or a biotin analogue and a biotinylating enzyme, resulting in the biotinylation of the chimeric peptide.

39. A method for biotinylating a chimeric peptide, the chimeric peptide comprising an MHC peptide and a biotinylation peptide which biotinylation peptide has a minimal sequence required for being biotinylated that is longer than 50 amino acids in length the method comprising (i) constructing a recombinant DNA expression vector that encodes the chimeric peptide, (ii) transforming a recombinant host cell with said vector, and (iii) culturing said host cell in the presence of biotin or a biotin analogue and under conditions such that said fusion protein and a biotinylating enzyme are expressed, resulting in the biotinylation of said chimeric peptide.