IMMUNOLOGICAL REAGENTS AND USES THEREFOR

• US 20150166542A1
• Filed: 07/05/2013
• Published: 06/18/2015
• Est. Priority Date: 07/06/2012
• Status: Active Grant

• Current Assignee: Monash University, The University of Queensland, University of Melbourne
• Original Assignee: University of Melbourne
• Inventors: Fairlie, David Paul, Liu, Ligong, Kjer-Nielsen, Lars, McCluskey, James, Corbett, Alexandra, Rossjohn, Jamie, Onisha, Patel
• Granted Patent: US 10,011,602 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/1774 Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
• CPC Class Codes: C07D 475/02 with an oxygen atom directly attached in position 4
• CPC Class Codes: C07D 475/04 with a nitrogen atom directly attached in position 2
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• CPC Class Codes: C12N 2500/46 Amines, e.g. putrescine
• CPC Class Codes: C12N 5/0636 T lymphocytes
• CPC Class Codes: G01N 2333/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: G01N 33/505 involving T-cells
• CPC Class Codes: G01N 33/56972 White blood cells

Abstract

The present invention provides a ligand which binds to MR1 wherein said binding results in binding of the MR1 to MAIT cells.

Claims

1. A ligand which binds to MR1 wherein said binding results in binding of the MR1 to MAIT cells.

2. The ligand according to claim 1, wherein said ligand binds in a binding cleft formed by the α

1 and α

2 domains of MR1.

3. The ligand according to claim 1, wherein when said ligand is bound to a MAIT cell the MAIT cell is stimulated or inhibited via its TCR.

4. The ligand according to claim 1, wherein said binding of the ligand to MR1 causes refolding of the MR1 into a form capable of binding to MAIT cells.

5. The ligand according to claim 1, wherein said ligand is represented by formula (I):

6. The ligand according to claim 5, wherein said ligand is 6-formyl pterin, acetyl 6-formyl pterin or a functional analogue thereof.

7. The ligand according to claim 5, wherein said ligand is rRL-6AM, rRL-6HM, RL-6M, RP-SPA, 6-methyl-7-hydroxy-8-ribityl lumazine, 6-,7-dimethyl-8-Ribityl Lumazine or a functional analogue thereof including oxidised and reduced forms thereof.

8. The ligand according to claim 1, wherein the MR1 polypeptide comprises all or part of SEQ ID NO:

1, SEQ ID NO;

4, or a functional derivative thereof having one or more amino acid substitutions, additions and/or deletions to SEQ ID NO;

1 or SEQ ID NO;

4.

9. The ligand according to claim 8, wherein said MR1 comprises at least one mutation selected from the list consisting of K43A, K43M, K43I, K43L, K43F, K43Q, Y7A, Y7W, R9K, R9A, S24F, Y62A, L66A, L66F, W69A, R94K, R94A, I96A, I96F and W156A.

10. The ligand according to claim 8, wherein said MR1 comprises one or more mutations in surface exposed groups selected from the list consisting of D57, R61, L65, M72, V75, R79, T138, Q141, N146, H148, L151, N155, E158, and R167.

11. An MR1-ligand subunit or multimeric complex thereof, wherein said subunit or multimeric complex binds to MAIT cells.

12. The MR1 ligand subunit or multimeric complex thereof according to claim 11, wherein said subunit or complex modulates MAIT cell activity.

13. The MR1 ligand subunit or multimeric complex thereof according to claim 12, wherein said modulation is down-regulation.

14. The MR1 ligand subunit or multimeric complex thereof according to claim 12, wherein said modulation is up-regulation.

15. The MR1 ligand subunit or multimeric complex thereof according to claim 11, wherein said MR1 is human or mouse MR1 and comprises all or part of SEQ ID NO:

1 or SEQ ID NO;

4 or a functional derivative thereof having one or more amino acid substitutions, additions and/or deletions to SEQ ID NO;

1 or SEQ ID NO;

4.

16. The MR1 ligand subunit or multimeric complex thereof according to claim 15, wherein said MR1 comprises at least one mutation selected from the list consisting of K43A, K43M, K43I, K43L, K43F, K43Q, Y7A, Y7W, R9K, R9A, S24F, Y62A, L66A, L66F, W69A, R94K, R94A, I96A, I96F and W156A.

17. The MR1 ligand subunit or multimeric complex thereof according to claim 15, wherein said MR1 comprises one or more mutations in surface exposed groups selected from the list consisting of D57, R61, L65, M72, V75, R79, T138, Q141, N146, H148, L151, N155, E158, and R167.

18. The MR1 ligand subunit or multimeric complex thereof according to claim 16, wherein said mutant is K43A.

19. The MR1 ligand subunit or multimeric complex thereof according to claim 16, wherein said MR1 is a) refolded and subsequently purified in the absence of any added ligand;

and b) the refolded MR1 is loaded with ligand.

20. The MR1 ligand subunit or multimeric complex thereof according to claim 11, wherein said ligand is rRL-6AM, rRL-6HM, RL-6M, RP-5PA, 6-methyl-7-hydroxy-8-ribityl lumazine, 6-,7-dimethyl-8-Ribityl Lumazine or a functional analogue thereof including oxidised and reduced forms thereof.

21. The MR1 ligand subunit or multimeric complex thereof according to according to claim 20, wherein said ligand is rRL-6HM,

22. The multimeric complex according to claim 11, wherein said multimeric complex comprises the formula [MR1-L]_n, wherein n≧

2 and ≦

10.

23. The multimeric complex according to claim 22, wherein n=4.

24. A method of modulating MAIT cell activity, said method comprising contacting said cell with an effective amount of the MR1 ligand subunit or multimeric complex thereof for a time and under conditions sufficient to modulate the interaction of MAIT cells with MR1 wherein promoting or otherwise agonizing said interaction up-regulates said MAIT cell activity and inhibiting or otherwise antagonizing said interaction down-regulates said MAIT cell activity.

25. The method according to claim 24, wherein said MR1 ligand subunit or multimeric complex thereof inhibits the interaction of MAIT cells with MR1.

26. The method according to claim 25, wherein said ligand is 6-formyl pterin, acetyl 6-formyl pterin or a functional analogue thereof.

27. The method according to claim 24, wherein said MR1 ligand subunit or multimeric complex thereof promotes the interaction of MAIT cells with MR1.

28. The method according to claim 27, wherein said ligand is rRL-6AM, rRL-6HM, RL-6M, RP-5PA, 6-methyl-7-hydroxy-8-ribityl lumazine, 6-,7-dimethyl-8-Ribityl Lumazine or a functional analogue thereof including oxidised and reduced forms thereof.

29. The method according to claim 28, wherein said ligand is rRL-6HM.

30. (canceled)

31. (canceled)

32. (canceled)

33. (canceled)

34. A method of detecting the presence of MAIT cells in a biological sample from a subject, the method comprising the steps of a) contacting the biological sample with antigen presenting cells expressing MR1 bound to a ligand or a soluble form thereof, under conditions that would allow binding of the MR1 with MAIT cells present in the sample;

and b) detecting the presence of MAIT cell bound MR1 in the biological sample.

35. The method according to claim 34, wherein said ligand is rRL-6HM, rRL-6AM, RL-6M, RP-SPA, 6-formyl pterin, acetyl 6-formyl pterin or a functional analogue thereof, or 6-methyl-7-hydroxy-8-ribityl lumazine, 6-,7-dimethyl-8-Ribityl Lumazine or a functional analogue thereof including oxidised and reduced forms thereof.

36. The method according to claim 35, wherein said ligand is rRL-6HM.

37. A method for the treatment and/or prophylaxis of a disease or condition in a mammal, said method comprising administering to said mammal an effective amount of an MR1 ligand subunit or multimeric complex thereof for a time and under conditions sufficient to modulate the interaction of MAIT cells with MR1, wherein promoting or otherwise agonizing said interaction up-regulates said MAIT cell activity and inhibiting or otherwise antagonizing said interaction down-regulates said MAIT cell activity.

40. The method according to claim 37, wherein said ligand is rRL-6HM, rRL-6AM, RL-6M, RP-SPA, 6-methyl-7-hydroxy-8-ribityl lumazine, 6-,7-dimethyl-8-Ribityl Lumazine or a functional analogue thereof including oxidised and reduced forms thereof, and said agent is administered for a time and under conditions sufficient to promote the interaction of MAIT cells with MR1.

41. The method according to claim 40, wherein said ligand is rRL-6HM.

44. The method according to claim 40, wherein said disease condition is selected from cancer, an infectious disease, an immune disease involving the mucosa, such as but not limited to Crohn'"'"'s Disease, ulcerative colitis, irritable bowel disease, Multiple Sclerosis, chronic fatigue syndrome, oral infections, peptic ulceration, intestinal helminth or bacterial infection, ocular disease such as Trachoma, pelvic inflammatory disease, sexually transmitted diseases, Chlamydia infection, candidiasis and other fungal infections at epithelial and mucosal sites, tuberculosis or Celiac disease.

38. (canceled)

39. (canceled)

42. (canceled)

43. (canceled)

45-54. -54. (canceled)