Method for integrin ligand discovery
This invention relates generally to integrin ligand discovery and to a method of integrin ligand discovery base upon induction of ligand-induced epitopes. Such ligands have the potential to be active agent as anti-inflammatory, anti-angiogenesis and/or anti-thrombotic agents and for the treatment of integrin mediated diseases and/or conditions.
1. A method for determining whether or not a compound of unknown integrin activity is an allosteric inhibitor of an integrin molecule selected from the group consisting of Very Late Antigen 4 (VLA-4, CD49d/CD29) and Leukocyte Function-Associated Antigen (LFA-1, CD18/CD11a) comprising exposing said integrin molecule to a known competitive ligand of said integrin molecule in the presence of an integrin-epitope binding antibody comprising a reporter moiety, measuring the binding of said antibody to the integrin molecule in the presence of the competitive ligand; thereafter exposing said bound integrin molecule in the presence of known competitive ligand and bound antibody to a compound of unknown activity and measuring the binding of the antibody to the integrin molecule, wherein the decrease in binding of antibody to integrin is evidence that the compound of unknown activity is an allosteric inhibitor of integrin, wherein said binding antibody for said VLA-4 integrin molecule is monoclonal antibody clone HUTS-21, said binding antibody for said LFA-1 integrin molecule is monoclonal antibody clone MEM-148, said competitive ligand of said VLA-4 integrin molecule is a compound according to the chemical structure:
where R″ is a peptide of SEQ ID NO:1 (compound LDV) or SEQ ID NO:2 (compound YLDV), N-(2,6-dichlorobenzoyl)-(L)-4-(2′,6′-bis-methoxyphenyl)phenylalanine (TR14035), 3-(adamantane-1-carbonylamino)-3-(4-ethoxyphenyl) propanoic acid (compound D) or 3-(adamantane-1-carbonylamino)-3-(4-propoxyphenyl) propanoic acid (compound E) and said competitive ligand of said LFA-1 integrin molecule is (2-[2-Chloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-succinamic acid) (compound 3 of FIG. 9 ) or (2-[2,6-Dichloro-4-(3-hydroxy-benzylcarbamoyl)-benzoylamino]-3-(3,5-dihydroxy-benzoylamino)-propionic acid) (XVA143), wherein said integrin molecule is expressed on a cell.
2. The method according to claim 1 wherein the decrease in binding of antibody to said integrin molecule is compared to a control wherein the control is a measurement of the non-specific binding of the antibody to the integrin molecule.
3. The method according to claim 1 wherein said integrin molecule is expressed on a wild type cell.
4. The method according to claim 1 wherein said integrin molecule is expressed on an engineered cell.
5. The method according to claim 4 wherein said cell expresses both VLA-4 and LFA-1.
6. The method according to claim 1 wherein said integrin molecule is expressed on a cell selected from the group consisting of human peripheral blood granulocytes, monocytes, lymphocytes and human platelet cells.
7. The method according to claim 2 wherein said integrin molecule is expressed on a cell selected from the group consisting of human peripheral blood granulocytes, monocytes, lymphocytes and human platelet cells.
8. The method according to claim 1 wherein said integrin is expressed on a cell selected from the group consisting of U937 cells, MOLT-4 cells, Jurkat cells, THP-1 cells, HL-60 cells, JY cells and MEG-01 cells.
9. The method according to claim 2 wherein said integrin is expressed on a cell selected from the group consisting of U937 cells, MOLT-4 cells, Jurkat cells, THP-1 cells, HL-60 cells, JY cells and MEG-01 cells.
10. The method according to claim 1 wherein said integrin molecule is VLA-4.
11. The method according to claim 2 wherein said integrin molecule is VLA-4.
12. The method according to claim 4 wherein said integrin molecule is VLA-4.
13. The method according to claim 1 wherein said integrin molecule is LFA-1.
14. The method according to claim 2 wherein said integrin molecule is LFA-1.
15. The method according to claim 4 wherein said integrin molecule is LFA-1.
16. The method according to claim 10 wherein said antibody is monoclonal antibody clone HUTS-21.
17. The method according to claim 11 wherein said antibody is monoclonal antibody clone HUTS-21.
18. The method according to claim 12 wherein said antibody is monoclonal antibody clone HUTS-21.
19. The method according to claim 13 wherein said antibody is monoclonal antibody clone MEM-148.
20. The method according to claim 14 wherein said antibody is monoclonal antibody clone MEM-148.
21. The method according to claim 15 wherein said antibody is monoclonal antibody clone MEM-148.
22. The method according to claim 16 wherein said ligand is, a compound according to the chemical structure:
where R″ is a peptide LDV or SEQ ID NO:1 (compound LDV) or SEQ ID NO:2 (compound YLDV), the compound TR14035, compound D or compound E.
23. The method according to claim 17 wherein said ligand is, a compound according to the chemical structure:
where R″ is a peptide of SEQ ID NO:1 (compound LDV) or SEQ ID NO:2 (compound YLDV), the compound TR14035, compound D or compound E.
24. The method according to claim 18 wherein said ligand is, a compound according to the chemical structure:
where R″ is a peptide of SEQ ID NO:1 (for compound LDV) or SEQ ID NO:2 (for compound YLDV), the compound TR14035, compound D or compound E.
25. The method according to claim 19 wherein said ligand is compound 3 or XVA143.
26. The method according to claim 20 wherein said ligand is compound 3 or XVA143.
27. The method according to claim 21 wherein said ligand is compound 3 or XVA143.
28. The method according to claim 16 wherein said reporter moiety is a fluorophore.
29. The method according to claim 19 wherein said reporter moiety is a fluorophore.
30. The method according to claim 19 wherein said reporter moiety is a fluorophore.
31. The method according to claim 20 wherein said reporter moiety is a fluorophore.
32. The method according to claim 28 wherein said flourophore is selected from the group consisting of fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde label, fluorescamine, tetramethylrhodamine, a dipyrrometheneboron difluoride dye, a near infrared dye and a lanthanide chelate.
33. The method according to claim 29 wherein said flourophore is selected from the group consisting of fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde label, fluorescamine, tetramethylrhodamine, a dipyrrometheneboron difluoride dye, a near infrared dye and a lanthanide chelate.
34. The method according to claim 30 wherein said flourophore is selected from the group consisting of fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde label, fluorescamine, tetramethylrhodamine, a dipyrrometheneboron difluoride dye, a near infrared dye and a lanthanide chelate.
35. The method according to claim 31 wherein said flourophore is selected from the group consisting of fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, ophthaldehyde label, fluorescamine, tetramethylrhodamine, a dipyrrometheneboron difluoride dye, a near infrared dye and a lanthanide chelate.
36. The method according to claim 28 wherein said fluorophore is fluorescein or phycoerythrin.
37. The method according to claim 30 wherein said fluorophore is fluorescein or phycoerythrin.
38. The method according to claim 1 adapted for use in high throughput flow cytometry.
39. The method according to claim 2 adapted for use in high throughput flow cytometry.
40. The method according to claim 16 adapted for use in high throughput flow cytometry.
41. The method according to claim 19 adapted for use in high throughput flow cytometry.