Claims
- 1. A method for screening for a molecule that is capable of antagonizing or agonizing RANK activity, wherein RANK is a polypeptide that comprises amino acids 1-616 of SEQ ID NO:2 or amino acids 1-625 of SEQ ID NO:4, said method comprising the steps of:
(a) contacting RANK responsive cells with one or more candidate molecules, said cells being cultured in a semi-solid medium; (b) determining whether the rate of colony formation or rate of colony growth in the contacted RANK responsive cells is enhanced or reduced as compared with the rate of colony formation or growth in one or more reference RANK responsive cells that are cultured in a semi-solid medium but not contacted with the candidate molecule(s); and (c) identifying the molecule as an antagonist if the rate of colony formation or colony growth in the contacted cells is comparatively enhanced and identifying the molecule as an agonist if the rate of colony formation or colony growth of the contacted cells is comparatively reduced.
- 2. The method of claim 1, said method further comprising the step of stimulating RANK activity in said RANK responsive cells by a method selected from the group consisting of:
(a) contacting the RANK responsive cells with a RANK-L polypeptide, wherein the RANK-L polypeptide comprises amino acids 162-317 of SEQ ID NO:6 or amino acids 161-316 of SEQ ID NO:8; (b) contacting the RANK responsive cells with agonistic anti-RANK antibodies; (c) contacting the RANK responsive cells with one or more cells that express a RANK-L polypeptide, said RANK-L polypeptide comprising amino acids 162-317 of SEQ ID NO:6 or amino acids 161-316 of SEQ ID NO:8; (d) overexpressing RANK in said RANK responsive cells; and (e) expressing in said RANK responsive cells a mutant form of RANK having an amino acid sequence as shown in SEQ ID NO: 10.
- 3. The method of claim 2, wherein the RANK responsive cells are contacted with a RANK-L polypeptide, and further wherein said polypeptide is selected from the group consisting of native RANK-L, soluble RANK-L, a leucine zipper fusion of RANK-L, and a FLAG™ polyHis fusion of RANK-L.
- 4. The method of claim 2, wherein RANK activity is stimulated by contacting the responsive cells with an agonistic anti-RANK antibody.
- 5. The method of claim 4, wherein the antibodies are selected from the group consisting of anti-human M330 antibodies, anti-human M331 antibodies, anti-mouse M395 antibodies and anti-mouse M396 antibodies.
- 6. The method of claim 1, wherein the step of determining the rate of colony formation or colony growth in the contacted RANK responsive cells comprises visually comparing the size of colonies formed in the contacted RANK responsive cells with the size of colonies formed in the one or more reference RANK responsive cells, wherein said visual comparison is done at least one day after contacting the RANK responsive cells with the candidate molecule.
- 7. The method of claim 1, wherein the step of contacting RANK responsive cells with a candidate molecule comprises expressing in said cells an introduced DNA molecule that encodes a nucleic acid molecule or a protein molecule that constitutes the candidate molecule.
- 8. The method of claim 7 wherein said introduced DNA molecule is a cDNA molecule.
- 9. The method of claim 7 wherein said introduced DNA molecule is integrated into the genome of said RANK responsive cells.
- 10. The method of claim 7 wherein said introduced DNA molecule is not integrated into the genome of said RANK responsive cells.
- 11. The method of claim 7 wherein said candidate molecule is a protein encoded by the introduced DNA molecule.
- 12. The method of claim 7 wherein said candidate molecule is a nucleic acid molecule encoded by the introduced DNA molecule, and said candidate molecule is selected from the group consisting of an antisense nucleic acid molecule and a nucleic acid molecule that possesses ribozyme activity.
- 13. The method of claim 8, further comprising isolating said introduced DNA molecule from a colony formed from said contacted RANK responsive cells.
- 14. The method of claim 1, wherein the candidate molecule is a protein and the step of contacting the RANK responsive cells with the candidate molecule comprises adding said protein to the semi-solid medium in which the cells are cultured.
- 15. The method of claim 1, wherein the contacting step comprises adding to the semi solid medium in which the cells are cultured a plurality of proteins that are candidate molecules.
- 16. The method of claim 1, wherein the RANK responsive cells are primary hematopoietic cells selected from the group consisting of hematopoietic precursor cells derived from bone marrow, spleen, fetal liver or peripheral blood; and primary hematopoietic cells derived from bone marrow, spleen, fetal liver or peripheral blood and enriched for osteoclast precursors.
- 17. The method of claim 1, wherein the RANK responsive cells are a cell line selected from the group consisting of RAW 264.7 cells; C7 cells; and BCL-Xl/Tag cells.
- 18. The method of claim 1 wherein the semi-solid medium comprises methylcellulose.
- 19. The method of claim 1, further comprising the step of purifying said candidate molecule from the contacted cells after detecting a comparatively enhanced or reduced rate of colony formation or colony growth in the contacted RANK responsive cells.
- 20. A method for screening for a molecule that modulates RANK activity, said method comprising the steps of:
(a) contacting cultured RANK responsive cells with a candidate molecule, said RANK responsive cells comprising a nucleic acid molecule encoding a reporter protein, said nucleic acid molecule being operably linked to a RANK-responsive regulatory nucleic acid sequence that is selected from the group consisting of a MMP-9 promoter and a TRAP promoter, wherein the RANK responsive cells are exposed to a RANK trigger prior to, during or after being contacted with said candidate molecule; (b) determining whether the level of reporter molecule expression in the contacted RANK responsive cells is enhanced or reduced as compared with the level of reporter molecule expression in a culture of reference RANK responsive cells that are not contacted with the candidate molecule; and (c) identifying the candidate molecule as a RANK agonist if the level of reporter molecule expression in the contacted cells is comparatively enhanced and identifying the candidate molecule as an RANK antagonist if the level of reporter molecule expression in the contacted cells is comparatively reduced.
- 21. The method of claim 20, wherein RANK is triggered in the RANK responsive cells by a method selected from the group consisting of:
(a) contacting the RANK responsive cells with a RANK-L polypeptide, wherein the RANK-L polypeptide comprises amino acids 162-317 of SEQ ID NO:6 or amino acids 161-316 of SEQ ID NO:8; (b) contacting the RANK responsive cells with an agonistic anti-RANK antibody; (c) contacting the RANK responsive cells with cells that express RANK-L, wherein RANK-L is a polypeptide that comprises amino acids 162-317 of SEQ ID NO:6 or amino acids 161-316 of SEQ ID NO:8; (d) overexpressing RANK in said RANK responsive cells; and (e) expressing in said RANK responsive cells a defective RANK polypeptide having an amino acid sequence as shown in SEQ ID NO: 10.
- 22. The method of claim 21, wherein RANK is triggered in the RANK responsive cells by contacting the cells with a RANK-L polypeptide comprising amino acids 162-317 of SEQ ID NO:6 or amino acids 161-316 of SEQ ID NO:8, and further wherein said RANK-L polypeptide is selected from the group consisting of native RANK-L, a leucine zipper fusion of RANK-L, and a FLAG polyHis fusion of RANK-L.
- 23. The method of claim 20, wherein the RANK responsive regulatory nucleic acid is a MMP-9 promoter.
- 24. The method of claim 23, wherein the MMP-9 promoter comprises nucleotides 1769-3591 of SEQ ID NO:11.
- 25. The method of claim 20 wherein the step of contacting RANK responsive cells with a candidate molecule comprises expressing in said RANK responsive cells an introduced DNA molecule that encodes a candidate nucleic acid molecule or a candidate protein molecule.
- 26. The method of claim 25 wherein said introduced DNA molecule is a cDNA molecule.
- 27. The method of claim 25 wherein said introduced DNA molecule is integrated into the genome of said RANK responsive cells.
- 28. The method of claim 25 wherein said introduced DNA molecule is not integrated into the genome of said RANK responsive cells.
- 29. The method of claim 25 wherein said candidate molecule is a protein encoded by the introduced DNA molecule.
- 30. The method of claim 25 wherein said candidate molecule is a nucleic acid molecule encoded by the introduced DNA molecule.
- 31. The method of claim 30 wherein said nucleic acid molecule possesses ribozyme activity.
- 32. The method of claim 25, further comprising isolating said introduced DNA molecule from a colony formed from said contacted RANK responsive cells.
- 33. The method of claim 20, wherein the step of contacting RANK responsive cells with a candidate molecule comprises culturing said RANK responsive cells in the presence of the candidate molecule, and further wherein said candidate molecule is a protein.
- 34. The method of claim 20, wherein the step of contacting RANK responsive cells with a candidate molecule comprises culturing said RANK responsive cells in the presence of a plurality of candidate proteins.
- 35. The method of claim 20, wherein said reporter molecule is selected from the group consisting of luciferase, green fluorescent protein, alkaline phosphatase and a heterologous surface protein.
- 36. The method of claim 35, wherein the reporter molecule is a heterologous surface protein that is selected from the group consisting of human IL-2 receptor, murine IL-4 receptor, human CD2 protein, human CD4 protein, human CD8 protein, luciferase protein, β-galactosidase, and green fluorescent protein.
- 37. The method of claim 20, wherein said RANK responsive cells express a defective RANK molecule and wherein said screening is for an agonist that complements said defective RANK activity.
- 38. The method of claim 20, wherein said RANK responsive cells are hematopoietic cells.
- 39. The method of claim 20, wherein said RANK responsive cells are RAW 264.7 cells.
- 40. The method of claim 20, further comprising the step of purifying the candidate molecule from cells in which the level of expressed reporter molecule is determined to be comparatively enhanced or reduced.
- 41. The method of claim 40, further comprising the step of demonstrating that the purified candidate molecule interacts with RANK.
- 42. The method of claim 20, wherein the level of reporter molecule expression is determined by an assay selected from the group consisting of a fluorescence-based assay, a solid phase assay, and an assay employing a radioactive compound.
- 43. The method of claim 20, wherein determining the level of expressed reporter molecule comprises physically isolating by fluorescence-based cell sorting the cells that are expressing said reporter molecule.
- 44. A method for screening for a molecule that antagonizes or agonizes RANK activity, said method comprising the steps of:
(a) contacting RANK responsive cells with a candidate molecule, wherein the RANK responsive cells are capable of differentiating into osteoclasts in response to the triggering of RANK in said cells; and (b) observing an enhanced level of c-src activity or F-actin formation in said contacted cells as compared to level of c-src activity or F-actin formation in reference RANK responsive cells that are not contacted with the candidate molecule.
- 45. A method for screening for a molecule that antagonizes or agonizes RANK activity, said method comprising the steps of:
(a) contacting RANK responsive cells with a candidate molecule, wherein the RANK responsive cells are capable of differentiating into osteoclasts in response to the triggering of RANK in said cells; (b) triggering RANK in said contacted cells; (c) culturing the contacted cells on a film of CaPO4; (d) determining the number of pits in said film caused by the contacted cultured cells as compared with the number of pits caused in a CaPO4 film by reference RANK responsive cells in which RANK is triggered but which are not contacted with the candidate molecule; and (e) identifying the candidate molecule as a RANK agonist if a greater number of pits is caused by the contacted cells than by the reference cells and as a RANK antagonist if the number of pits caused by the contacted cells is less than the number caused by the reference cells.
Parent Case Info
[0001] This application claims the benefit of priority from U.S. provisional patent application No. 60/235,157, filed Sep. 22, 2000, which is hereby incorporated by reference in its entirety.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60235157 |
Sep 2000 |
US |