Enzymatic activities in chemokine-mediated inflammation

• US 20060063223A1
• Filed: 08/04/2005
• Published: 03/23/2006
• Est. Priority Date: 08/04/2004
• Status: Active Grant

• Current Assignee: ChemoCentryx, Inc. (Amgen Inc.)
• Original Assignee: ChemoCentryx, Inc. (Amgen Inc.)
• Inventors: Schall, Thomas J., Miao, Zhenhua, Premack, Brett, Berahovich, Robert D.
• Granted Patent: US 7,572,600 B2
• Time in Patent Office: Days
• US Class Current: 435/23
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C07K 14/7158 for chemokines
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: G01N 2500/00 Screening for compounds of potential therapeutic value
• CPC Class Codes: G01N 2800/52 Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
• CPC Class Codes: G01N 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Abstract

Truncated chemokines lacking an N-terminal region that activate CCR1 and/or FPRL1 and compositions containing the truncated chemokines are provided. Methods of identifying agents that modulate CCR1 and/or FPRL1 activity either by modulating the production of the truncated chemokines or the ability of the truncated chemokines to activate CCR1 and/or FPRL1 are also disclosed. Methods using the truncated chemokines to inhibit or activate CCR1 and/or FPRL1 mediated biological activities are also disclosed.

Claims

1. A method for identifying an inhibitor of CCR1 activity, comprising:

(a) contacting protease with a chemokine substrate in the presence of a test compound;

(b) determining the activity of the protease in the presence of the test compound;

(c) comparing the activity of the protease in the presence of the test compound with the activity of the protease in the absence of the test compound;

(d) identifying the test compound as a potential inhibitor of CCR1 activity if the activity of the protease is inhibited in the presence of the test compound.

2. The method of claim 1, wherein the substrate is selected from the group consisting of CCL6, CCL9, CCL15 and CCL23.

3. The method of claim 2, wherein the protease is selected from the group consisting of chymase, cathepsin G, and elastase.

4. The method of claim 3, further comprising conducting an assay of CCR1 activity with the inhibitor identified in step (d) to determine whether the inhibitor inhibits an activity of CCR1.

5. A method for identifying an inhibitor of FPRL1 activity, comprising:

(a) contacting protease with a chemokine substrate in the presence of a test compound;

(b) determining the activity of the protease in the presence of the test compound;

(c) comparing the activity of the protease in the presence of the test compound with the activity of the protease in the absence of the test compound;

(d) identifying the test compound as a potential inhibitor of FPRL1 activity if the activity of the protease is inhibited in the presence of the test compound.

6. The method of claim 5, wherein the substrate is CCL23β

.

7. The method of claim 6, wherein the protease is selected from the group consisting of chymase, cathepsin G, and elastase.

8. The method of claim 7, further comprising conducting an assay of FPR1 activity with the inhibitor identified in step (d) to determine whether the inhibitor inhibits an activity of FPRL1.

9. A method for screening for a modulator of CCR1 activity, the method comprising assaying for an activity of a CCR1 receptor in the presence of a CCR1 ligand fragment and a test agent and comparing the activity level in the presence of the test agent with the activity level in the absence of the test agent, wherein a difference in the activity levels is an indication that the test agent is a modulator of the CCR1 activity.

10. The method of claim 9, wherein the CCR1 activity is binding between the CCR1 receptor and the CCR1 ligand fragment.

11. The method of claim 9, wherein the CCR1 activity is a biological activity selected from the group consisting of calcium mobilization, cell migration, and cell proliferation.

12. A method for screening for a modulator of FPRL1 activity, the method comprising assaying for an activity of an FPRL1 receptor in the presence of an FPRL1 ligand fragment and a test agent and comparing the activity level in the presence of the test agent with the activity level in the absence of the test agent, wherein a difference in the activity levels is an indication that the test agent is a modulator of the FPRL1 activity.

13. The method of claim 12, wherein the FPRL1 activity is binding between the FPRL1 receptor and the FPRL1 ligand fragment.

14. The method of claim 12, wherein the FPRL1 activity is a biological activity selected from the group consisting of calcium mobilization, cell migration, and cell proliferation.

15. A CCR1 ligand analogue, comprising:

(a) a CCL6 analogue, wherein said CCL6 analogue comprises a CCL6 amino acid sequence in which there is a modification which inhibits cleavage between residues 13 and 27 by a serine protease;

(b) a CCL9 analogue, wherein said CCL9 analogue comprises a CCL9 amino acid sequence in which there is a modification which inhibits cleavage between residues 13 and 26 by a serine protease;

(c) a CCL15 analogue, wherein said CCL15 analogue comprises a CCL15 amino acid sequence in which there is a modification which inhibits cleavage between residues 17 and 32 by a serine protease;

or

(d) a CCL23 analogue, wherein said CCL23 analogue comprises a CCL23 amino acid sequence in which there is a modification which inhibits cleavage between residues 17 and 33 by a serine protease.

16. The CCR1 ligand analogue of claim 15, wherein the serine protease is selected from the group consisting of chymase, cathepsin G, and elastase.

17. A pharmaceutical composition comprising (i) an inhibitory agent that inhibits a serine protease having capacity to cleave an N-terminal fragment from CCL6, CCL9, CCL15 and/or CCL23 to generate a CCR1 ligand fragment that can activate CCR1, and (2) a pharmaceutically effective carrier.

18. The pharmaceutical composition of claim 17, wherein the inhibitory agent is a CCR1 ligand analogue selected from the group consisting of:

(a) a CCL6 analogue, wherein said CCL6 analogue comprises a CCL6 amino acid sequence in which there is a modification which inhibits cleavage between residues 13 and 27 by a serine protease;

(b) a CCL9 analogue, wherein said CCL9 analogue comprises a CCL9 amino acid sequence in which there is a modification which inhibits cleavage between residues 13 and 26 by a serine protease;

(c) a CCL15 analogue, wherein said CCL15 analogue comprises a CCL15 amino acid sequence in which there is a modification which inhibits cleavage between residues 17 and 32 by a serine protease; and

(d) a CCL23 analogue, wherein said CCL23 analogue comprises a CCL23 amino acid sequence in which there is a modification which inhibits cleavage between residues 17 and 33 by a serine protease.

19. The pharmaceutical composition of claim 17, wherein the inhibitory agent is a small molecule inhibitor of a serine protease selected from the group consisting of chymase, cathepsin G, and elastase.

20. The pharmaceutical composition of claim 17, wherein the inhibitory agent is an antibody.

27. A medical device comprising the pharmaceutical composition as in one of claims 17-26.

28. A method of treating an inflammatory condition correlated with CCR1 activity, the method comprising administering an effective amount of a pharmaceutical composition of claim 17 or 22 to an individual in need thereof.

29. The method of claim 28, further comprising monitoring a symptom associated with the inflammatory condition of the individual to determine the efficacy of the treatment.

44. A method for inducing an immune response to an antigen in a subject, the method comprising administering a composition comprising a CCR1 ligand fragment to the subject, wherein the CCR1 ligand fragment is a polypeptide of claim 20, 23, 26 or 29.

45. A pharmaceutical composition comprising a CCR1 ligand fragment and a pharmaceutically acceptable carrier, wherein the CCR1 ligand fragment is a polypeptide of claim 20, 23, 26 or 29.

21. A pharmaceutical composition comprising (i) an inhibitory agent that inhibits a serine protease having capacity to cleave an N-terminal fragment from CCL23β

to generate an FPRL1 ligand fragment that can activate FPRL1, and (2) a pharmaceutically effective carrier.

30. A method of treating an inflammatory condition correlated with FPRL1 activity, the method comprising administering an effective amount of a pharmaceutical composition of claim 21 or 26 to an individual in need thereof.

31. The method of claim 30, further comprising monitoring a symptom associated with the inflammatory condition of the individual to determine the efficacy of the treatment.

22. A pharmaceutical composition comprising an inhibitory agent that inhibits a CCR1 ligand fragment from binding to CCR1 and a pharmaceutically effective carrier.

23. The pharmaceutical composition of claim 22, wherein the CCR1 ligand fragment is selected from the group of polypeptides having the amino acid sequence of SEQ ID NOs:

2-3, 5-8, 10-15 or 17-21.

24. The pharmaceutical composition of claim 22, wherein the inhibitory agent is a small organic molecule.

25. The pharmaceutical composition of claim 22, wherein the inhibitory agent is an antibody.

26. A pharmaceutical composition comprising an inhibitory agent that inhibits an FPRL1 ligand fragment from binding to FPRL1 and a pharmaceutically effective carrier.

32. An isolated polypeptide that is a fragment of CCL6 (SEQ ID NO:

1), has at least 90% sequence identity to SEQ ID NO;

2, and can bind CCR1.

33. An isolated polypeptide that (1) is a truncated form of CCL6 (SEQ ID NO:

1) in which the N terminal 13-27 amino acids of CCL6 are deleted, and (2) can bind CCR1.

34. The isolated polypeptide of claim 33, that has the amino acid sequence of SEQ ID NOs:

2 or 3.

46. The pharmaceutical composition of claim 34, further comprising an antigen.

35. An isolated polypeptide that is a fragment of CCL9 (SEQ ID NO:

4), has at least 90% sequence identity to SEQ ID NO;

5, and can bind CCR1.

36. An isolated polypeptide that (1) is a truncated form of CCL9 (SEQ ID NO:

4) in which the N terminal 13-26 amino acids of CCL9 are deleted, and (2) can bind CCR1.

37. The isolated polypeptide of claim 36, that has the amino acid sequence of SEQ ID NO:

5, 6, 7 or 8.

38. An isolated polypeptide that is a fragment of CCL15 (SEQ ID NO:

9), has at least 90% sequence identity to SEQ ID NO;

10, and can bind CCR1.

39. An isolated polypeptide that (1) is a truncated form of CCL15 (SEQ ID NO:

9) in which the N terminal 17-32 amino acids of CCL15 are deleted, and (2) can bind CCR1.

40. The isolated polypeptide of claim 39, that has the amino acid sequence of SEQ ID NO:

10, 11, 12, 13, 14 or 15.

41. An isolated polypeptide that is a fragment of CCL23 (SEQ ID NO:

16), has at least 90% sequence identity to SEQ ID NO;

17, and can bind CCR1.

42. An isolated polypeptide that (1) is a truncated form of CCL23 (SEQ ID NO:

16) in which the N terminal 17-33 amino acids of CCL23 are deleted, and (2) can bind CCR1.

43. The isolated polypeptide of claim 42, that has the amino acid sequence of SEQ ID NO:

17, 18, 19, 20 or 21.