Antibody that binds to LRP5 protein and method of use

Abstract

This invention provides an antibody that specifically binds to LRP5. [Solution] An antibody that specifically binds to LRP5 and a method of using the same are provided herein. TIFF2026102865000034.tif74165

Claims

1. An antibody that specifically binds to LRP5, comprising a light chain variable region and / or a heavy chain variable region, The heavy chain variable region comprises complementarity-determining regions CDR-H1, CDR-H2, and CDR-H3, and the light chain variable region comprises complementarity-determining regions CDR-L1, CDR-L2, and CDR-L3, and the amino acid sequence of the CDR comprises or consists of CDR sequences selected from the anti-LRP5 antibody CDR sequence set: LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6. antibody.

2. The amino acid sequence of the CDR includes or consists of sequences selected from the sequences described below. The CDR-H1 is Selected from the group consisting of, The CDR-H2 is Selected from the group consisting of, The CDR-H3 is Selected from the group consisting of, The CDR-L1 is SVSSA, CDR-L2 is SASSLYS, and CDR-L3 is Selected from the group consisting of, The antibody according to claim 1.

3. (i) Heavy chain amino acid sequences listed in Table 2, (ii) An amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the heavy chain amino acid sequences listed in Table 2, wherein the CDR sequence is the set of CDR sequences listed in Table 1, or (iii) A conservatively substituted amino acid sequence of (i), wherein the CDR sequence is one of the CDR sequence sets listed in the table. The antibody according to claim 2, comprising a heavy chain variable region.

4. (i) Light chain amino acid sequences listed in Table 2, (ii) An amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the light chain amino acid sequences listed in Table 2, wherein the CDR sequence is the set of CDR sequences listed in Table 1, or (iii) Conservatively substituted amino acid sequences of (i), wherein the CDR sequence is one of the CDR sequence sets listed in Table 1. The antibody according to any one of claims 2 to 3, comprising a light chain variable region including the above.

5. The antibody according to any one of claims 1 to 4, wherein the CDR sequence is a complete set of CDR sequences selected from the antibodies identified in Table 1.

6. An antibody according to any one of claims 1 to 5 that cross-reacts with LRP6.

7. The antibody according to claim 1, wherein the CDR sequence comprises a set of light chain CDR sequences or a set of heavy chain CDR sequences selected from the antibodies identified in Table 1.

8. An antibody according to any one of claims 1 to 7, which specifically binds to LRP5.

9. The antibody according to claim 8, wherein the CDR sequence is a set of CDR sequences of an antibody selected from the antibodies LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

10. The antibody according to any one of claims 1 to 9, which blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5.

11. The antibody according to any one of claims 1 to 9, which blocks the binding of a Wnt ligand to the non-Wnt3a binding site of LRP5.

12. The antibody according to any one of claims 1 to 11, which is a monoclonal antibody.

13. The antibody according to any one of claims 1 to 12, which is a humanized antibody.

14. The antibody according to any one of claims 1 to 13, which is a single-chain antibody.

15. The antibody according to any one of claims 1 to 14, which is an antibody-conjugated fragment selected from Fab, Fab', F(ab')2, scFv, dsFv, ds-scFv, dimer, nanobody, minibody, diabody, and polymers thereof.

16. The antibody according to any one of claims 1 to 14, which is a bispecific antibody.

17. The antibody according to any one of claims 1 to 14, which is a bispecific antibody that further binds to the FZD receptor.

18. The antibody according to any one of claims 1 to 17, comprising a non-natural glycosylation pattern.

19. For example, an antibody according to any one of claims 1 to 17, comprising substitution or addition of cysteine ​​to a constant region or framework region.

20. An immunoconjugate comprising the antibody according to any one of claims 1 to 17 and a detectable label or cytotoxic agent.

21. The immunoconjugate according to claim 20, comprising a cytotoxic agent selected from meitansinoids, auristatin, dorastatin, tubulicin, cryptophycin, pyrrolobenzodiazepine (PBD) dimers, indolinobenzodiazepine dimers, α-amanitin, trichoten, SN-38, duocalmycin, CC1065, calicheamicin, engine antibiotics, taxanes, doxorubicin derivatives, anthracyclines, and their stereoisomers, azanofide, isosteres, analogs, or derivatives.

22. A nucleic acid molecule encoding an antibody according to any one of claims 1 to 17.

23. The nucleic acid molecule according to claim 22, wherein one or more of the CDR sequences are encoded by the nucleic acids in Table 2.

24. The aforementioned antibody (i) Heavy chain nucleic acid sequences listed in Table 2, (ii) A nucleotide sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the heavy chain nucleic acid sequences listed in Table 2, wherein the CDR sequence is one of the CDR sequence sets listed in Table 1, or (iii) Codon-degenerate nucleic acid sequences of (i), wherein the CDR sequence is one of the CDR sequence sets listed in Table 1. The nucleic acid molecule according to claim 22, comprising a heavy chain variable region encoded by a nucleic acid containing the following:

25. The aforementioned antibody (i) Light chain nucleic acid sequences listed in Table 2, (ii) A nucleic acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with respect to the light chain nucleic acid sequences listed in Table 2, wherein the CDR sequence is a set of CDR sequences listed in Table 1, or (iii) Codon-degenerate nucleic acid sequences of (i), wherein the CDR sequence is one of the CDR sequence sets listed in Table 1. The nucleic acid molecule according to claim 22, comprising a light chain variable region encoded by a nucleic acid containing the following:

26. A vector comprising an expression control sequence functionally linked to a nucleic acid according to any one of claims 22 to 25.

27. A host cell comprising a recombinant nucleic acid molecule containing an expression regulatory sequence functionally linked to the nucleic acid according to any one of claims 22 to 26.

28. The host cells according to claim 27, which are Chinese hamster ovary (CHO) cells.

29. A host cell comprising the vector according to claim 26.

30. A method for producing an anti-LRP5 antibody, comprising the step of culturing host cells according to any one of claims 27 to 29.

31. A composition comprising, optionally with a suitable diluent, an antibody according to any one or more of claims 1 to 17, an immunoconjugate according to any one of claims 20 to 21, a nucleic acid molecule according to any one of claims 22 to 25, a vector according to claim 26, or a host cell according to claim 29.

32. The composition according to claim 31, comprising one or more antibodies or immunoconjugates, and optionally being a pharmaceutical composition.

33. A kit comprising an antibody according to any one or more of claims 1 to 17, an immunoconjugate according to any one of claims 20 to 21, a nucleic acid molecule according to any one of claims 22 to 25, a vector according to claim 26, or a host cell according to any one of claims 27 to 29.

34. A method for detecting LRP5 expression, comprising the steps of: contacting a sample containing one or more cells with one or more antibodies or immunoconjugates according to any one of claims 1 to 21 under conditions that allow for the formation of cell complexes; and detecting the presence of any antibody complex.

35. The method according to claim 34, wherein detection is performed by immunofluorescence.

36. The method according to claim 34, wherein the detection is performed by flow cytometry.

37. The method according to any one of claims 34 to 36, wherein the method is for detecting LRP4 expression, and the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

38. A method for inhibiting Wnt ligand binding to the LRP5 receptor, interfering with the Wnt signaling pathway, inhibiting Wnt-induced transcriptional activity, inhibiting disheveled activation, promoting the preservation of the β-catenin degradation complex, promoting the accumulation of β-catenin, or inhibiting cell proliferation, A method comprising the step of contacting cells expressing an LRP5 receptor with an antibody or immunoconjugate according to any one of claims 1 to 21.

39. The method according to claim 38, wherein the antibody or immunoconjugate blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5.

40. The method according to claim 38, wherein the antibody or immunoconjugate blocks the binding of a Wnt ligand to the non-Wnt3a binding site of LRP5.

41. The method according to claim 38, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from the group consisting of LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

42. A method for treating a cancer of a subject that requires such treatment, comprising the step of administering to the subject an effective amount of a pharmaceutical composition comprising an antibody or immunoconjugate according to any one of claims 1 to 21.

43. The method according to claim 42, wherein the cancer is selected from cancer cells of the colon, lung, breast, ovary, endometrium, pancreas, stomach, liver, adrenal cortex, and osteoblastoma.

44. The method according to claim 42, wherein the cancer is selected from acute myeloid leukemia, prostate cancer, glioblastoma, bladder cancer, and cervical cancer.

45. The process includes administering the first and second antibodies or antibody conjugates described in any one of claims 1 to 21 to the subject, The first method blocks the binding of Wnt ligand to the Wnt3a binding site of LRP5, and the second method blocks the binding of Wnt ligand to the non-Wnt3a binding site of LRP5. The method according to claim 42.

46. The method according to claim 45, wherein the first antibody or immunoconjugate comprises a set of CDR sequences selected from antibodies of epitope group 2.

47. The method according to claim 42, wherein the antibody or immunoconjugate specifically binds to LRP5 in at least one assay and inhibits Wnt3a-induced signaling in at least one assay, and optionally the antibody or immunoconjugate is the antibody or immunoconjugate described in any one of claims 1 to 21.

48. The method according to claim 42, wherein the antibody or immunoconjugate comprises a set of CDR sequences corresponding to an antibody selected from the group consisting of LRP5-A7, LRP5-A9, LRP5-C5, LRP5-C12, LRP5-D9, LRP5-E5, LRP5-G2, LRP5-G9, LRP5-G10, LRP5-G11, LRP5-H3, LRP5-H5, LRP5-H9, LRP5-R3O_D3, LRP5-R3_E8, and LRP5-R3O_G6.

49. A method for enhancing the signaling activity of a Wnt ligand that binds to the Wnt3a binding site of LRP5, comprising the step of contacting cells expressing LRP5 with an antibody that blocks the binding of a Wnt ligand to the non-Wnt3a binding site of LRP5.

50. A method for enhancing the signaling activity of a Wnt ligand that binds to the non-Wnt3a binding site of LRP5 by contacting cells expressing LRP5 with an antibody that blocks the binding of a Wnt ligand to the Wnt3a binding site of LRP5.

51. The method according to any one of claims 49 or 50, performed in vitro.

52. The method according to any one of claims 49 or 50, performed in vivo.

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