HETERODIMERIC IMMUNOGLOBULINS

• US 20140154254A1
• Filed: 11/21/2013
• Published: 06/05/2014
• Est. Priority Date: 11/21/2012
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Florio, Monica, Liu, Zhi, Yan, Wei, Kannan, Gunasekaran
• Granted Patent: US 10,233,237 B2
• Time in Patent Office: Days
• US Class Current: 424/136.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/526 CH3 domain
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/567 Framework region [FR]
• CPC Class Codes: C07K 2317/64 comprising a combination of variable region and constant region components
• CPC Class Codes: C07K 2317/72 Increased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/75 Agonist effect on antigen
• CPC Class Codes: C07K 2317/76 : Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C07K 2317/92 : Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 : Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide

Abstract

The present application is directed to heterodimeric antibodies and methods of use.

Claims

1. A heterodimeric antibody or fragment thereof comprising one or more substitutions in each of the following domains:

a first CH3-domain, a second CH3-domain, a CH1-domain, a CL-domain, a VH-domain and a VL-domain,

wherein the one or more substitutions introduce charged amino acids that are electrostatically unfavorable to homodimer formation and electrostatically favorable to heterodimer formation.

2. The heterodimeric antibody or fragment thereof of claim 1, wherein the first CH3-domain or the second CH3-domain comprises an amino acid sequence differing from wild-type IgG amino acid sequence such that (a) one or more positive-charged amino acids in a wild-type human IgG amino acid sequence are replaced with one or more negative-charged amino acids or (b) one or more negative-charged amino acids in a wild-type human IgG amino acid sequence are replaced with one or more positive-charged amino acids.

4. The heterodimeric antibody or fragment thereof of claim 1, wherein the CH1-domain or the CL-domain comprises an amino acid sequence differing from wild-type IgG amino acid sequence such that (a) one or more positive-charged amino acids in wild-type IgG amino acid sequence are replaced with one or more negative-charged amino acids or (b) one or more negative-charged amino acids in wild-type IgG amino acid sequence are replaced with one or more positive-charged amino acids.

6. The heterodimeric antibody or fragment thereof of claim 1, wherein the VH-domain or the VL-domain comprises an amino acid sequence differing from wild-type IgG amino acid sequence such that (a) one or more positive-charged amino acids in wild-type IgG amino acid sequence are replaced with one or more negative-charged amino acids or (b) one or more negative-charged amino acids in wild-type IgG amino acid sequence are replaced with one or more positive-charged amino acids.

8. The heterodimeric antibody or fragment thereof of claim 1, wherein the positive-charged amino acid is selected from the group consisting of lysine, histidine and arginine.

9. The heterodimeric antibody or fragment thereof of claim 1, wherein the negative-charged amino acid is selected from the group consisting of aspartic acid and glutamic acid.

10. The heterodimeric antibody or fragment thereof of claim 1, wherein the first CH3-domain comprises a replacement of a glutamic acid with a positive-charged amino acid.

12. The heterodimeric antibody or fragment thereof of claim 1, wherein the first CH3-domain comprises a replacement of an aspartic acid with a negative-charged amino acid.

14. The heterodimeric antibody or fragment thereof of claim 1, wherein the second CH3-domain comprises a replacement of a lysine with a negative-charged amino acid.

16. The heterodimeric antibody or fragment thereof of claim 1, wherein the CH1-domain comprises a replacement of a serine with a negative-charged amino acid.

18. The heterodimeric antibody or fragment thereof of claim 1, wherein the CL-domain comprises a replacement of a serine with a positive-charged amino acid.

20. The heterodimeric antibody or fragment thereof of claim 1, wherein the VH-domain comprises a replacement of a glutamine with a negative-charged amino acid.

22. The heterodimeric antibody or fragment thereof of claim 1, wherein the VL-domain comprises a replacement of a glutamine with a positive-charged amino acid.

24. The heterodimeric antibody or fragment thereof of claim 1, that is selected from the group consisting of an antibody, a bispecific antibody, a monospecific monovalent antibody, a bispecific maxibody, a monobody, a peptibody, a bispecific peptibody, a monovalent peptibody and a receptor fusion protein.

55. The heterodimeric antibody of claim 1, wherein the antibody binds to a region of sclerostin comprising amino acids 86-111 of SEQ ID NO:

1.

56. The heterodimeric antibody of claim 1, that is an IgG immunoglobulin.

57. A nucleic acid comprising a nucleotide sequence encoding the heterodimeric antibody according to claim 1.

58. A vector comprising the nucleotide sequence of claim 57.

59. An isolated host cell comprising the nucleic acid of claim 57.

60. A method of increasing bone mineral density in a mammalian subject comprising administering the heterodimeric antibody of claim 1 to the subject in an amount effective to increase bone mineral density in the subject.

61. A composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier, diluent or adjuvant.

62. The composition according to claim 61, wherein less than 5% of the antibody in the composition is in aggregate form after two weeks of storage at about 4°

C.

64. The composition according to claim 62, wherein an amount of antibody in the composition present in aggregate form in determined by Size Exclusion Chromatography.

3. (canceled)

5. (canceled)

7. (canceled)

11. (canceled)

13. (canceled)

15. (canceled)

17. (canceled)

19. (canceled)

21. (canceled)

23. (canceled)

25. A heterodimeric antibody or fragment thereof comprising a heavy chain comprising

(a) a first amino acid substitution at an AHo position selected from the group consisting of AHo positions 42-50 that introduces a charged amino acid at said position,

(b) a second amino acid substitution at a position selected from the group consisting of positions 126-213 (EU numbering) that introduces a charged amino acid at said position,

(c) a third amino acid substitution at a position selected from the group consisting of positions 352-360 (EU numbering) that introduces a charged amino acid at said position, and

(d) a fourth amino acid substitution at a position selected from the group consisting of positions 395-403 (EU numbering) that introduces a charged amino acid,

wherein the charged amino acid of (a) has the same charge as the charged amino acid of (b), and wherein the charged amino acids of (c) and (d) have an opposite charge of the charged amino acids of (a) and (b).

26. The heterodimeric antibody or fragment thereof of claim 25, wherein the first amino acid substitution is at AHo position 46, the second amino acid substitution is at EU position 183, the third amino acid substitution is at EU position 356 and the fourth amino acid substitution is at EU position 399.

27. The heterodimeric antibody or fragment thereof of claim 25, wherein glutamine at AHo position 46 is replaced with glutamic acid, serine at EU position 183 is replaced with glutamic acid, glutamic acid at EU position 356 is replaced with lysine, and aspartic acid at EU position 399 is replaced with lysine.

34. The heterodimeric antibody or fragment thereof of claim 25, wherein

(a) and (b) have a negative-charge and the charged amino acids of (c) and (d) have a positive charge.

36. The antibody or fragment thereof of claim 34, comprising a light chain comprising positive-charged amino acids at AHo position 46 and EU position 176.

40. The heterodimeric antibody of claim 26 further comprising a second heavy chain and a first light chain and a second light chain, wherein the second heavy chain comprises amino acid substitutions at AHo position 46 and EU positions 183, 392 and 409, and wherein the first and second light chains comprise an amino acid substitution at AHo position 46 and EU position 176, wherein the amino acid substitutions introduce a charged amino acid at said positions.

41. The heterodimeric antibody of claim 40, wherein glutamine at position AHo 46 of the first heavy chain is replaced with glutamic acid, wherein glutamine at position AHo 46 of the second heavy chain is replaced with lysine, wherein glutamine at position AHo 46 of the first light chain is replaced with lysine, wherein glutamine at position AHo 46 of the second light chain is replaced with glutamic acid, wherein serine at EU position 183 of the first heavy chain is replaced with glutamic acid, wherein glutamic acid at EU position 356 of the first heavy chain is replaced with lysine, wherein glutamic acid at EU position 399 of the first heavy chain is replaced with lysine, wherein serine at EU position 183 of the second heavy chain is replaced with lysine, wherein lysine at EU position 392 of the second heavy chain is replaced with aspartic acid, and wherein lysine at EU position 409 of the second heavy chain is replaced with aspartic acid.

53. The heterodimeric antibody of claim 40, wherein the heterodimeric antibody binds to a region of sclerostin comprising amino acids 86-111 of SEQ ID NO:

1, wherein the

first heavy chain and the first light chain comprise a sclerostin-binding portion and the second heavy chain and the second light chain comprise a DKK1-binding portion.

28. An antibody or fragment thereof comprising a heavy chain comprising

(a) a first amino acid substitution at an AHo position selected from the group consisting of AHo positions 35-43 that introduces a charged amino acid at said position,

(b) a second amino acid substitution at a position selected from the group consisting of positions 126-213 (EU numbering) that introduces a charged amino acid at said position,

(c) a third amino acid substitution at a position selected from the group consisting of positions 388-398 (EU numbering) that introduces a charged amino acid at said position, and

(d) a fourth amino acid substitution at a position selected from the group consisting of positions 404-413 (EU numbering) that introduces a charged amino acid,

wherein the charged amino acids of (c) and (d) have an opposite charge of the charged amino acids of (a) and (b).

29. The heterodimeric antibody or fragment thereof according to claim 28, wherein the first amino acid substitution is at AHo position 46, the second amino acid substitution is at EU position 183, the third amino substitution is at EU position 393 and the fourth amino acid substitution is at EU position 409.

30. The antibody or fragment thereof claim 29, wherein glutamine at AHo position 46 is replaced with lysine, serine at EU position 183 is replaced with lysine, lysine at EU position 392 is replaced with aspartic acid and lysine at EU position 409 is replaced with aspartic acid.

31. An heterodimeric antibody or fragment comprising a light chain comprising

(a) a first amino acid substitution at an AHo position selected from the group consisting of AHo positions 42-50 that introduces a charged amino acid at said position, and

(b) a second amino acid substitution at a position selected from the group consisting of positions 126-213 (EU numbering) that introduces a charged amino acid at said position.

32. The heterodimeric antibody or fragment thereof of claim 31, wherein the first amino acid substitution is at AHo position 46 and the second amino acid substitution is at EU position 176.

33. The heterodimeric antibody or fragment of claim 32, wherein glutamine at AHo position 46 is replaced with glutamic acid and serine at EU position 176 is replaced with glutamic acid.

35. (canceled)

37. A heterodimeric antibody or fragment thereof comprising a heavy chain comprising

(a) a first amino acid substitution at an AHo position selected from the group consisting of AHo positions 42-50 that introduces a charged amino acid at said position,

(b) a second amino acid substitution at a position selected from the group consisting of positions 126-213 (EU numbering) that introduces a charged amino acid at said position,

(c) a third amino acid substitution at a position selected from the group consisting of positions 388-397 (EU numbering) that introduces a charged amino acid at said position, and

(d) a fourth amino acid substitution at a position selected from the group consisting of positions 404-413 (EU numbering) that introduces a charged amino acid,

wherein the charged amino acids of (a) and (b) have a positive-charge and the charged amino acids of (c) and (d) have a negative-charge.

38. The heterodimeric antibody or fragment thereof of claim 37, wherein the first amino acid substitution is at AHo position 46, the second amino acid substitution is at EU position 183, the third amino acid substitution is at EU position 392 and the fourth amino acid substitution is at EU position 409.

39. The heterodimeric antibody or fragment thereof of claim 37, comprising a light chain comprising negative-charged amino acids at AHo position 46 and EU position 176.

42. A heterodimeric antibody that binds sclerostin and DKK-1, comprising

a first heavy chain comprising a variable region amino acid sequence selected from the group consisting of SEQ ID NOs:

378 and 366 and comprising amino acid substitutions at EU positions 183, 356 and 399 of the first heavy chain,

a second heavy chain comprising a variable region amino acid sequence selected from the group consisting of SEQ ID NOs;

1003 and 974 and comprising amino acid substitutions at EU positions 183, 392 and 409 of the second heavy chain,

a first light chain comprising a variable region amino acid sequence selected from the group consisting of SEQ ID NOs;

376 and 364 and comprising an amino acid substitution at EU position 176 of the first light chain, and

a second light chain comprising a variable region amino acid sequence selected from the group consisting of SEQ ID NOs;

1002 and 978 and comprising an amino acid substitution at EU position 176 of the second light chain;

wherein the amino acid substitutions introduce a charged amino acid at said positions.

43. (canceled)

44. An antibody that binds to a region of sclerostin comprising amino acids 86-111 of SEQ ID NO:

1, wherein the antibody comprises a heavy chain having a CH3 domain comprising one or more amino acid substitutions, wherein the one or more substitutions introduce charged amino acids that are electrostatically unfavorable to homodimer formation and electrostatically favorable to heterodimer formation.

45. The antibody of claim 44, wherein a negative charged amino acid in the CH3 domain is substituted with a positive charged amino acid.

46. The antibody of claim 45, wherein the negative charged amino acid is at EU position D399, E356, or E357.

48. The antibody of claim 45, wherein the positive charged amino acid is lysine.

49. The antibody of claim 44, wherein a positive charge amino acid in the CH3 domain is substituted with a negative charged amino acid.

50. The antibody of claim 49, wherein the positive charged amino acid is at EU position K370, K392 or K409.

52. The antibody of claim 49, wherein the negative charged amino acid is aspartic acid.

47. (canceled)

51. (canceled)

54. A heterodimeric antibody that binds to a region of sclerostin comprising amino acids 86-111 of SEQ ID NO:

1, the antibody comprising

a first heavy chain and a first light chain comprising a sclerostin-binding portion and a second heavy chain and a second light chain comprising a DKK1-binding portion, wherein the first heavy chain comprises amino acid substitutions at EU positions 183, 356 and 399, wherein the second heavy chain comprises amino acid substitutions at EU positions 183, 392 and 409, and wherein the first and second light chains comprise an amino acid substitution at EU position 176, wherein the amino acid substitutions introduce a charged amino acid at said positions.

63. (canceled)

65. (canceled)

66. (canceled)

67. (canceled)