Modified Fc molecules

• US 20070269369A1
• Filed: 08/10/2006
• Published: 11/22/2007
• Est. Priority Date: 08/12/2005
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Xiong, Fei, Gegg, Colin, Walker, Kenneth, Miranda, Leslie
• Granted Patent: US 8,008,453 B2
• Time in Patent Office: Days
• US Class Current: 424/1.410
• CPC Class Codes: A61K 38/02 Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61K 47/68 the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
• CPC Class Codes: A61K 47/6817 Toxins
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 37/02 Immunomodulators
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/02 Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/12 Antihypertensives
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 17/00 Carrier-bound or immobilised peptides carrier-bound or immobilised enzymes C12N11/00; Preparation thereof
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype

Abstract

Disclosed is a process for preparing a pharmacologically active compound, in which at least one internal conjugation site of an Fc domain sequence is selected that is amenable to conjugation of an additional functional moiety by a defined conjugation chemistry through the side chain of an amino acid residue at the conjugation site. An appropriate amino acid residue for conjugation may be present in a native Fc domain at the conjugation site or may be added by insertion (i.e., between amino acids in the native Fc domain) or by replacement (i.e., removing amino acids and substituting different amino acids). In the latter case, the number of amino acids added need not correspond to the number of amino acids removed from the previously existing Fc domain. This technology may be used to produce useful compositions of matter and pharmaceutical compositions containing them. A DNA encoding the inventive composition of matter, an expression vector containing the DNA, and a host cell containing the expression vector are also disclosed.

Claims

1. A composition of matter, which comprises a monomeric or multimeric Fc domain having at least one additional functional moiety that is covalently bound to one or more specifically selected conjugation site(s) in the Fc domain through the side chain of an amino acid residue at the conjugation site(s).

2. The composition of matter of claim 1, wherein the additional functional moiety is a pharmacologically active moiety.

3. The composition of matter of claim 2, wherein the pharmacologically active moiety is a polypeptide, a peptide, or a peptidomimetic moiety.

4. The composition of matter of claim 3, wherein the peptide is a toxin peptide.

5. The composition of matter of claim 3, wherein the peptide comprises a cyclic peptide.

6. The composition of matter of claim 2, wherein the pharmacologically active moiety is a non-peptide organic moiety.

7. The composition of matter of claim 1, wherein the conjugation site(s) is part of a loop region.

8. The composition of matter of claim 7, wherein the loop region comprises a CH2 or CH3 loop region.

9. The composition of matter of claim 1, wherein the additional functional moiety is a labeled moiety comprising a radioisotope, an enzyme, a biotinyl moiety, a fluorophore, or a chromophore.

10. The composition of matter of claim 1, wherein the additional functional moiety is an immobilized substrate.

11. The composition of matter of claim 1, wherein the additional functional moiety is a half-life extending moiety.

12. The composition of matter of claim 11, wherein the half-life extending moiety is a polyethylene glycol, a copolymer of ethylene glycol, a polypropylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an immunoglobulin Fc domain, an albumin, a transthyretin, a thyroxine-binding globulin, or a ligand that has an affinity for a long half-life serum protein, said ligand being selected from the group consisting of peptide ligands and small molecule ligands;

or a combination of any of these members.

13. The composition of matter of claim 1, wherein the one or more specifically selected conjugation site(s) is selected from the group consisting of underlined amino acid residue positions in FIG. 1, boldface amino acid residue positions in FIG. 2, highlighted amino acid residue positions in FIG. 3, or a combination of any of these.

14. The composition of matter of claim 1, wherein the one or more specifically selected conjugation site(s) is selected from the group consisting of underlined amino acid residue positions in FIG. 3.

15. The composition of matter of claim 1, wherein the one or more specifically selected conjugation site(s) corresponds to a site selected from the group consisting of position 1 of SEQ ID NO:

600, position 2 of SEQ ID NO;

600, position 3 of SEQ ID NO;

600, position 4 of SEQ ID NO;

600, position 5 of SEQ ID NO;

600, position 6 of SEQ ID NO;

600, position 9 of SEQ ID NO;

600, position 11 of SEQ ID NO;

600, position 13 of SEQ ID NO;

600, position 16 of SEQ ID NO;

600, position 17 of SEQ ID NO;

600, position 226 of SEQ ID NO;

600, and position 227 of SEQ ID NO;

600, or a combination of any of these.

16. The composition of matter of claim 1, wherein the amino acid residue at the specifically selected conjugation site is a cysteine residue added to the Fc domain by substitution at an Fc site selected from the group consisting of Leu139, Gln143, Ser145, and Ser196, relative to reference sequence SEQ ID NO:

599, or at a combination of any of these.

17. The composition of matter of claim 1, wherein the one or more specifically selected conjugation site(s) is a site selected from a loop region identified in Table 3.

18. The composition of matter of claim 1, wherein the amino acid residue at the specifically selected conjugation site is a cysteinyl residue.

19. The composition of matter of claim 18, wherein the cysteinyl residue is in a position of the amino acid sequence of the Fc domain, which in a native Fc domain sequence is not occupied by a cysteinyl residue.

20. The composition of matter of claim 1, wherein the additional functional moiety is a polymer.

21. The composition of matter of claim 1, wherein the additional functional moiety is polyethylene glycol.

22. The composition of matter of claim 1 of the formula

23. The composition of matter of claim 22 wherein the Fc domain comprises an IgG Fc domain.

24. The composition of matter of claim 1 wherein the Fc domain comprises an IgG1 Fc domain.

25. The composition of matter of claim 24, wherein the IgG1 Fc domain comprises SEQ ID NO:

600 or SEQ ID NO;

603.

26. The composition of matter of claim 22, wherein a=0 and b=1.

27. The composition of matter of claim 22, wherein a=1 and b=0.

28. The composition of matter of claim 22, wherein a=1 and b=1.

29. The composition of matter of claim 22, wherein a=0 and b=0.

30. The composition of matter of claim 22, wherein X³ comprises polyethylene glycol (PEG).

31. The composition of matter of claim 22, wherein X³ has the structure -(L¹)_c-P¹.

32. The composition of matter of claim 22, wherein X³ has the structure -(L¹)_c-P₁-(L²)_d-P².

33. The composition of matter of claim 22, wherein g=1 or 2.

34. The composition of matter of claim 1, wherein the Fc domain is an IgG1 Fc domain comprising an amino acid sequence SEQ ID NO:

603, and the one or more specifically selected conjugation site(s) is selected from an amino acid residue position contained in a loop region that comprises an amino acid sequence selected from the group consisting of SEQ ID NOS;

601, 602, 604, 605, 606, 607, 608, 609, 610, and 611.

35. The composition of matter of claim 1, comprising an angiotensin-2 (ang-2) binding peptide or polypeptide.

36. The composition of matter of claim 22, wherein any of X¹, X², or X³ comprises an angiotensin-2 (ang-2) binding peptide or polypeptide.

37. The composition of matter of claim 36, wherein the ang-2 binding peptide or polypeptide comprises an amino acid sequence selected from SEQ ID NOS:

100 to 189 and 612.

38. The composition of matter of claim 36, wherein the ang-2 binding peptide comprises SEQ ID NO:

147.

39. The composition of matter of claim 36, wherein F¹ comprises an IgG1 Fc domain.

40. The composition of matter of claim 1, comprising a myostatin binding peptide or polypeptide.

41. The composition of matter of claim 22, wherein any of X¹, X², or X³ comprises a myostatin binding peptide or polypeptide.

42. The composition of matter of claim 41, wherein the myostatin binding peptide or polypeptide comprises an amino acid sequence selected from SEQ ID NOS:

219 to 509, 613, and 616.

43. The composition of matter of claim 41, wherein the myostatin binding peptide comprises SEQ ID NO:

365.

44. The composition of matter of claim 41, wherein F¹ comprises an IgG1 Fc domain.

45. The composition of matter of claim 1, comprising an erythropoietin-mimetic (EPO-mimetic) peptide or polypeptide.

46. The composition of matter of claim 22, wherein any of X¹, X², or X³ comprises an erythropoietin-mimetic (EPO-mimetic) peptide or polypeptide.

47. The composition of matter of claim 46, wherein the EPO-mimetic peptide or polypeptide comprises an amino acid sequence selected from SEQ ID NOS:

1 to 27 and 614.

48. The composition of matter of claim 46, wherein the EPO-mimetic peptide comprises SEQ ID NO:

2.

49. The composition of matter of claim 46, wherein F¹ comprises an IgG1 Fc domain.

50. The composition of matter of claim 1 comprising a thrombopoietin-mimetic (TPO-mimetic) peptide or polypeptide.

51. The composition of matter of claim 22, wherein any of X¹, X², or X³ comprises a thrombopoietin-mimetic (TPO-mimetic) peptide or polypeptide.

52. The composition of matter of claim 51, wherein the TPO-mimetic peptide or polypeptide comprises an amino acid sequence selected from SEQ ID NOS:

28 to 99 and 615.

53. The composition of matter of claim 51, wherein the TPO-mimetic peptide comprises SEQ ID NO:

28.

54. The composition of matter of claim 51, wherein F¹ comprises an IgG1 Fc domain

55. The composition of matter of claim 1, comprising a nerve growth factor (NGF) binding peptide or polypeptide.

56. The composition of matter of claim 22, wherein any of X¹, X², or X³ comprises a nerve growth factor (NGF) binding peptide or polypeptide.

57. The composition of matter of claim 56, wherein the NGF binding peptide or polypeptide comprises an amino acid sequence selected from SEQ ID NOS:

190 to 218.

58. The composition of matter of claim 1, comprising a B cell activating factor (BAFF) binding peptide or polypeptide.

59. The composition of matter of claim 22, wherein any of X¹, X², or X³ comprises a B cell activating factor (BAFF) binding peptide polypeptide.

60. The composition of matter of claim 59, wherein the BAFF binding peptide or polypeptide comprises an amino acid sequence selected from SEQ ID NOS:

510 to 594.

61. A pharmaceutical composition, comprising the composition of matter of claim 1 and a pharmaceutically acceptable carrier.

62. A DNA encoding a composition of matter of claim 1.

63. An expression vector comprising the DNA of claim 62.

64. A host cell comprising the expression vector of claim 63.

65. The host cell of claim 64, wherein the cell is a bacterial or mammalian cell.

72. The composition of matter of claim 1, wherein:

(a) the composition of matter is an antibody modified so that it comprises at least one X³ additional functional moiety covalently bound to the Fc domain of the antibody through one or more specifically selected conjugation site(s) in the Fc domain selected from the group consisting of underlined residue positions in FIG. 1, boldface residue positions in FIG. 2, highlighted residue positions in FIG. 3, underlined residue positions in FIG. 3, and a cysteine residue added to the Fc domain by substitution at an Fc site selected from the group consisting of Leu139, Gln143, Ser145, and Ser196, relative to reference sequence SEQ ID NO;

599, or, if there is more than one X³, any combination of these members;

(b) X³ is selected from -(L¹)_c-P⁰, -(L¹)_c-P¹, -(L¹)_c-P¹-(L¹)_d-P², -(L¹)_c-P¹-(L²)_d-P²-(L³)_e-P³) and -(L¹)_c-P¹-(L²)_d-P²-(L³)_e-P³-(L⁴)_f-P⁴;

P⁰, P¹, P², P³, and P⁴ are each independently selected from the group consisting of;

i) a pharmaceutically acceptable polymer or dextran;

ii) a pharmacologically active polypeptide, peptide, peptidomimetic, or non-peptide organic moiety;

iii) a radioisotope, an enzyme, a biotinyl moiety, a fluorophore, or a chromophore; and

iv) an immobilized substrate, provided that in a chain comprising more than one additional functional moieties, the immobilized substrate is the moiety most distal from the Fc domain, and there can be no more than one immobilized substrate in the chain;

L¹, L², L³, and L⁴ are each independently linkers;

c, d, e, and f are each independently 0 or 1.

73. The composition of matter of claim 22, wherein any of X¹, X², or X³ comprises a polypeptide comprising SEQ ID NO:

617.

66. A process for preparing a pharmacologically active compound, which comprises:

a) selecting at least one internal conjugation site of an Fc domain sequence, said conjugation site being amenable to conjugation of an additional functional moiety by a defined conjugation chemistry through the side chain of an amino acid residue at the conjugation site; and

b) conjugating a predetermined functional moiety to the selected conjugation site by employing the defined conjugation chemistry.

67. The process of claim 66, wherein the internal conjugation site of the Fc domain is in a loop region.

68. The process of claim 66, wherein selecting the at least one internal conjugation site further comprises substituting a cysteinyl residue for an amino acid residue in the selected internal conjugation site, or inserting a cysteinyl residue into the selected internal conjugation site, whereby the additional functional moiety is conjugated through the side chain of said cysteinyl residue.

69. The process of claim 66, wherein selecting the at least one internal conjugation site further comprises substituting a non-canonical amino acid residue for an amino acid residue in the selected internal conjugation site, or inserting a non-canonical amino acid residue into the selected internal conjugation site, whereby the additional functional moiety is conjugated through the side chain of said non-canonical amino acid residue.

70. The process of claim 69, wherein the non-canonical amino acid residue is selected from the group consisting of azido-containing amino acid residues, keto-containing amino acid residues;

alkyne-containing amino acid residues, alkene-containing amino acid residues, aryl halide-containing amino acid residues; and

1,2-aminothiol-containing amino acid residues.

71. The process of claim 66, wherein a gene construct encoding the Fc domain sequence is expressed in a bacterial or a mammalian cell.