Aldehyde-Tagged Immunoglobulin Polypeptides and Methods of Use Thereof

• US 20120183566A1
• Filed: 01/13/2012
• Published: 07/19/2012
• Est. Priority Date: 01/14/2011
• Status: Active Grant

• Current Assignee: Redwood Bioscience, Inc (Catalent, Inc.)
• Original Assignee: Redwood Bioscience, Inc (Catalent, Inc.)
• Inventors: Breidenbach, Mark Alan, Rabuka, David, Barfield, Robyn M., deHart, Gregory W.
• Granted Patent: US 9,540,438 B2
• Time in Patent Office: Days
• US Class Current: 424/178.1
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 16/2803 against the immunoglobulin superfamily
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype

Abstract

The present disclosure provides aldehyde-tagged immunoglobulin (Ig) polypeptides that can be converted by a formylglycine-generating enzyme to produce a 2-formylglycine (FGly)-modified Ig polypeptide. An FGly-modified Ig polypeptide can be covalently and site-specifically bound to a moiety of interest to provide an Ig conjugate. The disclosure also encompasses methods of production of such aldehyde-tagged Ig polypeptides, FGly-modified Ig polypeptides, and Ig conjugates, as well as methods of use of same.

Claims

1. An isolated aldehyde-tagged immunoglobulin (Ig) polypeptide comprising an Ig constant region amino acid sequence comprising an amino acid sequence of a sulfatase motif,

wherein the sulfatase motif is positioned within or adjacent a solvent-accessible loop region of the Ig polypeptide constant region, and wherein the sulfatase motif is not at the C-terminus of the Ig polypeptide chain.

2. The isolated aldehyde-tagged Ig polypeptide of claim 1, wherein the sulfatase motif comprises an amino acid sequence of the formula X₁Z₁X₂Z₂X₃Z₃, wherein

Z₁ is cysteine or serine;

Z₂ is a proline or alanine residue;

Z₃ is an aliphatic amino acid or a basic amino acid;

X₁ is present or absent and, when present, is any amino acid, with the proviso that when the heterologous sulfatase motif is at an N-terminus of the polypeptide, X₁ is present;

X₂ and X₃ are each independently any amino acid,

3. The isolated Ig polypeptide of claim 2, wherein Z₃ is arginine (R).

4. The isolated Ig polypeptide of claim 2, wherein X₁, when present, X₂, and X₃ are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

5. The isolated Ig polypeptide of claim 2, wherein the X₁, when present, is L, M, V, S or T.

6. The isolated Ig polypeptide of claim 2, wherein X₂ and X₃ are each independently S, T, A, V, G, or C.

7. The isolated Ig polypeptide of claim 2, wherein the Ig polypeptide comprises an Ig heavy chain constant region.

8. The isolated Ig polypeptide of claim 7, wherein the Ig heavy chain constant region is an IgG1 heavy chain constant region.

9. The isolated Ig polypeptide of claim 1, wherein the Ig polypeptide comprises an Ig light chain constant region.

10. The isolated Ig polypeptide of claim 9, wherein the Ig light chain constant region is a kappa light chain constant region.

11. The isolated Ig polypeptide of claim 1, wherein the Ig polypeptide comprises two or more sulfatase motifs.

42. A recombinant nucleic acid comprising a nucleotide sequence encoding the aldehyde-tagged immunoglobulin (Ig) polypeptide of claim 1.

43. The nucleic acid of claim 42, wherein the Ig polypeptide comprises an Ig heavy chain constant region.

44. The nucleic acid of claim 43, wherein the Ig polypeptide further comprises an Ig variable region.

45. The nucleic acid of claim 42, wherein the Ig polypeptide comprises an Ig light chain constant region.

46. The nucleic acid of claim 45, wherein the Ig polypeptide further comprises an Ig variable region.

47. A recombinant expression vector comprising the nucleic acid of claim 42, wherein the aldehyde-tagged Ig polypeptide-encoding nucleotide sequence is operably linked to a promoter.

48. The recombinant expression vector of claim 47, wherein the Ig polypeptide comprises an Ig heavy chain constant region.

49. The recombinant expression vector of claim 48, wherein the vector comprises an insertion site for an Ig variable region 5′

of the aldehyde-tagged Ig polypeptide-encoding nucleotide sequence.

50. The recombinant expression vector of claim 47, wherein the Ig polypeptide comprises an Ig light chain constant region.

51. The recombinant expression vector of claim 50, wherein the vector comprises an insertion site for an Ig variable region 5′

of the aldehyde-tagged Ig polypeptide-encoding nucleotide sequence.

52. A library of recombinant expression vectors, the library comprising:

a population of the recombinant expression vectors of claim 47, wherein each member of the population comprises a nucleotide sequence encoding a differently aldehyde-tagged immunoglobulin polypeptide.

53. A library of aldehyde-tagged immunoglobulin (Ig) polypeptides, the library comprising:

a population of aldehyde-tagged Ig polypeptides according to claim 1, or FGly-modified immunoglobulin polypeptides according to claim 12, wherein the population comprises members having differently aldehyde-tagged Ig polypeptides.

58. An aldehyde-tagged antibody comprising an aldehyde-tagged immunoglobulin (Ig) polypeptide of claim 1.

59. The aldehyde-tagged antibody of claim 58, wherein the antibody comprises an aldehyde-tagged Ig heavy chain constant region.

60. The aldehyde-tagged antibody of claim 58, wherein the antibody comprises an aldehyde-tagged Ig light chain constant region.

61. The aldehyde-tagged antibody of claim 58, wherein the antibody comprises an aldehyde-tagged Ig heavy chain constant region and an aldehyde-tagged Ig light chain constant region

12. An isolated immunoglobulin (Ig) polypeptide modified to comprise 2-formylglycine (FGly) moiety, wherein the FGly-modified sulfatase motif is within or adjacent a solvent-accessible loop region of the Ig polypeptide, wherein the FGly-modified Ig presents the FGly group on a solvent-accessible surface when in a folded state, and wherein the sulfatase motif is not at the C-terminus of the Ig polypeptide chain.

13. The isolated Ig polypeptide of claim 12, wherein the FGly-converted sulfatase motif comprises the formula:

X₁(FGly)X₂Z₂X₃Z₃

wherein;

X₁ is present or absent and, when present, is any amino acid, with the proviso that when the sulfatase motif is at an N-terminus of the polypeptide, X₁ is present;

X₂ and X₃ are each independently any amino acid; and

Z₃ is a basic amino acid.

14. The FGly-modified Ig polypeptide of claim 12, wherein the FGly-modified Ig polypeptide comprises two or more FGly-converted sulfatase motifs.

15. The FGly-modified Ig polypeptide of claim 12, wherein the FGly-modified Ig polypeptide comprises an Ig heavy chain constant region.

16. The FGly-modified Ig polypeptide of claim 15, wherein the Ig heavy chain constant region is an IgG1 heavy chain constant region.

17. The FGly-modified Ig polypeptide of claim 12, wherein the FGly-modified Ig polypeptide comprises an Ig light chain constant region.

18. The FGly-modified Ig polypeptide of claim 17, wherein the Ig light chain constant region is a Ig kappa light chain constant region.

19. The FGly-modified Ig polypeptide of claim 13, wherein Z₃ is arginine (R).

20. The FGly-modified Ig polypeptide of claim 13, wherein X₁, when present, X₂, and X₃ are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

21. The FGly-modified Ig polypeptide of claim 13, wherein the X₁, when present, is L, M, V, S or T.

22. The FGly-modified Ig polypeptide of claim 13, wherein X₂ and X₃ are each independently S, T, A, V, G, or C.

55. A method of producing an immunoglobulin-drug conjugate, the method comprising

combining in a reaction mixture

an FGly-modified, aldehyde-tagged immunoglobulin (Ig) polypeptide of claim 12, wherein Z₁ is a 2-formyl-glycine (FGly) residue and

a drug for conjugation to the FGly-modified Ig polypeptide, wherein the drug comprises a aminooxy or hydrazide reactive group,

wherein the drug is provided in the reaction mixture in an amount sufficient to provide for a desired ratio of drug to Ig polypeptide, said combining being under conditions suitable to promote reaction between an aldehyde of the Ig polypeptide and reactive group of the drug to generate an Ig polypeptide-drug conjugate;

and

isolating the Ig polypeptide-drug conjugate from the reaction mixture.

62. An antibody comprising a formylglycine (FGly) moiety, wherein the antibody comprises an FGly-modified Ig polypeptide of claim 12.

63. The FGly-modified antibody of claim 62, wherein the antibody comprises an FGly-modified heavy chain constant region.

64. The FGly-modified antibody of claim 62, wherein the antibody comprises an FGly-modified light chain constant region.

65. The FGly-modified antibody of claim 62, wherein the antibody comprises an FGly-modified heavy chain constant region and an FGly-modified light chain constant region.

66. An antibody conjugate comprising an immunoglobulin (Ig) conjugate of claim 21.

67. The antibody conjugate of claim 66, wherein the antibody conjugate comprises a J¹ moiety covalently bound to an Ig heavy chain constant region.

68. The antibody conjugate of claim 66, wherein the antibody conjugate comprises a J¹ moiety covalently bound to an Ig light chain constant region.

69. The antibody conjugate of claim 66, wherein the antibody conjugate comprises a J¹ moiety covalently bound to an Ig heavy chain constant region and a J¹ moiety covalently bound to an Ig light chain constant region.

70. The antibody conjugate of claim 66, wherein the antibody specifically binds a tumor antigen on a cancer cell.

71. The antibody conjugate of claim 70, wherein the J¹ moiety is a cytotoxic agent.

72. The antibody conjugate of claim 66, wherein the antibody specifically binds an antigen on a cell infected by a virus.

73. The antibody conjugate of claim 72, wherein antigen is encoded by the virus.

74. The antibody conjugate of claim 72, wherein the J¹ moiety is a viral fusion inhibitor.

23. An immunoglobulin (Ig) conjugate comprising:

an Ig polypeptide and a covalently conjugated moiety, wherein the Ig polypeptide comprises a modified sulfatase motif comprising an FGly′

where FGly′

is of the formula;

24. The Ig conjugate of claim 23, wherein the sulfatase motif comprises the formula:

X₁(FGly′

)X₂Z₂X₃Z₃

wherein

Z₂ is a proline or alanine residue;

X₁ is present or absent and, when present, is any amino acid, with the proviso that when the sulfatase motif is at an N-terminus of the polypeptide, X₁ is present;

X₂ and X₃ are each independently any amino acid; and

Z₃ is an aliphatic amino acid or basic amino acid.

25. The Ig conjugate of claim 23, wherein J¹ is selected from a drug, a detectable label, a water-soluble polymer, and a synthetic peptide.

26. The Ig conjugate of claim 23, wherein the Ig conjugate comprises two or more modified sulfatase motifs.

27. The Ig conjugate of claim 25, wherein J¹ is a small molecule drug.

28. The Ig conjugate of claim 27, wherein the small molecule drug is a cancer chemotherapeutic agent.

29. The Ig conjugate of claim 28, wherein the cancer chemotherapeutic agent is an alkylating agent, a nitrosourea, an antimetabolite, an antitumor antibiotic, a vinca alkaloid, or a steroid hormone.

30. The Ig conjugate of claim 25, wherein J¹ is a water-soluble polymer.

31. The Ig conjugate of claim 30, wherein the water-soluble polymer is poly(ethylene glycol).

32. The Ig conjugate of claim 25, wherein J¹ is a detectable label.

33. The Ig conjugate of claim 32, wherein the detectable label is an imaging agent.

34. The Ig conjugate of claim 23, wherein the Ig polypeptide comprises an Ig heavy chain constant region.

35. The Ig conjugate of claim 34, wherein the Ig heavy chain constant region is an IgG1 heavy chain constant region.

36. The Ig conjugate of claim 23, wherein the FGly-modified Ig polypeptide comprises an Ig light chain constant region.

37. The Ig conjugate of claim 36, wherein the Ig light chain constant region is a Ig kappa light chain constant region.

38. The Ig conjugate of claim 24, wherein Z₃ is arginine (R).

39. The Ig conjugate of claim 24, wherein X₁, when present, X₂, and X₃ are each independently an aliphatic amino acid, a sulfur-containing amino acid, or a polar, uncharged amino acid.

40. The Ig conjugate of claim 24, wherein the X₁, when present, is L, M, V, S or T.

41. The Ig conjugate of claim 24, wherein X₂ and X₃ are each independently S, T, A, V, G, or C.

54. A formulation comprising:

a) an Ig conjugate of claim 23; and

b) a pharmaceutically acceptable excipient.

56. A method of delivering a drug to an individual in need thereof, the method comprising administering to the individual an effective amount of an Ig conjugate of claim 23.

57. A method of treating cancer in a subject, the method comprising:

administering to a subject having cancer an effective amount of an immunoglobulin-drug conjugate of claim 23, wherein said administering is effective to treat the cancer in the subject.