Antibodies having altered effector function and methods for making the same

• US 20070048300A1
• Filed: 02/22/2006
• Published: 03/01/2007
• Est. Priority Date: 08/22/2003
• Status: Active Grant

• Current Assignee: Biogen MA Inc. (Biogen, Inc.)
• Original Assignee: Biogen Idec MA Inc. (Biogen, Inc.)
• Inventors: Garber, Ellen, Taylor, Frederick
• Granted Patent: US 7,863,419 B2
• Time in Patent Office: Days
• US Class Current: 424/133.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• 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: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous 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 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/08 Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 16/2875 against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154 against NGF C07K16/22, against TNF C07K16/241
• CPC Class Codes: C07K 2317/21 : from primates, e.g. man
• CPC Class Codes: C07K 2317/41 : Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/52 : Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/71 : Decreased effector function due to an Fc-modification

Abstract

The invention provides a method of producing aglycosylated Fc-containing polypeptides, such as antibodies, having desired effector function. The invention also provides aglycosylated antibodies produced according to the method as well as methods of using such antibodies as therapeutics.

Claims

1. A variant polypeptide of a parent polypeptide comprising an Fc region, wherein the Fc region comprises a modified first amino acid residue, wherein the modified first amino acid is spatially positioned such that reduced glycosylation at a second amino acid is achieved, whereby the variant polypeptide has reduced effector function as compared to the parent polypeptide.

2. The polypeptide of claim 1, wherein the modified first amino acid is spatially positioned from the second amino acid by an interval selected from the group consisting of at least 1 amino acid position or more, at least 2 amino acid positions or more, at least 3 amino acid positions or more, at least 4 amino acid positions or more, at least 5 amino acid positions or more, at least 6 amino acid positions or more, at least 7 amino acid positions or more, at least 8 amino acid positions or more, at least 9 amino acid positions or more, and at least 10 amino acid positions or more.

3. The polypeptide of claim 1, wherein the modified first amino acid residue has a preferred side chain chemistry.

4. The polypeptide of claim 3, wherein the modified first amino acid residue comprises a cysteine thiol.

5. The polypeptide of claim 3, wherein the preferred side chain chemistry is of sufficient steric bulk such that the polypeptide displays reduced effector function.

6. The polypeptide of claim 5, wherein the preferred side chain chemistry of sufficient steric bulk is that of an amino acid residue selected from the group consisting of Phe, Trp, His, Glu, Gln, Arg, Lys, Met, and Tyr.

7. The polypeptide of claim 3, wherein the preferred side chain chemistry is of sufficient electrostatic charge such that the polypeptide displays reduced effector function.

8. The polypeptide of claim 7, wherein the preferred side chain chemistry is that of an amino acid residue selected from the group consisting of Asp, Glu, Lys, Arg, and His.

9. The polypeptide of claim 1, wherein the reduced effector function is reduced binding to an Fc receptor (FcR).

10. The polypeptide of claim 9, wherein the binding is reduced by a factor selected from the group consisting of about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, about 15-fold or more, about 50-fold or more, and about 100-fold or more.

11. The polypeptide of claim 9, wherein the Fc receptor (FcR) is selected from the group consisting of Fcγ

RI, Fcγ

RII, and Fcγ

RIII.

15. The polypeptide of claim 1, wherein the first amino acid residue and second amino acid residue are near or within an N-linked glycosylation motif.

16. The polypeptide of claim 15, wherein the N-linked glycosylation motif comprises the amino acid sequence NXT or NXS.

17. The polypeptide of claim 15, wherein the N-linked glycosylation motif comprises the amino acid sequence NXT.

18. The polypeptide of claim 1, wherein the first amino acid residue is modified by amino acid substitution.

19. The polypeptide of claim 18, wherein the amino acid substitution is selected from the group consisting of Gly, Ala, Val, Leu, Ile, Phe, Asn, Gln, Trp, Pro, Ser, Thr, Tyr, Cys, Met, Asp, Glu, Lys, Arg, and His.

20. The polypeptide of claim 18, wherein the amino acid substitution is a non-traditional amino acid residue.

21. The polypeptide of claim 1, wherein the first amino acid residue, having a preferred chain chemistry, is capable of being linked to a functional moiety.

22. The polypeptide of claim 21, wherein the functional moiety is selected from the group consisting of a blocking moiety, a detectable moiety, a diagnostic moiety, and a therapeutic moiety.

23. The polypeptide of claim 22, wherein the blocking moiety is selected from the group consisting of a cysteine adduct, mixed disulfide, polyethylene glycol, and polyethylene glycol maleimide.

24. The polypeptide of claim 22, wherein the detectable moiety is selected from the group consisting of a fluorescent moiety and isotopic moiety.

25. The polypeptide of claim 22, wherein the diagnostic agent is capable of revealing the presence of a disease or disorder.

26. The polypeptide of claim 22, wherein the therapeutic moiety is selected from the group consisting of an anti-inflammatory agent, an anticancer agent, an anti-neurodegenerative agent, and an anti-infective agent.

27. The polypeptide of claim 1, wherein the second amino acid residue is amino acid 297 according to the Kabat numbering.

28. The polypeptide of claim 1, wherein the modified first amino acid residue is amino acid 299 according to the Kabat numbering.

29. The polypeptide of claim 18, wherein the amino acid substitution is selected from the group consisting of T299A, T299N, T299G, T299Y, T299C, T299H, T299E, T299D, T299K, T299R, T299G, T2991, T299L, T299M, T299F, T299P, T299W, and T299V according to the Kabat numbering.

30. The polypeptide of claim 29, wherein the amino acid substitution is T299C.

31. The polypeptide of claim 1, wherein the polypeptide is pegylated at the modified first amino acid residue.

32. The polypeptide of claim 31, wherein the polypeptide is pegylated with PEG-maleimide.

33. The polypeptide of claim 1, wherein the modified first amino acid residue is a cysteine residue modified by a cysteine or mixed disulfide adduct.

34. The polypeptide of claim 1, wherein the polypeptide is an antibody.

35. The polypeptide of claim 1, wherein the Fc region is obtained from an antibody selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

36. The polypeptide of claim 1, wherein the polypeptide binds to an antigen selected from the group consisting of a ligand, cytokine, receptor, cell surface antigen, and cancer cell antigen.

37. A composition comprising the polypeptide of claim 1 in a suitable pharmaceutical carrier.

38. A method for treating or preventing a human disorder or disease comprising, administering a therapeutically-effective amount of the pharmaceutical composition of claim 37, such that therapy or prevention of the human disease or disorder is achieved.

39. An isolated nucleic acid encoding the polypeptide of claim 1.

40. A vector comprising the nucleic acid of claim 39.

41. A host cell comprising the nucleic acid of claim 39.

42. A method for producing an antigen binding polypeptide comprising culturing the host cell of claim 41 under conditions suitable for producing the polypeptide by the host cell.

12. The polypeptide of 1, wherein the reduced effector function is reduced binding to a complement protein.

13. The polypeptide of claim 12, wherein the complement protein is C1q.

14. The polypeptide of claim 12, wherein the reduced binding to a complement protein is by a factor selected from the group consisting of about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 6-fold or more, about 7-fold or more, about 8-fold or more, about 9-fold or more, about 10-fold or more, and about 15-fold or more.

43. A method of producing a modified antigen binding polypeptide having reduced glycosylation in an Fc region, the method comprising,

identifying a first amino acid residue in an original polypeptide and a second amino acid residue capable of being glycosylated in an Fc region of the original polypeptide wherein modification of the first amino acid will decrease glycosylation at a second amino acid;

selecting an amino acid for its preferred side chain chemistry, and

modifying said first amino acid residue to comprise said preferred side chain chemistry to produce a modified polypeptide, wherein glycosylation of said second amino acid residue of the Fc region is decreased in the modified polypeptide as compared to the original polypeptide.

44. The method of claim 43, further comprising the step of determining if the modified antigen binding polypeptide displays altered effector function.

45. The method of claim 43, wherein said identifying further comprises determining a spatial representation of the amino acids of the Fc region when said first amino acid is modified.

46. The method of claim 43, wherein one or more steps is computer-assisted.

47. A polypeptide produced by the method of claim 43.