Modified Fc molecules

• US 10,188,740 B2
• Filed: 07/10/2015
• Issued: 01/29/2019
• Est. Priority Date: 08/12/2005
• Status: Active Grant

• Current Assignee: Amgen Inc.
• Original Assignee: Amgen Inc.
• Inventors: Gegg, Colin V., Walker, Kenneth W., Miranda, Leslie P., Xiong, Fei
• Primary Examiner(s): Kim, Yunsoo
• Application Number:
  US14/796,502
• Publication Number:
  US 20160000932A1
• Time in Patent Office: 1,299 Days
• Field of Search: None
• 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 comprising:

(i) a monomeric or multimeric Fc domain having a cysteine or non-canonical amino acid substitution at one or more specifically selected conjugation site(s) selected from D46, S48, H49, E50, E53, K55, D61, G62, Q76, Y81, K107, K121, G122, Q123, E126, R136, D137, T140, K141, N142, E169, N170, N171, K173, L179, S181, G183, D194, K195, R197, Q199, Q200, G201, N202, or S223, relative to reference sequence SEQ ID NO;

599; and

(ii) at least one additional functional moiety, wherein the functional moiety is conjugated to the Fc domain through the side chain of the cysteine residue or non-canonical amino acid residue substituted at said one or more 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 additional functional moiety is a labeled moiety comprising a radioisotope, an enzyme, a biotinyl moiety, a fluorophore, or a chromophore.

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

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

10. The composition of matter of claim 9, 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.

11. The composition of matter of claim 1, wherein the functional moiety is conjugated to the Fc domain through the side chain of the cysteine residue substituted at said one or more conjugation site(s).

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

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

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

14. A composition of matter of the formula

15. The composition of matter of claim 14, wherein the Fc domain comprises an IgG Fc domain.

16. The composition of matter of claim 15, wherein the Fc domain comprises an IgG1 Fc domain.

17. The composition of matter of claim 16, wherein the IgG1 Fc domain comprises SEQ ID NO:

600 or SEQ ID NO;

603.

18. The composition of matter of claim 14, wherein a=0 and b=1, or a=1 and b=0, or a=1 and b=1, or a=0 and b=0.

19. The composition of matter of claim 14, wherein X³ comprises polyethylene glycol (PEG).

20. The composition of matter of claim 14, wherein X³ has the structure -(L¹)_c-P¹ or -(L¹)_c-P¹-(L²)_d-P².

21. The composition of matter of claim 14, wherein g=1 or 2.

22. The composition of matter of claim 14, wherein any of X¹, X², or X³ comprises a pharmacologically active polypeptide, peptide, or peptidomimetic.

23. The composition of matter of claim 22, wherein the peptide is a toxin peptide.

24. The composition of matter of claim 22, wherein the peptide comprises a cyclic peptide.

27. A pharmaceutical composition, comprising the composition of matter of claim 14 and a pharmaceutically acceptable carrier.

26. A composition of matter comprising:

(a) an antibody comprising a Fc domain having a cysteine or non-canonical amino acid substitution at one or more specifically selected conjugation site(s) selected from D46, S48, H49, E50, E53, K55, D61, G62, Q76, Y81, K107, K121, G122, Q123, E126, R136, D137, T140, K141, N142, E169, N170, N171, K173, L179, S181, G183, D194, K195, R197, Q199, Q200, G201, N202, or S223, relative to reference sequence SEQ ID NO;

599; and

(b) at least one X³ additional functional moiety, wherein the X³ additional functional moiety is conjugated to the Fc domain of the antibody through the side chain of the cysteine residue or non-canonical amino acid residue substituted at said one or more specifically selected conjugation site(s) in the Fc domain; and

wherein

X³ is selected from -(L¹)_c-P⁰, -(L¹)_c-P¹, -(L¹)_c-P¹-(L²)_d-P², -(L²)_c-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 moiety, 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.

28. The composition of matter of claim 26, wherein the X³ additional functional moiety is conjugated to the Fc domain of the antibody through the side chain of the cysteine residue substituted at said one or more conjugation site(s).

29. The composition of matter of claim 26, wherein X³ comprises a pharmacologically active polypeptide, peptide, or peptidomimetic.

30. The composition of matter of claim 26, wherein the Fc domain is an IgG Fc domain.

31. The composition of matter of claim 30, wherein the Fc domain is an IgG1 Fc domain.

32. The composition of matter of claim 31, wherein the IgG1 Fc domain comprises SEQ ID NO:

600 or SEQ ID NO;

603.

33. A pharmaceutical composition, comprising the composition of matter of claim 26 and a pharmaceutically acceptable carrier.