Modified Fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use

• US 9,732,161 B2
• Filed: 06/26/2013
• Issued: 08/15/2017
• Est. Priority Date: 06/26/2012
• Status: Active Grant

• Current Assignee: Sutro Biopharma, Inc.
• Original Assignee: Sutro Biopharma, Inc.
• Inventors: McEvoy, Leslie, Yin, Gang, Penta, Kalyani, Baliga, Ramesh, Bajad, Sunil, Pollitt, Sonia, Murray, Chris, Steiner, Alex, Gill, Avinash, Thanos, Christopher D.
• Primary Examiner(s): HUYNH, PHUONG N
• Application Number:
  US13/928,182
• Publication Number:
  US 20140051836A1
• Time in Patent Office: 1,511 Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• 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: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• CPC Class Codes: C07K 16/2878 against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 16/46 Hybrid immunoglobulins hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype

Abstract

Provided herein are modified Fc proteins comprising non-natural amino acid residues at site-specific positions, conjugates of the modified Fc proteins for therapy or diagnosis, compositions comprising the modified Fc proteins and conjugates thereof, methods of their production and methods of their use. The modified Fc proteins and conjugates are useful for methods of treatment and prevention, methods of detection and methods of diagnosis.

Claims

1. An antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having a non-natural amino acid residue at heavy chain residue 404 according to the EU index of Kabat, or a post-translationally modified variant or an aglycosylated variant thereof,

wherein the non-natural amino acid residue is selected from the group consisting of:

ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine.

5. The antibody fragment of claim 1, wherein the Fc protein is aglycosylated.

6. The antibody fragment of claim 5, wherein the Fc protein has a higher thermal stability (T_m1) compared to the corresponding wild-type Fc protein.

7. A composition comprising the antibody fragment of claim 5, wherein said composition is substantially pure.

8. A composition comprising the antibody fragment of claim 5 wherein said antibody fragment is at least 95% by mass of the total protein mass of said composition.

9. The antibody fragment of claim 5, wherein the Fc protein does not comprise a variable domain or a light chain.

10. The antibody fragment of claim 5, wherein the Fc protein corresponds to a subclass selected from the group consisting of Ig1, IgG2, IgG3, and IgG4.

11. The antibody fragment of claim 5, wherein said non-natural amino acid residue comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl.

12. The antibody fragment of claim 5, wherein each non-natural amino acid residue is according to the formula

13. The antibody fragment of claim 5, wherein the non-natural amino acid residue is selected from the group consisting of:

p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p-azidomethyl-L-phenylalanine.

14. The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azido-L-phenylalanine.

15. The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azidomethyl-L-phenylalanine.

16. An Fc protein conjugate comprising the antibody fragment of claim 5, wherein the Fc protein is linked to one or more therapeutic moieties or labeling moieties.

17. The Fc protein conjugate of claim 16, wherein the Fc protein is linked to one or more drugs or polymers.

18. The Fc protein conjugate of claim 16, wherein the Fc protein is linked to one or more labeling moieties.

19. The Fc protein conjugate of claim 16, wherein said Fc protein is linked to one or more single chain binding domains (scFv).

20. The Fc protein conjugate of claim 16, wherein said one or more therapeutic moieties or labeling moieties is linked to the non-natural amino acid, or a residue thereof, via one or more linkers.

2. An antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having a non-natural amino acid at heavy chain residue 404 according to the EU index of Kabat, or a post-translationally modified variant or an aglycosylated variant thereof, wherein each non-natural amino acid residue is according to the formula

3. The antibody fragment of claim 2 wherein each R is a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl.

4. The antibody fragment of claim 2 wherein each L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene and substituted heteroarylene.