Antibodies comprising site-specific non-natural amino acid residues, methods of their preparation and methods of their use

• US 9,738,724 B2
• Filed: 06/07/2013
• Issued: 08/22/2017
• Est. Priority Date: 06/08/2012
• Status: Active Grant

• Current Assignee: Sutro Biopharma, Inc.
• Original Assignee: Sutro Biopharma, Inc.
• Inventors: Thanos, Christopher D., McEvoy, Leslie, Yin, Gang, Penta, Kalyani, Baliga, Ramesh, Bajad, Sunil, Pollitt, Sonia, Murray, Chris, Steiner, Alex, Gill, Avinash
• Primary Examiner(s): HUYNH, PHUONG N
• Application Number:
  US13/913,345
• Publication Number:
  US 20140046030A1
• Time in Patent Office: 1,537 Days
• CPC Class Codes: A61K 47/6817 Toxins
• CPC Class Codes: A61K 47/6855 the tumour determinant being from breast cancer cell
• 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 16/30 from tumour cells
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2317/73 Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C07K 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance

Abstract

Provided herein are antibodies comprising non-natural amino acid residues at site-specific positions, compositions comprising the antibodies, methods of their production and methods of their use. The antibodies are useful for methods of treatment and prevention, methods of detection and methods of diagnosis.

Claims

1. An isolated antibody of the IgG class comprising a heavy chain and a light chain wherein:

a. the light chain has one or more non-natural amino acid residues at specific sites selected from the group consisting of light chain residues 7 and 22 according to the Kabat numbering scheme; and

/or

b. the heavy chain has one or more non-natural amino acid residues at specific sites selected form the group consisting of heavy chain residues 19, 25, 40, and 70 according to the Kabat numbering scheme and heavy chain residues 121, 180, and 190 according to the EU index of Kabat;

or an aglycosylated variant thereof,

wherein each non-natural amino acid residue is independently selected from the group consisting of ortho-substituted tyrosine, meta substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine.

2. The antibody of claim 1 wherein the antibody is aglycosylated.

20. The antibody of claim 2 wherein the one or more non-natural amino acid residues are at specific sites selected from the group consisting of heavy chain residues 25 and 40 according to the Kabat numbering scheme, heavy chain residues 121, 180, and 190 according to the EU index of Kabat, and light chain residues 7 and 22, according to the Kabat numbering scheme.

21. The antibody of claim 2 wherein the one or more non-natural amino acid residues are at specific sites selected from the group consisting of light chain residues 7 and 22 according to the Kabat numbering scheme and heavy chain residues 121 and 180, according to the EU index of Kabat.

22. The antibody of claim 2, wherein the light chain is selected from λ

and κ

.

23. The antibody of claim 2, wherein the heavy chain is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

24. The antibody of claim 2, wherein said one or more non-natural amino acid residue is substituted with a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, and alkynyl.

25. The antibody of claim 2, wherein each of the one or more non-natural amino acid residues is according to the formula

26. The antibody of claim 2, wherein the one or more non-natural amino acid residues are 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-A 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.

27. The antibody of claim 2, wherein at least one non-natural amino acid residue is p-azido-L-phenylalanine.

28. The antibody of claim 2, wherein at least one non-natural amino acid residue is p-azidomethyl-L-phenylalanine.

29. An antibody conjugate comprising the antibody of claim 2 linked to one or more therapeutic moieties or labeling moieties, wherein at least one therapeutic moiety or labeling moiety is linked to one of the non-natural amino acids, or a residue thereof.

30. The antibody conjugate of claim 29, wherein said antibody is linked to one or more drugs or polymers.

31. The antibody conjugate of claim 29, wherein said antibody is linked to one or more labeling moieties.

32. The antibody conjugate of claim 29, wherein said antibody is linked to one or more single chain binding domains (scFv).

33. The antibody conjugate of claim 29, wherein said at least one therapeutic or labeling moiety is linked to one of the non-natural amino acids, or a residue thereof, via one or more linkers.

34. A composition comprising the antibody of claim 2, wherein said composition is substantially pure.

35. A composition comprising the antibody of claim 2 wherein said antibody is at least 95% by mass of the total antibody mass of said composition.

3. An isolated antibody of the IgG class comprising a heavy chain and a light chain wherein

the heavy chain has a non-natural amino acid residue at site 404 according to the EU index of Kabat, or an aglycosylated variant thereof,

wherein the non-natural amino acid residue is independently selected from the group consisting of ortho-substituted tyrosine, meta substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine.

4. The antibody of claim 3, wherein the antibody is aglycosylated.

5. The antibody of claim 4, wherein the antibody has a higher thermal stability (T_m1) compared to the corresponding wild-type antibody following conjugation of a linker-drug to the non-natural amino acid.

6. The antibody of claim 4, wherein the light chain is selected from λ

and κ

.

7. The antibody of claim 4, wherein the heavy chain is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

8. The antibody of claim 4, wherein the non-natural amino acid residue is substituted with a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, and alkynyl.

9. The antibody of claim 4, wherein the non-natural amino acid residues is according to the formula

10. The antibody of claim 4, wherein the non-natural amino acid residue is A 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-A benzoyl-L-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p-azidomethyl-L-phenyl alanine.

11. The antibody of claim 4, wherein the non-natural amino acid residue is p-azido-L-phenylalanine.

12. The antibody of claim 4, wherein the non-natural amino acid residue is p-azidomethyl-L-phenyl alanine.

13. An antibody conjugate comprising the antibody of claim 4 linked to one or more therapeutic moieties or labeling moieties, wherein at least one therapeutic moiety or labeling moiety is linked to the non-natural amino acid, or a residue thereof.

14. The antibody conjugate of claim 13, wherein said antibody is linked to one or more drugs or polymers.

15. The antibody conjugate of claim 13, wherein said antibody is linked to one or more labeling moieties.

16. The antibody conjugate of claim 13, wherein said antibody is linked to one or more single chain binding domains (scFv).

17. The antibody conjugate of claim 13, wherein said at least one therapeutic or labeling moiety is linked to the non-natural amino acid, or a residue thereof, via one or more linkers.

18. A composition comprising the antibody of claim 4, wherein said composition is substantially pure.

19. A composition comprising the antibody of claim 4, wherein said antibody is at least 95% by mass of the total antibody mass of said composition.