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

• US 9,764,039 B2
• Filed: 07/10/2014
• Issued: 09/19/2017
• Est. Priority Date: 07/10/2013
• Status: Active Grant

• Current Assignee: Sutro Biopharma, Inc.
• Original Assignee: Sutro Biopharma, Inc.
• Inventors: Thanos, Christopher D., Baliga, Ramesh, Penta, Kalyani, Gill, Avinash, Yin, Gang, Zimmerman, Erik
• Primary Examiner(s): HUYNH, PHUONG N
• Application Number:
  US14/328,532
• Publication Number:
  US 20150017187A1
• Time in Patent Office: 1,167 Days
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 47/6803 Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
• CPC Class Codes: A61K 47/68031 the drug being an auristatin
• CPC Class Codes: A61K 47/68033 the drug being a maytansine
• CPC Class Codes: A61K 47/68035 the drug being a pyrrolobenzodiazepine
• CPC Class Codes: A61K 47/68037 the drug being a camptothecin [CPT] or derivatives
• CPC Class Codes: A61K 47/6813 the drug being a peptidic cytokine, e.g. an interleukin or interferon
• CPC Class Codes: A61K 47/6851 the antibody targeting a determinant of a tumour cell
• CPC Class Codes: A61K 47/6855 the tumour determinant being from breast cancer cell
• CPC Class Codes: A61K 47/6869 the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• 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 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/51 Complete heavy chain or Fd fragment, i.e. VH + CH1
• CPC Class Codes: C07K 2317/515 Complete light chain, i.e. VL + CL
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/56 variable (Fv) region, i.e. VH and/or VL
• 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 2317/94 Stability, e.g. half-life, pH, temperature or enzyme-resistance
• CPC Class Codes: C12N 15/09 : Recombinant DNA-technology

Abstract

Provided herein are antibodies comprising multiple 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 antibody of the IgG class comprising six or fewer site-specific non-natural amino acid residues, wherein at least one of said site-specific non-natural amino acid residues comprises an azide moiety and at least one of said site-specific non-natural amino acid residues comprises a tetrazine moiety, wherein at least one of the said site-specific non-natural amino acid residues comprising the azide moiety or the tetrazine moiety is at heavy chain site 404 according to the EU index of Kabat.

2. The antibody of claim 1, wherein the six or fewer site-specific non-natural amino acid residues are in a single heavy chain or heavy chain variable domain.

3. The antibody of claim 1, wherein at least one site-specific non-natural amino acid is in a light chain or light chain variable domain.

4. The antibody of claim 1, wherein at least one site-specific non-natural amino acid is in a light chain or light chain variable domain and a site-specific non-natural amino acid at site 404 is in each of two heavy chains.

5. The antibody of claim 1, wherein at least one site-specific non-natural amino acid is in each of two light chains or light chain variable domains and a site-specific non-natural amino acid at site 404 is in a heavy chain.

6. The antibody of claim 1, wherein at least one site-specific non-natural amino acid is in each of two light chain or light chain variable domains.

7. The antibody of claim 1, wherein said site-specific non-natural amino acid residues are not one of the 20 common amino acids or pyrrolysine or selenocysteine, or post-translationally modified variants thereof.

8. The antibody of claim 1 comprising two to six site-specific non-natural amino acid residues, wherein said site-specific non-natural amino acid residues are not one of the 20 common amino acids or pyrrolysine or selenocysteine, or post-translationally modified variants thereof.

9. The antibody of claim 1, wherein at least one of said site-specific non-natural amino acid residue is at one or more sequence positions selected from the group consisting of light chain residues 7 and 22 according to the Kabat numbering scheme, light chain residue 152 according to the EU index of Kabat, heavy chain residues 19, 25, 40, 52, 70, and 110 according to the Kabat numbering scheme, and heavy chain residues 119, 121, 136, 180, 190, 221, and 222 according to the EU index of Kabat.

10. The antibody of claim 1, wherein the antibody comprises a heavy chain according to SEQ ID NO:

1.

11. The antibody of claim 1, wherein the antibody comprises a light chain according to SEQ ID NO:

2.

12. The antibody of claim 1, comprising a light chain of a type selected from λ

, and κ

.

13. The antibody of claim 1, that is of a class or subclass selected from the group consisting of IgG1, IgG2, IgG3 and IgG4.

14. The antibody of claim 9, wherein each further site-specific non-natural amino acid residue comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, and alkynyl, wherein said site-specific non-natural amino acid residue is not one of the 20 amino acids or pyrrolysine or selenocysteine, or post-translationally modified variant thereof.

15. The antibody of claim 1, wherein said antibody is a full-length antibody.

16. The antibody of claim 1, wherein each site-specific non-natural amino acid residue is according to the formula

17. The antibody of claim 16 wherein each further non-natural amino acid residue comprises an R that comprises a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido, alkynyl, and tetrazine.

18. The antibody claim 16 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.

19. An antibody conjugate comprising the antibody of claim 1 linked to one or more therapeutic moieties or labeling moieties.

20. The antibody conjugate of claim 19 wherein said antibody is linked to one or more drugs or polymers.

21. The antibody conjugate of claim 19 wherein said antibody is linked to one or more labeling moieties.

22. The antibody conjugate of claim 19 wherein said antibody is linked to one or more single-chain binding domains (scFv).

23. The antibody conjugate of claim 19 wherein at least one of said therapeutic moieties or labeling moieties is linked to said antibody via a site-specific non-natural amino acid comprising an azide moiety, or a residue thereof.

24. The antibody conjugate of claim 19 wherein at least one of said therapeutic moieties or labeling moieties is linked to said antibody via a site-specific non-natural amino acid comprising a tetrazine moiety, or a residue thereof.

25. The antibody conjugate of claim 19 wherein at least one of said therapeutic moieties or labeling moieties is linked to said antibody via a site-specific non-natural amino acid comprising an azide moiety, or residue thereof, and at least one of said therapeutic moieties or labeling moieties is linked to said antibody via a site-specific non-natural amino acid comprising a tetrazine moiety, or residue thereof.

26. The antibody conjugate of claim 25 wherein a first therapeutic moiety is linked to said antibody via a site-specific non-natural amino acid comprising an azide moiety, or residue thereof, and a second therapeutic moiety is linked to said antibody via a site-specific non-natural amino acid comprising a tetrazine moiety, or a residue thereof.

27. The antibody conjugate of claim 25 wherein a first labeling moiety is linked to said antibody via a site-specific non-natural amino acid comprising an azide moiety and a second labeling moiety is linked to said antibody via a site-specific non-natural amino acid comprising a tetrazine moiety, or a residue thereof.

28. The antibody conjugate of claim 25 wherein a therapeutic moiety is linked to said antibody via a site-specific non-natural amino acid comprising an azide moiety and a labeling moiety is linked to said antibody via a site-specific non-natural amino acid comprising a tetrazine moiety, or a residue thereof.

29. The antibody conjugate of claim 25 wherein a labeling moiety is linked to said antibody via a site-specific non-natural amino acid comprising an azide moiety, or a residue thereof, and a therapeutic moiety is linked to said antibody via a site-specific non-natural amino acid comprising a tetrazine moiety, or a residue thereof.

30. The antibody conjugate of claim 19, wherein said antibody is linked to said one or more therapeutic moieties or labeling moieties via one or more linkers.

31. The antibody conjugate of claim 19, wherein said antibody conjugate has a melting temperature within about five degrees Celsius of the antibody without the site-specific non-natural amino acids.

32. The antibody conjugate of claim 19, wherein said antibody conjugate has a melting temperature that is at least about three degrees Celsius greater than that of the antibody without the site-specific non-natural amino acids.

33. The antibody of claim 1, wherein the six or fewer site-specific non-natural amino acid residues are independently selected from the group consisting of:

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

34. The antibody of claim 9, wherein the further site-specific non-natural amino acid residue or residues are selected from the group consisting of:

p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 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, phosphonotyrosine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p-azidomethyl-L-phenylalanine.

35. The antibody of claim 1, wherein at least one of the two or more site-specific non-natural amino acid residues is p-azido-L-phenylalanine.

36. The antibody of claim 1, wherein at least one of the two or more site-specific non-natural amino acid residues is p-azidomethyl-L-phenylalanine.