METHOD FOR MAKING ANTIBODY FC-REGION CONJUGATES COMPRISING AT LEAST ONE BINDING ENTITY THAT SPECIFICALLY BINDS TO A TARGET AND USES THEREOF

• US 20150232541A1
• Filed: 12/22/2014
• Published: 08/20/2015
• Est. Priority Date: 06/27/2012
• Status: Active Grant

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: KOPETZKI, ERHARD, FENN, SEBASTIAN
• Granted Patent: US 11,421,022 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 47/6889 Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• 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 16/18 against material from animals or humans
• 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 2317/55 Fab or Fab'
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/90 containing a motif for post-translational modification
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 9/52 derived from bacteria or Archaea
• CPC Class Codes: C12Y 304/2207 Sortase A (3.4.22.70)

Abstract

Herein is reported a method for producing an antibody Fc-region conjugate comprising as first component an antibody Fc-region and as second component at least one binding entity that specifically binds to a target using a transpeptidase for enzymatic conjugation of the antibody Fc-region to at least one binding entity.

Claims

1. A method for producing an antibody Fc-region conjugate, which comprises as a first component an antibody Fc-region and as a second component at least one binding entity that specifically binds to a target, wherein the method co rises conjugating the antibody Fc region to the binding entity b using a sortase A.

2. The method according to claim 1, comprising:

incubating

i) the binding entity comprising the amino acid sequence G_nSLPX1TG (SEQ ID NO;

02, n=1, 2, or 3, wherein X1 is any amino acid residue) within its 20 C-terminal amino acid residues, and

ii) the antibody Fc-region, which comprises at one of its N-termini an oligoglycine G_m (m=2, 3, 4, or

     5) with

iii) the enzyme Sortase A,

and thereby producing the antibody Fc-region conjugate.

3. The method of claim 1, wherein the binding entity comprises the amino acid sequence G_nSLPX1TGGSGS (SEQ ID NO:

03, n=1, 2 or 3 and wherein X1 is any amino acid residue) within its 20 C-terminal amino acid residues.

4. The method of claim 1, wherein the binding entity comprises the amino acid sequence X2GSLPX1TGGSGS (SEQ ID NO:

04, wherein X1 is any amino acid residue, and X2 is any amino acid residue except glycine) within its 20 C-terminal amino acid residues.

5. The method of claim 1, wherein the binding entity comprises the amino acid sequence G_nSLPX1TGGSGSX3 (SEQ ID NO:

05, n=1, 2 or 3 and wherein X1 is any amino acid residue, and X3 is an amino acid sequence tag) within its 20 C-terminal amino acid residues.

6. The method of claim 1, wherein the binding entity is selected from the group consisting Fv, Fab, Fab′

, Fab′

-SH, F(ab′

)₂, diabody, linear antibody, scFv, scFab, and dsFv.

7. The method of claim 1, wherein the antibody Fc-region comprises at one of its N-termini two glycine residues.

8. The method of claim 1, wherein the antibody Fc-region comprises at one of its N-termini the amino acid sequence GGCPX4C (SEQ ID NO:

07), wherein X4 is either S or P.

9. The method of claim 1, wherein the antibody Fc-region comprises at both of its N-termini the amino acid sequence GGCPX4C (SEQ ID NO:

07), wherein X4 is either S or P.

10. An antibody Fc-region conjugate obtained by the method of claim 1.

15. The method of claim 1 comprising

(i) culturing a first host cell comprising a nucleic acid encoding the antibody Fc-region component of the conjugate under conditions suitable for expression of the antibody Fc-region;

(ii) culturing a second host cell comprising a nucleic acid encoding the binding entity component of the conjugate under conditions suitable for expression of the binding entity; and

(iii) conjugating the antibody Fc-region component and the binding entity component enzymatically using Sortase A mediated transpeptidation.

11. An antibody Fc-region conjugate comprising the amino acid sequence G_nSLPX1TGG (SEQ ID NO:

09, n=1, 2 or 3 and wherein X1 is any amino acid residue).

12. A pharmaceutical formulation comprising the antibody Fc-region conjugate according to claim 11 and a pharmaceutically acceptable carrier.

22. A method of treatment comprising administering the antibody Fc-region conjugate of claim 11 to a patient to treat a chronic disease or disorder.

23. The antibody Fc-region conjugate of claim 11, wherein X1 is E.

24. The antibody Fc-region conjugate of claim 11 comprising two polypeptide chains wherein each is an antibody heavy chain that comprises the amino acid sequence GnSLPX1TGGHTCPX4C (SEQ ID NO:

12, n=1, 2 or

     3), wherein the two polypeptide chains are covalently linked by at least one disulfide bond, wherein X1 is any amino acid residue, and wherein X4 is either S or P.

13-14. -14. (canceled)

16. A binding entity that specifically binds to a target, wherein the binding entity comprises the amino acid sequence G_nSLPX1TG (SEQ ID NO:

02, n=1, 2, or 3, wherein X1 is any amino acid residue) within its 20 C-terminal amino acid residues.

18. Nucleic acid encoding the binding entity of claim 16.

20. A host cell comprising the nucleic acid of claim 18.

17. An antibody Fc-region, which comprises at one of its N-termini an oligoglycine G_m (m=2, 3, 4, or 5).

19. Nucleic acid encoding the antibody Fc-region of claim 17.

21. A host comprising the nucleic acid of claim 19.