HOMOGENOUS ANTIBODY DRUG CONJUGATES VIA ENZYMATIC METHODS

• US 20180360793A1
• Filed: 05/22/2018
• Published: 12/20/2018
• Est. Priority Date: 06/12/2014
• Status: Active Grant

• Current Assignee: CSPC Megalith Biopharmaceutical Company Limited
• Original Assignee: CSPC Dophen Corporation (CSPC Pharmaceutical Group Limited)
• Inventors: HU, Sean, ALLEN, Lisha
• Granted Patent: US 10,639,291 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 31/255 of sulfoxy acids or sulfur analogues thereof
• CPC Class Codes: A61K 38/16 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof enzyme inhibitors A61K38/005
• CPC Class Codes: A61K 38/164 from bacteria
• CPC Class Codes: A61K 38/45 Transferases (2)
• 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 47/6855 the tumour determinant being from breast cancer cell
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 1/00 General methods for the preparation of peptides , i.e. processes for the organic chemical preparation of peptides or proteins of any length
• CPC Class Codes: C07K 14/195 from bacteria
• 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 16/2863 against receptors for growth factors, growth regulators
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/40 characterized by post-translational modification
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/524 CH2 domain
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: C12N 9/1044 : Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
• CPC Class Codes: C12Y 203/02013 : Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Abstract

The present application in one aspect provide Fc-containing polypeptide conjugates comprising an Fc-containing polypeptide conjugated to a conjugate moiety, wherein the Fc-containing polypeptide comprises an N-glycosylated Fc region comprising an acceptor glutamine residue flanked by an N-glycosylation site and wherein the conjugate moiety is conjugated to the Fc-containing polypeptide via the acceptor glutamine residue. Also provided are methods of making such Fc-containing polypeptide conjugates by using a wildtype or engineered transglutaminases. Further provided are engineered transglutaminases specifically designed for carrying out such reactions.

Claims

1. A method of making an antibody drug conjugate comprising an antibody specifically conjugated to a conjugate moiety comprising:

contacting an antibody composition comprising the antibody with the conjugate moiety in the presence of a transglutaminase under a condition sufficient to generate the antibody drug conjugate, wherein at least about 50% of the antibody in the antibody composition is glycosylated in the Fc-region, and wherein the conjugate moiety is conjugated to an endogenous acceptor glutamine residue on the antibody.

2. The method of claim 1, wherein the transglutaminase is a wildtype transglutaminase.

3. The method of claim 1, wherein the transglutaminase is derived from Streptomyces Ladakanum or Streptomyces mobaraensis.

4. The method of claim 1, wherein the transglutaminase comprises an amino acid sequence having at least about 80% identity to SEQ ID NO:

16.

5. The method of claim 1, wherein the transglutaminase is an engineered transglutaminase.

6. The method of claim 5, wherein the engineered transglutaminase comprises an amino acid sequence having at least about 80% identity to SEQ ID NO:

16, and wherein the engineered transglutaminase comprises one or more mutations selected from the group consisting of;

deletion of D1-E4;

deletion of P244-P247;

deletion of N282-L285;

substitution of H279-A287 with a G; and

substitution of A280-H289 with a G.

7. The method of claim 5, wherein the engineered transglutaminase comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:

17, and wherein the engineered transglutaminase comprises one or more mutations selected from the group consisting of;

deletion of P1-E5;

deletion of P245-P248;

deletion of N283-L286;

substitution of H280-A288 with a G; and

substitution of A281-H290 with a G.

8. The method of claim 1, wherein the transglutaminase has a purity of at least about 90%.

9. The method of claim 1, wherein the molar ratio of the transglutaminase and the antibody composition is about 10:

1 to about 1;

10.

10. The method of claim 1, wherein the transglutaminase is immobilized on a solid support.

11. The method of claim 1, wherein the antibody is immobilized on a solid support.

12. The method of claim 1, where the antibody is a human or humanized antibody.

13. The method of claim 1, wherein both heavy chains of the antibody are conjugated to the conjugate moiety.

14. The method of claim 1, wherein the conjugate moiety comprises an active moiety selected from the group consisting of a moiety that improves the pharmacokinetic property of the antibody composition, a therapeutic moiety, and a diagnostic moiety.

15. The method of claim 1, wherein the conjugate moiety comprises a toxin.

16. The method of claim 1, wherein the antibody is N-glycosylated in the Fc-region.

17. The method of claim 1, wherein the concentration of the transglutaminase in the reaction mixture is about 0.05 mg/ml to about 1 mg/ml.

18. The method of claim 1, wherein the concentration ratio between the conjugate moiety and the antibody is from about 2:

1 to about 800;

1.

19. A method of making an antibody drug conjugate comprising an antibody specifically conjugated to a conjugate moiety comprising a small molecule handle and an active moiety comprising:

a) contacting an antibody composition comprising the antibody with the small molecule handle in the presence of a transglutaminase under a condition sufficient to generate an intermediate conjugate comprising the antibody specifically conjugated to the small molecule handle, and

b) contacting the intermediate conjugate with the active moiety thereby obtaining the antibody drug conjugate, wherein at least about 50% of the antibody in the antibody composition is glycosylated in the Fc-region, and wherein the conjugate moiety is conjugated to an endogenous acceptor glutamine residue on the antibody.

20. The method of claim 19, wherein the small molecule handle has the structure of NH₂—

R, wherein R is a moiety that allows the attachment of the activate moiety.