# US 10,639,291 B2 · Homogenous antibody drug conjugates via enzymatic methods

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- Number: US 10,639,291 B2
- Document ID: US10639291B2
- Status: Granted
- Kind: B2
- Application number: 15/986,665
- Filed: 2018-05-22
- Published: 2018-12-20
- Granted: 2020-05-05
- Earliest claimed: 2014-06-12
- Assignees: CSPC Dophen Corporation
- Applicants: CSPC Dophen Corporation
- Inventors: Sean Hu; Lisha Allen
- Web page: https://patentgrep.com/patent/US10639291B2

## CPC

- A61K 31/255: of sulfoxy acids or sulfur analogues thereof
- A61K 38/16: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof {(enzyme inhibitors A61K38/005)}
- A61K 38/164: {from bacteria}
- A61K 38/45: Transferases (2)
- A61K 47/6803: {Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates}
- A61K 47/68031: {the drug being an auristatin}
- A61K 47/68033: {the drug being a maytansine}
- A61K 47/6855: {the tumour determinant being from breast cancer cell}
- A61P 35/00: Antineoplastic agents
- 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}
- C07K 14/195: from bacteria
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C07K 16/2863: {against receptors for growth factors, growth regulators}
- C07K 16/30: from tumour cells
- C07K 16/32: against translation products of oncogenes
- C12N 9/1044: {Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII}
- C12Y 203/02013: Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/40: characterized by post-translational modification
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/524: CH2 domain
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## 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

### Claim 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, wherein the conjugate moiety is conjugated to an endogenous acceptor glutamine residue on the antibody, and wherein the transglutaminase is a wildtype transglutaminase and the concentration of the transglutaminase in the reaction mixture is about 0.1 mg/ml to about 1 mg/ml.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

The method of claim 1, wherein the transglutaminase consists of the amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 18.

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 1)

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

### Claim 10 (depends on 1)

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.

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 1)

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

### Claim 13 (depends on 1)

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.

### Claim 14

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, wherein the transglutaminase is a wildtype transglutaminase and the concentration of the transglutaminase in the reaction mixture is about 0.1 mg/ml to about 1 mg/ml, 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, wherein the conjugate moiety is conjugated to an endogenous acceptor glutamine residue on the antibody, and wherein the active moiety is selected from the group consisting of a moiety that improves the pharmacokinetic property of the antibody composition, a therapeutic moiety, and a diagnostic moiety.

### Claim 15 (depends on 14)

The method of claim 14, wherein the small molecule handle has the structure of NH2—R, wherein R is a moiety that allows the attachment of the active moiety.

### Claim 16

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, wherein the conjugate moiety is conjugated to an endogenous acceptor glutamine residue on the antibody, and wherein the transglutaminase is a wildtype transglutaminase with an added proline residue at the N-terminus, and optionally a purification tag, and the concentration of the transglutaminase in the reaction mixture is about 0.1 mg/ml to about 1 mg/ml.