# US 10,471,037 B2 · Homogenous antibody drug conjugates via enzymatic methods

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- Number: US 10,471,037 B2
- Document ID: US10471037B2
- Status: Granted
- Kind: B2
- Application number: 15/986,668
- Filed: 2018-05-22
- Published: 2018-12-20
- Granted: 2019-11-12
- Earliest claimed: 2014-06-12
- Assignees: CSPC Dophen Corporation
- Applicants: CSPC Dophen Corporation
- Inventors: Sean Hu; Lisha Allen
- Web page: https://patentgrep.com/patent/US10471037B2

## 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

An engineered transglutaminase comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:16, 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.

### Claim 2

An engineered transglutaminase comprising an amino acid sequence having at least about 80% identity to SEQ ID NO:17, 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.

### Claim 3 (depends on 2)

The engineered transglutaminase of claim 2, wherein the engineered transglutaminase comprises a deletion of P1-E5.

### Claim 4 (depends on 2)

The engineered transglutaminase of claim 2, wherein the engineered transglutaminase comprises a deletion of P245-P248.

### Claim 5 (depends on 2)

The engineered transglutaminase of claim 2, wherein the engineered transglutaminase comprises a deletion of N283-L286.

### Claim 6 (depends on 2)

The engineered transglutaminase of claim 2, wherein the engineered transglutaminase comprises a deletion of P1-E5 and a deletion of N283-L286.

### Claim 7 (depends on 2)

The engineered transglutaminase of claim 2, wherein the engineered transglutaminase comprises a deletion of P1-E5, a deletion of P245-P248, and a deletion of N283-L286.

### Claim 8 (depends on 2)

The engineered transglutaminase of claim 2, wherein the engineered transglutaminase comprises a deletion of P1-E5 and a substitution of H280-A288 with a G.

### Claim 9 (depends on 2)

The engineered transglutaminase of claim 2, wherein the engineered transglutaminase comprises a deletion of P1-E5 and a substitution of A281-H290 with a G.

### Claim 10 (depends on 2)

The engineered transglutaminase of claim 2, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 37-43.

### Claim 11 (depends on 2)

The engineered transglutaminase of claim 2, wherein the engineered transglutaminase is immobilized on a solid support.

### Claim 12 (depends on 2)

An isolated nucleic acid encoding the engineered transglutaminase of claim 2.

### Claim 13 (depends on 12)

A recombinant vector comprising the isolated nucleic acid of claim 12.

### Claim 14 (depends on 13)

A host cell comprising the recombinant vector of claim 13.

### Claim 15 (depends on 2)

A method of preparing the engineered transglutaminase of claim 2, comprising culturing a host cell comprising a vector comprising a nucleic acid encoding the engineered transglutaminase and isolating the engineered transglutaminase from the host cell.