# US 12,110,338 B2 · Site-specific antibody-drug conjugation through glycoengineering

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- Number: US 12,110,338 B2
- Document ID: US12110338B2
- Status: Granted
- Kind: B2
- Application number: 17/404,412
- Filed: 2021-08-17
- Published: 2022-03-24
- Granted: 2024-10-08
- Earliest claimed: 2013-03-11
- Assignees: Genzyme Corporation
- Applicants: Genzyme Corporation
- Inventors: Clark Pan; Qun Zhou; James Stefano; Pradeep Dhal; Bo Chen; Diego Gianolio; Robert Miller; Huawei Qiu
- Web page: https://patentgrep.com/patent/US12110338B2

## CPC

- C07K 16/2893: {against CD52}
- A61K 47/549: {Sugars, nucleosides, nucleotides or nucleic acids}
- A61K 47/6803: {Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates}
- A61K 47/68031: {the drug being an auristatin}
- A61K 47/6869: {the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate}
- 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}
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 16/2809: {against the T-cell receptor (TcR)-CD3 complex}
- C07K 16/2851: {against the lectin superfamily, e.g. CD23, CD72}
- C07K 16/32: against translation products of oncogenes
- C07K 16/40: against enzymes
- C07K 2317/40: characterized by post-translational modification
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/522: CH1 domain
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/71: Decreased effector function due to an Fc-modification
- C07K 2317/732: Antibody-dependent cellular cytotoxicity [ADCC]
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- C07K 2319/00: Fusion polypeptide
- 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)
- 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

## Abstract

The current disclosure provides binding polypeptides (e.g., antibodies), and effector moiety conjugates thereof (e.g., antibody-drug conjugates or ADCs), comprising a site-specifically engineered drug-glycan linkage within native or engineered glycans of the binding polypeptide. The current disclosure also provides nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. Methods of using the antigen-binding polypeptides disclosed herein to treat disease are also provided.

## Claims

### Claim 1

A method of treating a HER2+ cancer in a subject, the method comprising administering to the subject an effective amount of a binding polypeptide comprising:
at least one modified glycan comprising at least one moiety of Formula (IV):
-Gal-Sia-C(H)=N-Q-CON-X Formula (IV),
wherein:
A) Q is NH or O;
B) CON is a connector moiety;
C) X is a therapeutic effector moiety;
D) Gal is a galactose moiety; and
E) Sia is a sialic acid moiety
wherein the subject has a HER2+ tumor, and
wherein the binding polypeptide is an anti-HER2 antibody.

### Claim 2 (depends on 1)

The method of claim 1, wherein the binding polypeptide comprises a human Fc domain.

### Claim 3 (depends on 1)

The method of claim 1, wherein the modified glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 297 of the Fc domain, according to EU numbering.

### Claim 4 (depends on 1)

The method of claim 1, wherein the modified glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 298 of the Fc domain, according to EU numbering.

### Claim 5 (depends on 1)

The method of claim 1, wherein the ratio of therapeutic effector moiety to binding polypeptide is less than 4.

### Claim 6 (depends on 1)

The method of claim 1, wherein the ratio of therapeutic effector moiety to binding polypeptide is about 2.

### Claim 7 (depends on 1)

The method of claim 1, wherein the therapeutic effector moiety comprises Monomethyl Auristatin E (MMAE) or Dolastatin 10 (Dol10).

### Claim 8 (depends on 7)

The method of claim 7, wherein the therapeutic effector moiety comprises PEG8-Dol10.

### Claim 9

A method of treating a HER2+ cancer in a subject, the method comprising administering to the subject an effective amount of a binding polypeptide comprising:
at least one modified glycan comprising at least one moiety of Formula (IV):
-Gal-Sia-C(H)═N-Q-CON-X Formula (IV),
wherein:
A) Q is NH or O;
B) CON is a connector moiety;
C) X comprises MMAE;
D) Gal is a galactose moiety; and
E) Sia is a sialic acid moiety
wherein the subject has a HER2+ tumor, and
wherein the binding polypeptide is an anti-HER2 antibody.

### Claim 10 (depends on 9)

The method of claim 9, wherein CON comprises MC-VC-PABC, wherein MC is a component derived from maleimide, VC is a component derived from valine coupled with citruline, and PABC is a component derived from 4-aminobenzyl carbamate.

### Claim 11

A method of treating a HER2+ cancer in a subject, the method comprising administering to the subject an effective amount of a binding polypeptide comprising:
at least one modified glycan comprising at least one moiety of Formula (IV):
-Gal-Sia-C(H)═N-Q-CON-X Formula (IV),
wherein:
A) Q is NH or O;
B) CON is a connector moiety;
C) X comprises Dol10;
D) Gal is a galactose moiety; and
E) Sia is a sialic acid moiety
wherein the subject has a HER2+ tumor, and
wherein the binding polypeptide is an anti-HER2 antibody.

### Claim 12 (depends on 11)

The method of claim 11, wherein CON comprises MC-VC-PABC, wherein MC is a component derived from maleimide, VC is a component derived from valine coupled with citruline, and PABC is a component derived from 4-aminobenzyl carbamate.

### Claim 13 (depends on 11)

The method of claim 11, wherein the therapeutic effector moiety comprises PEG8-Dol10.

### Claim 14 (depends on 1)

The method of claim 1, wherein the modified glycan is a biantennary glycan.

### Claim 15 (depends on 1)

The method of claim 1, wherein the biantennary glycan is fucosylated.

### Claim 16 (depends on 1)

The method of claim 1, wherein said modified glycan comprises at least two moieties of Formula (IV), and wherein Sia is present in either the connector moiety or the therapeutic effector moiety of Formula (IV).

### Claim 17 (depends on 15)

The method of claim 15, wherein said modified glycan comprises at least two moieties of Formula (IV), and wherein Sia is present in both the connector moiety and the therapeutic effector moiety of Formula (IV).

### Claim 18 (depends on 1)

The method of claim 1, wherein the modified glycan is N-linked to the binding polypeptide.

### Claim 19 (depends on 1)

The method of claim 1, wherein the modified glycan is N-linked to the binding polypeptide.

### Claim 20 (depends on 1)

The method of claim 1, wherein the connector moiety comprises a pH-sensitive linker, disulfide linker, enzyme-sensitive linker or other cleavable linker moiety.

### Claim 21 (depends on 1)

The method of claim 1, wherein the effector moiety is a cytotoxin.

### Claim 22 (depends on 21)

The method of claim 21, wherein the cytotoxin is selected from the group consisting of the cytotoxins listed in the following tables:
or

### Claim 23 (depends on 1)

The method of claim 1, where the HER2 antibody has a heavy chain sequence selected from SEQ ID Nos 12-16.

### Claim 24 (depends on 9)

The method of claim 9, wherein the effector moiety is a cytotoxin.

### Claim 25 (depends on 24)

The method of claim 24, wherein the cytotoxin is selected from the group consisting of the cytotoxins listed in the following tables:
and

### Claim 26 (depends on 9)

The method of claim 9, where the HER2 antibody has a heavy chain sequence selected from SEQ ID Nos 12-16.

### Claim 27 (depends on 11)

The method of claim 11, wherein the effector moiety is a cytotoxin.

### Claim 28 (depends on 27)

The method of claim 27, wherein the cytotoxin is selected from the group consisting of the cytotoxins listed in the following tables:
and

### Claim 29 (depends on 11)

The method of claim 11, where the HER2 antibody has a heavy chain sequence selected from SEQ ID Nos 12-16.