# US 10,118,966 B2 · Anti-EGFR monoclonal antibody, methods of making, and uses thereof in the treatment of cancer

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- Number: US 10,118,966 B2
- Document ID: US10118966B2
- Status: Granted
- Kind: B2
- Application number: 15/541,291
- Filed: 2016-01-04
- Published: 2018-02-01
- Granted: 2018-11-06
- Earliest claimed: 2015-01-07
- Assignees: Shanghai Biomabs Pharmaceuticals Co., Ltd.
- Applicants: Shanghai Biomabs Pharmaceuticals Co., Ltd.
- Inventors: Weizhu Qian
- Web page: https://patentgrep.com/patent/US10118966B2

## CPC

- C07K 16/2863: {against receptors for growth factors, growth regulators}
- A61K 31/4745: condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines (yohimbine derivatives, vinblastine A61K31/475; ergoline derivatives A61K31/48)
- A61K 31/513: having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
- A61K 31/519: ortho- or peri-condensed with heterocyclic rings
- A61K 39/39558: {against tumor tissues, cells, antigens}
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61P 35/00: Antineoplastic agents
- A61P 35/04: specific for metastasis
- C07K 16/30: from tumour cells
- C07K 16/3046: {Stomach, Intestines}
- A61K 2039/505: {comprising antibodies}
- A61K 2039/545: {characterised by the dose, timing or administration schedule}
- C07K 2317/14: Specific host cells or culture conditions, e.g. components, pH or temperature
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/51: Complete heavy chain or Fd fragment, i.e. VH + CH1
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/55: Fab or Fab'
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2319/02: containing a signal sequence
- 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 present invention provides a method for producing an anti-EGFR monoclonal antibody and the applications thereof. The method comprises the steps of: designing and synthesizing the light chain and heavy chain according to the codons preferred by Chinese hamster, transfecting GS knockout host cells CHO-CR-GS−/−, culturing cells using serum-free technology, isolating and purifying the antibody, and obtaining the low immunogenicity CMAB009 antibody.

## Claims

### Claim 1

A composition comprising an antibody, and a pharmaceutically acceptable carrier,
wherein the antibody comprises a light chain comprising an amino acid sequence set forth in SEQ ID NO: 2 and a heavy chain comprising an amino acid sequence set forth in SEQ ID NO: 4, and
wherein the antibody has a z-average (z-avg) of 10-25 nm as determined by a dynamic light scattering (DLS) analysis,
wherein the Fab segments of the antibody have a sialic acid NANA glycan chain structure, and
wherein the antibody does not comprise an N-glycolylneuraminic acid (NGNA) and does not comprise a Gal-α(1,3)-Gal glycan,
wherein the antibody binds epidermal growth factor receptor (EGFR).

### Claim 2 (depends on 1)

The composition of claim 1, wherein the antibody is produced by a method comprising:
a) providing an anti-EGFR monoclonal antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 4;
b) constructing a recombinant plasmid using a nucleic acid encoding the light chain and the heavy chain,
c) transfecting CHO cells with the recombinant plasmid;
d) screening for a high-expressing clone;
e) culturing in large scale, the high-expressing clone, wherein a cell culture temperature is 33° C.˜36° C., a pH of a cell culture growth media is 6.5˜6.9, and an osmotic pressure of the cell culture growth media is 290 mOsm/kg˜350 mOsm/kg;
f) isolating the cultured clone of e); and
g) purifying the isolated clone of f) to obtain the anti-EGFR monoclonal antibody.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the cell culture temperature is 34° C.

### Claim 4 (depends on 2)

The composition of claim 2, wherein the pH of the cell culture growth media is pH 6.6.

### Claim 5 (depends on 2)

The composition of claim 2, wherein the osmotic pressure of the cell culture growth media is 340 mOsm/kg.

### Claim 6

A liquid pharmaceutical composition comprising water and an anti-EGFR antibody, wherein the anti-EGFR antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 4,
wherein the antibody has a z-average (z-avg) of 10-25 nm as determined by dynamic light scattering (DLS) analysis, and the anti-EGFR antibody does not comprise an N-glycolylneuraminic acid (NGNA), and does not comprise a Gal-α(1,3)-Gal glycan.

### Claim 7 (depends on 6)

The composition of claim 6, wherein the z-avg of the antibody is 15-20 nm.

### Claim 8 (depends on 6)

The composition of claim 6, wherein the antibody is produced by a method comprising:
a) providing an anti-EGFR monoclonal antibody, the anti-EGFR monoclonal antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 4;
b) constructing a recombinant plasmid using a nucleic acid encoding the light chain and the heavy chain,
c) transfecting CHO cells with the recombinant plasmid;
d) screening for a high-expressing clone;
e) optimizing cell culture conditions, culturing in large scale, the high-expressing clone from d), wherein the cell culture growth media is maintained at a temperature of 33° C.˜36° C. at a pH of 6.5˜6.9 and wherein the osmotic pressure of the cell culture growth media is 290 mOsm/kg˜350 mOsm/kg;
f) isolating the cultured clone of e); and
g) purifying the isolated clone of f) to obtain the antibody.

### Claim 9 (depends on 8)

The composition of claim 8, wherein the cell culture temperature is 34° C.

### Claim 10 (depends on 8)

The composition of claim 8, wherein the pH of the cell culture growth media is pH 6.6.

### Claim 11 (depends on 8)

The composition of claim 8, wherein the osmotic pressure of the cell culture growth media is 340 mOsm/kg.

### Claim 12

A liquid pharmaceutical composition comprising water and an anti-EGFR antibody,
wherein the anti-EGFR antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 4,
wherein the anti-EGFR antibody is produced in a Chinese Hamster Ovary (CHO) cell,
and wherein the composition does not comprise a polysorbate and/or a saccharobiose.

### Claim 13

A liquid pharmaceutical composition consisting essentially of water, an anti-EGFR antibody, sodium chloride, sodium dihydrogen phosphate dihydrate, and disodium phosphate dihydrate,
wherein the anti-EGFR antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2 and a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 4, and
wherein the anti-EGFR antibody does not comprise an N-glycolylneuraminic acid (NGNA) glycan, does not comprise a Gal-α(1,3)-Gal glycan, and/or does comprise a Gal-α(2, 3/6)-Gal glycan.

### Claim 14

A method of producing an anti-EGFR monoclonal antibody, said method comprising:
a) an anti-EGFR monoclonal antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 2 and a heavy chain comprising the amino acid sequence of SEQ ID NO: 4;
b) constructing a recombinant plasmid using a nucleic acid encoding the light chain and the heavy chain;
c) transfecting CHO cells with the recombinant plasmid;
d) screening for a high-expressing clone;
e) culturing in large scale, the high-expressing clone, wherein a cell culture temperature is 33° C.˜36° C., a pH of a cell culture growth media is 6.5˜6.9, and an osmotic pressure of the cell culture growth media is 290 mOsm/kg˜350 mOsm/kg;
f) isolating the cultured clone of e); and
g) purifying the isolated clone of f) to obtain the anti-EGFR monoclonal antibody.

### Claim 15 (depends on 14)

The method of claim 14, wherein the coding sequences for the light chain and heavy chain of the anti-EGFR monoclonal antibody are designed and synthesized according to the codons preference of Chinese hamster ovary cells.

### Claim 16 (depends on 14)

The method of claim 14, wherein said host cell is a mammalian CHO cell.

### Claim 17 (depends on 14)

The method of claim 14, wherein said cell culture temperature is 33° C.˜36° C., preferably 34° C.

### Claim 18 (depends on 14)

The method of claim 14, wherein said pH of the cell culture growth media is 6.5˜6.9.

### Claim 19 (depends on 14)

The method of claim 14, wherein said osmotic pressure of the cell culture growth media is 290 mOsm/kg˜350 mOsm/kg.

### Claim 20 (depends on 17)

The method according to claim 17 wherein the cell culture growth media is serum-free cell culture growth media, and the host cell is cultured in serum-free conditions.

### Claim 21 (depends on 14)

The antibody of claim 14 wherein said antibody has lower immunogenicity than the antibodies produced with currently existing.

### Claim 22 (depends on 14)

A method according to claim 14 further comprising the step of administering in combination with other drugs for treating tumors expressing epidermal growth factor receptor (EGFR).

### Claim 23

A method of inhibiting progression of cancer in a human subject, said method comprising administering the composition of claim to the subject, such that progression is inhibited.

### Claim 24 (depends on 6)

A method of treating a human subject having cancer, said method comprising administering the composition of claim 6 to the subject, such that the cancer is treated.

### Claim 25 (depends on 24)

The method of claim 24 wherein the cancer is squamous cell carcinoma of the head and neck (SCCHN) or colorectal cancer.

### Claim 26 (depends on 25)

The method of claim 25, wherein the colorectal cancer is K-Ras wild-type, EGFR-expressing colorectal cancer.

### Claim 27 (depends on 26)

The method of claim 26, wherein the antibody is administered in combination with FOLFIRI (Folinic Acid, Fluorouracil and Irinotecan Hydrochloride).

### Claim 28 (depends on 26)

The method of claim 26, wherein the antibody is administered in combination with irinotecan.

### Claim 29 (depends on 24)

The method of claim 24 wherein the subject has recurrent or metastatic squamous cell carcinoma of the head and neck and has failed prior platinum-based therapy.

### Claim 30 (depends on 24)

The method of claim 24 wherein the subject has locally or regionally advanced squamous cell carcinoma of the head and neck.

### Claim 31 (depends on 30)

The method of claim 30, wherein the antibody is administered in combination with radiation therapy for the initial treatment of the cancer.

### Claim 32 (depends on 24)

The method of claim 24 wherein the subject has recurrent locoregional disease or metastatic squamous cell carcinoma of the head and neck.

### Claim 33 (depends on 29)

The method of claim 29, wherein the antibody is administered in combination with platinum-based therapy with 5-FU.

### Claim 34 (depends on 24)

The method of claim 24 wherein the antibody is administered in combination with an additional therapeutic agent.

### Claim 35 (depends on 34)

The method of claim 34, wherein the additional therapeutic agent is a chemotherapeutic agent.

### Claim 36 (depends on 24)

The method of claim 24 wherein the subject has failed oxaliplatin- and irinotecan-based chemotherapy.

### Claim 37 (depends on 24)

The method of claim 24 wherein the subject is intolerant to irinotecan.

### Claim 38

A method of treating or inhibiting progression of colorectal cancer in a subject having colorectal cancer, said method comprising administering an anti-EGFR antibody and irinotecan, such that colorectal cancer is treated, wherein the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2, comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 4, and contains a Gal-α 2, 3/6)-Gal glycan.

### Claim 39

A method of treating or inhibiting progression of colorectal cancer in a subject having colorectal cancer, said method comprising administering an anti-EGFR antibody and irinotecan, such that colorectal cancer is treated, wherein the antibody comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 2, comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 4, and does not contain either an N-glycolylneuraminic acid (NGNA) glycan or a Gal-α(1,3)-Gal glycan.

### Claim 40 (depends on 39)

The method of claim 39, wherein the colorectal cancer is advanced colorectal cancer.

### Claim 41 (depends on 39)

The method of claim 39, wherein the antibody is administered via infusion to the subject at an initial dose of 400 mg/m2 followed by a weekly dose of 250 mg/m2.

### Claim 42 (depends on 39)

The method of claim 39 wherein the antibody is produced in a Chinese Hamster Ovary (CHO) cell.