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Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro.

Acta pharmacologica Sinica (2014-09-30)
Chang-hong Yi, Can-ping Ruan, Hao Wang, Xin-yun Xu, Yun-peng Zhao, Meng Fang, Jun Ji, Xing Gu, Chun-fang Gao
ABSTRACT

To evaluate the biochemical features and activities of a glyco-engineered form of the anti-human epidermal growth factor receptor monoclonal antibody (EGFR mAb) cetuximab in vitro. The genes encoding the Chinese hamster bisecting glycosylation enzyme (GnTIII) and anti-human EGFR mAb were cloned and coexpressed in CHO DG44 cells. The bisecting-glycosylated recombinant EGFR mAb (bisec-EGFR mAb) produced by these cells was characterized with regard to its glycan profile, antiproliferative activity, Fc receptor binding affinity and cell lysis capability. The content of galactose-α-1,3-galactose (α-Gal) in the bisec-EGFR mAb was measured using HPAEC-PAD. The bisec-EGFR mAb had a higher content of bisecting N-acetylglucosamine residues. Compared to the wild type EGFR mAb, the bisec-EGFR mAb exhibited 3-fold higher cell lysis capability in the antibody-dependent cellular cytotoxicity assay, and 1.36-fold higher antiproliferative activity against the human epidermoid carcinoma line A431. Furthermore, the bisec-EGFR mAb had a higher binding affinity for human FcγRIa and FcγRIIIa-158F than the wild type EGFR mAb. Moreover, α-Gal, which was responsible for cetuximab-induced hypersensitivity reactions, was not detected in the bisec-EGFR mAb. The glyco-engineered EGFR mAb with more bisecting modifications and lower α-Gal content than the approved therapeutic antibody Erbitux shows improved functionality in vitro, and requires in vivo validations.

MATERIALS
Product Number
Brand
Product Description

Galactose, European Pharmacopoeia (EP) Reference Standard
Sigma-Aldrich
D-(+)-Galactose, meets analytical specification of Ph. Eur., BP
Millipore
D-(+)-Galactose, suitable for microbiology, ≥99.0%
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D-(+)-Galactose, ≥98%
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D-(+)-Galactose, powder, anhydrous, BioReagent, suitable for cell culture, suitable for insect cell culture
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D-(+)-Galactose, ≥99%
Supelco
Galactose, Pharmaceutical Secondary Standard; Certified Reference Material
SAFC
Galactose, plant-derived
USP
Galactose, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
D-(+)-Galactose, BioXtra, pH 5.0-7.0 (20 °C, 1 M in H2O), ≥99%