title: Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Laboratory Medicine, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
authors.forename: Chang-hong
authors.initials: CH
authors.lastname: Yi
authors.affiliation: Department of General Surgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors.forename: Can-ping
authors.initials: CP
authors.lastname: Ruan
authors.affiliation: Department of Laboratory Medicine, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Department of General Surgery, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
authors.forename: Xin-yun
authors.initials: XY
authors.lastname: Xu
authors.affiliation: Department of Laboratory Medicine, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
authors.forename: Yun-peng
authors.initials: YP
authors.lastname: Zhao
authors.affiliation: Department of Laboratory Medicine, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
authors.forename: Meng
authors.initials: M
authors.lastname: Fang
authors.affiliation: Department of Laboratory Medicine, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Ji
authors.affiliation: Department of Laboratory Medicine, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Gu
authors.affiliation: Department of Laboratory Medicine, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200438, China.
authors.forename: Chun-fang
authors.initials: CF
authors.lastname: Gao
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology* / Q000633:toxicity; D000920:Antibody-Dependent Cell Cytotoxicity; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D016466:CHO Cells; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000068818:Cetuximab; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004342:Drug Hypersensitivity / Q000209:etiology / Q000517:prevention & control; D066246:ErbB Receptors / Q000037:antagonists & inhibitors* / Q000276:immunology / Q000378:metabolism; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D017351:N-Acetylglucosaminyltransferases / Q000096:biosynthesis / Q000235:genetics; D011485:Protein Binding; D015202:Protein Engineering; D047428:Protein Kinase Inhibitors / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D011499:Protein Processing, Post-Translational; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; D017452:Receptors, IgG; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase; C512478:EGFR protein, human; D066246:ErbB Receptors; D000068818:Cetuximab