title: Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.
abstract: BACKGROUND
  The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested.
  
  
  METHODS
  The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes.
  
  
  RESULTS
  The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model.
  
  
  CONCLUSIONS
  The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.
authors.affiliation: Cancer and Immunogenetics Laboratory, Department of Medical Oncology, Weatherall Institute of Molecular Medicine, Oxford, UK.
authors.forename: S Q
authors.initials: SQ
authors.lastname: Ashraf
authors.forename: P
authors.initials: P
authors.lastname: Umana
authors.forename: E
authors.initials: E
authors.lastname: Mössner
authors.forename: T
authors.initials: T
authors.lastname: Ntouroupi
authors.forename: P
authors.initials: P
authors.lastname: Brünker
authors.forename: C
authors.initials: C
authors.lastname: Schmidt
authors.forename: J L
authors.initials: JL
authors.lastname: Wilding
authors.forename: N J
authors.initials: NJ
authors.lastname: Mortensen
authors.forename: W F
authors.initials: WF
authors.lastname: Bodmer
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D000920:Antibody-Dependent Cell Cytotoxicity / Q000187:drug effects / Q000276:immunology; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000276:immunology / Q000628:therapy; D005434:Flow Cytometry; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D010587:Phagocytosis / Q000187:drug effects / Q000276:immunology*; D015202:Protein Engineering / Q000379:methods; D017452:Receptors, IgG / Q000037:antagonists & inhibitors / Q000276:immunology; D014162:Transfection
chemical_list: D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; C496124:FCGR3A protein, human; D007074:Immunoglobulin G; D017452:Receptors, IgG