title: Deglycosylation of mAb by EndoS for improved molecular imaging.
abstract: OBJECTIVE
  Monoclonal antibodies (mAbs) have been shown preclinically as reliable targeting moieties for antigen imaging using near-infrared fluorescence (NIRF) molecular imaging. However, crystallizable fragment-gamma receptor (FcγRs) expressed on immune cells also bind mAbs through defined epitopes on the constant fragment (Fc) of IgG. Herein, we evaluate the potential impact Fc interactions have on mAb agent imaging specificity.
  
  
  METHODS
  Through the removal of conserved glycans within the Fc domain, shown to have Fc/FcγR interactions, we evaluate their impact on non-specific binding/accumulation of a NIRF-labeled mAb-based imaging agent in lymph nodes (LNs) in inflamed animals and in an orthotopic prostate cancer animal model of LN metastasis.
  
  
  RESULTS
  Deglycosylation of a murine mAb against the human epithelial cell adhesion marker using endoglycosidase EndoS significantly reduced non-specific binding in the LNs of inflamed animals and in cancer-negative LNs of tumor-bearing animals. Sensitivity remained unchanged while improvement in imaging specificity increased imaging accuracy.
  
  
  CONCLUSIONS
  The reduction of non-specific binding through deglycosylation of a mAb-based imaging agent shows that reducing Fc/FcγR interactions can improve imaging accuracy.
authors.affiliation: Center for Molecular Imaging, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center, 1825 Pressler Street, Houston, TX, 77030, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Gao
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Pinkston
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Wilganowski
authors.forename: Holly
authors.initials: H
authors.lastname: Robinson
authors.forename: Ali
authors.initials: A
authors.lastname: Azhdarinia
authors.forename: Banghe
authors.initials: B
authors.lastname: Zhu
authors.forename: Eva M
authors.initials: EM
authors.lastname: Sevick-Muraca
authors.forename: Barrett R
authors.initials: BR
authors.lastname: Harvey
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000951:Antigens, Neoplasm / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000276:immunology; D002460:Cell Line; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D000071858:Epithelial Cell Adhesion Molecule; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007249:Inflammation / Q000473:pathology; D008198:Lymph Nodes / Q000473:pathology; D008264:Macrophages / Q000378:metabolism; D008297:Male; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods*; D011485:Protein Binding; D012372:ROC Curve; D017452:Receptors, IgG / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D000071858:Epithelial Cell Adhesion Molecule; C485701:Fcgr1 protein, mouse; D017452:Receptors, IgG; D006026:Glycoside Hydrolases