title: Kinetic analysis of novel mono- and multivalent VHH-fragments and their application for molecular imaging of brain tumours.
abstract: OBJECTIVE
  The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications.
  
  
  METHODS
  An anti-EGFR/EGFRvIII llama single-domain antibody (EG(2)) and two higher valency format constructs, bivalent EG(2)-hFc and pentavalent V2C-EG(2) sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies.
  
  
  RESULTS
  Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG(2) to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG(2) and EG(2)-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG(2), V2C-EG(2) and EG(2)-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG(2)-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG(2)-hFc selective accumulation/retention in anatomically defined tumour regions.
  
  
  CONCLUSIONS
  Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma.
authors.affiliation: Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada.
authors.forename: U
authors.initials: U
authors.lastname: Iqbal
authors.forename: U
authors.initials: U
authors.lastname: Trojahn
authors.forename: H
authors.initials: H
authors.lastname: Albaghdadi
authors.forename: J
authors.initials: J
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: O'Connor-McCourt
authors.forename: D
authors.initials: D
authors.lastname: Stanimirovic
authors.forename: B
authors.initials: B
authors.lastname: Tomanek
authors.forename: G
authors.initials: G
authors.lastname: Sutherland
authors.forename: A
authors.initials: A
authors.lastname: Abulrob
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism; D000915:Antibody Affinity; D001932:Brain Neoplasms / Q000098:blood supply / Q000175:diagnosis* / Q000378:metabolism / Q000648:ultrastructure; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000276:immunology / Q000378:metabolism*; D005909:Glioblastoma / Q000098:blood supply / Q000175:diagnosis* / Q000378:metabolism / Q000648:ultrastructure; D006207:Half-Life; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000276:immunology / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D051379:Mice; D008819:Mice, Nude; D057054:Molecular Imaging / Q000379:methods; D008970:Molecular Weight; D041622:Tomography, Optical / Q000379:methods; D051598:Whole Body Imaging / Q000379:methods; D055114:X-Ray Microtomography / Q000379:methods
chemical_list: D000906:Antibodies; D055767:Immobilized Proteins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; C410723:epidermal growth factor receptor VIII; C512478:EGFR protein, human; D066246:ErbB Receptors