title: Humanization by variable domain resurfacing and grafting on a human IgG4, using a new approach for determination of non-human like surface accessible framework residues based on homology modelling of variable domains.
abstract: Many antithrombotic agents have only a small therapeutic window, frequently leading to bleeding problems. However, interfering with platelet adhesion through the collagen-VWF-GPIbalpha axis is expected to cause less bleeding problems. Our group developed a monoclonal antibody, 82D6A3, directed against the von Willebrand factor (VWF) A3-domain, which inhibits the VWF-interaction to fibrillar collagen. 82D6A3 has antithrombotic effects in vivo without bleeding time prolongation. To further investigate the promising features of 82D6A3, we have humanized it by variable domain resurfacing and grafting on the constant regions of a human IgG4. First, the sequence of the variable domains was determined and the murine scFv was constructed. The expressed scFv had a comparable activity as the IgG of 82D6A3, and its DNA was thus used in subsequent humanization procedures. For this, a new approach was introduced to identify non-human like framework surface residues, since the general distribution of accessible residues described for human and murine heavy and light chain variable domains showed several discrepancies with the homology modelled Fv of 82D6A3. Identification of non-human like framework residues and evaluation of their surface accessibility within the context of the homology modelled Fv of 82D6A3, revealed 10 residues that need to be humanized without influencing the conformation of the CDR loops. Indeed, the humanized scFv of 82D6A3, obtained by mutating all 10 residues to their human counterpart, was still binding with high affinity to VWF and retained the inhibitory properties of the murine scFv. Next, in order to increase its half life and to decrease its immunogenicity, the humanized variable domains were grafted on the constant regions of a human IgG4, resulting in h82D6A3 with an in vitro activity comparable to that of the murine IgG.
authors.affiliation: Laboratory for Thrombosis Research, IRC, KU Leuven Campus Kortrijk, E. Sabbelaan 53, 8500 Kortrijk, Belgium. Stephanie.Staelens@kulak.ac.be
authors.forename: Stephanie
authors.initials: S
authors.lastname: Staelens
authors.forename: Johan
authors.initials: J
authors.lastname: Desmet
authors.forename: Thu Hoa
authors.initials: TH
authors.lastname: Ngo
authors.forename: Stephan
authors.initials: S
authors.lastname: Vauterin
authors.forename: Inge
authors.initials: I
authors.lastname: Pareyn
authors.forename: Philippe
authors.initials: P
authors.lastname: Barbeaux
authors.forename: Isabel
authors.initials: I
authors.lastname: Van Rompaey
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Stassen
authors.forename: Hans
authors.initials: H
authors.lastname: Deckmyn
authors.forename: Karen
authors.initials: K
authors.lastname: Vanhoorelbeke
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D016466:CHO Cells; D003094:Collagen / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D003094:Collagen