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Title: Humanization by variable domain resurfacing and grafting on a human IgG(4), using a new approach for determination of non-human like surface accessible framework residues based on homology modelling of variable domains
Authors: Staelens, Stephanie
Desmet, J
Ngo, Thu Hoa
Vauterin, S
Pareyn, I
Barbeaux, Philippe
Van Rompaey, Isabel
Stassen, Jean-Marie
Deckmyn, Hans
Vanhoorelbeke, Karen #
Issue Date: Mar-2006
Publisher: Pergamon-elsevier science ltd
Series Title: Molecular immunology vol:43 issue:8 pages:1243-1257
Abstract: Mary antithrombotic agents have only a small therapeutic window, frequently leading to bleeding problems. However, interfering with platelet adhesion through the collagen-VWF-GPIb alpha 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 IgG(4). 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 IgG(4), resulting in h82D6A3 with an in vitro activity comparable to that of the murine IgG. (c) 2005 Elsevier Ltd. All rights reserved.
ISSN: 0161-5890
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Electrical Engineering - miscellaneous
Molecular and Vascular Biology
Vesalius Research Centre (-)
Chemistry, Campus Kulak Kortrijk
Interdisciplinary Research Facility Life Sciences, Campus Kulak Kortrijk
# (joint) last author

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