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
| Autor: | Karen Vanhoorelbeke, S Vauterin, Philippe Barbeaux, Isabel Van Rompaey, Inge Pareyn, Jean-Marie Stassen, Hans Deckmyn, Thu Hoa Ngo, Stephanie Staelens, Johan Desmet |
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| Rok vydání: | 2006 |
| Předmět: |
Models
Molecular medicine.drug_class Recombinant Fusion Proteins Molecular Sequence Data Immunology Immunoglobulin Variable Region CHO Cells Plasma protein binding Immunoglobulin light chain Monoclonal antibody Homology (biology) Mice Cricetulus Protein structure Cricetinae von Willebrand Factor medicine Animals Humans Amino Acid Sequence Binding site Molecular Biology Peptide sequence Base Sequence Sequence Homology Amino Acid Chemistry Immunogenicity Antibodies Monoclonal Molecular biology Protein Structure Tertiary Immunoglobulin G Biophysics Binding Sites Antibody Collagen Protein Binding |
| Zdroj: | Molecular Immunology. 43:1243-1257 |
| ISSN: | 0161-5890 |
| DOI: | 10.1016/j.molimm.2005.07.018 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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