Combined Fc-protein- and Fc-glyco-engineering of scFv-Fc fusion proteins synergistically enhances CD16a binding but does not further enhance NK-cell mediated ADCC
| Autor: | Paul W. H. I. Parren, Michael Dechant, Thomas Valerius, Jan G. J. van de Winkel, Matthias Staudinger, Patrick Van Berkel, Dalia Akramiene, Christian Kellner, Anja Muskulus, Martin Gramatzki, Pia Glorius, Georg H. Fey, Matthias Peipp, Sahar Mohseni Nodehi, Roland Repp |
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| Rok vydání: | 2010 |
| Předmět: |
Immunology
Blotting Western Antibody Affinity chemical and pharmacologic phenomena Plasma protein binding Antigens CD7 CHO Cells Receptors Fc CD16 Biology Protein Engineering Immunoglobulin G Cell Line Cricetulus Cell Line Tumor Cricetinae Immunology and Allergy Animals Humans Fucose Glycoproteins Antibody-dependent cell-mediated cytotoxicity HLA-D Antigens Binding protein Immunoglobulin Fc Fragments Receptors IgG Antibody-Dependent Cell Cytotoxicity Protein engineering Antigens CD20 Flow Cytometry Molecular biology Fusion protein Killer Cells Natural Mutagenesis biology.protein Protein Binding Single-Chain Antibodies |
| Zdroj: | Journal of immunological methods. 373(1-2) |
| ISSN: | 1872-7905 |
| Popis: | Protein- or glyco-engineering of antibody molecules can be used to enhance Fc-mediated effector functions. ScFv-Fc fusion proteins (scFv-Fc) represent interesting antibody derivatives due to their relatively simple design and increased tissue penetration. Here, the impact of protein- and glyco-engineering on ADCC potency of a panel of human IgG1-based scFv-Fc was tested. Three matched sets of scFv-Fc variants targeting CD7, CD20 or HLA class II and optimized for CD16a binding by mutagenesis, lack of core-fucose, or their combination, were generated and functionally tested in comparison to the corresponding wild type scFv-Fc. Antigen binding activity was not compromised by altered glycosylation or Fc mutagenesis, whereas Fc binding to CD16a was significantly enhanced in the order: non-core fucosylated/Fc-mutated double-engineered≫Fc-mutated≥non-core-fucosylated>wild-type IgG1-Fc. All engineered variants triggered potent ADCC with up to 100-fold reduced EC50 values compared to non-engineered variants. Interestingly, double-engineered variants were similarly effective in triggering ADCC compared to single-engineered variants irrespective of their 1 log greater CD16a binding affinity. Thus, these data demonstrate that protein- and glyco-engineering enhances NK-cell mediated ADCC of scFv-Fc similarly and show that enhancing CD16a affinity beyond a certain threshold does not result in a further increase of NK-cell mediated ADCC. |
| Databáze: | OpenAIRE |
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