title: 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.
abstract: 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.
authors.affiliation: Division of Stem Cell Transplantation and Immunotherapy, 2nd Department of Medicine, Christian-Albrechts-University Kiel, Kiel, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Repp
authors.forename: Christian
authors.initials: C
authors.lastname: Kellner
authors.forename: Anja
authors.initials: A
authors.lastname: Muskulus
authors.forename: Matthias
authors.initials: M
authors.lastname: Staudinger
authors.forename: Sahar Mohseni
authors.initials: SM
authors.lastname: Nodehi
authors.forename: Pia
authors.initials: P
authors.lastname: Glorius
authors.forename: Dalia
authors.initials: D
authors.lastname: Akramiene
authors.forename: Michael
authors.initials: M
authors.lastname: Dechant
authors.forename: Georg H
authors.initials: GH
authors.lastname: Fey
authors.forename: Patrick H C
authors.initials: PH
authors.lastname: van Berkel
authors.forename: Jan G J
authors.initials: JG
authors.lastname: van de Winkel
authors.forename: Paul W H I
authors.initials: PW
authors.lastname: Parren
authors.forename: Thomas
authors.initials: T
authors.lastname: Valerius
authors.forename: Martin
authors.initials: M
authors.lastname: Gramatzki
authors.forename: Matthias
authors.initials: M
authors.lastname: Peipp
mesh_terms: D000818:Animals; D000915:Antibody Affinity / Q000276:immunology; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D018951:Antigens, CD20 / Q000276:immunology / Q000378:metabolism; D019011:Antigens, CD7 / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D005643:Fucose / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D006681:HLA-D Antigens / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism; D016296:Mutagenesis; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods; D011961:Receptors, Fc / Q000276:immunology / Q000378:metabolism; D017452:Receptors, IgG / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
chemical_list: D018951:Antigens, CD20; D019011:Antigens, CD7; C496124:FCGR3A protein, human; D006023:Glycoproteins; D006681:HLA-D Antigens; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011961:Receptors, Fc; D017452:Receptors, IgG; D057127:Single-Chain Antibodies; D005643:Fucose