title: Enhanced ADCC activity of affinity maturated and Fc-engineered mini-antibodies directed against the AML stem cell antigen CD96.
abstract: CD96, a cell surface antigen recently described to be preferentially expressed on acute myeloid leukemia (AML) leukemic stem cells (LSC) may represent an interesting target structure for the development of antibody-based therapeutic approaches. The v-regions from the CD96-specific hybridoma TH-111 were isolated and used to generate a CD96-specific single chain fragment of the variable regions (scFv). An affinity maturated variant resulting in 4-fold enhanced CD96-binding was generated by random mutagenesis and stringent selection using phage display. The affinity maturated scFv CD96-S32F was used to generate bivalent mini-antibodies by genetically fusing an IgG1 wild type Fc region or a variant with enhanced CD16a binding. Antibody dependent cell-mediated cytotoxicity (ADCC) experiments revealed that Fc engineering was essential to trigger significant effector cell-mediated lysis when the wild type scFv was used. The mini-antibody variant generated by fusing the affinity-maturated scFv with the optimized Fc variant demonstrated the highest ADCC activity (2.3-fold enhancement in efficacy). In conclusion, our data provide proof of concept that CD96 could serve as a target structure for effector cell-mediated lysis and demonstrate that both enhancing affinity for CD96 and for CD16a resulted in mini-antibodies with the highest cytolytic potential.
authors.affiliation: Division of Stem Cell Transplantation and Immunotherapy, Department of Medicine II, Zoological Institute, Christian-Albrechts-University, Kiel, Germany.
authors.forename: Sahar
authors.initials: S
authors.lastname: Mohseni Nodehi
authors.forename: Roland
authors.initials: R
authors.lastname: Repp
authors.forename: Christian
authors.initials: C
authors.lastname: Kellner
authors.forename: Joachim
authors.initials: J
authors.lastname: Bräutigam
authors.forename: Matthias
authors.initials: M
authors.lastname: Staudinger
authors.forename: Natalie
authors.initials: N
authors.lastname: Schub
authors.forename: Matthias
authors.initials: M
authors.lastname: Peipp
authors.forename: Martin
authors.initials: M
authors.lastname: Gramatzki
authors.forename: Andreas
authors.initials: A
authors.lastname: Humpe
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology*; D015703:Antigens, CD / Q000737:chemistry / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000276:immunology; D045744:Cell Line, Tumor; D004306:Dose-Response Relationship, Immunologic; D006801:Humans; D006825:Hybridomas; D007074:Immunoglobulin G / Q000276:immunology; D015470:Leukemia, Myeloid, Acute / Q000276:immunology* / Q000473:pathology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D014411:Neoplastic Stem Cells / Q000276:immunology*; D011485:Protein Binding; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000276:immunology*; D016415:Sequence Alignment; D057127:Single-Chain Antibodies / Q000276:immunology
chemical_list: D015703:Antigens, CD; D000951:Antigens, Neoplasm; C116131:CD96 antigen; D007074:Immunoglobulin G; D050505:Mutant Proteins; D011961:Receptors, Fc; D057127:Single-Chain Antibodies