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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Antibody Affinity / immunology*
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Antibody Specificity / immunology
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Antibody-Dependent Cell Cytotoxicity / immunology*
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Antigens, CD / chemistry
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Antigens, CD / immunology*
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Antigens, Neoplasm / immunology
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Cell Line, Tumor
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Dose-Response Relationship, Immunologic
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Humans
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Hybridomas
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Immunoglobulin G / immunology
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Leukemia, Myeloid, Acute / immunology*
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Leukemia, Myeloid, Acute / pathology
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Models, Molecular
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Molecular Sequence Data
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Mutant Proteins / chemistry
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Mutant Proteins / metabolism
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Mutation / genetics
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Neoplastic Stem Cells / immunology*
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Protein Binding
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Protein Engineering*
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Protein Structure, Tertiary
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Receptors, Fc / immunology*
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Sequence Alignment
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Single-Chain Antibodies / immunology
Substances
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Antigens, CD
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Antigens, Neoplasm
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CD96 antigen
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Immunoglobulin G
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Mutant Proteins
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Receptors, Fc
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Single-Chain Antibodies
Grants and funding
This study was supported by research grant DJCLS R 08/01 from the Deutsche José Carreras Leukaemie Stiftung e.V. (Munich, Germany;
http://www.carreras-stiftung.de/) and research grant 2007.065.1 from the Wilhelm Sander-Stiftung (Neustadt, Germany;
http://www.sanst.de/cms/front_content.php), SMN was supported by the FAZIT-Stiftung (Frankfurt am Main, Germany;
http://www.fazit-stiftung.de/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.