Abstract
We constructed a single-chain antibody fragment (scFv) of murine monoclonal antibody, MABL, which specifically bound to human CD47 (hCD47) and induced apoptosis of the leukemic cells. The scFv of MABL antibody with a 15-residue linker (MABL scFv-15) formed both dimer (Mr 50 kDa) and monomer (Mr 25 kDa). Both MABL scFv-15 dimer and monomer had binding activity for hCD47. MABL scFv-15 dimer strongly induced apoptosis of hCD47-introduced mouse leukemic cells in vitro and exhibited anti-tumor effect in a myeloma transplanted mice model. However, MABL scFv-15 monomer scarcely exhibited these activities. These results strongly demonstrate that the ligation of CD47 antigen by two antigen-binding sites of MABL dimer is needed for inducing apoptosis. The parent MABL antibody caused hemagglutination due to the CD47 expressed on erythrocytes. Interestingly, MABL scFv-15 dimer did not cause hemagglutination. This apoptosis-inducing dimer appears to be a lead candidate for novel leukemic therapy.
MeSH terms
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Animals
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Antibodies, Monoclonal / chemistry
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Antigens, CD / immunology*
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Antigens, CD / metabolism
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Apoptosis / drug effects*
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Binding Sites
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CD47 Antigen
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CHO Cells
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Carrier Proteins / immunology*
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Carrier Proteins / metabolism
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Cell Line, Tumor
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Cell Survival / drug effects
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Cricetinae
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Dimerization
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Flow Cytometry / methods
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Hemagglutination Tests
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Humans
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Immunoglobulin Fragments / genetics
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Immunoglobulin Fragments / immunology*
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Immunoglobulin Fragments / metabolism
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Immunoglobulin Fragments / pharmacology*
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Immunoglobulin G / blood
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Immunoglobulin Variable Region / genetics
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Immunoglobulin Variable Region / immunology
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Immunoglobulin Variable Region / metabolism
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Immunoglobulin Variable Region / pharmacology
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Leukemia L1210 / immunology
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Leukemia L1210 / pathology*
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Leukemia L1210 / therapy
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Male
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Mice
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Mice, SCID
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Multiple Myeloma / immunology
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Multiple Myeloma / pathology
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Multiple Myeloma / therapy
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Neoplasm Transplantation / immunology
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Recombinant Proteins / genetics
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Recombinant Proteins / immunology
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Recombinant Proteins / metabolism
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Recombinant Proteins / pharmacology
Substances
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Antibodies, Monoclonal
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Antigens, CD
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CD47 Antigen
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CD47 protein, human
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Carrier Proteins
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Cd47 protein, mouse
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Immunoglobulin Fragments
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Immunoglobulin G
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Immunoglobulin Variable Region
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Recombinant Proteins