Abstract
Since the disialoganglioside GD2 is abundantly present on the surface of neuroblastoma cells, we constructed a new recombinant immunotoxin for possible clinical use in patients with neuroblastoma. A functional 14.18 scFv-phage was obtained by selection of an anti-GD2 hybridoma derived phage antibody mini-library on the neuroblastoma-derived, GD2-expressing cell line IMR5. By insertion into the bacterial expression vector pBM1.1 the selected scFv was fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). Periplasmically expressed 14.18(scFv)-ETA' bound to the GD2 expressing cell line IMR5, but not to the GD2 negative Hodgkin-derived cell line L540Cy as documented by ELISA and flow cytometry. The recombinant immunotoxin (rIT) inhibited cell viability of IMR5 cells by 50% at concentrations (IC(50)) of 0.326 microg/ml. This recombinant immunotoxin will be further investigated in vivo for its value as a new immunotherapeutic agent for the treatment of patients with neuroblastoma.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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ADP Ribose Transferases*
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Antibodies, Monoclonal / genetics
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Antibodies, Monoclonal / pharmacology*
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Bacterial Toxins*
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Binding, Competitive
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Cell Membrane / metabolism
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Cell Survival / drug effects
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Cell Survival / immunology
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Cloning, Molecular
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Cytotoxicity, Immunologic
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Dose-Response Relationship, Drug
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Exotoxins / genetics
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Gangliosides / immunology*
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Humans
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Immunoglobulin Fragments / genetics
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Immunoglobulin Fragments / metabolism
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Immunoglobulin Fragments / pharmacology*
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Immunoglobulin Variable Region / genetics
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Immunotoxins / genetics
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Immunotoxins / isolation & purification
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Immunotoxins / pharmacology*
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Neuroblastoma / drug therapy*
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Neuroblastoma / immunology
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Protein Binding
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Pseudomonas aeruginosa Exotoxin A
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / pharmacology
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Tumor Cells, Cultured / drug effects
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Tumor Cells, Cultured / immunology
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Virulence Factors*
Substances
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Antibodies, Monoclonal
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Bacterial Toxins
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Exotoxins
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Gangliosides
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Immunoglobulin Fragments
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Immunoglobulin Variable Region
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Immunotoxins
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Recombinant Fusion Proteins
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Virulence Factors
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immunoglobulin Fv
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ganglioside, GD2
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ADP Ribose Transferases