title: Design, expression, purification, and characterization, in vitro and in vivo, of an antimelanoma single-chain Fv antibody fused to the toxin gelonin.
abstract: We constructed a single-chain anti-gp240 antibody (designated MEL sFv) and fused this to the recombinant toxin gelonin (rGel). MEL sFv-rGel was produced in bacterial expression plasmid (pET-32), and the protein composition was confirmed by both DNA sequencing and Western analysis. Inhibition of cell-free protein synthesis by the fusion construct demonstrated an IC(50) of 100 pM, comparable with that for native gelonin (104 pM). The MEL sFv-rGel fusion toxin bound to antigen-positive but not antigen-negative cells as assessed by ELISA. Internalization into A-375 target cells was demonstrable by 1 h after exposure. Against A-375 cells, MEL sFv-rGel demonstrated an IC(50) of approximately 8 nM, which was 250-fold lower than that for free rGel (2000 nM). The cytotoxic effects of the construct did not involve apoptosis because terminal deoxynucleotidyl transferase-mediated nick end labeling assays of treated cells were negative. (125)I-labeled MEL sFv-rGel demonstrated biphasic clearance of the construct from plasma (t(1/2) alpha and t(1/2) beta were 0.46 and 7.2 h, respectively). At 72 h after administration, xenograft studies showed that the tissue:blood ratio was highest for tumor followed by spleen, kidney, and liver. Groups of tumor-bearing nude mice were treated with fusion toxin at either 2 or 20 mg/kg. Compared with saline-treated controls, for which mean tumor burden increased 6-fold, the groups treated with the high and low doses of fusion construct showed no increase or only a 2-fold increase, respectively. These studies suggest that this recombinant fusion construct has potent cytotoxic activity both in vitro and in vivo and is an excellent candidate for clinical development.
authors.affiliation: Department of Bioimmunotherapy, Section of Immunopharmacology and Targeted Therapy, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. mrosenbl@notes.mdacc.tmc.edu
authors.forename: Michael G
authors.initials: MG
authors.lastname: Rosenblum
authors.forename: Lawrence H
authors.initials: LH
authors.lastname: Cheung
authors.forename: Yuying
authors.initials: Y
authors.lastname: Liu
authors.forename: John W
authors.initials: JW
authors.lastname: Marks
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D001483:Base Sequence; D003602:Cytotoxicity, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D000922:Immunotoxins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D020287:In Situ Nick-End Labeling; D007457:Iodine Radioisotopes; D008545:Melanoma / Q000188:drug therapy* / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology*; D010957:Plasmids / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D007457:Iodine Radioisotopes; D010940:Plant Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; C025674:GEL protein, Gelonium multiflorum