Design, expression, purification, and characterization, in vitro and in vivo, of an antimelanoma single-chain Fv antibody fused to the toxin gelonin.
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.
Rosenblum
Michael G
MG
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
Cheung
Lawrence H
LH
Liu
Yuying
Y
Marks
John W
JW
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
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