• Michael G Rosenblum, Lawrence H Cheung, Yuying Liu, and John W Marks.
• 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
• Cancer Res. 2003 Jul 15; 63 (14): 3995-4002.

AbstractWe 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.

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