PMID: 9439650Jan 24, 1998Paper

Construction and characterization of a fusion protein of single-chain anti-carcinoma antibody 323/A3 and human beta-glucuronidase

Cancer Immunology, Immunotherapy : CII
H J HaismaE Boven

Abstract

We report the construction and expression of a fusion protein between a single-chain antibody specific for human carcinomas and human beta-glucuronidase by recombinant DNA technology. The sequences encoding the murine monoclonal antibody 323/A3 light- and heavy-chain variable genes were joined by a synthetic sequence encoding a 15-amino-acid linker and combined with human beta-glucuronidase by a synthetic sequence encoding a 6-amino-acid linker. The construct was placed under the control of the cytomegalovirus promotor and expressed in COS-7 cells. The yield of active fusion protein was 10 ng/ml transfectoma supernatant. Antibody affinity, antibody specificity and enzyme activity were fully retained by the fusion protein. Biochemical characterization of the fusion protein by sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed a molecular mass of 100 kDa under denaturing conditions. Gel-filtration analysis indicated that the enzymatically active form is a tetramer of approximately 400 kDa. The non-toxic prodrug N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate was activated to the cytotoxic drug doxorubicin by the fusion protein with a hydrolysis rate similar to that of human beta-glucuronida...Continue Reading

Citations

Feb 7, 2001·The Journal of Gene Medicine·M J FonsecaH J Haisma
Sep 5, 2003·Cancer Biotherapy & Radiopharmaceuticals·Barbara H BielaAlan L Epstein

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