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

Hidde J. Haisma, Rudd H. Brakenhoff, Ida V.D. Meulen-Muileman, Herbert M. Pinedo, Epie Boven

Research output: Contribution to journalArticlepeer-review

15 Scopus citations


We report the construction and expression of a fusion protein between a single-chain antibody specific for human carcinomas and human β- 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 β-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-β-glucuronyl carbamate was activated to the cytotoxic drug doxorubicin by the fusion protein with a hydrolysis rate similar to that of human β-glucuronidase. The growth inhibition of tumor cells coated with the fusion protein and exposed to prodrug was similar to that obtained with doxorubicin. This study shows the feasibility of constructing eukaryotic fusion proteins consisting of a single-chain antibody and human β-glucuronidase for use in the specific activation of anticancer prodrugs.

Original languageEnglish (US)
Pages (from-to)266-272
Number of pages7
JournalCancer Immunology Immunotherapy
Issue number5
StatePublished - 1997
Externally publishedYes


  • β-glucuronidase
  • Antibody-directed enzyme prodrug therapy (ADEPT)
  • Antibody-enzyme immunoconjugate
  • Prodrug activation

ASJC Scopus subject areas

  • Cancer Research
  • Immunology
  • Oncology


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