Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human β-glucuronidase for antibody-directed enzyme prodrug therapy

Hidde J. Haisma, M. Fleur Sernee, Erik Hooijberg, Ruud H. Brakenhoff, Ida H V D Meulen-Muileman, Herbert M. Pinedo, Epie Boven

Research output: Contribution to journalArticle

Abstract

The CD20 antigen is an attractive target for specific treatment of B- cell lymphoma. Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a nontoxic prodrug at the tumor site by an enzyme targeted by a tumor-specific antibody such as anti-CD20. We constructed a fusion protein of the single-chain Fv anti-CD20 mouse monoclonal antibody (MoAb) 1H4 and human β-glucuronidase for the activation of the nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-β-glucuronyl carbamate to doxorubicin at the tumor site. The cDNAs encoding the light- and heavy-chain variable regions of 1H4 were cloned, joined by a synthetic sequence encoding a 15-amino acid linker and fused to human β-glucuronidase by a synthetic sequence encoding a 6-amino acid linker. An antibody-enzyme fusion protein-producing cell line was established by transfection of the construct into human embryonic kidney 293/EBNA cells. The yield of active fusion protein was 100 ng/mL transfectoma supernatant. Antibody affinity, antibody specificity, and enzyme activity were fully retained by the fusion protein. Immunoprecipitation and analysis by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) showed that the fusion protein has a relative molecular weight (Mw) of 100 kD under denaturing conditions. Gel filtration analysis indicated that the enzymatically active form of the fusion protein is a tetramer with an Mw of approximately 400 kD. The nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-β-glucuronyl carbamate was hydrolyzed by the fusion protein at a hydrolysis rate similar to that of human β-glucuronidase. When the fusion protein was specifically bound to Daudi lymphoma cells, the prodrug induced similar antiproliferative effects as doxorubicin. Thus, it is feasible to construct a eukaryotic fusion protein consisting of a single-chain anti-CD2O antibody and human β- glucuronidase for future use in the activation of anticancer prodrugs in B- cell lymphoma.

Original languageEnglish (US)
Pages (from-to)184-190
Number of pages7
JournalBlood
Volume92
Issue number1
StatePublished - Jul 1 1998
Externally publishedYes

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Enzyme Therapy
Single-Chain Antibodies
Glucuronidase
Prodrugs
Anti-Idiotypic Antibodies
Fusion reactions
Antibodies
Enzymes
Proteins
Tumors
Chemical activation
Cells
B-Cell Lymphoma
Doxorubicin
CD20 Antigens
Molecular Weight
Molecular weight
Neoplasm Antibodies
Amino Acids
Antibody Affinity

ASJC Scopus subject areas

  • Hematology

Cite this

Haisma, H. J., Fleur Sernee, M., Hooijberg, E., Brakenhoff, R. H., Meulen-Muileman, I. H. V. D., Pinedo, H. M., & Boven, E. (1998). Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human β-glucuronidase for antibody-directed enzyme prodrug therapy. Blood, 92(1), 184-190.

Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human β-glucuronidase for antibody-directed enzyme prodrug therapy. / Haisma, Hidde J.; Fleur Sernee, M.; Hooijberg, Erik; Brakenhoff, Ruud H.; Meulen-Muileman, Ida H V D; Pinedo, Herbert M.; Boven, Epie.

In: Blood, Vol. 92, No. 1, 01.07.1998, p. 184-190.

Research output: Contribution to journalArticle

Haisma, HJ, Fleur Sernee, M, Hooijberg, E, Brakenhoff, RH, Meulen-Muileman, IHVD, Pinedo, HM & Boven, E 1998, 'Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human β-glucuronidase for antibody-directed enzyme prodrug therapy', Blood, vol. 92, no. 1, pp. 184-190.
Haisma HJ, Fleur Sernee M, Hooijberg E, Brakenhoff RH, Meulen-Muileman IHVD, Pinedo HM et al. Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human β-glucuronidase for antibody-directed enzyme prodrug therapy. Blood. 1998 Jul 1;92(1):184-190.
Haisma, Hidde J. ; Fleur Sernee, M. ; Hooijberg, Erik ; Brakenhoff, Ruud H. ; Meulen-Muileman, Ida H V D ; Pinedo, Herbert M. ; Boven, Epie. / Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human β-glucuronidase for antibody-directed enzyme prodrug therapy. In: Blood. 1998 ; Vol. 92, No. 1. pp. 184-190.
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