Construction and Characterization of a Fusion Protein of Single-Chain
Anti-CD20 Antibody and Human 
-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, and
Epie Boven
From the Departments of Medical Oncology and Otolaryngology/Head and
Neck Surgery, Academic Hospital Vrije Universiteit, Amsterdam; and the
Department of Immunology, The Netherlands Cancer Institute, Antoni van
Leeuwenhoek Huis, Amsterdam, The Netherlands.
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 
-glucuronidase for the
activation of the nontoxic prodrug
N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O--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
-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--glucuronyl
carbamate was hydrolyzed by the fusion protein at a hydrolysis rate
similar to that of human -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-CD20 antibody and human -glucuronidase for future use in the
activation of anticancer prodrugs in B-cell lymphoma.
Blood, Vol. 92 No. 1 (July 1), 1998:
pp. 184-190
© 1998 by The American Society of Hematology.
