Construction and characterization of a fusion protein of single-chain anti-CD20 antibody and human beta-glucuronidase for antibody-directed enzyme prodrug therapy.
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 beta-glucuronidase for the activation of the nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) phenyl] O-beta-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 beta-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-beta-glucuronyl carbamate was hydrolyzed by the fusion protein at a hydrolysis rate similar to that of human beta-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 beta-glucuronidase for future use in the activation of anticancer prodrugs in B-cell lymphoma.
Haisma
H J
HJ
Departments of Medical Oncology and Otolaryngology/Head and Neck Surgery, Academic Hospital Vrije Universiteit, Amsterdam, The Netherlands. hj.haisma.oncol@med.vu.nl
Sernee
M F
MF
Hooijberg
E
E
Brakenhoff
R H
RH
vd Meulen-Muileman
I H
IH
Pinedo
H M
HM
Boven
E
E
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology*; D000918:Antibody Specificity; D018951:Antigens, CD20 / Q000276:immunology*; D001483:Base Sequence; D005966:Glucuronidase / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology* / Q000494:pharmacology*; D007135:Immunoglobulin Variable Region / Q000276:immunology / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D011355:Prodrugs / Q000494:pharmacology*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology
D000906:Antibodies; D018951:Antigens, CD20; D007140:Immunoglobulin Fab Fragments; D007135:Immunoglobulin Variable Region; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D005966:Glucuronidase