CONSTRUCTION AND CHARACTERIZATION OF A FUSION PROTEIN OF SINGLE-CHAINANTI-CD20 ANTIBODY AND HUMAN BETA-GLUCURONIDASE FOR ANTIBODY-DIRECTEDENZYME PRODRUG THERAPY

The CD20 antigen is an attractive target for specific treatment of B-c ell 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 co nstructed a fusion protein of the single-chain Fv anti-CD20 mouse mono clonal antibody (MoAb) 1H4 and human beta-glucuronidase for the activa tion of the nontoxic prodrug N-[4-doxorubicin-N-carbonyl(-oxymethyl) p henyl] O-beta-glucuronyl carbamate to doxorubicin at the tumor site. T he cDNAs encoding the light- and heavy-chain variable regions of 1H4 w ere cloned, joined by a synthetic sequence encoding a 18-amino acid li nker and fused to human beta-glucuronidase by a synthetic sequence enc oding a 6-amino acid linker. An antibody-enzyme fusion protein-produci ng cell line was established by transfection of the construct into hum an embryonic kidney 293/EBNA cells. The yield of active fusion protein was 100 ng/mL transfectoma supernatant. Antibody affinity, antibody s pecificity, and enzyme activity were fully retained by the fusion prot ein. Immunoprecipitation and analysis by sodium dodecyl sulfate-polyac rylamide gel electrophoresis (SDS PAGE) showed that the fusion protein has a relative molecular weight (Mw) of 100 kD under denaturing condi tions. 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) p henyl] O-beta-glucuronyl carbamate was hydrolyzed by the fusion protei n at a hydrolysis rate similar to that of human beta-glucuronidase. Wh en 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 consisti ng of a single chain anti-CD20 antibody and human beta-glucuronidase f or future use in the activation of anticancer prodrugs in B-cell lymph oma. (C) 1998 by The American Society of Hematology.