TY - JOUR AB - Antibody-directed enzyme-prodrug therapy (ADEPT) aims at improving the specificity of conventional chemotherapy by employing artificial antibody-enzyme constructs to convert a non-toxic prodrug into a cytotoxic agent specifically localized to the tumor site. The gpA33 antigen is a promising target for ADEPT in colon cancer, as it is expressed by >95% of human colon cancers, but is absent in all non-gastrointestinal tissues. We designed a recombinant fusion construct of a phage display-generated anti-gpA33 single chain fragment, A33scFv, with cytosine deaminase from yeast (CDy), which converts 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU). The resulting construct, A33scFv::CDy, was overexpressed in Pichia pastoris and secreted into culture supernatant. The fusion protein was purified by affinity chromatography on protein L. Silver-staining after SDS-polyacrylamide gel electrophoresis confirmed molecular mass and purity. Antibody binding and specificity were quantified by flow cytometry. The complete ADEPT system was applied in vitro on gpA33-positive LIM1215 cells, assessing cell survival by a fluorescein diacetate assay. Cytotoxicity of the prodrug 5-FC after A33scFv::CDy binding was equimolar to that of 5-FU, and this effect depended specifically on both antibody and enzyme function. These results demonstrate bifunctional activity of the heterogeneous Pichia-produced A33scFv::CDy fusion protein and proof of principle for the ADEPT system proposed herein. AD - CharitéCentrum für Tumormedizin, Medizinische Klinik mit Schwerpunkt Hämatologie und Onkologie CBF, 12200 Berlin, Germany null AU - Coelho,Vânia AU - Dernedde,Jens AU - Petrausch,Ulf AU - Panjideh,Hossein AU - Fuchs,Hendrik AU - Menzel,Christoph AU - Dübel,Stefan AU - Keilholz,Ulrich AU - Thiel,Eckhard AU - Deckert,P.,Markus DA - 2007/10/01 DO - 10.3892/ijo.31.4.951 EP - 957 IS - 4 JO - Int J Oncol PY - 2007 SN - 1019-6439 1791-2423 SP - 951 ST - Design, construction, and in vitro analysis of A33scFv::CDy, a recombinant fusion protein for antibody-directed enzyme prodrug therapy in colon cancer T2 - International Journal of Oncology TI - Design, construction, and in vitro analysis of A33scFv::CDy, a recombinant fusion protein for antibody-directed enzyme prodrug therapy in colon cancer UR - https://doi.org/10.3892/ijo.31.4.951 VL - 31 ER -