title: Design, construction, and in vitro analysis of A33scFv::CDy, a recombinant fusion protein for antibody-directed enzyme prodrug therapy in colon cancer.
abstract: 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.
authors.affiliation: CharitéCentrum für Tumormedizin, Medizinische Klinik mit Schwerpunkt Hämatologie und Onkologie CBF, 12200 Berlin, Germany.
authors.forename: Vânia
authors.initials: V
authors.lastname: Coelho
authors.forename: Jens
authors.initials: J
authors.lastname: Dernedde
authors.forename: Ulf
authors.initials: U
authors.lastname: Petrausch
authors.forename: Hossein
authors.initials: H
authors.lastname: Panjideh
authors.forename: Hendrik
authors.initials: H
authors.lastname: Fuchs
authors.forename: Christoph
authors.initials: C
authors.lastname: Menzel
authors.forename: Stefan
authors.initials: S
authors.lastname: Dübel
authors.forename: Ulrich
authors.initials: U
authors.lastname: Keilholz
authors.forename: Eckhard
authors.initials: E
authors.lastname: Thiel
authors.forename: P Markus
authors.initials: PM
authors.lastname: Deckert
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics*; D000918:Antibody Specificity; D019677:Artificial Gene Fusion; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D043525:Cytosine Deaminase / Q000276:immunology / Q000494:pharmacology*; D005434:Flow Cytometry; D005437:Flucytosine / Q000494:pharmacology*; D005472:Fluorouracil / Q000378:metabolism; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000276:immunology* / Q000494:pharmacology; D010843:Pichia / Q000201:enzymology; D011355:Prodrugs / Q000494:pharmacology*; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000494:pharmacology*; D014407:Tumor Cells, Cultured