title: Genetic construction, expression, and characterization of a single chain anti-CEA antibody fused to cytosine deaminase from yeast.
abstract: We report the genetic construction and expression of a fusion protein between a single chain fragment variable (scFv) human antibody (E8) specific for CEA cell surface antigen and yeast cytosine deaminase (yCD). Sequences encoding for the scFvE8 human monoclonal antibody recognizing an epitope shared by CEACAM1, CEACAM3 and CEACAM5 isoforms were assembled with a monomer of yCD. The construct was placed under the transcriptional regulation of the lac promoter, and in frame with 6xHis tag for protein purification. After transformation and induction of E. coli, the protein was recovered from cell lysates and processed for purification. The scFvE8:yCD fusion protein possessed the binding specificity for melanoma (Mel P5) and colon carcinoma (LoVo) cell lines similar to its cognate human scFv antibody. The scFv8:yCD system showed the ability to render tumor cells susceptible to the far less toxic substrate 5-fluorocytosine (5-FC) by its enzymatic conversion into 5-fluorouracil (5-FU). In vitro pre-treatment of Mel P5 and LoVo cell lines with scFvE8:yCD followed by cell washing and incubation with 5-FC, resulted in significant cell killing supporting the utility of this fusion protein as an agent for tumor-selective prodrug activation. This study shows the feasibility of constructing fusion proteins in a prokaryotic cell based system consisting of a human scFv antibody and yCD to convert the antifungal agent 5-FC to 5-FU, one of the widely used anticancer agent.
authors.affiliation: Section of Pharmacogenetics, Drug Resistance and Experimental Therapeutics, Department of Drug Research and Evaluation, Istituto Superiore di Sanità, I-00161 Rome, Italy. silvia.zamboni@iss.it
authors.forename: Silvia
authors.initials: S
authors.lastname: Zamboni
authors.forename: Alessandra
authors.initials: A
authors.lastname: Mallano
authors.forename: Michela
authors.initials: M
authors.lastname: Flego
authors.forename: Alessandro
authors.initials: A
authors.lastname: Ascione
authors.forename: Maria Luisa
authors.initials: ML
authors.lastname: Dupuis
authors.forename: Mara
authors.initials: M
authors.lastname: Gellini
authors.forename: Stefano
authors.initials: S
authors.lastname: Barca
authors.forename: Maurizio
authors.initials: M
authors.lastname: Cianfriglia
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002272:Carcinoembryonic Antigen / Q000276:immunology*; D015179:Colorectal Neoplasms / Q000378:metabolism / Q000473:pathology*; D043525:Cytosine Deaminase / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005437:Flucytosine / Q000494:pharmacology; D005472:Fluorouracil / Q000378:metabolism; D005818:Genetic Engineering; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D014407:Tumor Cells, Cultured / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D002272:Carcinoembryonic Antigen; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D005437:Flucytosine; D043525:Cytosine Deaminase; D005472:Fluorouracil