Genetic construction, expression, and characterization of a single chain anti-CEA antibody fused to cytosine deaminase from yeast

  • Authors:
    • Silvia Zamboni
    • Alessandra Mallano
    • Michela Flego
    • Alessandro Ascione
    • Maria Luisa Dupuis
    • Mara Gellini
    • Stefano Barca
    • Maurizio Cianfriglia
  • View Affiliations

  • Published online on: June 1, 2008     https://doi.org/10.3892/ijo.32.6.1245
  • Pages: 1245-1251
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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.

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June 2008
Volume 32 Issue 6

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Spandidos Publications style
Zamboni S, Mallano A, Flego M, Ascione A, Dupuis ML, Gellini M, Barca S and Cianfriglia M: Genetic construction, expression, and characterization of a single chain anti-CEA antibody fused to cytosine deaminase from yeast. Int J Oncol 32: 1245-1251, 2008
APA
Zamboni, S., Mallano, A., Flego, M., Ascione, A., Dupuis, M.L., Gellini, M. ... Cianfriglia, M. (2008). Genetic construction, expression, and characterization of a single chain anti-CEA antibody fused to cytosine deaminase from yeast. International Journal of Oncology, 32, 1245-1251. https://doi.org/10.3892/ijo.32.6.1245
MLA
Zamboni, S., Mallano, A., Flego, M., Ascione, A., Dupuis, M. L., Gellini, M., Barca, S., Cianfriglia, M."Genetic construction, expression, and characterization of a single chain anti-CEA antibody fused to cytosine deaminase from yeast". International Journal of Oncology 32.6 (2008): 1245-1251.
Chicago
Zamboni, S., Mallano, A., Flego, M., Ascione, A., Dupuis, M. L., Gellini, M., Barca, S., Cianfriglia, M."Genetic construction, expression, and characterization of a single chain anti-CEA antibody fused to cytosine deaminase from yeast". International Journal of Oncology 32, no. 6 (2008): 1245-1251. https://doi.org/10.3892/ijo.32.6.1245