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

Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Ascione, Alessandro; Dupuis, Maria Luisa; Gellini, Mara; Barca, Stefano; Cianfriglia, Maurizio

Zamboni, Silvia; Mallano, Alessandra; Flego, Michela; Ascione, Alessandro; Dupuis, Maria Luisa; Gellini, Mara; Barca, Stefano; Cianfriglia, Maurizio.

Afiliación

Zamboni S; 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

Int J Oncol ; 32(6): 1245-51, 2008 Jun.

Article en En | MEDLINE | ID: mdl-18497986

RESUMEN

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.

Asunto(s)

Anticuerpos Monoclonales/genética; Apoptosis/efectos de los fármacos; Antígeno Carcinoembrionario/inmunología; Neoplasias Colorrectales/patología; Citosina Desaminasa/genética; Melanoma/patología; Proteínas Recombinantes de Fusión/farmacología; Secuencia de Aminoácidos; Western Blotting; Neoplasias Colorrectales/metabolismo; Citosina Desaminasa/metabolismo; Electroforesis en Gel de Poliacrilamida; Ensayo de Inmunoadsorción Enzimática; Flucitosina/farmacología; Fluorouracilo/metabolismo; Ingeniería Genética; Humanos; Región Variable de Inmunoglobulina/genética; Melanoma/metabolismo; Datos de Secuencia Molecular; Saccharomyces cerevisiae/enzimología; Células Tumorales Cultivadas/efectos de los fármacos

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Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Proteínas Recombinantes de Fusión / Neoplasias Colorrectales / Antígeno Carcinoembrionario / Apoptosis / Citosina Desaminasa / Melanoma / Anticuerpos Monoclonales Límite: Humans Idioma: En Revista: Int J Oncol Asunto de la revista: NEOPLASIAS Año: 2008 Tipo del documento: Article País de afiliación: Italia Pais de publicación: Grecia

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