Production and characterization of a colon cancer-specific immunotoxin based on the fungal ribotoxin α-sarcin

• Published in: Protein engineering, design and selection design and selection, 2012-08, Vol.25 (8), p.425-435
• Main Authors: Carreras-Sangrà, Nelson, Tomé-Amat, Jaime, García-Ortega, Lucía, Batt, Carl A., Oñaderra, Mercedes, Martínez-del-Pozo, Álvaro, Gavilanes, José G., Lacadena, Javier
• Format: Article
• Language: English
• Subjects: Cell Line, Tumor; Colonic Neoplasms - drug therapy; Colonic Neoplasms - immunology; Colonic Neoplasms - metabolism; Endoribonucleases - chemistry; Endoribonucleases - genetics; Endoribonucleases - metabolism; Flow Cytometry; Fungal Proteins - chemistry; Fungal Proteins - genetics; Fungal Proteins - metabolism; Humans; Immunotoxins - chemistry; Immunotoxins - genetics; Immunotoxins - metabolism; Membrane Glycoproteins - chemistry; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Microscopy, Fluorescence; Pichia - genetics; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Single-Chain Antibodies - chemistry; Single-Chain Antibodies - genetics; Single-Chain Antibodies - metabolism
• ISSN: 1741-0126; 1741-0134
• DOI: 10.1093/protein/gzs032

A single-chain fusion protein that directed the cytolytic activity of α-sarcin to A33 tumor antigen expressing cells was constructed and shown to effectively kill targeted cells. Glycoprotein A33 (GPA33) is a well-known colon cancer marker and a humanized antibody against it was used to target the α-sarcin. The fungal ribotoxin α-sarcin is one of the most potent and specific toxins known. It is small, protease resistant, thermostable and highly efficient towards the inactivation of ribosomes. This work describes the production and characterization of an immunotoxin resulting from fusing the single-chain variable fragment (scFv) of the monoclonal antibody that targets GPA33 to fungal α-sarcin. This chimeric protein (scFvA33αsarcin), produced in Pichia pastoris and purified in high yield was proven to be properly folded, active, specific and stable. It showed high specific toxicity against GPA33-positive tumoral cell lines providing scientific evidence to sustain that scFvA33αsarcin is a good immunotherapeutic candidate against GPA33-positive colon carcinomas.