Preparation of an engineered safer immunotoxin against colon carcinoma based on the ribotoxin hirsutellin A.
Immunotoxins are chimeric proteins composed of an antibody domain that specifically directs the action of the toxic domain, resulting in the death of the targeted cells. Over recent years, immunotoxins have been widely studied and the number of different constructions has increased exponentially. Protein engineering has allowed the design of optimized versions of immunotoxins with an improved tumor binding affinity, stability or cytotoxic efficacy, although sometimes this has compromised the safety of the patient in terms of undesirable adverse secondary reactions. A triple mutant at three Trp residues (HtA3ΔW) of the ribotoxin hirsutellin A retains its specific ribonucleolytic activity, although cell internalization capacity is lacking. This toxin variant has been fused to the single chain variable fragment A33 (scFvA33). This immunoconjugate (IMTXA33HtA3ΔW) was produced in the methylotrophic yeast Pichia pastoris and purified using nickel-nitrilotriacetic acid affinity chromatography. Both target and toxic domains were characterized. The immunotoxin showed an exquisite specific binding against GPA33-positive culture cells, which results in the death of the targeted cells because of specific ribonucleolytic activity against ribosomes of the engineered hirsutellin A variant. IMTXA33HtA3ΔW represents a promising structure in the search for an improved immunotoxin without compromising the safety of patients.
Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain.|Department of Food Science, Cornell University, Ithaca, NY, USA.
Jaime
J
Tomé-Amat
Department of Vascular Biology and Inflammation, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain.
Elías
E
Herrero-Galán
Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain.
Mercedes
M
Oñaderra
Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain.
Álvaro
Á
Martínez-Del-Pozo
Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain.
José G
JG
Gavilanes
Departamento de Bioquímica y Biología Molecular I, Universidad Complutense, Madrid, Spain.
Javier
J
Lacadena
D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000302:isolation & purification; D003110:Colonic Neoplasms / Q000188:drug therapy; D004354:Drug Screening Assays, Antitumor; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D019073:HT29 Cells; D006801:Humans; D000922:Immunotoxins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D000900:Anti-Bacterial Agents; D005656:Fungal Proteins; C104258:GPA33 protein, human; C121692:Hirsutellin A protein, Hirsutella thompsonii; D000922:Immunotoxins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins
GPA33; colon cancer; hirsutellin A; immunotoxin; ribotoxin