title: Highly efficient production of anti-HER2 scFv antibody variant for targeting breast cancer cells.
abstract: The human epidermal growth factor receptor 2 (HER2) is a transmembrane tyrosine kinase receptor overexpressed in 30% of human breast cancers. One of the mechanisms by which tumor cell proliferation can be inhibited consists in hampering HER2 dimerization by targeting its extracellular domain with specific antibodies. In recent clinical practice, a valuable alternative to entire IgGs resides in the use of smaller molecules, such as single-chain variable fragments (scFv), developed for selective molecular targeting. In this paper, we report on the production and purification of a soluble anti-HER2 scFv antibody secreted by Pichia pastoris. The gene encoding scFv800E6 with an additional 6× His-tag at the 3'-end was inserted into the expression vector pPICZα and transformed in P. pastoris. The highest expression level was obtained in presence of 0.5% methanol and 0.8% glycerol in the culture medium after 48 h of induction. The use of P. pastoris proved very valuable as an expression system, allowing the isolation of 10 mg/L of highly purified antibody, remarkably higher than previously reported data. The functionality of purified anti-HER2 scFv was assessed by cytofluorimetry and immunofluorescence on HER2-positive MCF7 breast cancer cells, showing good affinity and high selectivity for the target membrane receptor. These findings confirm that P. pastoris is a suitable host for high level expression of antibody fragments and highlight the potential role of scFv800E6 in diagnostic and therapeutic application.
authors.affiliation: Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, 20126 Milan, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Sommaruga
authors.forename: Alessio
authors.initials: A
authors.lastname: Lombardi
authors.forename: Agnese
authors.initials: A
authors.lastname: Salvadè
authors.forename: Serena
authors.initials: S
authors.lastname: Mazzucchelli
authors.forename: Fabio
authors.initials: F
authors.lastname: Corsi
authors.forename: Patrizia
authors.initials: P
authors.lastname: Galeffi
authors.forename: Paolo
authors.initials: P
authors.lastname: Tortora
authors.forename: Davide
authors.initials: D
authors.lastname: Prosperi
mesh_terms: D001943:Breast Neoplasms / Q000276:immunology*; D045744:Cell Line, Tumor; D005260:Female; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006801:Humans; D018613:Microscopy, Confocal; D010843:Pichia / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D057127:Single-Chain Antibodies / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D018719:Erb-b2 Receptor Tyrosine Kinases; D057127:Single-Chain Antibodies; C508053:ERBB2 protein, human