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.

Engineering production of functional scFv antibody in E. coli by co-expressing the molecule chaperone Skp

“…scFv specific to any particular antigen may be easily generated by Phage display (Wang et al, 2006), and this scFv have been developed to select molecular targets in cancer research such as in lymphatic invasion vessels, colon cancer and hepatocarcinoma (Rinderknecht et al, 2010; Sakai et al, 2010; Sommaruga et al, 2011). Furthermore, scFv has been extensively used to generate ligands for detecting pathogenic germs in vitro or in vivo (Wang et al, 2007, 2008a, b; Zhang et al, 2010; Cattepoel et al, 2011).…”

Screening for a Single-Chain Variable-Fragment Antibody That Can Effectively Neutralize the Cytotoxicity of the Vibrio parahaemolyticus Thermolabile Hemolysin

“…It has also been used for selective molecular targeting in cancer research for conditions such as lymphatic invasion vessels, colon cancer, and hepatocarcinoma (27,29,33,43). Furthermore, scFv antibody generation has been used extensively to generate ligands for detecting pathogenic germs in vitro and in vivo (8,22,38,39).…”

Novel Bifunctional Single-Chain Variable Antibody Fragments to Enhance Virolysis by Complement: Generation and Proof-of-Concept

“…The methylotrophic yeast Pichia pastoris provides several advantages for heterologous protein expression including their posttranslational protein processing, notably glycosylation and disulphide-bond formation. Single-chain antibodies have already been successfully produced in Pichia pastoris [29,38].…”

“…Our initial attempts (i.e., 0.5% methanol/24 h) revealed proper overexpression of scFv. We further investigated the influence of different methanol concentrations on the expression rate, supplementing the media with casamino acids (CA) to serve as protease-"prey" or addition of glycerol, which facilitates the carbon source adaption process [29]. Samples were periodically analyzed by western-blotting.…”