[Preparation and identification of single-chain fragment variable against epidermal growth factor receptor 3].

Objective To express, purify and identify the single-chain fragment variable (scFv) against human epidermal growth factor receptor 3 (HER3). Methods We searched NCBI for the light chain sequence and heavy chain sequence of anti-HER3 mAb LJM716 to construct the gene of scFv against HER3. The recombinant expression vector pGAPZαA-anit-HER3-scFv was constructed using the constitutive expression vector pGAPZαA and then electro-transformed into Pichia Pastoris X-33 to screen the strains with high expression of the protein of interest. After shaking flask fermentation, the supernatant was purified by hydrophobic chromatography and metal ion affinity chromatography. The purified product was identified by Western blotting and ELISA. Results The anti-HER3-scFv gene was successfully constructed and the strains with high expression of anti-HER3-scFv were obtained. The anti-HER3-scFv was purified to a purity of more than 95% by two-step chromatography, and the purified yield was 192 mg/L. Western blotting showed that the anti-HER3-scFv was correctly expressed and ELISA indicated that anti-HER3-scFv could specifically recognize HER3. Conclusion The anti-HER3-scFv has been successfully prepared.

Preparation and characterization of humanized nanobodies targeting the dimer interface of epidermal growth factor receptor (EGFR).

Epidermal growth factor receptor (EGFR) is an effective target for the treatment of many epithelial cancers. However, EGFR inhibitors have low clinical response rates and are prone to drug resistance arising from mutations and heterodimerization of EGFR. Therefore, targeting the highly conserved dimer interface of EGFR may be an effective strategy for improving the clinical response of anti-EGFR therapies. Nanobodies have significant advantages over conventional antibodies in terms of size, solubility, stability and cost-effectiveness. To investigate the feasibility of nanobodies targeting the dimer interface of EGFR as novel anticancer drugs, four nanobodies were screened from a commercial humanized nanobody phage antibody library using the EGFR237-267 peptide from the ß-hairpin loop of the dimer interface of EGFR as the antigen. A nanobody with an isoelectric point (pI) of 8.6, named EGFR dimer Nb77, was selected for further analysis of anticancer activities. EGFR dimer Nb77 was expressed in Escherichia coli Shuffle T7-B as a soluble (His)6-tagged protein and purified by a CM Sepharose column and a nickel-nitrilotriacetic acid (Ni-NTA) column. Purified EGFR dimer Nb77 could specifically bind to the surface of EGFR-overexpressing A431 cells in a dose-dependent and ligand-dependent manner, and this nanobody could effectively inhibit the growth of the tumour cells, with an inhibition rate similar to that of the monoclonal antibody EGFR dimer 5G9, which also targets the dimer interface of EGFR. This work is the first to prove that nanobodies targeting the dimer interface of EGFR have promising prospects as anticancer agents.