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Updated: Jun 2, 2026
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
Highly efficient production of anti-HER2 scFv antibody variant for targeting breast cancer cells
Silvia Sommaruga1, Alessio Lombardi, Agnese Salvadè
1Dipartimento di Biotecnologie e Bioscienze, Università di Milano-Bicocca, 20126 Milan, Italy.
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.
Insights
Researchers produced a highly purified anti-HER2 single-chain variable fragment (scFv) antibody using Pichia pastoris. This antibody shows high affinity and selectivity for HER2-positive breast cancer cells, indicating potential diagnostic and therapeutic applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Oncology
Background:
- Human Epidermal Growth Factor Receptor 2 (HER2) is overexpressed in 30% of breast cancers.
- Targeting HER2 dimerization with antibodies can inhibit tumor cell proliferation.
- Single-chain variable fragments (scFv) offer a smaller, targeted alternative to full antibodies.
Purpose of the Study:
- To produce and purify a soluble anti-HER2 scFv antibody using Pichia pastoris.
- To evaluate the expression levels and functionality of the anti-HER2 scFv.
- To assess the potential diagnostic and therapeutic applications of the scFv antibody.
Main Methods:
- The gene for scFv800E6 with a 6× His-tag was inserted into the Pichia pastoris expression vector pPICZα.
- Pichia pastoris was cultured and induced for scFv expression using methanol and glycerol.
- Purified scFv was analyzed for functionality using cytofluorimetry and immunofluorescence on HER2-positive MCF7 cells.
Main Results:
- Pichia pastoris yielded 10 mg/L of highly purified anti-HER2 scFv, a significantly higher level than previously reported.
- The purified scFv demonstrated good affinity and high selectivity for the HER2 receptor on cancer cells.
- Optimal expression conditions involved 0.5% methanol and 0.8% glycerol after 48 hours of induction.
Conclusions:
- Pichia pastoris is a highly effective host for high-level expression of antibody fragments.
- The anti-HER2 scFv800E6 shows promise for diagnostic and therapeutic applications in HER2-positive breast cancers.
- This study validates the use of scFv antibodies for targeted cancer therapy.
