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Updated: Oct 10, 2025
Construction of Synthetic Phage Displayed Fab Library with Tailored Diversity
Published on: May 1, 2018
Generation of a nanobody against HER2 tyrosine kinase using phage display library screening for HER2-positive breast
Thomanai Lamtha1, Lueacha Tabtimmai2, Kunan Bangphoomi1
1Department of Biochemistry, Faculty of Sciences, Kasetsart University, Laboratory of Protein Engineering and Bioinformatics, Chatuchak, Bangkok 10900, Thailand.
Abstract:
Human epidermal growth factor receptor 2 (HER2) protein overexpression is found in ~30% of invasive breast carcinomas and in a high proportion of noninvasive ductal carcinomas in situ. Targeted cancer therapy is based on monoclonal antibodies and kinase inhibitors and reflects a new era of cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. Furthermore, there are many disadvantages with the current anti-HER2 drug, including drug resistance and adverse effects. Nanobodies (15 kDa), single-domain antibody (sdAb) fragments, can overcome these limitations. This study produced the recombinant sdAb against the HER2-tyrosine kinase (HER2-TK) domain using phage display technology. Three specific anti-HER2-TK sdAbs were selected for further characterization. Hallmark VHH residue identification and amino acid sequence analysis revealed that clone numbers 4 and 22 were VH antibodies, whereas clone number 17 was a VH H antibody (nanobody). The half-maximal inhibitory concentration of VHH17 exhibited significantly greater HER2 kinase-inhibition activity than the other clones. Consistent with these results, several charges and polar residues of the HER2-TK activation loop that were predicted based on mimotope analysis also appeared in the docking result and interacted via the CDR1, CDR2 and CDR3 loops of VHH17. Furthermore, the cell-penetrable VHH17 (R9 VHH17) showed cell-penetrability and significantly decreased HER2-positive cancer cell viability. Thus, the VH H17 could be developed as an effective therapeutic agent to treat HER2-positive breast cancer.
Insights
Researchers developed a novel nanobody (VHH17) targeting the HER2-tyrosine kinase domain. This smaller, potent nanobody shows promise for treating HER2-positive breast cancer, overcoming limitations of larger antibodies.
Area of Science:
- Biotechnology
- Oncology
- Molecular Biology
Background:
- HER2 protein overexpression occurs in ~30% of invasive breast carcinomas and non-invasive ductal carcinomas.
- Conventional anti-HER2 antibodies face challenges in tumor cell delivery due to their large size (150 kDa).
- Existing anti-HER2 therapies can lead to drug resistance and adverse effects.
Purpose of the Study:
- To produce and characterize recombinant single-domain antibodies (sdAbs) targeting the HER2-tyrosine kinase (HER2-TK) domain.
- To evaluate the therapeutic potential of these sdAbs, particularly nanobodies, for HER2-positive breast cancer.
Main Methods:
- Phage display technology was employed to generate anti-HER2-TK sdAbs.
- Three specific anti-HER2-TK sdAbs were selected and characterized, including sequence analysis and VHH residue identification.
- Kinase inhibition assays, mimotope analysis, docking studies, and cell viability assays were performed.
Main Results:
- Three anti-HER2-TK sdAbs were identified, with VHH17 showing significantly higher HER2 kinase-inhibition activity.
- VHH17 interacted with key residues in the HER2-TK activation loop.
- A cell-penetrable version (R9 VHH17) demonstrated significant reduction in HER2-positive cancer cell viability.
Conclusions:
- The nanobody VHH17 exhibits potent HER2 kinase inhibition and cell-penetrating capabilities.
- VHH17 holds potential as an effective therapeutic agent for HER2-positive breast cancer.
- Nanobodies offer an alternative to conventional antibodies for targeted cancer therapy.
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