Production and characterization of single-chain variable fragment antibodies targeting the breast cancer tumor marker nectin-4

Front Immunol. 2024 Jan 15:14:1292019. doi: 10.3389/fimmu.2023.1292019. eCollection 2023.

Abstract

Background: Nectin-4 is a novel biomarker overexpressed in various types of cancer, including breast cancer, in which it has been associated with poor prognosis. Current literature suggests that nectin-4 has a role in cancer progression and may have prognostic and therapeutic implications. The present study aims to produce nectin-4-specific single-chain variable fragment (scFv) antibodies and evaluate their applications in breast cancer cell lines and clinical specimens.

Methods: We generated recombinant nectin-4 ectodomain fragments as immunogens to immunize chickens and the chickens' immunoglobulin genes were amplified for construction of anti-nectin-4 scFv libraries using phage display. The binding capacities of the selected clones were evaluated with the recombinant nectin-4 fragments, breast cancer cell lines, and paraffin-embedded tissue sections using various laboratory approaches. The binding affinity and in silico docking profile were also characterized.

Results: We have selected two clones (S21 and L4) from the libraries with superior binding capacity. S21 yielded higher signals when used as the primry antibody for western blot analysis and flow cytometry, whereas clone L4 generated cleaner and stronger signals in immunofluorescence and immunohistochemistry staining. In addition, both scFvs could diminish attachment-free cell aggregation of nectin-4-positive breast cancer cells. As results from ELISA indicated that L4 bound more efficiently to fixed nectin-4 ectodomain, molecular docking analysis was further performed and demonstrated that L4 possesses multiple polar contacts with nectin-4 and diversity in interacting residues.

Conclusion: Overall, the nectin-4-specific scFvs could recognize nectin-4 expressed by breast cancer cells and have the merit of being further explored for potential diagnostic and therapeutic applications.

Keywords: breast cancer; nectin-4; phage display; single-chain variable fragment antibody; tumor marker.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers, Tumor
  • Chickens
  • Molecular Docking Simulation
  • Nectins
  • Neoplasms*
  • Single-Chain Antibodies* / genetics

Substances

  • Single-Chain Antibodies
  • Nectins
  • Biomarkers, Tumor

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The study was supported by funding from the Ministry of Science and Technology of Taiwan (MOST109-2622-B-038-005 to S-JL; MOST109-2320-B-038-053 to Y-YY; MOST107-2320-B-038-034-MY3 and MOST110-2320-B-038-041-MY3 to L-TL), the Ministry of Health and Welfare of Taiwan (MOHW110-TDU-B-212-144014 to Y-YY), the Aim for the Top University Project – Cancer Translational Center of Taipei Medical University (TMUTOP103005-4 to L-TL), and the 2023 Neuro TMULille Program (111-3800-014-400 to L-TL). The funders have no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.