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Synthesis of the scFv fragment of anti-Frizzled-7 antibody and evaluation of its effects on triple-negative breast cancer in vitro study

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Abstract

Objectives

Among the most promising antibody formats in terms of inhibiting carcinogenesis are single-stranded variable fragments, whose targeted binding to the Fzd7 receptor has been proven effective at suppressing tumorigenesis. In this study, we investigated the effectiveness of an anti-Fzd7 antibody fragment against both tumor growth and metastasis of breast cancer cells.

Methods

To develop anti-Fzd7 antibodies, bioinformatics approaches were used and the antibodies were expressed recombinantly in E. coli BL21 (DE3). The expression of anti-Fzd7 fragments was verified by Western blotting. Analysis of the antibody's binding capacity to Fzd7 was conducted by flow cytometry. Cell death and apoptosis were assessed by MTT and Annexin V/PI assays. The transwell migration and invasion assays, as well as the scratch method, were used to evaluate cell motility and invasiveness.

Results

The anti-Fzd7 antibody was expressed successfully as a single band of 31 kDa. It could bind to 21.5% of MDA-MB-231 cells, as opposed to only 0.54% of SKBR-3 cells as negative control. According to MTT assay, induced apoptosis was 73.7% in MDA-MB-231 cells, compared with 29.5% in SKBR-3 cells. Also, the antibody exerted a significant inhibitory effect of 76% and 58% on migration and invasion of MDA-MB-231 cells, respectively.

Conclusion

The recombinantly developed anti-Fzd7 scFv of this study could exhibit significant antiproliferative and antimigratory properties, along with a high apoptosis-inducing potential, making it suitable for the immunotherapy of triple negative breast cancer.

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Data availability

The authors confirm that the data justifying the findings of this study are available within the manuscript.

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Acknowledgements

None.

Funding

This study was financially supported by a grant (grant number: 1656) of the Research and Technology Council of Semnan University of Medical Sciences.

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Authors and Affiliations

  1. Department of Medical Biotechnology, Faculty of Medicine, Semnan University of Medical Sciences, Semnan, Iran

    Ebrahim Khodaverdi, Ali Akbar Shabani & Hamid Madanchi

  2. Student Research Committee, Semnan University of Medical Sciences, Semnan, Iran

    Ebrahim Khodaverdi

  3. Nervous System Stem Cells Research Center, Semnan University of Medical Sciences, Semnan, Iran

    Hamid Madanchi

  4. Drug Design and Bioinformatics Unit, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

    Hamid Madanchi

  5. Recombinant Proteins Department, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran

    Leila Farahmand

Authors
  1. Ebrahim Khodaverdi
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  2. Ali Akbar Shabani
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  4. Leila Farahmand
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Contributions

LF and AS developed the methodology. EKh conducted the experiments, contributed to the data analysis and wrote the manuscript. HM contributed to scientific assist and revised the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Ali Akbar Shabani or Leila Farahmand.

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Conflict of Interest

The authors declare that they have no conflict of interests.

Ethical approval

This project was in accordance with the national norms, the ethical principles and standards for conducting medical research in Iran and evaluated by Motamed Cancer Institute-Academic Centre for Education, Culture and Research. This institution performed its reviews based on the United States Public Health Service (USPHS) regulations and applicable federal and local laws. Approved ID: IR.SEMUMS.REC.1398.252.

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Khodaverdi, E., Shabani, A.A., Madanchi, H. et al. Synthesis of the scFv fragment of anti-Frizzled-7 antibody and evaluation of its effects on triple-negative breast cancer in vitro study. Clin Transl Oncol 26, 231–238 (2024). https://doi.org/10.1007/s12094-023-03242-1

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