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Production and Conjugation of Truncated Recombinant Diphtheria Toxin to VEGFR-2 Specific Nanobody and Evaluation of its Cytotoxic Effect on PC-3 Cell Line

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Abstract

Immunotoxins have represented a great potency in targeted therapeutics to encounter tumors. They consist of a protein toxin conjugated to a targeting moiety, which recognizes a specific antigen on surface of cancer cells and accordingly induces cell death by toxin segment. The targeting part could be a nanobody, which is a group of antibodies composed of an only functional single variable heavy chain (VHH).Therefore, this study was done to produce an immunotoxin (VGRNb-DT) by chemical conjugation of a truncated diphtheria toxin moiety to an anti-vascular endothelial growth factor receptor 2(VEGFR-2) nanobody, and to identify effectiveness of immunotoxin in recognizing the VEGFR-2- positive cancer cells and inhibiting cell growth and survival. Diphtheria toxin was expressed and purified by nickel affinity chromatography, and accordingly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis confirmed its expression. Function of heterobifunctional crosslinkers, Sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate), and SATP (N-succinimidyl-S- acetylthiopropionate) for bioconjugation purposes was acknowledged by cation exchange high-performance liquid chromatography (HPLC). Cytotoxicity of immunotoxin was evaluated on the VEGFR-2 positive PC-3 cell line by MTT assay. Overexpression of VEGFR-2 in the PC-3 cell line allowed immunotoxin to recognize them by anti-VEGFR-2 nanobodies. The concentrations above 5 μg/ml represented a significant decrease in cell survival rate in PC-3 cells compared to HEK293 cells (VEGFR-2 negative cells) as controls.VGRNb-DT demonstrated a successful bioconjugation; furthermore, variable concentrations were correlated with cell death in prostate cancer PC-3 cells.

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Data Availability

The data used to support the findings of this study are included within the article.

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Funding

This work was supported by the Iran University of Medical sciences, under grant number 33778.

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Authors and Affiliations

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

    Samira Shajari, Mohammad Morad Farajollahi & Parastoo Tarighi

  2. Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

    Mahdi Behdani

Authors
  1. Samira Shajari
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  2. Mohammad Morad Farajollahi
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  3. Mahdi Behdani
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  4. Parastoo Tarighi
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Contributions

PT and MF: were responsible for the conception and design of the research. Methodology was done by all authors. Material preparation was performed by PT, MF, and MB. SS: performed the experiments and collected data. PT and MF: interpreted the results. SS: drafted manuscript and all the authors approved the draft of the submitted manuscript.

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Correspondence to Mohammad Morad Farajollahi or Parastoo Tarighi.

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Shajari, S., Farajollahi, M.M., Behdani, M. et al. Production and Conjugation of Truncated Recombinant Diphtheria Toxin to VEGFR-2 Specific Nanobody and Evaluation of its Cytotoxic Effect on PC-3 Cell Line. Mol Biotechnol 64, 1218–1226 (2022). https://doi.org/10.1007/s12033-022-00485-1

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  • DOI: https://doi.org/10.1007/s12033-022-00485-1

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