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Design and Production of a Novel Anti-PD-1 Nanobody by CDR Grafting and Site-Directed Mutagenesis Approach

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Abstract

Programmed cell death protein-1 (PD-1) is a membrane protein expressed on the surface of activated T-cells, B-cells, natural killer cells, dendritic cells, macrophages, and monocytes. Inhibition of the PD-1/PD-L1 interaction by monoclonal antibodies (mAbs) has many therapeutic benefits and has led to a major advance in the treatment of various types of tumors. Due to the large size and immunogenicity of the antibodies (Abs), using small molecules such as nanobodies (nanobodies or VHH) is more appropriate for this purpose. In this research, the complementarity determining regions (CDR) grafting method was used to produce anti-PD-1 nanobody. For producing the grafted anti-PD-1 nanobody, CDRs from the tislelizumab mAb were grafted into the frameworks of a nanobody whose sequence is similar to the tislelizumab mAb. Also, the site-directed mutagenesis method was used to produce two mutated anti-PD-1 nanobodies which increased the affinity of grafted anti-PD-1 nanobodies. Two amino acid substitutions (Tyr97Arg and Tyr102Arg) in the VHH-CDR3 were used to improve grafted nanobody affinity and the binding capacity of the mutated nanobodies. The binding of the anti-PD-1 nanobodies and PD-1 antigen (Ag) was confirmed by Dot blot, western blot, and indirect ELISA analysis. According to the results of these in silico and in vitro studies, the binding between grafted and mutated nanobodies with PD-1 was confirmed. Also, our findings show that site-directed mutagenesis can increase the affinity of nanobodies.

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Data Availability

All experimental data generated or analyzed during this study are included in the article.

Abbreviations

mAb:

Monoclonal antibodies

PD-1:

Programmed cell death protein-1

Ab:

Antibody

CDR:

Complementarity determining regions

Ag:

Antigen

ICI:

Immune checkpoint inhibitor

VH:

Heavy chain

PVDF:

Polyvinylidene fluoride

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Acknowledgements

The authors thank the Yazd University Research Council for the support of this research.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. Department of Biology, Yazd University, Yazd, Iran

    Mahsa Mirzaei, Shima Mirhoseini, Mohammad Mehdi Heidari & Mehri Khatami

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  1. Mahsa Mirzaei
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  2. Shima Mirhoseini
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  3. Mohammad Mehdi Heidari
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Contributions

Conceptualization, M.M.H. and M.K.; methodology, M.M. and S.M.; formal analysis and investigation, M.M.H., M.K., M.M. and S.M; writing—original draft preparation, M.M.; writing—review and editing, J M.M.H. and M.K.; supervision, M.M.H. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Mohammad Mehdi Heidari.

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Mirzaei, M., Mirhoseini, S., Heidari, M.M. et al. Design and Production of a Novel Anti-PD-1 Nanobody by CDR Grafting and Site-Directed Mutagenesis Approach. Mol Biotechnol (2024). https://doi.org/10.1007/s12033-024-01162-1

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