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Development of a Functional Nanobody Targeting Programmed Cell Death Protein-1 as Immune Checkpoint Inhibitor
Mohammad Hosseininejad-Chafi1,2, Zahra Kianmehr1, Kamran Pooshang-Bagheri2
1Department of Biochemistry, Faculty of Biological Sciences, Islamic Azad University, North Tehran Branch, Tehran, Iran.
Background:
Programmed cell death protein 1 (PD-1) is a membrane receptor that is expressed on the surface of various immune cells, such as T cells, B cells, monocytes, natural killer T cells, and dendritic cells. In cancer, the interaction between PD-1 and its ligand PD-L1 suppresses the activation and function of T lymphocytes, leading to the impairment and apoptosis of tumor-specific T cells. This mechanism allows cancer cells to evade the immune response and promotes tumor progression.
Methods:
Recombinant PD-1 protein was produced and used to immunize a camel. A nanobody library was generated from the camel's peripheral blood lymphocytes and screened for PD-1 binding. A specific nanobody (3PD9) was selected and characterized by affinity measurement, western blotting, and flow cytometry analysis. The ability of the selected nanobody to block the inhibitory signal of PD-1 in peripheral blood mononuclear cells (PBMCs) was evaluated by measuring the level of interleukin-2 (IL-2).
Results:
The selected nanobody showed high specificity and affinity for human PD-1. Western blot and flow cytometry analysis confirmed that 3PD9 could recognize and bind to human PD-1 on the cell surface. It was demonstrated that the level of IL-2 was significantly increased in PBMCs treated with 3PD9 compared to the control group, indicating that the nanobody could enhance the T cell response by disrupting the PD-1/PD-L1 interaction.
Conclusion:
The results suggested that the anti-PD-1 nanobody could be a promising candidate for cancer immunotherapy.
Insights
A novel nanobody targeting Programmed Cell Death protein 1 (PD-1) was developed. This anti-PD-1 nanobody effectively enhances T cell responses by blocking the PD-1/PD-L1 interaction, showing promise for cancer immunotherapy.
Area of Science:
- Immunology
- Molecular Biology
- Biotechnology
Background:
- Programmed Cell Death protein 1 (PD-1) is a receptor on immune cells that suppresses T cell activation.
- In cancer, PD-1/PD-L1 interaction allows tumor cells to evade immune responses.
- This immune evasion mechanism impairs anti-tumor T cell function and promotes tumor growth.
Purpose of the Study:
- To develop and characterize a novel nanobody targeting human PD-1.
- To evaluate the potential of this nanobody in blocking PD-1 mediated immunosuppression.
- To assess its efficacy in enhancing T cell responses for cancer immunotherapy.
Main Methods:
- Recombinant human PD-1 protein was used to immunize a camel.
- A nanobody library was generated and screened for PD-1 binding, leading to the selection of nanobody 3PD9.
- Characterization involved affinity measurements, western blotting, flow cytometry, and IL-2 level assessment in PBMCs.
Main Results:
- The selected nanobody (3PD9) demonstrated high specificity and affinity for human PD-1.
- 3PD9 successfully recognized and bound to human PD-1 on cell surfaces.
- Treatment with 3PD9 significantly increased IL-2 levels in PBMCs, indicating enhanced T cell response via PD-1/PD-L1 blockade.
Conclusions:
- The anti-PD-1 nanobody 3PD9 effectively disrupts the PD-1/PD-L1 inhibitory signaling pathway.
- This nanobody enhances T cell activation and function.
- The findings suggest that 3PD9 is a promising candidate for developing novel cancer immunotherapies.
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