Design and Synthesis of A PD-1 Binding Peptide and Evaluation of Its Anti-Tumor Activity
Abdul Baset Abbas1,2, Bingjing Lin3, Chen Liu4
1The Engineering Research Center of Synthetic Polypeptide Drug Discovery and Evaluation of Jiangsu Province, China Pharmaceutical University, Nanjing 210009, China. abduabbas04@gmail.com.
Abstract:
Immune-checkpoint blockades, suchas PD-1 monoclonal antibodies, have shown new promising avenues to treat cancers. Failure responsesof many cancer patients to these agents have led to a massive need for alternative strategies to optimize tumor immunotherapy. Currently, new therapeutic developments involve peptide blocking strategies, as they have high stability and low immunogenicity. Here, we have designed and synthesized a new peptide FITC-YT-16 to target PD-1. We have studied FITC-YT-16 by various experiments, including Molecular Operating Environment MOE modeling, purification testing by HPLC and LC mass, peptide/PD-1 conjugation and affinity by microscale thermophoresis (MST), and T cell immune-fluorescence imaging by fluorescence microscopy and flow cytometry. The peptide was tested for its ability to enhanceT cell activity against tumor cell lines, including TE-13, A549, and MDA-MB-231. Lastly, we assessed T cell cytotoxicity under peptide treatment. YT-16⁻PD-1 interaction showed a high binding affinity as a low energy complex that was confirmed by MOE. Furthermore, the peptide purity and molecular weights were 90.96% and 2344.66, respectively. MST revealed that FITC-YT-16 interacted with PD-1 at a Kd value of 17.8 ± 2.6 nM. T cell imaging and flow cytometry revealed high affinity of FITC-YT-16 to PD-1. Interestingly, FITC-YT-16 efficiently blocked PD-1 signaling pathways and promoted T cell inflammatory responses by elevating IL-2 and INF-γ levels. Moreover, FITC-YT-16 has the ability to activate T cell cytotoxicity. Therefore, FITC-YT-16 significantly enhanced T cell anti-tumor activity by blocking PD-1⁻PD-L1 interactions.
Insights
A novel peptide, FITC-YT-16, effectively targets programmed cell death protein 1 (PD-1) to enhance anti-tumor immunity. This peptide blocks PD-1 signaling, boosting T cell activity and cytotoxicity against cancer cells.
Area of Science:
- Immunology
- Oncology
- Biochemistry
Background:
- Immune-checkpoint inhibitors like PD-1 monoclonal antibodies show promise in cancer treatment.
- Many patients do not respond to current therapies, necessitating alternative strategies for tumor immunotherapy.
- Peptide-based blocking strategies offer high stability and low immunogenicity for therapeutic development.
Purpose of the Study:
- To design, synthesize, and evaluate a novel peptide, FITC-YT-16, targeting PD-1 for cancer immunotherapy.
- To assess the binding affinity and efficacy of FITC-YT-16 in blocking PD-1 signaling pathways.
- To determine the impact of FITC-YT-16 on T cell activity, inflammatory responses, and cytotoxicity against various cancer cell lines.
Main Methods:
- Molecular Operating Environment (MOE) modeling for structural analysis.
- High-Performance Liquid Chromatography (HPLC) and LC mass spectrometry for peptide purification and characterization.
- Microscale Thermophoresis (MST) to determine binding affinity between FITC-YT-16 and PD-1.
- Immune-fluorescence imaging, flow cytometry, and assessment of cytokine levels (IL-2, INF-γ) to evaluate T cell responses.
- In vitro cytotoxicity assays against TE-13, A549, and MDA-MB-231 tumor cell lines.
Main Results:
- MOE modeling confirmed a stable, low-energy complex between YT-16 and PD-1.
- Peptide purity was 90.96% with a molecular weight of 2344.66.
- MST analysis showed a high binding affinity of FITC-YT-16 to PD-1 with a Kd value of 17.8 ± 2.6 nM.
- FITC-YT-16 demonstrated effective PD-1 blockade, enhancing T cell inflammatory responses (increased IL-2 and INF-γ) and activating T cell cytotoxicity.
- The peptide significantly boosted T cell anti-tumor activity by inhibiting PD-1/PD-L1 interactions.
Conclusions:
- FITC-YT-16 is a potent peptide inhibitor of PD-1 with high binding affinity.
- This peptide effectively enhances T cell-mediated anti-tumor immunity by blocking the PD-1 pathway.
- FITC-YT-16 represents a promising alternative therapeutic strategy for optimizing cancer immunotherapy.
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