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.
Highlights
Cancer is a major cause of public health problems worldwide
They are activated through the connection of the T cell receptor (TCR) with major histocompatibility complex (MHC) in antigen presenting cells (APC) and interaction of the co-stimulatory molecules
We have demonstrated the interactions of fluorescein isothiocyanate (FITC)-YT-16/PD-1 by microscale thermophoresis (MST) analysis, cellular imaging, and flow cytometry analysis
Summary
Cancer is a major cause of public health problems worldwide. Its incidence is associated with an increase in annual mortality [1,2,3,4]. T cells have potential to be stimulated by tumor cells, triggering immune responses. They are activated through the connection of the T cell receptor (TCR) with major histocompatibility complex (MHC) in antigen presenting cells (APC) and interaction of the co-stimulatory molecules. Tumors avoid the immune system by down regulating both co-stimulatory molecules and MHC, and upregulate co-inhibitory molecules [5,6]. Expression and engagement of PD-1 and CTLA-4 with their ligands are associated with tumorigenesis prognosis and massiveness [7]. PD-1 is an immune checkpoint molecule expressed on T cells. It is a member of the CD28/B7/CTLA-4 family and is encoded by PD-1 gene placed on chromosome number
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