Development and Evaluation of Novel Aptamers Specific for Human PD1 Using Hybrid Systematic Evolution of Ligands by Exponential Enrichment Approach

ABSTRACT

Background

Programmed cell death protein 1 (PD1; also known as CD279) is an inhibitory receptor on T lymphocytes interacting with PD1-ligand 1 and PD1-ligand 2 in the synapse of T cells and antigen presenting cells (APC) resulting in the suppression of T cell activity. Systematic evolution of ligands by exponential enrichment (SELEX) is a method for generating aptamers which can bind specifically to the target of interest. PD-1 antagonistic aptamers could introduce an attractive alternative over the antibody-based treatments due to the distinguished advantages of aptamers including small size and efficient tissue penetration, low cost, lack of immunogenicity, and ease of manufacturing. Methods: Here, we developed single-stranded DNA aptamers which bind specifically to the human extracellular domain of PD-1. We performed hybrid SELEX, a combination of targeting of recombinant proteins and cell membrane expressed PD1 to select and identify specific aptamers and for the first time, homology of aptamer sequences selected from protein and cell SELEX pool have been evaluated in this study. Results: C42–aptamer, one of the selected aptamers, could specifically bind to human PD1 with dissociation constant in the nanomolar range. Although the developed aptamer inhibited binding of PD1 to PD-L1 but it was not able to restore the cell proliferation and cytokine production of the CD8+ CD279+ T cells. Conclusion: Further studies are required to assess the therapeutic potential of C42 aptamer and other aptamers developed in this study. The introduced PD1 specific aptamers can be used for specific detection of PD1 in diagnostic assay such as immunohistochemistry and targeted drug delivery to PD+ T cells.

Graphical abstract

Using hybrid SELEX for generating ssDNA aptamer against PD1. (a) In the initial round of selection, ssDNA library was added to protein-loaded Ni-NTI magnetic beads, unbound aptamer was then eluted and PCR amplification performed. ssDNA was generated from PCR-amplified dsDNA and protein-free beads was used for counter selection. After 13 rounds, SELEX process was screened by the flow cytometry of beads. The selected pool of round 11 was transferred to the next phase (cell SELEX) and cloned for sequencing. (b) Stimulated Jurkat cells were used as PD1 positive cell and A2780 used as PD1 negative cell in the process of cell SELEX. Selected pool of round 1 and 2 was screened for the enrichment of aptamer by flow cytometry. Pool aptamers from the 1st round of cell SELEX were also selected for cloning and sequencing. (c) Aptamers with the ability to bind PD1 were selected for further analysis using blocking assay. PD-L1 with IgG fc-tag was added to PD1-expressing cells then binding of PD-L1 to PD1 was detected using FITC-conjugated antibodies against IgG fc-tag in either the presence or absence of aptamer.

KEYWORDS:

• Programmed cell death protein 1
• hybrid SELEX
• aptamer

Acknowledgments

The authors would like to thank Dr. M. Sankian, P. Bayat and M. Shahdordizadeh for their valuable comments and Dr. P. Lavaee for synthesizing of biotinylated aptamers. The results described in this paper were part of the Ph.D. thesis of M. Khedri.

Declaration of Interest

The authors declare no conflicts of interest.

Supplementary Material

Supplementary data for this article can be accessed here.

Additional information

Funding

This work was financially supported by vice president of research of Mashhad University of Medical Sciences (Grant # 930611).