Screen for inhibitors of PD-1 signalling with a complete cellular assay system

T cell - Cancer cell CAR-T Cells, Cancer Immuno-therapy, Immune-checkpoints

Immunotherapy represents a field in Drug Discovery which is quickly developing and leading to significant progress in treatments of a number of diseases, especially cancer. The approach is based on inducing, enhancing, or surpressing an immune response. Therapeutic manipulation of immunopathways has led to promising clinical results . The first therapeutic antibodies directed against the checkpoint receptor PD-1 have been already brought to the market (Nivolumab, Pembrolizumab) by Bristol Myers Squibb and Merck/MSD respectively, and approved for the treatment of diverse cancer types.

Today, I would like to review tools to build up a comprehensive assay set up for  cell based inhibitor screening on PD-1 / PD-L1/PD-L2 binding.

PD1 - PD-L1 - PD-L2

Fig 1: Immuno checkpoint proteins on T cell interact with ligands on APCs or cancer cell

PD-1:PD-L1/2 pathway

T-cell activation and inactivation require the coordination of various co-inhibitory and co-stimulatory signals which are modulated by most immunotherapeutic approaches. The binding of Programmed Cell Death Protein 1 (PD-1), a receptor expressed on activated T-cells, to its ligands, PD-L1 and PD-L2, negatively regulates immune responses. The PD-1 ligands are found on most cancers, and PD-1:PD-L1/2 interaction inhibits T cell activity and allows cancer cells to escape immune surveillance. The PD-1:PD-L1/2 pathway is also involved in regulating autoimmune responses, making these proteins promising therapeutic targets for a number of cancers, as well as multiple sclerosis, arthritis, lupus, and type I diabetes. Further research projects in this field are rapidly evolving as scientists seek to identify the next generation of therapies, especially developing therapies combining different therapeutic approaches.

After having presented a number of  biochemical binding assays to measure the inhibitory effects of potential drugs on e.g. PD-1/PD-L1/PD-L2 binding and the binding of other checkpoint proteins to their respective ligands, such as CTLA-4/B7-1/B-7.2 and CD28/B7-1/B-7.2 (see Figure 1), I will concentrate on cell lines developed by BPS Biosciences which allow for setting up a cell based screening of compounds inhibiting PD-1 – PD-L1/PD-L2 binding.

Tools to run cell based screening assays on PD-1 / PD-L1/PD-L2 binding

BPS Biosciences has developed two stable cell line to set up inhibitor screening assays:

The first cell line is a Jurkat T cell expressing firefly luciferase gene under the control of NFAT (Nuclear Factor of Activated T-cells) response elements with constitutive expression of human PD-1. Furthermore, it expresses TCR (T cell receptor).


Fig 2: Principle of the cell based assay using PD-1/ NFAT Reporter/Jurkat T cells and TCR activator / PD-L1 CHO cells

The second cell line is a CHO cell expressing TCR activator and PD-L1.

The principle of the assay which can be run by co-culturing both cell lines is shown in Fig 2. In this assay, PD-1/ NFAT Reporter – Jurkat cells are used as effector cells while the TCR activator / PD-L1 – CHO cells are used as target cells.  When these two cells are co-cultivated, TCR complexes on effector cells are activated by TCR activator on target cells, resulting in expression of the NFAT luciferase reporter. However, PD1 and PD-L1 ligation prevents TCR activation and suppresses the NFAT-responsive luciferase activity. This inhibition can be specifically reversed by anti-PD-1 or anti-PD-L1 antibodies. PD-1/PD-L1 neutralizing antibodies block PD-1:PD-L1 interaction and promote T cell activation, resulting in reactivation of the NFAT responsive luciferase reporter.

What can you use these cell lines for?

In combination with…

…this cell line pair represents a complete co-culture model to…

  • Screen for activators or inhibitors of PD-1 signaling in a cellular context
  • Characterize the biological activity of PD-1 and its interactions with ligands

Growth arrested PD-1 cells as a cost-effective alternative

If you do not want to immediately invest in stable cell lines, the PD-1:PD-L1/PD-L2 Cell-Based Inhibitor Screening Assay Kit might be an alternative starting point for you.


Figure 3: Characterization of biological activity of anti-PD-1 neutralizing antibody in PD-1/PD-L1 cell-based assay using the PD-1/NFAT Reporter-Jurkat cells (A) and the NFAT Reporter-Jurkat control cells (B).

This kits contains PD-1/NFAT reporter – Jurkat cells in a thaw-and-use format that does not require cell propagation. Furthermore it comes with expression vectors for TCR activator, human PD-L1, and human PD-L2 which are used to transfect cells to create the target cells that overexpress PD-L1 or PD-L2 and an engineered cell surface T cell receptor (TCR) activator. Thus you can even extend your experiments to PD-1 : PD-L2 inhibition screenings.

A typical characterization of biological activity of anti-PD-1 neutralizing antibody in a PD-1/PD-L1 cell-based assay using the PD-1/NFAT Reporter-Jurkat cells and HEK293 cells transiently transfected with human PD-L1 and an the TCR activator is shown in Fig 3.The Figure also shows control results with NFAT Reporter-Jurkat cells not expressing PD-1. Very similar results could be obtained using HEK293 cells  transiently transfected with human PD-L2 and an engineered T cell receptor (TCR) activator (data not shown, but shown in the manual of the PD-1 / NFAT Reporter – Jurkat Cell Line).

Any questions, or interested in learning more? Get in touch via the comments form below! 

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Written by Ali El Baya, PhD
Ali el Bayâ is the Sales Manager at tebu-bio for the North of Europe.