The PathHunter® PD-1 Checkpoint Signaling cell line is a stable, engineered cell line for use in studying drug candidates (small molecules or biologics) targeting inhibitory checkpoints. This cell line enables assessment of ligand (e.g. PD-L1, PD-L2) based activation of PD-1 receptor via detection of SH2 domain protein recruitment. This product uses EFC technology. The included cell line overexpresses PK-tagged PD-1 target protein and EA-tagged SH2 domain protein. Upon activation of the receptor by its ligand or an agonist antibody, recruitment of SH2 domain protein to the phosphorylated receptor tail leads to production of chemiluminescent signal.

This cell line can be used to evaluate both agonist and antagonist antibodies targeting PD-1 receptor. Running the assay in agonist mode may require the use of Fcg Receptors expressing cells to enable clustering.

This stable engineered cell line is provided as two vials of cryopreserved cells.

Background

PD-1 is an immune checkpoint receptor on activated T and B cells, NK cells, and myeloid cells. It maintains immune tolerance by suppressing T cell activity. Tumors exploit PD-1 by binding PD-L1/PD-L2, leading to T cell exhaustion and immune evasion. Blocking this pathway with immune checkpoint inhibitors (ICIs) restores T cell function and boosts anti-tumor responses. PD-1 is also a promising target for inflammatory diseases, as it is selectively upregulated on activated immune cells. Activating PD-1 with agonists may suppress pathogenic effector cells while sparing PD-1-negative cells, offering therapeutic potential in autoimmunity.

PathHunter PD-1 (SHP1) Signaling Assay is a simple, homogenous assay that can be used in both agonist and antagonist mode and is suitable for implementation in characterization, QC lot release, stability, and detection of neutralizing antibodies.

Product Highlights
  • Designed for interrogating PD1/PD-L1 signaling analysis
  • Detects and measures SHP1 recruitment to activated PD-1 receptor
  • Can be used for evaluating anti-PD-1 antibodies with either antagonist or agonist activity
  • Suitable for rank ordering, potency measurement, stability studies and neutralizing antibody detection
  • Applicable across various stages from characterization and QC lot-release

Assay Principle

The PathHunter Jurkat PD-1 Signaling Assay measures SHP1 recruitment to PD-1 ITIM motifs. A. Co-culture of U2OS PD-L1 Ligand Cells with Jurkat PD-1 Signaling Cells activates PD-1, recruiting SHP1/2 SH2-EA fusion protein to PD-1 tagged with ED. Anti-PD-1 antibody blocks PD-1/PD-L1 interaction, inhibiting signaling and reducing the EFC-derived chemiluminescent signal. B. In antagonist mode, PathHunter Jurkat PD-1 (SHP1) Signaling Assay exhibits dose-dependent blocking of PD-1 activation by anti-PD-1 antibody (NAT105). C. Including FcγR clustering cells in the assay design elicits enhanced agonistic activity driven by anti-PD1 antibodies like pembrolizumab in the Jurkat PD-1 signaling assay. D. FcyR-mediated clustering significantly enhanced anti-PD-1 therapeutic, pembrolizumab's agonistic activity in the same assay.

PathHunter Jurkat PD-1 Signaling Assay  - Assay Principle