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PathHunter® Checkpoint Signaling Assays

Accelerate Cancer Immunotherapy Drug Development with Simple Cell-based Assays

Regulation of immune responses is tightly controlled through a balance of co-stimulatory and inhibitory checkpoint receptors, often exploited by many cancers. Therefore, therapeutics that block inhibitory receptors or activate immunostimulatory checkpoint receptors have proved to be powerful agents to restore anti-tumor immune responses, such as anti-PD1 therapy.  However, developing drugs targeting these checkpoint proteins has proved to be quite challenging as cell-based assays used to screen for functional drugs are often difficult to create, involve the use of primary cells, and have long, complicated protocols.  The PathHunter Checkpoint assay portfolio solves this bottleneck with an expanding menu of cell-based assays that can enable screening & potency assays for immunotherapy drugs. These biologically relevant cell-based assays do not require primary cells, have a simple protocol, and provide a highly sensitive response in less than 5 hours.

Learn more about the updated Cell Culture and Handling Procedure in the new Technical Bulletin.

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Advantages Of The Checkpoint Assays

  • No primary cells – Get biologically relevant responses without the use of difficult and costly primary cells
  • Simple Protocol, Fast Results – Increase efficiency with an add and read protocol and results in less than 5 hours
  • Highly Sensitive Response – 15X more sensitive than other assays
  • Multiple Applications – Monitor checkpoint signaling for biologic and small molecule drug development

Subtract primary cells. Add reproducibility and ease of use.

The PathHunter PD-1 Checkpoint Assay was performed with Pembrolizumab (anti-PD-1 antibody) and PD-L1 ligand presenting cell line. Data show consistent EC50’s and S/B on three different runs of the assay within the same plate with less than 4% relative standard deviation (%RSD). For more details on how the assay works, please visit the ‘Assay Principle & Data’ tab above.

Complete full assay in less than 5 hours with simple, add and read protocol.

15X more sensitive than other reporter gene assays

Using the same anti-PD-1 antibody (BioLegend Catalog #329912), we were able to compare the assay performance of our PathHunter PD-1 signaling assay to a commercially available reporter gene assay, and observed that the PathHunter PD-1 assay demonstrated more than 15-fold better sensitivity than the reporter gene assay, with a slightly better assay window as well.

Screen and identify both small molecules and biologic drugs

PD-1 is known to be phosphorylated with an as yet unidentified src family kinase, which causes the recruitment of the SHP-1 protein to the phosphorylated PD-1 receptor. Testing of the PathHunter PD-1 assay with small molecule inhibitors that are known to inhibit several known src family kinases has shown inhibition of the SHP-1 recruitment to PD-1, demonstrating that the PathHunter PD-1 assay can be used to identify novel small molecule inhibitors of this exciting immunotherapy target.
Available Assays

Cell-based assays based on the target biology to accelerate your immuno-oncology drug development programs.

Target Description Cell Line Ready-to-Use Bioassay Kits
(2- / 10-Plate Kits)
Ready-to-Use eXpress Kits
(1- / 2- / 10-Plate Kits)
BTLA  Jurkat BTLA Signaling Assay 93-1112C19    
CD137 U2OS CD137 Signaling Assay 93-1089C3    
CD200 Ligand U2OS CD200 Ligand Cell Line 93-1137C3    
CD200R Jurkat CD200R Signaling Cell Line 93-1136C19    
CD28 Jurkat CD28 Signaling Assay 93-1126C19    
CD86 Ligand U2OS CD86 Ligand Cell Line 93-1093C3    
CTLA4 Jurkat CTLA4 Signaling Assay 93-1094C19    
FcyRIIb Clustering U2OS FcyRIIb Clustering Cell Line 93-1133C3    
FcγR1A Clustering U2OS FcγR1A Clustering Cell Line 93-1138C3    
FcγRIIa Clustering U2OS FcγRIIa Clustering Cell Line 93-1140C3    
HVEM Ligand U2OS HVEM Ligand Cell Line 93-1116C3    
ICOS Jurkat ICOS Signaling Assay 93-1127C19    
NIK U2OS NIK Signaling Assay 93-1059C3    
OX40 U2OS OX40 Signaling Assay 93-1080C3    
PD-1 (SHP1)* Jurkat PD-1 (SHP1) Signaling Assay 93-1104C19 93-1104Y19-00117 / -00118  
PD-1 (SHP2) Jurkat PD-1 (SHP2) Signaling Assay 93-1106C19    
PD-L1 U2OS PD-L1 Ligand Cell Line 93-1066C3 93-1104Y19-00117 / -00118  
PD-L2 U2OS PD-L2 Ligand Cell Line 93-1076C3    
SIRPa Jurkat SIRPa Signaling Cell Line 93-1135C19    
TIM3/TIM3** U2OS TIM3/TIM3 Dimerization Cell Line 93-1082C3   93-1082E3CP5S / M / L
VISTA/VISTA** U2OS VISTA/VISTA Dimerization Cell Line 93-1079C3   93-1079E3CP5S / M / L

Early Access Assays

To request access to the following assays, please email

Target Description Cell Line
PD-1/TIM3 PD-1/TIM3 Receptor Dimerization Cell Line 83-0008C3
PD-1/LAG3 PD-1/LAG3 Receptor Dimerization Cell Line 83-0009C3
PD-1/CTLA4 PD-1/CTLA4 Receptor Dimerization Cell Line 83-0010C3
PD-1/TIGIT PD-1/TIGIT Receptor Dimerization Cell Line 83-0011C3
PD-L1/TIM3 PD-L1/TIM3 Receptor Dimerization Cell Line 83-0012C3
PD-1/PD-L1 PD-1/PD-L1 Receptor Dimerization Cell Line 83-0013C3
PD-L1/CTLA4 PD-L1/CTLA4 Receptor Dimerization Cell Line 83-0014C3
PD-1/CEACAM1 PD-1/CEACAM1 Receptor Dimerization Cell Pool 83-0015C3
TIM3/CEACAM1 TIM3/CEACAM1 Receptor Dimerization Cell Pool 83-0016C3
mPD-1/mLAG3 mPD-1/mLAG3 Receptor Dimerization Cell Line 83-0017C3
mPD-1/mTIGIT mPD-1/mTIGIT Receptor Dimerization Cell Line 83-0018C3
mPD-1/mCTLA4 mPD-1/mCTLA4 Receptor Dimerization Cell Line 83-0019C3
SHP2 InCELL Hunter™ Jurkat SHP2 Protein Binding Cell Line 83-0023C19
CD27 CD27 Signaling Cell Line 83-0021C3