Targets / Checkpoint Receptors
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Checkpoint 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 immune-stimulatory checkpoint receptors have proved to be powerful agents to restore anti-tumor immune responses, such as anti-PD1 therapy. PathHunter® checkpoint assays are cell-based functionally relevant assays for screening, potency testing and lot release of immunotherapy drugs without involving primary cells or complex protocols and provides a highly sensitive response in a few hours for some targets.
 

Advantages of Checkpoint Assays

PathHunter checkpoint assays are MOA-reflective assays for quantifying a functional response as a result of direct ligand binding to its checkpoint receptor.  These cell-based assays enable rapid screening and characterization of large and small molecules including analyzing agonists, antagonists, and clustering/cross-linking of agonist antibodies. These qualified assays as cell lines or bioassay kits decrease assay development and validation times, reduce costs, and facilitate a simplified global method transfer and ultimately accelerate potency testing and lot release. Generating highly sensitive and biologically relevant responses, these assays use stable clonal cells — in contrast to primary cell lines – thus eliminating the need for maintaining optimizing cell culture conditions or worrying about donor pool variability.
 
  • Biologically-Relevant — MOA-reflective, functional assays for monitoring checkpoint signaling and testing of small molecule or biologic drugs
  • Comprehensive Portfolio — Largest menu of off-the-shelf assays for accelerating assay development and implementation time
  • Robust Assays – Highly reproducible assays for potency and lot release applications for immunotherapy drug discovery and development
  • Simple Protocol, Fast Results — Easy-to-run, rapid homogeneous protocol amenable to implementation in multiple labs and high-throughput format for increased efficiency

Key Resources

 

PathHunter checkpoint assays are available as complete ready-to-use bioassay kits and stable cell lines formats for signaling and dimerization assessment. Additional cell lines are available for several checkpoint receptor targets not shown below.  Please contact custom assay development for more information.

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Ready-to-Use Co-Culture Bioassay Kits


Target Description Cat. No.
PD-1 PathHunter® PD-1 (SHP1) Signaling Bioassay Kit 93-1104Y19-00117
PD-1 PathHunter® PD-1 (SHP1) Signaling Bioassay Kit 93-1104Y19-00118
SIRPα PathHunter® Jurkat SIRPa Signaling Bioassay Kit 93-1135Y19-00129
SIRPα PathHunter® Jurkat SIRPa Signaling Bioassay Kit 93-1135Y19-00130

Cell Lines


Signaling Receptor Cell Lines
Target Description Cat. No.
OX40 PathHunter® U2OS OX40 Signaling Cell Line 93-1080C3
CD137 PathHunter® U2OS CD137 Signaling Cell Line 93-1089C3
CTLA4 PathHunter® Jurkat CTLA4 Signaling Cell Line 93-1094C19
PD-1 PathHunter® Jurkat PD-1 (SHP2) Signaling Cell Line 93-1106C19
PD-1 PathHunter® Jurkat PD-1 (SHP1) Signaling Cell Line 93-1104C19
BTLA PathHunter® Jurkat BTLA Signaling Cell Line 93-1112C19
CD28 PathHunter® Jurkat CD28 Signaling Cell Line 93-1126C19
ICOS PathHunter® Jurkat ICOS Signaling Cell Line 93-1127C19
SIRPα PathHunter® Jurkat SIRPa Signaling Cell Line 93-1135C19
CD200R PathHunter® Jurkat CD200R Signaling Cell Line 93-1136C19
mPD-1 PathHunter® Jurkat mPD-1 Signaling Cell Line 93-1158C19
mCTLA4 PathHunter® Jurkat mCTLA4 Signaling Cell Line 93-1161C19

Ligand-Presenting Cell Lines
Target Description Cat. No.
CD47 PathHunter® Jurkat CD47-Presenting Cell Line 93-1149C19

Clustering Cell Lines
Target Description Cat. No.
FCGR2B PathHunter® U2OS FcgRIIb Clustering Cell Line 93-1133C3
FCGR1A PathHunter® U2OS FcgRIa Clustering Cell Line 93-1138C3
FCGR2A PathHunter® U2OS FcgRIIa Clustering Cell Line 93-1140C3
mFCGR2B PathHunter® U2OS mFCGR2B Clustering Cell Line 93-1139C3

Receptor Dimerization Cell Lines
Target Description Cat. No.
VISTA/VISTA PathHunter® U2OS VISTA/VISTA Dimerization Cell Line 93-1079C3
TIM3/TIM3 PathHunter® U2OS TIM3/TIM3 Dimerization Cell Line 93-1082C3
PD-1/PD-1 PathHunter® U2OS PD-1/PD-1 Dimerization Cell Line 93-1130C3

Receptor Internalization Cell Lines
Target Description Cat. No.
CD33 PathHunter® U2OS CD33 Internalization Cell Line 93-1148C3
BCMA PathHunter® U2OS BCMA Internalization Cell Line 93-1145C3
Siglec9 PathHunter® U2OS Siglec9 Internalization Cell Line 93-1147C3

Early Access Checkpoints Cell Lines (for Bispecific Antibody Development)
Target Description Cat. No.
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
TIGIT/LAG3 TIGIT/LAG3 Receptor Dimerization Cell Line 83-0024C3
PD-1/CD28 PD-1/CD28 Receptor Dimerization Cell Line 83-0026C3
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
 
Please contact custom assay development to obtain our Early Access Checkpoints Cell Lines.
 


A full-length checkpoint receptor (e.g. PD-1, SIRPα, or other) is engineered with a small β galactosidase (β-gal) fragment (PK) fused to its C-terminus, and an SH2-domain of a relevant scaffold/signaling protein (e.g. SHP-1) is engineered with the complementing β-gal fragment (EA). These constructs are stably co-expressed in Jurkat cells, while untagged full-length ligand (e.g PD-L1 or PD-L2 for PD-1 assay; CD47 for SIRPα assay) is stably expressed in ligand-presenting cells (representing the tumor cell). [A] Ligand engagement, through co-culture with ligand-presenting cells, results in phosphorylation of the checkpoint receptor-PK fusion protein, leading to the recruitment of SHP-1-EA, which forces complementation of the EFC components to create an active β-gal enzyme. This active enzyme hydrolyzes substrate to create a chemiluminescence signal as a measure of receptor activity. [B] In the presence of an anti-receptor or anti-ligand antibody, or a checkpoint receptor-FC fusion protein, the ligand-receptor engagement and EFC complementation are both disrupted and no chemiluminescence can be measured.

Versatile Assays for Both Biologic & Small Molecules Therapeutics



[A] Jurkat PD-1 cells were treated with serial dilutions of Keytruda® (blue) or Opdivo® (green) for 1 hour prior to stimulation with U2OS PD-L2 cells for 2 hours at room temperature. Expected rank order of the two therapeutic antibodies is observed in the assay with low ng/mL sensitivity for the two marketed drugs. Keytruda and Opdivo are registered trademarks of Merck and BMS, respectively. [B] The PathHunter PD-1 assay, when treated with small molecules known to inhibit Src family kinases, has exhibited reduced activity (Dasatinib). Additionally, a low molecular weight inhibitor of PD-L1 interaction with PD-1 (PD-1/PD-L1 inhibitor) also reduces the assay signal in a dose-dependent manner, although not as potently as the anti-PD-1 antibody. These data demonstrate that the PathHunter PD-1 Signaling Assay can be used to identify novel small molecule inhibitors of PD-1, including kinase inhibitors and disruptors of PD-1 interaction with its ligands, PD-L1 and PD-L2.

 

Highly Sensitive Response 
 



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.
 
 

Exceptional Specificity




Specificity of the SIRPα Signaling Assay. Antibodies to CD47 and three other immune checkpoint receptor ligands (PD-L1, PVR and CD80) were pre-incubated with CD47-presenting cells (30K) for 1 hour at 37°C. Jurkat Signaling cells (20K) were added to the CD47-presenting cells and incubated at 37°C for 5 hours prior to addition of detection reagent. Only the anti-CD47 antibody produced a robust dose-dependent decrease in signal in the assay.

 

Reproducible Assays with Excellent Lot-to-Lot Consistency and Intermediate Precision




The PathHunter PD-1 Checkpoint Assay was performed with an anti-PD-1 antibody and stimulated with the 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).



[A] Preliminary assay qualification data for the Jurkat SIRPα signaling assay. Intermediate precision of the assay was evaluated by two analysts who compared a 100% anti-CD47 test sample to a 100% reference standard on 10 different plates over 3 days. Excellent repeatability (3.8%) and intermediate precision (3.34%) was obtained with this preliminary dataset. [B] The Jurkat SIRPα signaling assay is stability-indicating. A 100% sample of anti-CD47 antibody was incubated at 65°C for 17 hours prior to testing a dose response curve of the heat treated antibody in the Jurkat SIRPα signaling assay relative to the 100% reference standard. A noticeable shift in potency was observed for the heat-treated sample.

 

Large Response Window




[A] Response of the Jurkat SIRPα signaling assay to cell-presented ligand. Increasing numbers of CD47-presenting cells were co-cultured with SIRPα signaling cells for 5 hours at 37°C prior to addition of detection reagent. In this experiment, assay window (S/B) obtained was greater than 20-fold. [B] Inhibition of SIRPα signaling with a commercial anti-CD47 antibody. A fixed concentration of CD47-presenting cells (30K) were pre-incubated with a dose response of anti-CD47 antibody for 1 hour at 37°C. SIRPα signaling cells (20K) were added to the CD47-presenting cells and incubated at 37°C for 5 hours prior to addition of detection reagent. A robust dose-dependent decrease in signal, with an assay window of 28-fold was observed.

 

Clustering Cell Lines for Cross-Linking (Testing Potency)




Agonist antibody-mediated stimulation of OX40 signaling in the PathHunter OX40 Signaling Cell Line with the OX40 agonist antibody Pogalizumab. As has been demonstrated previously, soluble Pogalizumab is unable to activate OX40 signaling (purple curve). However, when OX40 Signaling cells are co-cultured with human FcγRIIB cells (at a ratio of 2:1 (blue curve) or 4:1 (green curve)) in the presence of a dose response of Pogalizumab, a robust dose-dependent response to Pogalizumab is observed with both ratios of cells.
 

Rapid, Homogeneous Assays with Easy-to-Use Protocol



Example workflow based on the Jurkat PD-1 (SHP1) Signaling Bioassay Kit protocol.