Checkpoint Receptor Assays

Accelerate Cancer Immunotherapy Drug Development with Easy-to-use, Functional 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, such as PD-1 receptor, have proven to be powerful agents to restore anti-tumor immune responses.

Eurofins DiscoverX® offers a large, diverse selection of assays for characterization, screening, and potency QC lot-release testing of immunotherapy therapeutics targeting both inhibitory and co-stimulatory checkpoint receptors. Utilize PathHunter® functional, mechanism of action (MOA)-reflective cell-based checkpoint receptor assays to analyze signaling, dimerization, internalization, and clustering/crosslinking of checkpoint receptors. These simple, rapid assays provide a highly sensitive response in a few hours for most targets without primary cells or complex protocols.

Product Highlights

  • Biologically-relevant — MOA-reflective, functional assays for monitoring checkpoint signaling and testing of small molecule or biologic therapeutics
  • Comprehensive Portfolio — Large 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
Consider Eurofins DiscoverX’s custom development capabilities for custom cell lines, assays, & enzyme development.

PathHunter checkpoint assays are available as complete ready-to-use bioassay kits and stable cell lines formats for checkpoint receptor dimerization, signaling, clustering, and internalization assessment. For additional checkpoint receptor cell lines not shown below, please contact custom assay development (CAD) for more information.

TargetDescriptionCat. No.
PD-1PathHunter® PD-1 (SHP1) Signaling Bioassay Kit93-1104Y19-00117
PD-1PathHunter® PD-1 (SHP1) Signaling Bioassay Kit93-1104Y19-00118
SIRPαPathHunter® Jurkat SIRPa Signaling Bioassay Kit93-1135Y19-00129
SIRPαPathHunter® Jurkat SIRPa Signaling Bioassay Kit93-1135Y19-00130
TargetDescriptionCat. No.
BCMAPathHunter® BCMA Signaling Reporter Cell Line (K562)93-1183C042
OX40PathHunter® U2OS OX40 Signaling Cell Line93-1080C3
CD137PathHunter® U2OS CD137 Signaling Cell Line93-1089C3
CTLA4PathHunter® Jurkat CTLA4 Signaling Cell Line93-1094C19
PD-1PathHunter® Jurkat PD-1 (SHP2) Signaling Cell Line93-1106C19
PD-1PathHunter® Jurkat PD-1 (SHP1) Signaling Cell Line93-1104C19
BTLAPathHunter® Jurkat BTLA Signaling Cell Line93-1112C19
CD28PathHunter® Jurkat CD28 Signaling Cell Line93-1126C19
ICOSPathHunter® Jurkat ICOS Signaling Cell Line93-1127C19
SIRPαPathHunter® Jurkat SIRPa Signaling Cell Line93-1135C19
CD200RPathHunter® Jurkat CD200R Signaling Cell Line93-1176C19
mPD-1PathHunter® Jurkat mPD-1 Signaling Cell Line93-1158C19
mCTLA4PathHunter® Jurkat mCTLA4 Signaling Cell Line93-1161C19
PD-1 NFATPathHunter® Jurkat PD1-NFAT Reporter Cell Line93-1153C19
TargetDescriptionCat. No.
CD33PathHunter® U2OS CD33 Internalization Cell Line93-1148C3
BCMAPathHunter® U2OS BCMA Internalization Cell Line93-1145C3
Siglec9PathHunter® U2OS Siglec9 Internalization Cell Line93-1147C3
TargetDescriptionCat. No.
VISTA/VISTAPathHunter® U2OS VISTA/VISTA Dimerization Cell Line93-1079C3
TIM3/TIM3PathHunter® U2OS TIM3/TIM3 Dimerization Cell Line93-1082C3
PD-1/PD-1PathHunter® U2OS PD-1/PD-1 Dimerization Cell Line93-1130C3
VISTA/VISTAPathHunter® eXpress VISTA/VISTA Dimerization Assay93-1079E3CP5M
VISTA/VISTAPathHunter® eXpress VISTA/VISTA Dimerization Assay93-1079E3CP5L
TIM3/TIM3PathHunter® eXpress TIM3/TIM3 Dimerization Assay93-1082E3CP5M
TIM3/TIM3PathHunter® eXpress TIM3/TIM3 Dimerization Assay93-1082E3CP5L
PD-1/LAG3PathHunter® PD-1/LAG3 Dimerization Cell Lineinquire
PD-1/CTLA4PathHunter® PD-1/CTLA4 Dimerization Cell Lineinquire
PD-1/TIGITPathHunter® PD-1/TIGIT Dimerization Cell Lineinquire
PD-L1/TIM3PathHunter® PD-L1/TIM3 Dimerization Cell Lineinquire
PD-1/PD-L1PathHunter® PD-1/PD-L1 Dimerization Cell Lineinquire
PD-L1/CTLA4PathHunter® PD-L1/CTLA4 Dimerization Cell Lineinquire
PD-1/CEACAM1PathHunter® PD-1/CEACAM1 Dimerization Cell Poolinquire
TIM3/CEACAM1PathHunter® TIM3/CEACAM1 Dimerization Cell Poolinquire
TIGIT/LAG3PathHunter® TIGIT/LAG3 Dimerization Cell Lineinquire
PD-1/CD28PathHunter® PD-1/CD28 Dimerization Cell Lineinquire
mPD-1/mLAG3PathHunter® mPD-1/mLAG3 Dimerization Cell Lineinquire
mPD-1/mTIGITPathHunter® mPD-1/mTIGIT Dimerization Cell Lineinquire
mPD-1/mCTLA4PathHunter® mPD-1/mCTLA4 Dimerization Cell Lineinquire
TargetDescriptionCat. No.
FCGR2BPathHunter® U2OS FcgRIIb Clustering Cell Line93-1133C3
FCGR1APathHunter® U2OS FcgRIa Clustering Cell Line93-1138C3
FCGR2APathHunter® U2OS FcgRIIa Clustering Cell Line93-1140C3
mFCGR2BPathHunter® U2OS mFcgRIIb Clustering Cell Line93-1139C3
TargetDescriptionCat. No.
CD47PathHunter® Jurkat CD47-Presenting Cell Line93-1149C19
CD200PathHunter® U2OS CD200 Ligand Cell Line93-1137C3
CD86PathHunter® U2OS CD86 Ligand Cell Line93-1093C3
HVEMPathHunter® U2OS HVEM Ligand Cell Line93-1116C3
mCD80PathHunter® U2OS mCD80 Ligand Cell Line93-1162C3
mPD-L1PathHunter® U2OS mPD-L1 Ligand Cell Line93-1159C3
PD-L1PathHunter® U2OS PD-L1 Ligand Cell Line93-1066C3
PD-L2PathHunter® U2OS PD-L2 Ligand Cell Line93-1076C3
Checkpoint Receptors Critical for Communication and Regulation

Checkpoint receptors are critical for communication and regulation between tumor and immune cells. T-cell receptors (TCRs) recognize tumor antigens that are presented by antigen presenting cells (APCs) like dendritic cells to T-cells. The type and duration of responses by the T cell depends on the type of antigen being presented by the APCs. T-cell receptor-ligand interactions can lead to activation (through co-stimulatory signals) and amplification of effector T cells such as OX40–OX40L, ICOS-ICOSL, CD137−4-1BBL (or CD137L), and CD28-CD80/86 interactions. While interactions that suppress (through co-inhibitory signals) effector T-cell responses include CD200-CD200R, PD-1-PD-L1/PD-L2, BTLA-HVEM, and CTLA4-CD80/86. The latter interaction type helps to limit the overall duration of the effector T-cell response to minimize damage to host tissue. These responses are often used by tumor cells to evade the host immune system either by suppressing T-cell activation or by suppressing phagocytosis by macrophages.

High Passage Stability

Eurofins DiscoverX stable cell line generation and stability testing. A. The process outline for cell line generation. After ascertaining the assay’s MOA, the gene of interest is tagged with an appropriate enzyme fragment complementation (EFC) enzyme tag and introduced in the cells using retroviral transduction followed by pool and clone selection. The assay is optimized with the reference ligand, and the selected clone is tested for passage stability. B. Cell surface expression of SIRPα receptor was analyzed up to 45 passages and was found to be highly consistent. C. Passage stability of the SIRPα cell line was analyzed functionally. Inhibition of SIRPα signaling with a commercial anti-CD47 antibody presented a robust dose-dependent decrease in signal, where a consistent assay window with <19% relative standard deviation (RSD) over 45 passages was observed.

Superior Assay Sensitivity and Specificity

PathHunter checkpoint receptor assays provide superior assay sensitivity and specificity. A. The PathHunter PD-1 signaling assay performance demonstrates a 15-fold better sensitivity compared to another commercially available reporter gene assay B. The PathHunter SIRPα signaling assay exhibits exceptional specificity. As demonstrated, this assay responds specifically to the anti-CD47 antibody alone when treated with three other immune checkpoint receptor ligands (PD-L1, PVT, and CD80).

Large and Robust Dynamic Range

PathHunter checkpoint assays exhibit a wide dynamic range or signal-to-background (S/B) ratio. A. Increasing numbers of CD47-presenting cells where co-cultured with SIRPα signaling cells in the assay and results show an assay window of greater than 20-fold. B. Inhibition of SIRPα signaling with a commercial anti-CD47 antibody. Addition of SIRPα signaling cells demonstrated a robust dose-dependent 28-fold decrease in signal.

Excellent Intermediate Precision and Lot-to-Lot Consistency

PathHunter checkpoint assays produce excellent replicate reproducibility and lot-to-lot consistency. A. 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 shows consistent EC50 and S/B ratios for three different runs of the assay within the same plate with less than 4% relative standard deviation. Representative control charts show S/B ratios (B.) and potency (EC50) (C.) for an anti-PD1 antibody over 10 sequential lots using the PathHunter PD-1 Signaling Bioassay Kit. The two datasets demonstrate high level of lot-to-lot consistency with both the S/B ratios and potency for all 10 lots being within 2-standard deviations from the mean, thereby resulting in consistent assay performance across lots generated at different times.

Phase-Appropriate Formats: Continuous Culture vs. Cryopreserved Cells

Checkpoint receptor cells are available in both stable cell line and ready-to-use (RTU) bioassay formats.  This example shows comparable IC50 and S/B values between continuous culture and RTU thaw-and-plate cells. RTU bioassays eliminate the need for continuous culture and includes additional qualifications for improved assay reproducibility and intermediate precision. For more information, please refer to the Bioassays for Biologics webpage and Checkpoint White Paper.

Example workflow based on the PathHunter Jurkat PD-1 SHP1 Signaling Assay co-culture model system

Example workflow based on the PathHunter Jurkat PD-1 (SHP1) Signaling Assay (co-culture model system).

PathHunter Checkpoint Receptor assays are based on the Eurofins DiscoverX’s proprietary Enzyme Fragment Complementation (EFC) technology. Assay principles for checkpoint receptor dimerization, internalization, and ligand-presenting signaling are shown below. View assay principles associated with signaling reporter checkpoint receptor cell line.

Checkpoint signaling cell line assay principle. A full-length checkpoint receptor (e.g., PD-1, SIRPα, or other) is engineered with a small β galactosidase (β-gal) donor fragment called ProLink (PK) fused to its C-terminus. Additionally, an SH2-domain of a relevant scaffold/signaling protein (e.g. SHP1) is engineered with the complementing larger β-gal enzyme acceptor (EA) fragment in Jurkat cells (representing immune cells). 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 the recruitment of SHP1-EA, which forces the complementation of the EFC components to create an active β-gal enzyme. This active enzyme hydrolyzes substrate to create a chemiluminescent signal as a measure of receptor activity. B. In the presence of an anti-receptor, anti-ligand antibody, or a checkpoint receptor-FC fusion protein, the ligand-receptor engagement and EFC complementation are both disrupted and a decrease in chemiluminescent signal results that can be measured in a dose dependent manner.

PathHunter receptor dimerization and internalization assay principles

PathHunter receptor dimerization and internalization assay principles. A. Dimerization assays detect ligand-induced dimerization of two subunits of a receptor-dimer pair. The cells have been engineered to co-express one receptor subunit fused to the β-gal enzyme donor (ED) and a second dimer partner fused to the larger β-gal enzyme acceptor (EA) fragment – both tagged at their cytoplasmic tail. Binding of biologic (e.g., antibody) or small molecule agonist to one receptor subunit induces it to interact with its dimer partner, forcing the complementation of the two enzyme fragments. This results in the formation of a functional enzyme that hydrolyzes a substrate to generate a chemiluminescent signal. B. Internalization assays detect membrane receptor translocation to the endosome. Cell lines are engineered to co-express ED-tagged membrane receptors and an EA tag localized to the endosome. Small molecules or biologics (e.g., antibodies, antibody-drug conjugate toxins) that induce activation of the receptor-ED fusion protein leads to internalization of the receptor to the EA-tagged endosomes, thus forcing the complementation of the two β-gal enzyme fragments. The resulting functional β-gal enzyme hydrolyzes a substrate to generate a chemiluminescent signal.

Checkpoint receptor assays have broad applications for cell-based screening, functional characterization studies, and QC lot-release assays. They can be implemented in anti-receptor, anti-ligand, co-stimulatory, and inhibitory phase-appropriate assessment of biologics and small molecule therapeutics. View applications associated with checkpoint receptor signaling reporter.

Anti-Receptor and Anti-Ligand Application

PathHunter checkpoint assays can be used to study anti-receptor or anti-ligand activity. The PathHunter PD-1 Signaling Assay was used to demonstrate anti-PD-1 and anti-PD-L1 inhibitory and shows inhibition dose response curves for A. anti-receptor and anti-ligand antibodies using the FDA approved anti-PD-1 antibodies Keytruda®, Opdivo®, and B. an anti-PD-L1 antibody. Keytruda® and Opdivo® are registered trademarks of Merck and BMS, respectively.

Co-Stimulatory and Co-Inhibitory Response

PathHunter assays can be applied to screen agonist and antagonist responses. This is seen in the anti-receptor and anti-ligand application for an antagonist example, and here, for an agonistic example using the PathHunter ICOS signaling assay with an anti-ICOS antibody to quantitate the agonistic activity of the antibody targeting the ICOS receptor.

Characterizing Small Molecule Inhibitors

PathHunter assays are fit-for-purpose for determining the potency of biologics and small molecule therapeutics. In this example, the PathHunter PD-1 Signaling Assay was used to assess small molecule receptor tyrosine kinase inhibitors, anti-PD-1 (antibody), and a low molecular inhibitor of anti-PD-L1.

Antibody-Mediated Cross-Linking by FcγR Clustering

Cross-linking of Fcγ receptors through clustering enables greater access to checkpoint receptors to improve assay performance. Agonistic antibody Pogalizumab-mediated stimulation of OX40 using the PathHunter OX40 signaling assay. Soluble Pogalizumab was unable to activate OX40 cells alone. However co-culturing with human FcγRIIb clustering cells (Cat No. 93-1133C3), enables access to more receptors on the cell surface thereby allowing Pogalizumab to exhibit a dose-dependent increase in NIK stabilization as opposed to none-to-marginal response with the soluble antibody.

Checkpoint Receptor Dimerization

PathHunter dimerization cell lines can be used to assess ligand-induced checkpoint receptor dimerization. Human VISTA antibody (an agonist) was used to dimerize VISTA/VISTA receptors resulting in a robust dose-dependent increase in signal reflecting an EC50 of 4.31 ng/mL and an assay window (S/B) of 4.9.

Study Antibody-Drug Conjugates ADCs  Analyze Antibodies That Activate Checkpoint Receptor Internalization

Checkpoint receptor antibodies activate receptor internalizationA. PathHunter internalization assay for the CD33 checkpoint receptor was tested with a commercial antibody to CD33. CD33 antibody was pre-incubated with a secondary antibody to cluster the CD33 receptor and then added to cells to monitor internalization. With higher cross-linked antibody concentrations, greater amounts of PK-tagged CD33 complemented EA-tagged protein localized to the endosome. A number of ADCs for CD33 are in clinical trials for the treatment of acute myeloid leukemia. B. B-cell maturation antigen (BCMA) receptor internalization was activated with a commercial BCMA antibody. Several BCMA ADCs are in development as a therapeutic for multiple myeloma.

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Target Lists Products Target List

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Target Lists Cell-based Assays for Biologics Development Programs

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White Papers Accelerating Immune Checkpoint Drug Discovery through Functional Cell-Based Assays

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Screening cell-based assays for analyzing the movement of proteins to multiple membrane compartments within the cell

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Custom Capabilities

Custom cell lines, kits, assays, & protein development capabilities optimized to fit your requirements

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