CHECKPOINT RECEPTOR ASSAYS

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.

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.

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).

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.

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.

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.

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. 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.

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.

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.

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.

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.

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.

Checkpoint receptor antibodies activate receptor internalization. A. 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.