CYTOKINE RECEPTOR PRODUCT SOLUTIONS

PathHunter cytokine receptor assays are based on the Enzyme Fragment Complementation (EFC) technology. These assays allow for the measurement of receptor activation, dimerization, SH2-recruitment, IκB degradation, and STAT3 transcription through various assay modalities, ultimately allowing for therapeutic candidate screening, potency, and stability testing. Select assay principles for receptor dimerization and IκB protein degradation assays are shown below. For additional assay principles, refer to receptor kinase, signaling pathway reporter, targeted protein degradation, and GPCR target and application pages.

PathHunter Tocilizumab Bioassay Assay Principle

Measure IL-1β induced heterodimerization of IL-1R1 and IL-1RAP receptors using the PathHunter Anakinra Bioassay (Cat. No. 93-1032Y3-00106) qualified with the FDA approved therapeutic Kineret (anakinra, an IL-1R antagonist being evaluated as potential therapeutic). The assay principle involves the complementing of PK and EA EFC fragments tagged to IL-1RAP and IL-R1, respectivitly. In presence of IL-1β, the receptors dimerize and there is an increase in signal detected upon substrate addition. However, in the presence of anakinra, the binding of IL-1β is inhibited and the signal decreases. Kineret^® is a registered trademark, licensed by Swedish Orphan Biovitrum AB and marketed by Sobi, Inc.

PathHunter Anakinra Bioassay Assay Principle

Measure IL-1β induced heterodimerization of IL-1R1 and IL-1RAP receptors using the PathHunter Anakinra Bioassay (Cat. No. 93-1032Y3-00106) qualified with the FDA approved therapeutic Kineret (anakinra, an IL-1R antagonist being evaluated as potential therapeutic). The assay principle involves the complementing of PK and EA EFC fragments tagged to IL-1RAP and IL-R1, respectivitly. In presence of IL-1β, the receptors dimerize and there is an increase in signal detected upon substrate addition. However, in the presence of anakinra, the binding of IL-1β is inhibited and the signal decreases. Kineret^® is a registered trademark, licensed by Swedish Orphan Biovitrum AB and marketed by Sobi, Inc.

PathHunter Sargramostim Bioassay Assay Principle

Measure GM-CSF induced heterodimerization of CSF2RA with CSF2RB PathHunter Sargramostim Bioassay Kit (Cat. No. 93-1078Y3-00112) qualified with the FDA approved therapeutic Leukin (sargramostim), which is being evaluated as potential therapeutic. The assay principle involves the complementing of PK and EA EFC fragments tagged to CSF2RB and CSF2RA, respectivitly. In presence of GM-CSF or sargramostim, the receptors dimerize and there is an increase in signal detected upon substrate addition. Leukine^® is a registered trademark licensed to Genzyme Corporation.

PathHunter Adalimumab Bioassay Assay Principle

Detect IkB protein degradation as a result of TNFα-mediated activation of the NF-kB signaling pathway using PathHunter Adalimumab Bioassay Kit (Cat. No. 93-0538B15-00132) qualified with the FDA approved therapeutic Humira (adalimumab), which is being evaluated as potential therapeutic. The assay principle involves the enzyme donor ProLabel^® (PL) tagged to phosphorylated IkB, and the IκB levels can then be measured by the addition of EA, which forces complementation of the two EFC fragments. The resulting active enzyme hydrolyzes the substrate to generate the signal that is proportional to the degree of IκB stabilization. In presence of TNFα, which activates the NF-kB signaling pathway, proteasome-mediated IκB degradation occurs and there is a loss of signal. In the presence of both TNFα and adalimumab, which blocks the binding of TNFα, degradation of IκB is avoided, and the EFC complementation occurs leading to an increase in signal upon substrate addition. Humira^® is a registered trademark AbbVie, Inc.

Accurately Measure Receptor Activation with Highly Reproducable Bioassays for QC Lot Release

Tocilizumab Bioassay (Cat. No. 93-1045B3-00110) assessment of intra-day plate-to-plate and inter-day variability. Three experimental replicates of the assay were prepared as 11-point dose-response curves and run on the same plate with duplicate wells per dose on the same day by one operator. Results indicate 978, 960 and 997 ng/mL mean IC₅₀’s for plates 1-3, respectively, and an overall 9.7% intermediate precision IC₅₀. Similar experiments were repeated again to obtain data from three individual days (data not shown) for inter-day variability assessment with results showing an overall 1330 ng/mL mean IC₅₀ and 24.6% intermediate precision IC₅₀ for the 3 days. Results from both experiments for inter-plate and inter-day variability analysis show excellent uniformity demonstrating high reproducibility required for bioassay implementation in a QC environment for drug release

Analyze Multiple Interleukin Receptor Famillies with Highly Specific Assays

Representative examples of assays for interleukin receptors from 6 different interleukin receptor families. Each plot shows a dose response for the relevant ligand(s) in a given assay from the indicated family. Data plotted are mean RLU and standard deviation from at least triplicate wells for each dose. These assays are characterized by robust assay windows and low CVs.

Quantify Anti-Receptor or Anti-Ligand Therapeutic Antibodies with Receptor Dimerization Assays

Reproducibly measure ligand-mediated receptor dimerization for potency and stability testing of therapeutics such as Kineret^® (anakinra) implemented in QC lot release programs.

The PathHunter Anakinra Bioassay (Cat. No. 93-1032Y3-00106) was used to quantify an inhibitory response from Kineret (anakinra, an IL-1R antagonist being evaluated as potential therapeutic).
– [A.] In the presence of anakinra, there is a dose dependent inhibition (signal-to-background (S/B) 4.2; IC₅₀ 302 ng/mL) of IL-R1 and IL-1RAP dimerization. In comparison with agonist IL-1β, the receptors dimerize and there is an increase in signal detected (S/B 5.3; EC₅₀ 4.2 ng/mL).

– [B.] Dilutional linearity derived from the relative potency experiments performed over a range of 50-150%. Overall, the bioassay measures a robust response to both anakinra and IL-1β with a high accurate potency of over 99.4%. Kineret is a registered trademark, licensed by Swedish Orphan Biovitrum AB and marketed by Sobi, Inc.

Measure TNFα-Mediated Activation of the NF-κB Signaling Pathway with MOA-Reflective, IkB Protein Degradation Bioassays

The PathHunter Adalimumab Bioassay Kit (Cat. No. 93-0538B15-00132) was used to measure IκB protein degradation as a result of TNFα-mediated activation of the NF-kB signaling pathway.
– [A.] In the presence of adalimumab (Humira^®), there is a dose dependent inhibition of TNFα-mediated activation IkB protein degradation and an increased signal (S/B 6.5; IC₅₀ 5.45 ng/mL). In comparison with TNFα alone, a decrease in signal detected (S/B 10.3; IC₅₀ 53.9 ng/mL).

– [B.] Dilutional linearity derived from the relative potency experiments performed over a range of 50-150%. Overall, the bioassay measures a robust response to both Humira and TNFα with an excellent accurate potency of over 99.99%. Humira is a registered trademark AbbVie, Inc.