Accelerate Your COVID-19 Drug Discovery with Qualified Cell-Based Assays for Proinflammatory Cytokines
Gaurav Agrawal, Ph.D. Scientific Development Manager, Eurofins DiscoverX COVID-19: Therapeutic Approaches for Disease Management
The pathophysiology of COVID-19 in severe cases shows an aggressive inflammatory responses. Thus the disease severity is not only due to the viral infection but also the host’s immune response to the infection.
The pathology of COVID-19 in severe cases shows an aggressive inflammatory responses from the host’s immune system against the infected tissue. Such acute inflammatory response causes long term organ damage. Therefore, several ongoing clinical programs are evaluating therapeutics targeting the inflammatory signals as a treatment strategy for COVID-19.
Cytokine release syndrome (CRS), commonly referred to as “cytokine storm” is the major cause of death in fatal COVID-19 cases. It is caused by a dysfunctional hyper-release of proinflammatory cytokines by the host immune system and may cause Acute respiratory distress syndrome (ARDS). This can lead directly to respiratory failure.
Proinflammatory cytokine levels are significantly elevated in patients with ARDS, and the degree of Image source www.fda.gov increase is positively correlated with mortality rate.
Cerep | DiscoverX | Panlabs | EID | Villapharma 2 Proinflammatory Cytokine Pathogenesis of COVID-19
Host Factors Healthy immune response Viral Clearance - Age
ACE2 - Smoking CD8+ Alveolar T-cell - Pre-existing Epithelial conditions Cells - IL-15 T - Cell - Clearing of infected cells by CD8+ - IFNα/β T-cell - IFNγ - Virus inactivated by Nabs and cleared - IL-12 - Minimal inflammation and lung Inflammatory - IL-22 damage Signals Dendritic Cells Dysfunctional immune response Hyperinflamation
CCL3 CCL2 - IL-6 - Excessive infiltration of immune cells CCL4 CXCL10 Monocyte - IL1β - Systemic cytokine storm - TNFα - Edema and pneumonia - GM-CSF - Widespread inflammation and multi- - IL17A organ damage Macrophages - G-CSF
Cytokine Storm High Mortality
Cerep | DiscoverX | Panlabs | EID | Villapharma 3 Implications of Cytokine Storm in COVID-19 Patients
IL-1β IL-6 IL-17 TNFα GM-CSF Cytokine IL-2 CCL3 Storm G-CSF CCL2 CXCL10 IL-10 IL-8
Immune ARDS Clotting Lung Injury Cardiac Arrest Renal Failure Paralysis
Immune Failure, Multiple Organ Failure, Secondary Infections
Death
Cerep | DiscoverX | Panlabs | EID | Villapharma 4 Withstanding the Cytokine Storm in COVID-19: Therapeutic Approach
Eurofins DiscoverX Cell-Based Functional Assay Available!
Cerep | DiscoverX | Panlabs | EID | Villapharma 5 Analytical Assays for Therapeutics Targeting Key Proinflammatory Cytokines
1. Qualified Bioassays: • Case Study with PathHunter® Tocilizumab Bioassay - Qualified with for therapeutics targeting IL-6 pathway
• IL-1β - PathHunter® Anakinra Bioassay - Qualified with
• GM-CSF - PathHunter® Sargramostim Bioassay – Qualified with
• TNFα - PathHunter® Adalimumab Bioassay - Qualified with
2. Signaling Reporter Assays • IL-6 • TNFα • RANK (TNFSF11)
3. Functional Cell-Based Assays for Small Molecule Inhibitors • JAK1, JAK2 and JAK3
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Cerep | DiscoverX | Panlabs | EID | Villapharma 6 Technology Overview Enzyme Fragment Complementation
Cerep | DiscoverX | Panlabs | EID | Villapharma 7 Enzyme Fragment Complementation (EFC) Platform
Enabling Technologies with a Flexible Platform based on a Split β-Galactosidase Enzyme
Inactive Fragments Active Enzyme EFC Assay Principle: • Complementation between two inactive enzyme fragments called the Enzyme Donor (ED) and Enzyme acceptor (EA) results in formation of an active β-gal enzyme. • The active functional β-gal enzyme hydrolyzes its Enzyme Donor Enzyme Acceptor substrate to produce chemiluminescent signal. ED EA
• Homogenous Format — Mix-and-read assay format that does not require washing, centrifugation, or filtration • Robust — Enzymatically-amplified assays with a large signal-to-background ratio and high precision with Z' factors >0.7 and lot-to-lot reproducibility • Qualified and Validated — Extensively optimized for hundreds of targets used for screening in billions of data points, and thousands of peer-reviewed publications • Scalable — Easily scalable and HTS-friendly from 96- to 1536-well microplate format
Cerep | DiscoverX | Panlabs | EID | Villapharma 8 Enzyme Fragment Complementation (EFC) Versatile and Robust Platform for Cell-Based assays
ExampleExample 2:1: ReceptorSHP recruitment dimerization
FGFR4 Agonist IL-1β FGFR4 FGF IL-1R1 IL-1RAP IL-1R1 IL-1RAP Agonist Anakinra IL-1β IL-1R1 IL-1RAP or Anti-IL-1β Enzyme fragment complementationAgonist Enzyme donor Enzyme fragment(active β -gal enzymeIL-)1β fragment SHP1 SHP1 complementation Enzyme donor Enzyme donor (active β-gal enzyme) fragment Enzyme acceptorfragment Enzyme acceptor Enzyme acceptor fragment fragment fragment
Antagonist Agonist Signal
[Compound] Highly quantitative responses
Cerep | DiscoverX | Panlabs | EID | Villapharma 9 Enzyme Fragment Complementation (EFC) Versatile and Robust Platform for Cell-Based Assays
Protein-Protein Quantitation and Downstream Response Secretion and Interaction Degradation Evaluation Translocation
Internalization and Compound Target Quantify Cytotoxicity Analyte Detection Trafficking Engagement
Cerep | DiscoverX | Panlabs | EID | Villapharma 10 Flexibility of Formats Phase-Appropriate Solutions for Every Development Program
From Continuous Culture to Qualified Bioassays
Continuous Cell Lines Culture (Discovery to QC)
Qualified Target (Marketed Drug)
Bioassays Ready-to-Use (Designed for QC Lot Release) MOA-Based (Reference Ligand)
Eurofins DiscoverX Qualified Bioassays are designed for QC Lot Release Programs
Cerep | DiscoverX | Panlabs | EID | Villapharma 11 EFC Cell-Based Assays Protocol
Typical assay protocol for stable cell lines and assay ready kits
Incubate 1 to 3 hours
Incubate 1 hour Add overnight Add Dark compound Read assay plate Detection in a luminometer Plate cells in assay plate reagents Simplified, universal assay format Everything is included! • Easy-to-follow, detailed user manuals • Rapid, single addition protocol • Homogeneous, no wash format Sensitive detection with large S:B ratio Easily quantified luminescence read-out • Dose-response curve Bioassay Kit includes all reagents needed for the assay. No need to worry • Utilizes any standard plate luminometer about external sourcing of media reagents that may introduce variability
Cerep | DiscoverX | Panlabs | EID | Villapharma 12 Case Study: PathHunter® Tocilizumab Bioassay - Qualified with Actemra® for Therapeutics Targeting IL-6 Activity
Cerep | DiscoverX | Panlabs | EID | Villapharma 13 PathHunter® Tocilizumab Bioassay - Qualified with Actemra®
MOA-reflective Assay Design: IL-6 Receptor Dimerization IL-6ST IL-6ST The assay is designed to detect IL-6 – induced interaction of the IL-6 receptor (IL-6R) with the IL-6 Signal Transducer protein (IL-6ST) Enzyme Fragment IL-6R EA Light Complementation Tocilizumab IL-6 IL-6ST IL-6ST Substrate (gp130) (gp130) IL-6
IL-6 Signal Tocilizumab [Compound] PK EA IL-6ST IL-6ST IL-6 +
Tocilizumab
EA
Actemra® is a registered trademark of Chugai Seiyaku Kabushiki Kaisha Corp., a member of the Roche Group
Cerep | DiscoverX | Panlabs | EID | Villapharma 14 Assay Parameters Assessed for Qualification
• Assay consistency (%CV) between eight 11-pt DRCs
• Plate uniformity: EC80 and IC80 (of IL-6 and Tocilizumab) across entire plate • Plate-to-Plate variability: 3 plates with 11-pt DRCs run on 3 days • Slope consistency • Relative potency analysis: Accuracy, precision and linearity of the assay over a range of 50-150% preformed by two operators • Parallel line analysis using PLA3.0 or SoftMax Pro 7.1
Cerep | DiscoverX | Panlabs | EID | Villapharma 15 Assessing Intra-Day Plate-to-Plate Variability
Experiments depicting 3 assay plates with full-plate DRC - Same Day – One operator
Plate 1 Plate 2 Plate 3
Cerep | DiscoverX | Panlabs | EID | Villapharma 16 Assay Robustness Analysis
Repeatability and Intermediate Precision (Inter-Plate) Intermediate Precision (Inter-Day)
Plate Sample S/B % RSD, IC50, Mean IC50, % RSD, Day IC50, Mean IC50, %RSD, S/B ng/mL ng/mL IC50 ng/mL ng/mL IC50 R1 12.1 868 1 1250
1 R2 13.7 6.4 935 978 13.9 2 1690 1330 24.6 R3 12.6 1130 3 1050
R1 13.2 966 Intermediate Precision (Inter-Plate): 9.7% 2 R2 15.2 7.1 943 960 1.59 Intermediate Precision (Inter-Day): 24.6% R3 14.1 972
R1 14.5 850
3 R2 12.8 6.2 1080 997 12.8 R3 13.8 1060
Cerep | DiscoverX | Panlabs | EID | Villapharma 17 Parallelism and Relative Potency Estimation
Relative Potency: Parallelism and Potency Estimation (PLA) 50% 75% 125% 150%
Analyst 1
Analyst 2
Reference
Sample Note: RP values calculated in PLA 3.0
Cerep | DiscoverX | Panlabs | EID | Villapharma 18 Tocilizumab Bioassay: Qualification with Actemra®
Summary: Accuracy, Precision and Dilutional Linearity (Single Analyst)
Dilutional Linearity: 99.27% Accuracy: 105.1% Precision: 5.4%
Tocilizumab Bioassay is highly accurate, precise and linear!
Cerep | DiscoverX | Panlabs | EID | Villapharma 19 Case Study 2: IL-1β PathHunter® Anakinra Bioassay - Qualified with Kineret®
MOA-reflective Assay Design: Receptor Dimerization The assay is designed to detect IL-1β induced heterodimerization of the IL1R1 and IL1RAP receptors units
Light
Substrate
Kineret® is a registered trademark, licensed by Swedish Orphan Biovitrum AB and marketed by Sobi, Inc.
Cerep | DiscoverX | Panlabs | EID | Villapharma 20 Case Study 2: IL-1β PathHunter® Anakinra Bioassay - Qualified with Kineret®
Functional Data Bioassay Qualification Data
Dilutional Linearity: 96.7%
S/B EC50 / IC50 Accuracy: 99.4% IL-1β 5.5 4.2 ng/mL Precision: 11.4% Anakinra 4.2 302 ng/mL
Bioassay measures a robust response to IL1β and Anakinra. Anakinra Bioassay is highly accurate, precise and linear
Cerep | DiscoverX | Panlabs | EID | Villapharma 21 Case Study 3: GM-CSF PathHunter® Sargramostim Bioassay - Qualified with Leukin®
MOA-reflective Assay Design: Receptor Dimerization Bioassay Qualification Data The assay is designed to detect GM-CSF- induced heterodimerization of the CSF2RA and CSF2RB receptors
Sargramostim
CSF2RA CSF2RB Sargramostim or CSF2RA CSF2RB GM-CSF
Enzyme donor Dilutional Linearity: 99.56% fragment Enzyme acceptor fragment Accuracy: 99.1% Precision: 3% Sargramostim Bioassay is highly Substrate Light accurate, precise and linear
Cerep | DiscoverX | Panlabs | EID | Villapharma Leukine® is a registered trademark licensed to Genzyme Corporation 22 Case Study 4: TNFα PathHunter® Adalimumab Bioassay - Qualified with Humira®
MOA-reflective Assay Design: Protein Degradation TNFα The assay is designed to measure TNFα IκB degradation as a result of Adalimumab TNFα-mediated activation of the TNFR TNFR NF-κB signaling pathway.
IкB P PL Phosphorylated IкB PL NFкB NFкB IкB TNFα EA P Adalimumab NFкB Polyubiquitination PL Light Signal NFкB Proteasome-mediated Substrate IкB degradation [Compound] Humira® is a registered trademark AbbVie, Inc
Cerep | DiscoverX | Panlabs | EID | Villapharma 23 Case Study 4: TNFα PathHunter® Adalimumab Bioassay - Qualified with Humira®
Functional Data Bioassay Qualification Data
Dilutional Linearity: 99.99% S/B EC50 / IC50 TNFα 10.3 53.9 pM Accuracy: 105.1% Humira 6.5 5.45 ng/mL Precision: 5.65% Bioassay measures a robust response to TNFα and Humira Adalimumab Bioassay is highly accurate, precise and linear
Humira® is a registered trademark AbbVie, Inc
Cerep | DiscoverX | Panlabs | EID | Villapharma 24 Accelerating Implementation Phase for QC Lot Release with Qualified Bioassays Typical Bioassay Development Timeline Time to Market >12 months
Testing Method Characterize Assay Assay Lot/s Assay QC Lot Innovator Transfer Cell Line Optimization Qualification Manufacture Validation Drug (Client/CRO) Release
• Choose assay • Optimize assay • Refine • Establish • Generate lots for • Method review • ICH Validation format that best parameters conditions robustness, bioassay • On-site transfer (GMP) reflects MOA of (thaw-and-use with innovator precision, validation runs drug cells) drug accuracy, and linearity of assay
Eurofins DiscoverX Bioassay Development Timeline Time to market <3 months
Testing Method Characterize Assay Assay Lot/s Assay Innovator Transfer QC Lot Cell Line Optimization Qualification Manufacture Validation Drug (Client/CRO) Release
DONE DONE DONE DONE DONE • Method review • ICH Validation • On-site transfer (GMP) Cell Line Bioassay Bioassay Qualification Lots available runs established developed optimized with complete for immediate innovator drug use
Eurofins DiscoverX Ready-To-Plate Bioassay Kits Save 9 Months of Assay Development Time
Cerep | DiscoverX | Panlabs | EID | Villapharma 25 Signaling Pathway Reporter Assays for Cytokines
PathHunter® HepG2 PathHunter U2OS Assay Design : Signaling Reporter STAT3 Pathway Reporter Assay NF-κB Pathway Reporter Assay The assay is designed for quantifying the activation or inhibition 600000 500000
of signaling pathways to provide a downstream read-out that is 400000 400000 complementary to upstream receptor-based assays to gain a IL-6 300000
RLU Tocilizumab
comprehensive understanding of drug molecule’s MOA RLU 200000 200000 TNFα 100000 Humira Ligand Target Receptor 0 0 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -14 10 -13 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 (Endogenous or Heterologous) [Tocilizumab], g/mL + 0.3 ng/mL IL-6 [TNFα], g/ml or [Humira], g/mL + 11.4 ng/mL TNFα
EC /IC S/B Cell Membrane 50 50 TNF EC50Humira/IC50 +S/B 11.4ng/mL TNFα IL-6 0.33 ng/mL 6.2 Cytosol HillSlope TNF0.8773α 1.7 ng/mL-1.444 72.3 Tocilizumab 44 ng/mL 11.6 EC50 Humira1.702e-00913 pg1.385e-011/mL 64 S/B 72.3 64
Signaling Pathway PathHunter U2OS RANK NF-κB Pathway Reporter Assay
500000 RANKL Nucleus 400000 Prolia
300000
Transcription RLU Factor 200000 ePL-tagged Reporter 100000
Protein 0 EC50/IC50 S/B Regulatory ePL-tagged 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 RANKL 7.7 ng/mL 27 Element Reporter Gene Light [Prolia], g/mL +10 ng/mL RANKL Prolia 23 ng/mL 21
Cerep | DiscoverX | Panlabs | EID | Villapharma 26 PathHunter® Cell-Based Functional Assays for Small Molecule JAK Inhibitors
• There are several active, randomized, controlled trials evaluating the therapeutic potential of JAK inhibitors for treatment of COVID-19 (e.g. Ruxolitinib, Baricitinib) • JAK inhibitors can have a distinct advantage over other immunomodulatory strategies in COVID-19, as they can exert a dual effects: anti-inflammatory (blockade of multiple, pro-inflammatory cytokines at the same time) and anti-viral effects (inhibiting cellular viral endocytosis)
PathHunter U2OS CSF3R-JAK1 JAK2 PathHunter U2OS TYK2 Activity Assay JAK3 PathHunter U2OS JAK3 Activity Assay JAK1 Functional Assay 8000 12000 Ruxolitinib Tofacitinib hGH 6000 Prolactin 8000 4000 RLU RLU 4000 2000
0 0 10-12 10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10 -12 10 -11 10 -10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 [hGH], g/mL / [Ruxolitinib], M + hGH 0.5 µg/mL [Prolactin], g/mL / [Tofacitinib], M + 25 ng/mL Prolactin
EC50/IC50 S/B EC50/IC50 S/B EC50/IC50 S/B G-CSF 9 ng/mL 13 hGH 1.3 ng/mL 2.8 Prolactin 3.7 ng/mL 7.4 Lestaurtinib 0.2 μg/mL 12 Ruxolitinib 1.4 μg/mL 3.1 Ruxolitinib 0.7 μg/mL 5.2 Pyridone 6 0.25 μg/mL 4.5
Cerep | DiscoverX | Panlabs | EID | Villapharma 27 Custom Assay Services
Your Target Biology, Our Expertise — Building a Better Assay Together
• Development Expertise — Decades of cell-based assay development, cell line engineering, and recombinant enzyme development expertise CAD Services Capabilities • Cell Line Engineering Capability — Exogenous expression approaches (constitutive vs inducible) or gene editing (e.g. KO/KI with CRISPR/Cas9) • Collaborative — Consultative assay development with regular updates through a dedicated project manager • Complete Solution — Customized assay development with screening and profiling services within the same team
Cerep | DiscoverX | Panlabs | EID | Villapharma 28 Conclusions
Accelerate your drug discovery program with qualified, ready-to-use cell-based interleukin and cytokine assays for Covid-19 research • Target-Specific – MOA-reflective, functional assays for drugs targeting pro-inflammatory cytokines, such as IL-1, IL-6, IL-7, GM-CSF and TNFα, all of which are implicated in COVID-19 pathology • Qualified Assays – Robust, reproducible off-the-shelf assays ACE2 qualified with innovator drugs TMPRSS • Complete Solutions – Custom Assay Development enabling overexpression of other COVID-19-relevant receptors such as ACE2, TMPRSS2, etc.
Visit discoverx.com/covid-19 to learn more
Cerep | DiscoverX | Panlabs | EID | Villapharma 29 Thank You! Stable Cell Lines Qualified Bioassays MOA-based Bioassays Analytical Cell Banks Custom Assay Development GPCRs Checkpoint Receptors Cytokine Receptors Kinases Signaling Pathways TGFβ Superfamily Learn more about our available cell-based assays Products: www.discoverx.com ADCC Assays Services: www.eurofinsdiscoveryservices.com ADCP Assays CDC Assays T – Cell Redirection CD16 Effector Cells
Cerep | DiscoverX | Panlabs | EID | Villapharma 30