Validated Cell-Based Assays for Rapid Screening and Functional Characterization of Therapeutic Monoclonal Antibodies Abhishek Saharia, Ph. D. Senior Product Manager
Validation Assays Drug Discovery Evolution and Diversification Natural Extracts Small Molecules Fragment Based Peptide Monoclonal Antibodies Antibody Conjugates Ligand Binding Assays Cell-Based Assays Human Primary Cells 2
DiscoveRx Platform Technologies Quantitative Binding Enzyme Complementation Primary Human Cell Profiling Products & Services Portfolio Kits and Cell Lines LeadHunter Services 3 Assay Development
Depth of Coverage 284 389 21 23 28 Custom Development DiscoveRx Assays for Drug Discovery 1,118 assays covering 741 druggable targets All major drug target classes covered Multiple assays for single target 0% GPCRs Kinases NHRs Pathways Epigenetics Other Targets 100% Primary Human Cell Profiling 4
Bioassays Needed for Therapeutic MAbs Good bioassays needed for druggable receptors (GPCRs, RTKs, etc.) Difficult to develop bioassays for novel receptor targets Bioassays to identify antibodies causing receptor internalization for antibody-drug conjugates (ADCs) Difficult to develop and validate bioassays for QC and Immunogenicity testing 5
Agenda Functional bioassays for MAb development GPCRs RTKs Novel receptor targets Bioassays to identify internalizing antibodies (for ADCs) Key benefits of PathHunter bioassays Validation of assays for QC and immunogenicity testing Availability of assays from DiscoveRx 6
DiscoveRx EFC Technology Substrate 7
Functional Assays for GPCRs and RTKs GPCR Assays Arrestin Recruitment RTK Assays SH2 Recruitment 2 nd Messenger Signaling Assays Available camp Detection Calcium Flux* IP3* 8
Mean RLU RLU RLU Validated for Antibody Development Anti-Ligand Antibody Anti-Receptor Antibody 5000 4000 CCR6 12500 10000 CCR5 GPCRs 3000 2000 7500 5000 1000 2500 0 10-8 10-7 10-6 10-5 10-4 10-3 Anti-CCL20 mab [g/ml] + 10nM CCL20 0 10-10 10-9 10-8 10-7 10-6 10-5 10-4 Anti-CCR5 mab [g/ml] + 8 nm CCL3 EGFR Receptor Tyrosine Kinases TrkA 10000 8000 6000 4000 EGFR 111.6 528 225 2000 10-11 10-10 10-9 10-8 10-7 10-6 10-5 Blocking Ab (g/ml) 9
Prioritize Leads Based on Potency Data generated with BioTek Instrumentation 10
Industry Leading Menu GPCR, RTK & Cytokine Receptor Assays 88% of the 207 GPCRs with known ligands validated Largest collection of orthologs Receptor Tyrosine Kinases c-kit c-met c-ret-gfrα2 DDR1 DDR2 EphA4 EphA5 EphA7 EphB1 EphB2 EphB3 EphB4 ErbB1 ErbB2-ErbB3 ErbB4 FGFR1 IGF1R INSR PDGFRa PDGFRb TrkA TrkA-P75 TrkB TrkB-P75 TrkC TrkC-P75 Cytokine Receptor Assays CSF3R-JAK1 EpoR-JAK2 GHR-JAK1 PRLR-JAK1 TNFa Receptor 11
Highly Specific Assays for Receptor-Receptor Interaction 12
Kinase Receptor Assays for Antibody Characterization 13
IL-17R Dimerization Using PathHunter * * 14
Added Value!! Whole Cell Antibody Binding Assays * Cells stained with 20uL of anti- CXCR4 PE (R&D Systems) Receptor Number: CHO-K1 CXCR4 ~ 106,494 15 Data kindly provided by Hassan Issafras, Xoma LTD
Agenda Functional bioassays for MAb development GPCRs RTKs Novel receptor targets Bioassays to identify internalizing antibodies (for ADCs) Key benefits of PathHunter bioassays Validation of assays for QC and immunogenicity testing Availability of assays from DiscoveRx 16
Identify Antibodies for Antibody Drug Conjugates Receptor Internalization Assays 17
Activated Total Simple Assays for Receptor Internalization RTKs GPCRs 18
Validated Assays for Receptor Internalization GFP-Labeled GPCR - agonist + agonist EGFR INSR 19
Agenda Functional bioassays for MAb development GPCRs RTKs Novel receptor targets Bioassays to identify internalizing antibodies (for ADCs) Key benefits of PathHunter bioassays Validation of assays for QC and immunogenicity testing Availability of assays from DiscoveRx 20
Bioassay Needs For Antibody Discovery and Development Assays in Current Use Binding Assays (ELISA and Cellular) camp Assays Protein Secretion Assays Reporter Gene Assays Cytotoxicity/Proliferation Assays Features of an Ideal Assay Full-length Native Receptors Target-Specific Functional Response Broad Range of Sample Tolerance Challenges Downstream response of assay High Reproducibility & Miniaturizable Validated for Antibody Development 21 Assay output may not be specific Assay not tolerant of complex samples Assay may be lengthy and complex May not be sensitive enough
Target- Specific Signal Tagged Full Length Receptor No background from other receptors Tagged Response Protein Only specific protein-protein interaction can provide signal Receptor Protein 1 Signal Receptor Protein 3 Protein 2 +++++ Signal 22
-12-11 -10-9 -8-7 RLU Beta-arrestin activity Arbitrary units Broad Range of Sample Compatibility Various Antibody Sources Semi-purified/Purified MAbs Polyclonal Sera Bacterial Supernatant Mammalian Supernatant Complex biological samples Data generated by Gemma White and kindly provided by Dr. David Greaves High Tolerance to Serum CCR6 Serum Tolerance with agonist 25000 20000 15000 10000 5000 0 10-11 10-10 10-9 10-8 10-7 10-6 CCL20 [M] CCL20 20% FBS 40% FBS 60% FBS 80% FBS High Tolerance to Plasma CCR5 Neat heparinized plasma 70000 60000 50000 40000 30000 20000 10000 0 0 [hccl5] M Plasma Media 23
Run 1 Simple Protocols with Reproducible Results One-Step, Plate-Treat-Read Protocol Highly Reproducible Results RTK Assays Run 1 vs. Run 2 with 45-degree Line GPCR Assays 100 10 1 0.1 Correlation Coefficient of LOG-LOG P = 0.991 11 points below the 45-degre 8 points above the 45-degre 0.01 24 0.001 0.001 0.01 0.1 1 10 100 Run 2 No validation requirements exist for t
Key Benefits of PathHunter Assays for Antibody Development Validated for antibody development Full-length receptors Target-specific functional response Broad range of sample tolerance Highly reproducible & can be miniaturized Can be used for early functional screens Industry leading menu 25
Agenda Functional bioassays for MAb development GPCRs RTKs Novel receptor targets Bioassays to identify internalizing antibodies (for ADCs) Key benefits of PathHunter bioassays Validation of assays for QC and immunogenicity testing Availability of assays from DiscoveRx 26
Well Suited for QC and Immunogenicity Testing Good S:B ratios No interference from human serum Detect Neutralizing Antibody NAb sensitivity 250-500ng/ml Excellent lot-to-lot reproducibility Highly specific assay Intra-assay Inter-assay 3-7 %CV Inter-analyst Strong correlation between Live cells and Frozen cells. Ab Hunter EC 50 * Experiment was performed with 100 targets 27
Agenda Functional bioassays for MAb development GPCRs RTKs Novel receptor targets Bioassays to identify internalizing antibodies (for ADCs) Key benefits of PathHunter bioassays Validation of assays for QC and immunogenicity testing Availability of assays from DiscoveRx 28
Technology Available as Products AbHunter Kits - Frozen Cells (Growth Arrested) - Detection Reagents - Cell Plating Media - 384-well plate - Antibody dilution buffer* - Agonist* 29 Assay Cell Lines - Frozen Cells (Unlimited Culture) Synergy Products - Culture Media - Freezing and Thawing Media - Plating Reagents - Cell Detachment Reagent - Detection Reagent
LeadHunter Services Portfolio MAX Service Target Classes Description ELECT GPCR, TK, NHR, PATH GPCR, TK, NHR, PATH Profile against entire panel for separate Target Class, including the full GPCR panel and orphan GPCRs Create personalized or custom panels using any Target Class and any readout. EC50 ELECT GPCR, TK, NHR, PATH Quantitative affinity constants against any assay IC50 ELECT GPCR, TK, NHR, PATH Quantitative affinity constants against any assay. PANEL GPCR 15 therapeutically relevant disease panels DUO GPCR Study the same target using any 2 assay readouts or compare pharmacology of the human & ortholog receptors. TRIO GPCR Study the same target using any 3 assay readouts. LIBRARY GPCR, TK, NHR, PATH Compound library screening against a single target or against the full panel 30
DiscoveRx Custom Assay Development Capabilities Custom assay provider for past 6 years ~30 projects per year Flexible structure based on milestone targets and deliverables Access to all technologies Master service agreement or single custom projects Matrixed into current R&D structure 31
Summary Largest Commercial GPCR and TK assay menu Develop Assays for Novel Receptors Study Mechanism of Action Validated for Antibody Characterization and Identification Key Benefits of PathHunter Assays Target-Specific Functional Response Broad Range of Sample Tolerance Rapid, Simple Protocol Miniaturizable & High Reproducibility Flexibility of Options for Purchase 32