Validated Cell-Based Assays for Rapid Screening and Functional Characterization of Therapeutic Monoclonal Antibodies
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1 Validated Cell-Based Assays for Rapid Screening and Functional Characterization of Therapeutic Monoclonal Antibodies Abhishek Saharia, Ph. D. Senior Product Manager
2 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
3 DiscoveRx Platform Technologies Quantitative Binding Enzyme Complementation Primary Human Cell Profiling Products & Services Portfolio Kits and Cell Lines LeadHunter Services 3 Assay Development
4 Depth of Coverage 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
5 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
6 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
7 DiscoveRx EFC Technology Substrate 7
8 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
9 Mean RLU RLU RLU Validated for Antibody Development Anti-Ligand Antibody Anti-Receptor Antibody CCR CCR5 GPCRs Anti-CCL20 mab [g/ml] + 10nM CCL Anti-CCR5 mab [g/ml] + 8 nm CCL3 EGFR Receptor Tyrosine Kinases TrkA EGFR Blocking Ab (g/ml) 9
10 Prioritize Leads Based on Potency Data generated with BioTek Instrumentation 10
11 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
12 Highly Specific Assays for Receptor-Receptor Interaction 12
13 Kinase Receptor Assays for Antibody Characterization 13
14 IL-17R Dimerization Using PathHunter * * 14
15 Added Value!! Whole Cell Antibody Binding Assays * Cells stained with 20uL of anti- CXCR4 PE (R&D Systems) Receptor Number: CHO-K1 CXCR4 ~ 106, Data kindly provided by Hassan Issafras, Xoma LTD
16 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
17 Identify Antibodies for Antibody Drug Conjugates Receptor Internalization Assays 17
18 Activated Total Simple Assays for Receptor Internalization RTKs GPCRs 18
19 Validated Assays for Receptor Internalization GFP-Labeled GPCR - agonist + agonist EGFR INSR 19
20 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
21 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
22 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 Signal 22
23 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 CCL20 [M] CCL20 20% FBS 40% FBS 60% FBS 80% FBS High Tolerance to Plasma CCR5 Neat heparinized plasma [hccl5] M Plasma Media 23
24 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 Correlation Coefficient of LOG-LOG P = points below the 45-degre 8 points above the 45-degre Run 2 No validation requirements exist for t
25 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
26 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
27 Well Suited for QC and Immunogenicity Testing Good S:B ratios No interference from human serum Detect Neutralizing Antibody NAb sensitivity ng/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
28 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
29 Technology Available as Products AbHunter Kits - Frozen Cells (Growth Arrested) - Detection Reagents - Cell Plating Media 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
30 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
31 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
32 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
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