High-Throughput Screening for Antibody Discovery Using Mirrorball

Transcription

1 High-Throughput Screening for Antibody Discovery Using Mirrorball Christyne Kane Senior Scientist I AbbVie Bioresearch Center Antibody Engineering & Therapeutics December 12, 2017

2 Christyne Kane is an employee of AbbVie. Assessment of equipment is the opinion of the author. AbbVie does not endorse any of the platforms described in this presentation. Disclaimer High-Throughput Screening for Antibody Discovery using Mirrorball Date

3 Overview Antibody Screening Assays ELISA Alternative: Bead Size Discrimination Antibody Discovery: Mirrorball HTS Isotype Determination: Bead Color Discrimination Cell Surface Binding Singleplex Multiplex Cell Killing Assay High-Throughput Screening for Antibody Discovery using Mirrorball Date

4 High-Throughput Screening for Antibody Discovery Numbers Immunizations In Vivo Antibody Discovery Workflow 5 x x Hybridoma Generation (Fusion) Screening Sub-cloning Purification & Characterization Desired Antibody Properties Soluble Target Binding Cell Surface Antigen Binding Species Cross-reactivity Target Specificity Functionality Epitope Binning Isotype Use of Mirrorball Technology High-Throughput Screening for Antibody Discovery using Mirrorball Date

5 Mirrorball Technology Mirrorball Mirrorball Instrument Instrument Configuration Benefits of Mirrorball HOMOGENEOUS ASSAY - No wash steps Ability to MULTIPLEX for multiple antigens, cross-reactivity, or counter-screening Compatible with CONDITIONED MEDIA SENSITIVITY (adequate for low concentration supernatants) 3 LED lasers 405nm 488nm 640nm 4 Fluorescent Channels FL-1 ( nm) FL-2 ( nm) FL-3 ( nm) FL-4 ( nm) 1 Scatter Channel (SC-1) Automation: Thermo Scientific Orbitor RS +/- HIT ASSESSMENT, flexible data analysis Integrates with AUTOMATION, miniaturization for HTS TIME SENSITIVE (24-hour turnaround) PLUG-AND-PLAY assay for multiple targets High-Throughput Screening for Antibody Discovery using Mirrorball Date

6 Case Study: Target A Target Biology TNF family member Transmembrane glycoprotein Forms a homotrimer Extracellular Membrane Autoimmune indications Goal: To generate a panel of antibodies binding cell-surface Target A. Desired Binding Properties Binding to specific epitope Binding to cell surface protein Immunization Strategy Peptide immunizations in mice High-Throughput Screening for Antibody Discovery using Mirrorball Date

7 Multiplexed Screening Enabled by Bead Size Discrimination Mix-and-Read Assay Setup Beads are Distinguishable Compare to ELISA 384 well plate format No washing steps Shorter incubation times Smaller volumes of reagents Multiplex-able BV421 Anti-Species Target-Specific Ab 7μm beads 16μm beads Non-specific Antigen (optional) Target Antigen SA/Biotin Specific Binding Detectable 7μm Bead 16μm Bead BV421 Bead Size High-Throughput Screening for Antibody Discovery using Mirrorball Date

8 Hybridoma HTS by Mirrorball Completely Correlates with ELISA Mirrorball vs. ELISA Mirrorball (Median FL2 Mean) Threshold ELISA (OD 450nm) Hit by Mirrorball & ELISA Hit by neither 100% Correlation Singleplex assay using 7μm beads No false positives No false negatives High-Throughput Screening for Antibody Discovery using Mirrorball Date

9 Screening Methods are in Agreement for Hit Identification Screening Assay Number of mabs Primary Screening (ELISA) 153 Secondary Screening ELISA 38 Flow 38 Mirrorball 38 Binding to Target A OD Binding to Target A Peptide (ELISA) nm mab 1 mab 2 mab 3 mab 4 mab 6 mab 7 mab 8 mab 5 Neg. Control Summary Bead-based binding assay correlated with ELISA methods 100% for early supernatant screening. 8 antibodies identified bound specifically to Target A by flow cytometry. GeoMean Ratio TNF-293H/wt-293-H Binding to Target A Cells (Flow) nm mab 1 mab 2 mab 3 mab 4 mab 6 mab 7 mab 8 mab 5 Neg. Control High-Throughput Screening for Antibody Discovery using Mirrorball Date

10 Case Study: Target B Target Biology Soluble protein Neurology and cardiovascular indications Extracellular Membrane Goal: To generate a panel of antibodies binding Target B. Desired Binding Properties Binding to target B protein +/- Binding to target protein family members Immunization Strategy Protein immunizations in mice High-Throughput Screening for Antibody Discovery using Mirrorball Date

11 Direct Isotype Determination Enabled by Sixplex Bead-based Assay Mix-and-Read Assay Setup Beads are Distinguishable AF488 Anti-Species Light Chain AF405/BV421 *LC-specificity (optional) sol-r2 sol-r3 sol-r4 sol-r5 Test Antibody Anti- Species HC SA/Biotin Specific Binding Detectable sol-r2 sol-r3 sol-r4 sol-r5 AF488 IgG2b IgG3 IgG2a IgG1 FL-4 High-Throughput Screening for Antibody Discovery using Mirrorball Date

12 HC & LC Isotypes are Clearly Detected in Hybridoma Supernatant Antibody HC Isotype LC Isotype Kappa Lambda mab 1 IgG3 Kappa mab 2 IgG1 Kappa IgG2b IgG3 IgG2a IgG1 IgG2b IgG3 IgG2a IgG1 mab 3 IgG2a Kappa mab 4 IgG2a Kappa mab 5 IgG1 Lambda IgG2b IgG3 IgG2a IgG1 IgG2b IgG3 IgG2a IgG1 mab 6 IgG2a Kappa mab 7 IgG2a Kappa Able to detect multiple isotypes per sample mab 8 IgG2a Kappa mab 9 IgG2a Kappa IgG2b IgG3 IgG1 IgG2b IgG3 IgG2a IgG1 mab 10 IgG3 Kappa High-Throughput Screening for Antibody Discovery using Mirrorball Date

13 HT Isotype Determination Improves Upon Traditional ELISA ELISA Isotyping Mirrorball Isotyping OD (450nm) IgG1 IgG2a IgG2b IgG3 Isotype Antibody Isotype IgG2a mab 1 IgG3 IgG2a mab 2 IgG2a IgG2a mab 3 IgG2a IgG1 IgG2a IgG2b IgG3 OD (450nm) IgG1 IgG2a IgG2b IgG3 Non-specific signals have potential to dilute or overpower specific signals, leading to incorrect isotype conclusions. IgG2a mab 4 IgG2a IgG1 mab 5 IgG3 IgG2a mab 6 IgG2a IgG2a mab 7 IgG2a IgG2a mab 8 IgG2a IgG2a mab 9 IgG2a IgG3 mab 10 IgG3 IgG1 IgG2a IgG2b IgG3 Specific signals are greater than all background levels, allowing for accurate determination of isotype results. High-Throughput Screening for Antibody Discovery using Mirrorball Date

14 Case Study: Target C Target Biology 7-Transmembrane GPCR with short extracellular loops. Extracellular NH2 EL1 EL2 EL3 Small molecule antagonists and agonists are available. Unprecedented for targeting by biologics Membrane M1 M2 M3 M4 M5 M6 M7 COOH Goal: To generate a panel of antibodies binding cell surface Target C which can be internalized. Desired Binding Properties Binding to cell surface Cross reactivity to cyno Cross reactivity to rat Desired Biological Activity Cell Killing Immunization Strategy cdna immunizations in rats High-Throughput Screening for Antibody Discovery using Mirrorball Date

15 Singleplex Cell Binding Assay Enables HT Supernatant Screening Mix-and-Read Assay Setup GFP+ Cells are Detectable AF647 Anti-Species Target- Specific Ab Target - Expressing Cell Line (GFP+) Target Antigen GFP AF647 Merge AF647 Specific Binding Detectable (-) Binding (+) Binding GFP High-Throughput Screening for Antibody Discovery using Mirrorball Date

16 Target C Campaign Enabled by Mirrorball Antibody Screening Target C Species Summary Mirrorball Cell Killing Number of mabs Human 65 Cyno 59 Rat 39 22/28 tested Single-plex cell binding assay enabled all stages of supernatant screening. Antibodies identified bound specifically to Target C by flow cytometry and had a range of functional potencies. GMFI Ratio (Trans/WT) GMFI Ratio (Trans/WT) Flow Cytometry Binding to Target C Human nm Rat 1 nm High-Throughput Screening for Antibody Discovery using Mirrorball Date GMFI Ratio (Trans/WT) Cyno nm 1 10 Bio-activity

17 Case Study: Target D Target Biology TNF family member Transmembrane glycoprotein Forms a homotrimer Extracellular Membrane Goal: To generate a panel of antibodies binding Target D which blocks interaction with receptor. Desired Binding Properties Binding to cell-surface protein Cross-reactivity to Cyno Desired Biological Activity Block ligand/receptor interactions Immunization Strategy Whole cell immunizations in mice High-Throughput Screening for Antibody Discovery using Mirrorball Date

18 Multiplexed Cell Binding Assay Enables HT Supernatant Screening Mix-and-Read Assay Setup AF488 Anti-Species Target- Specific Ab Target Antigen Compare to Flow Cytometry 384 well plate format No washing steps Shorter incubation times Smaller volumes of reagents Fewer cells required Multiplex-able Cell Types are Distinguishable Transfected Parental Specific Binding Detectable Target- Expressing Cell Line Parental Cell Line AF nM Far Red 1000nM Far Red Far Red High-Throughput Screening for Antibody Discovery using Mirrorball Date

19 Hybridoma HTS by Mirrorball: 89% Correlation with Flow Cytometry Mirrorball vs. Flow Cytometry Mirrorball (Trans Median FL2 Mean/WT Median FL2 Mean) Flow Cytometry (GMFI Ratio: Trans/WT) Hit by Mirrorball & Flow Cytometry Hit by Flow Cytometry only Hit by neither 89% Correlation Mirrorball missed 5/45 hits by Flow Cytometry (false negatives) No false positives High-Throughput Screening for Antibody Discovery using Mirrorball Date

20 Multiplexed Cell Binding Assay is Implemented for Sub-clone Screening Summary Screening Assay Number of mabs Primary Screening (Flow) 95 Secondary Screening Flow 56 Blocking (Flow) 16 Multiplexed cell binding assay demonstrated an 89% correlation with Flow cytometry methods. All downstream sub-clone screening was performed using the Mirrorball multiplexed cell binding assay, enabling the delivery of 8 antibodies. Binding & Blocking Assays GMFI (Trans/WT) GMFI Binding to Target D Cells Conc. (nm) Target D Ligand Blocking (Flow) Conc. (nm) mab 1 mab 2 mab 3 mab 4 mab 5 mab 6 mab 7 mab 8 Neg.Control mab 1 mab 2 mab 3 mab 4 mab 5 mab 6 mab 7 mab 8 Neg Control Pos Control High-Throughput Screening for Antibody Discovery using Mirrorball Date

21 Case Study: Target E Target Biology GPI-linked, glycosylated, membrane protein. Modulates T cell and macrophage activation. Oncology indications Extracellular Membrane GPI GPI Goal: To generate a panel of antibodies binding cell surface Target E which can be internalized. Desired Binding Properties Binding to cell surface target E Cross-reactivity to Cyno Cross-reactivity to Mouse Desired Biological Activity Internalization Immunization Strategy cdna immunizations in mice High-Throughput Screening for Antibody Discovery using Mirrorball Date

22 High Throughput Cell Killing Assay Mix-and-Read Assay Setup SMi Anti-Species Ab Target- Specific Ab Target Antigen ~72-96hrs Detection Live Apoptotic* Dead Hoechst Annexin V- AF488* TO-PRO-3 *Optional Well View Target- Expressing Cell Line Cells & Media Cells & Ab Complex High-Throughput Screening for Antibody Discovery using Mirrorball Date

23 Target-specific Cell Killing is Detectable Plate View Example Data 100 Cell Killing 80 % Viability Cells & Media Anti-Target C mab Neg. Control Conc. (nm) 1 10 % Viability = # Dead Cells + # Apoptotic Cells # Total Cells Summary Target-specific killing is detectable. No non-specific killing is observed. High-Throughput Screening for Antibody Discovery using Mirrorball Date

24 Cell Killing Assay Enables HT Detection of Internalizing Antibodies Screening of Hybridoma Supernatants % SN 2% SN 0.4% SN 0.08% SN Cell Viability Hybridoma clones (-)(+) Platform Advantages Compatible with early hybridoma screening. Automated for high-throughput assay set-up and data reporting. Streamlined process to identify functional leads. High-Throughput Screening for Antibody Discovery using Mirrorball Date

25 Antibody Screening Assays: Saving Time, Labor & Reagents Antibody Discovery: Mirrorball HTS Antibody Screening Assays ELISA Alternative: Bead Size Discrimination Plug-and-play assay is easily established, requiring smaller amounts of reagents and significantly less time, compared to traditional ELISA. Isotype Determination: Bead Color Discrimination Color-coded sol-r beads enable the establishment of a robust multiplex assay able to identify 4-8 unique antibody properties with increased sensitivity compared to traditional methods. Format may be applied to cross-reactivity and specificity assays. Cell Surface Binding No wash assays require 1/10 th the cells of traditional flow cytometry screening and may be automated at all steps, including data processing. Cell Killing High throughput cell killing assay allows for functional screening of hybridoma supernatants using a small amount of material and a variety of secondaryconjugated toxins. High-Throughput Screening for Antibody Discovery using Mirrorball Date

26 Acknowledgements Amanda Horowitz Kwasi Ofori Mary Abdalla Jane Seagal William Stine Julie Hugunin Shawn Jennings Chung-Ming Hsieh Diana Caracino Richard Kim Paul Wylie Ben Schenker Doug Mann Aline Cagwin Daniela Ionescu Jenny Buttaro Mirrorball Support High-Throughput Screening for Antibody Discovery using Mirrorball Date