Challenging Targets: How to identify Antibodies against G-Protein Coupled Receptors
Markus Enzelberger, Senior Vice President R&D MorphoSys AG Challenging Targets: How to identify Antibodies against G-Protein Coupled Receptors GPCRs Offer Only Small Extracellular Area Accessible to Antibodies Hutchings C. et al. 2010, mAbs 2, 594-606 G-protein coupled receptors (GPCRs) comprise a huge class of proteins From the known ~800 GPCRs ~350 are considered relevant targets for drug discovery High potential as antibody targets: - High specificity: Binding to extracellular regions - Limited access through BBB → no CNS side effects - Long in vivo half life - Possibility of utilizing effector functions Technically challenging to drive antibodies to small extracellular GPCR surface © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 2 An Integrated Platform for GPCR Targeting The generation of specific, high quality and functionally active antibodies against GPCRs is a very complex, technically challenging task No all-in-one solution possible Instead: Portfolio of many techniques and different materials required − Large, high quality antibody library covering broad structural antibody repertoire: YLANTHIA − Presentation of target antigen in various formats − Elaborate selection strategies − High throughput screening, e.g., in flow cytometry − Broad assay portfolio MorphoSys has all the expertise, technologies and materials required in-house to make GPCR antibody programs a success © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 3 Challenges in Generating AntiGPCR Antibodies © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 „Standard“ Antibody Targets EITHER: Soluble (cytokine, chemokine, complement factors etc.) OR: Receptors with relatively large, hydrophilic extracellular domains (e.g., receptor tyrosine kinases, RTKs) Stable proteins, easy to target Well accessible during antibody selections and characterization Composite image of EGF-activated EGFR generated from known structures (no structural information for regions that link the extracellular and intracellular domains). From Bessmann & Lemmon: Finding the missing links in EGFR (2012). Nature Structural & Molecular Biology 19, p. 1-3 © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 5 The Challenging Antibody Targets: GPCRs Often only 25-30% of the protein (~100 aa) are theoretically accessible for antibodies All extracellular residues are very close to the membrane Glycosylation etc. further shield the GPCR surface Many GPCR antibodies lack specificity (reviewed by Michel et al., Naunyn Schmiedebergs Arch Pharmacol 2009; 379, p. 385-388) Mukhopadyay & Huber: Molecular model of an opsintransducin complex in a lipid bilayer From: The Sakmar Lab, Rockefeller University Sophisticated approaches are needed to drive antibodies to the small, partially hidden extracellular GPCR regions © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 6 MorphoSys‘ Solution to Anti-GPCRs Antibodies Phage display Fab library Fully human antibodies with superior biophysical properties GPCR peptides GPCR overexpressing cell lines Purified, stabilized GPCR in detergent Purified GPCR in lipid vesicles Heptares Andrew Dore, Structure 2011 Antibodies against: No binding to structured GPCR loops („3D“) Cell surface protein Binding to proteins other than target GPCR StaR® Binding to TM or intracellular portion Lipid-embedded GPCR Binding to intracellular portion; hydrophobic, sticky - Combination of different antigen materials for selections (cells, peptides, StaR etc.) - HT flow cytometry to screen for native conformation, extracellular loops & specificity - Antibody library producing „manufacturable“ antibodies: © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 RISKS SOLUTION SOLUTION RISKS Peptide Antibodies against: 7 Showcase: CXCR2 © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 Showcase: CXCR2 Target: CXCR2 is a class A GPCR belonging to the chemokine receptor family Size: 360 amino acids (N-terminus: 48 aa) Ligands: CXCR2 is the only high-affinity receptor for all proangiogenic chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, CXCL8/IL-8). Physiological roles: − Mobilization and recruitment of leukocytes (especially neutrophils) from the bone marrow to sites of inflammation − Migration of endothelial cells in angiogenesis Expression: − Mainly on neutrophils, but also on monocytes − Various cancers and cancer cell lines (NSCLC, pancreas, breast etc.) © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 9 CXCR2 Over-Expressing CHO Cell Line: Quantification of Receptor Density BD QuantibriteTM PE Beads conjugated with four levels of PE were used to assess receptor density (antibodies bound per cell) of CXCR2 on over-expressing CHO cell line by flow cytometry Anti-CXCR2 antibody MOR-CXCR2-D: Labeled with 1 phycoerythrin (PE) molecule per mAb Titration of anti-CXCR2 antibody on CXCR2 CHO cells 700.000 1.0×10 6 PE GeoMean 8.0×10 5 6.0×10 5 4.0×10 5 2.0×10 5 0 0.001 0.01 0.1 1 10 100 1000 MOR-CXCR2-D – PE [nM] MOR-CXCR2-D – PE [nM] Calibration curve with Quantibrite beads: x PE molecules correspond to y fluorescence signal About 700.000 molecules of anti-CXCR2 PE labeled antibody MOR-CXCR2-D bound per cell → ~700.000 CXCR2 molecules expressed on each CXCR2 CHO cell © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 10 CXCR2: Material and Antibody Selections Material: − Stable CXCR2 over-expressing CHO cell line − Stable CXCR2 over-expressing HEK293 cell line − Virus-like particles (Integral Molecular; HEK based) − N-terminal and ECL3 peptides Cell line CHO-CXCR2 + MOR-CXCR2-F GPCR material used through different rounds of antibody selections: − Over-expressing CXCR2 cell lines only − Combination of CXCR2 CHO cells with peptides − Combination of CXCR2 CHO cells with virus-like particles © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 11 CXCR2: Antibody Selection and Characterization Hundreds of CXCR2-specific fully human, cell binding hits were identified applying various selection strategies − Peptide pannings (mainly N-terminus) − Pannings on CXCR2 over-expressing cells − Selections using virus-like particles … and any combinations thereof Sensitive and high-throughput flow cytometry assays in combination with overexpressing GPCR cell lines built up for antibody screening So far, six ‘front runner’ antibodies characterized in detail in Fab and IgG1 formats. Further antibodies selected to be characterized in functional and binding assays Also antibodies were identified binding to epitopes other than the N-terminus by selections on full-length antigen and affinity maturation © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 12 Characterization of anti-CXCR2 Antibodies Specific Cell Binding MFI Titration of anti-CXCR2 antibodies (IgG1) CXCR2 CHO cells MIF CXCR2 150000 MOR022714 MOR-CXCR2-A MOR022715 MOR-CXCR2-B MOR022716 MOR-CXCR2-C MOR022717 MOR-CXCR2-D MOR-CXCR2-E MOR022718 MOR-CXCR2-F MOR022719 MOR-negative MOR003207 control 100000 50000 0 0.0001 MFI 0.01 1 100 Antibody Concentration [nM] Evaluation of specific cell binding Anti-CXCR2 antibodies were titrated on CXCR2 overexpressing CHO cells and cell binding was determined by flow cytometry 10000 Titration of anti-CXCR2 antibodies (IgG1) GPCR CHO cells MIF Irrelevant GPCR 150000 MOR022714 MOR-CXCR2-A MOR022715 MOR-CXCR2-B MOR022716 MOR-CXCR2-C MOR022717 MOR-CXCR2-D MOR-CXCR2-E MOR022718 MOR-CXCR2-F MOR022719 MOR-negative control MOR003207 100000 50000 0 0.0001 0.01 1 100 Antibody Concentration [nM] 10000 © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 Numerous antibodies bind selectively to CXCR2 overexpressing CHO cells No binding to CHO cells transfected with unrelated GPCR detectable 13 Characterization of anti-CXCR2 Antibodies Induction of ADCC http://www.promega.com/dede/products/biologics/bioassays-forbiologics/adcc-reporter-bioassays/ RLU 600000 Titration of anti-CXCR2 antibodies (IgG1) CXCR2 CHO cells MOR022714 MOR-CXCR2-A MOR022715 MOR-CXCR2-B MOR022716 MOR-CXCR2-C MOR022717 MOR-CXCR2-D MOR-CXCR2-E MOR022718 MOR-CXCR2-F MOR022719 MOR-negative MOR003207control 400000 200000 0 0.0001 0.01 1 Evaluation of antibodydependent cellular cytotoxicity (ADCC) Standard ADCC assay with human PBMCs and flow cytometry read out might be hampered by expression of CXCR2 on PBMCs leading to substantial cell death of PBMCs Alternative: Luciferase reporter assay with FcgRIIIa expressing cell line (Promega) Good correlation to ADCC with human PBMCs shown 100 Concentration IgG1 [nM] Efficient stimulation of FcγRIIIa signaling as a measure for ADCC detected with most anti-CXCR2 antibodies © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 14 Characterization of anti-CXCR2 Antibodies Induction of CDC Evaluation of complementdependent cytotoxicity (CDC) Standard CDC assay Absolutely specific for CXCR2expressing cells (data not shown) Titration of anti-CXCR2 antibodies (IgG1) CXCR2 CHO cells 100 % Dead cells 80 60 MOR-CXCR2-A MOR022714 MOR-CXCR2-C MOR022716 MOR-CXCR2-D MOR022717 MOR-CXCR2-E MOR022718 MOR-CXCR2-F MOR022719 MOR-negative MOR003207 control 40 20 0 0.01 0.1 1 10 Concentration IgG1 [nM] 100 Anti-CXCR2 antibodies mediate efficient complement-dependent cell killing of CXCR2 overexpressing CHO cells using human serum Maximum cell killing almost 100% with IC50 values in low nM range © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 15 Characterization of anti-CXCR2 Antibodies Efficient Internalization Titration of anti-CXCR2 antibodies (IgG1) CXCR2 CHO cells RLU 5.0x106 MOR-CXCR2-C MOR022716 MOR-CXCR2-D MOR022717 MOR-CXCR2-E MOR022718 MOR-CXCR2-F MOR022719 1.0×10 6 5.0x105 1.0×10 5 0.0001 0.01 1 100 Antibody Concentration [nM] Evaluation of internalization Fab-ZAP is a monovalent saporin conjugate binding to human antibodies Once the Fab-ZAP is internalized together with the primary anti-CXCR2 antibody, free saporin inactivates the ribosomes that leads to the inhibition of protein synthesis and cell death Useful as measure for internalization and first filter for potential of primary antibody as antibody-drug conjugate Anti-CXCR2 antibodies internalize efficiently and specifically © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 16 Characterization of anti-CXCR2 Antibodies β-Arrestin Recruitment Blockade of β-arrestin recruitment (PathHunter® cells, DiscoveRx) Titration of anti-CXCR2 antibodies (Fab) CXCR2 CHO ß-arrestin cells (PathHunter®) 800000 RLU 600000 400000 200000 Fab 1000 nM Fab 100 nM Fab 10 nM 0 + - + + + - + + MOR-CXCR2-G MOR-CXCR2-H MOR-CXCR2-I - + + - + - - + + - MOR-neg.contr. 8 + + 8 + - ] IL-8 Peptide Whole cell, functional assay that measures CXCR2 activity by detecting the interaction of βarrestin with the activated CXCR2 Read-out: Chemiluminescence (β-galactosidase substrate) N-terminal CXCR2 peptide added to show specificity Concentration dependent inhibition of IL-8 induced β-arrestin recruitment detected with several anti-CXCR2 Fab and IgG1 © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 17 Characterization of anti-CXCR2 Antibodies Binding to Endogenous CXCR2 4000 RFU Antibody binding 6000 2000 0 10000 RFU IL8 ligand binding MOR-CXCR2-D MOR-negative control Control anti-CXCR2 15000 5000 0 IL8 + anti-IL8 non-stimulated U937 71 h 95 h Stimulation 119 h 142 h 167 h CHO irr.GPCR1 CHO huCxCR2 CHO irr.GPCR2 Evaluation of binding to endogenous CXCR2 Stimulation of promonocytic U937 cell line with PMA* induces differentiation into macrophages and (among others) upregulation of CXCR2 expression Specific binding of antiCXCR2 antibodies was detected in FACS * phorbol myristate acetate Anti-CXCR2 recognize endogenous CXCR2 expressed on differentiated U937 cells © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 18 Characterization of anti-CXCR2 Antibodies Binding to CXCR2 on Ovarian Tumor Cells anti-EpCAM anti-EpCAM Tumor cells isolated from an ovarian cancer sample Tumor specimen was digested to obtain isolated cells Target expression was assessed by flow cytometry measurement using anti-CXCR2 antibody MOR-CXCR2-D MOR-CXCR2-D MOR-negative control ab Antibody MOR-CXCR2-D recognizes endogenous CXCR2 on cells isolated from an ovarian tumor © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 19 Summary CXCR2 Stable CXCR2 over-expressing mammalian cell lines were generated Recombinant CXCR2 can be produced, solubilized and purified in sufficient amounts and quality Anti-CXCR2 antibodies have been generated by various selection methods: Combinations of pannings using CXCR2 over-expressing cell lines, virus like particles and peptides representing the GPCR N-terminus The resulting antibodies show specific CXCR2 cell surface binding on recombinant cell lines, differentiated cells with endogenous expression as well as primary tumor cells The antibodies mediate ADCC and CDC as well as targetdependent internalization Several antibodies antagonize IL-8 signaling as shown by inhibition of β-arrestin recruitment © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 20 Antibody Generation with Detergent Solubilized, Stabilized GPCR © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 MorphoSys Teams up with Heptares © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 22 Heptares: The StaR® Company Stabilized Receptor StaR® = GPCR contains a small number of point mutations that improve its thermostability (also in detergent), while retaining its functional and drug-binding characteristics StaR proteins are locked in the conformation derived from the pharmacology of the ligand used in their creation The stabilization make a GPCR perfectly suitable for all processes during antibody generation, such as phage display selections and screening The MorphoSys – Heptares collaboration: Heptares to generate StaRs for a set of GPCR disease targets proposed by MorphoSys MorphoSys to apply its Ylanthia antibody library to discover & develop antibody therapeutics 1) Structure of GPCR thermostabilized in the agonist conformation and in complex with endogenous agonist ligand (grey). 2) Electron density around the cyanopindolol ligand binding site in b1AR. Acknowledgements: Warne et al, Nature. 2008. © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 23 First Collaboration Project Class A GPCR; validated target in inflammatory diseases Stabilized in antagonist conformation After generation and analysis of >900 receptor mutants: Heptares delivered a stabilized (StaR) form of the first GPCR target selected by MorphoSys No stabilizing mutations in parts accessible to antibody (N-terminus, ECLs) Fully capable of binding small molecule antagonist ligand WT Var. 1 Var. 2 Var. 3 Var. 4 Melting temperature Tm increased by 26°C, from ~18°C to ~44°C Half life increased from 1 hr to 18 hrs at 10°C → sufficient to survive antibody selections © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 24 Antibody Selections on StaR Three rounds of phage display antibody selections were performed on: − StaR immobilized on plates − StaR immobilized on magnetic beads − StaR in solution − StaR and target GPCR-CHO cell line in alternating selection rounds After antibody selections: More than 5100 clones were screened in high-throughput flow cytometry on the target GPCR CHO cell line versus a CHO cell line expressing an irrelevant GPCR using Fabs expressed from bacterial lysates © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 25 Screening for Anti-GPCR Antibodies High Throughput Flow Cytometry “The HTFC™ Screening System is a flow-cytometry-based platform designed to perform high-throughput, multiplexed, single-cell analysis on cells or particles (beads) in suspension” Cytometer (Accuri C6 from BD) HTFC Sampler HyperCytPRO Software 96-well / 384-well plates Standard and deep well © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 26 Screening in High-Throughput Flow Cytometry Flow cytometry screening on target GPCR-CHO cell line to select for primary hits: >10 fold over background 5-10 fold 2-5 fold background Flow cytometry screen on CHO cell line expressing an irrelevant GPCR to deselect non-target or unspecific antibodies: Output of antibody selections on StaR (solubilized and purified GPCR): - Primary screening in flow cytometry yielded many antibodies binding the cell surface expressed GPCR specifically - This indicates correct folding of StaR GPCR used for the antibody selections - Almost no antibodies binding to cells expressing irrelevant GPCR © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 27 Summary: Antibody Generation with Detergent Solubilized, Stabilized GPCR Stabilized and detergent solubilized GPCR (= StaR generated by Heptares) is suitable for phage display antibody selections − High hit rate on cell surface expressed GPCR − High binding signals with no detectable background binding: high specificity − Identification of a diverse set of antibodies Next steps: − Antibody characterization (binding, functional activity, biophysical properties etc) © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 28 MorphoSys‘ Track Record of GPCR Targeting MorphoSys‘ comprehensive GPCR portfolio: − Reliable generation of stable GPCR over-expressing mammalian cell lines − Advanced antibody selection and screening methods combining various GPCRderived antigens − In depth antibody characterization, also for biophysical properties − Broad assay portfolio (β-arrestin, pERK1/2, internalization, ADCC, CDC, ligand binding inhibition, etc.) − For difficult GPCRs (instable, low expressing) where stabilization might be needed: Partnership with Heptares © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 29 Summary Generation of specific and functionally active antibodies against GPCRs − Broad range of GPCR-focused antigen presentation and selection methods − Comprehensive assay portfolio − Biophysical antibody characterization Shown for three class A and one Frizzledtype GPCR Absolutely important for generating diverse, functional and high quality antibodies: Best in class antibody library YLANTHIA as unique antibody source MorphoSys is offering the complete package required for successfully generating anti-GPCR antibodies © MorphoSys, BIT's 6th Annual Congress of Antibodies; 25th-28th April 2014 Ailanthus spec: Tree of the Gods, Tree of Heaven 30 Thank you! HuCAL®, HuCAL GOLD®, HuCAL PLATINUM®, CysDisplay®, RapMAT®, arYla® , Ylanthia® and 100 billion high potentials® are registered trademarks of MorphoSys AG. Slonomics® is a registered trademark of Sloning BioTechnology GmbH, a subsidiary of MorphoSys AG.
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