Stefanie Urlinger, Director R&D, MorphoSys AG Antibodies against difficult targets: How to tackle G-protein coupled receptors 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, September 2013: Discovery on Target 2 Introduction: GPCRs & Antibody Technologies © MorphoSys, September 2013: Discovery on Target 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, September 2013: Discovery on Target 4 „Standard“ Antibody Targets EITHER: Soluble (cytokine, chemokine, complement factors etc.) OR: Receptors with relatively large, hydrophilic extracellular domains (e.g., receptor tyrosine kinases, RTKs) 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, September 2013: Discovery on Target 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, September 2013: Discovery on Target 6 The Principle of Ylanthia Phage Display Fab antibody phage are exposed to immobilized target molecule Target-specific antibodies bind to immobilized antigen Amplification: Antibody phage production w/ helper phage Unbound phage are washed away REPEAT TWO ROUNDS Screening of 1000s of individual Fab-expressing E. coli clones; hit picking and sequence analysis Phage infection of E. coli Elution of targetspecific phage recovers genetic antibody information Adapted from: Grönwall et al. 2009, J. Biotechnol. 140, 254 © MorphoSys, September 2013: Discovery on Target 7 Anti-GPCR Antibodies by Phage Display © MorphoSys, September 2013: Discovery on Target 8 Showcase: CXCR2 © MorphoSys, September 2013: Discovery on Target 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 pro-angiogenic 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, September 2013: Discovery on Target 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 + MOR022719 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, September 2013: Discovery on Target 11 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 MOR022717: Labeled with 1 phycoerythrin (PE) molecule per mAb Titration of MOR022717-PE CXCR2 CHO cells 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 Concentration MOR022717-PE [nM] = Antibodies Bound per Cell Titration of MOR022717-PE CXCR2 CHO cells 1.0×10 6 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 Concentration MOR022717-PE [nM] About 700.000 molecules of anti-CXCR2 PE labeled antibody MOR022717 bound per cell → ~700.000 CXCR2 molecules expressed on each CXCR2 CHO cell © MorphoSys, September 2013: Discovery on Target 12 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, September 2013: Discovery on Target 13 Characterization of anti-CXCR2 Antibodies: Specific Cell Binding Titration of anti-CXCR2 antibodies (IgG1) CXCR2 CHO cells Evaluation of specific cell binding: MOR022714 MOR022715 MOR022716 MOR022717 MOR022718 MOR022719 MOR003207 100000 MIF Anti-CXCR2 antibodies were titrated on CXCR2 overexpressing CHO cells and cell binding was determined by flow cytometry 150000 50000 0 0.0001 MOR003207: Negative control antibody 0.01 1 100 Antibody Concentration [nM] 10000 Titration of anti-CXCR2 antibodies (IgG1) GPCR CHO cells 150000 Numerous antibodies bind selectively to CXCR2 overexpressing CHO cells 100000 MIF No binding to CHO cells transfected with unrelated GPCR detectable 50000 0 0.0001 © MorphoSys, September 2013: Discovery on Target MOR022714 MOR022715 MOR022716 MOR022717 MOR022718 MOR022719 MOR003207 MOR003207: Negative control antibody 0.01 1 100 Antibody Concentration [nM] 10000 14 Characterization of anti-CXCR2 Antibodies: Induction of ADCC Evaluation of antibody-dependent 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 FcγRIIIa expressing cell line (Promega; good correlation to ADCC with human PBMCs shown) 600000 Titration of anti-CXCR2 antibodies (IgG1) CXCR2 CHO cells MOR022714 MOR022715 MOR022716 MOR022717 MOR022718 MOR022719 MOR003207 RLU 400000 200000 0 0.0001 http://www.promega.com/de-de/products/biologics/bioassays-for-biologics/adcc-reporter-bioassays/ MOR003207: Negative control antibody 0.01 1 100 Concentration IgG1 [nM] Stimulation of FcγRIIIa signaling as a measure for ADCC detected with most antiCXCR2 antibodies © MorphoSys, September 2013: Discovery on Target 15 Characterization of anti-CXCR2 Antibodies: Induction of CDC Evaluation of complement-dependent cytotoxicity (CDC): Titration of anti-CXCR2 antibodies (IgG1) CXCR2 CHO cells 100 % Dead cells 80 60 MOR022714 MOR022716 MOR022717 MOR022718 MOR022719 MOR003207 40 20 0 0.01 0.1 1 10 Concentration IgG1 [nM] MOR003207: 100 Negative control antibody 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 Absolutely specific for CXCR2-expressing cells (data not shown) © MorphoSys, September 2013: Discovery on Target 16 Characterization of anti-CXCR2 Antibodies: Efficient Internalization Evaluation of internalization: Titration of anti-CXCR2 antibodies (IgG1) CXCR2 CHO cells 5.0x106 RLU 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 MOR022716 MOR022717 MOR022718 MOR022719 1.0×10 6 5.0x105 1.0×10 5 0.0001 0.01 1 100 Antibody Concentration [nM] Anti-CXCR2 antibodies internalize efficiently and specifically © MorphoSys, September 2013: Discovery on Target 17 Characterization of anti-CXCR2 Antibodies: β-Arrestin Recruitment Blockade of β-arrestin recruitment (PathHunter® cells, DiscoveRx): Whole cell, functional assay that measures CXCR2 activity by detecting the interaction of β-arrestin with the activated CXCR2: Titration of anti-CXCR2 antibodies (Fab) CXCR2 CHO β-Arrestin cells (PathHunter®) Stimulation with IL-8 800000 RLU 600000 Fab 1000nM Fab 100nM Fab 10nM 400000 200000 M O R 02 11 79 M O R 02 11 + 79 aa 148 pe pt M O id R e M 02 O 23 R 02 15 23 + 15 aa 148 pe pt M O id R e M 02 O 23 R 02 16 23 + 16 aa 148 pe pt m M M I id L O O -8 ed e R R 00 00 M on ium 32 32 O ly o 07 07 R0 [1 nl 0 .4 y + aa + a 320 9n 1- a1 7 M] 48 -4 + pe 8 p ILpt ep 8 id tid e + e IL -8 0 MOR003207: Negative control antibody Concentration dependent inhibition of IL-8 induced β-arrestin recruitment detected with several anti-CXCR2 Fab and IgG1 © MorphoSys, September 2013: Discovery on Target 18 Affinity Maturation Leads to Epitope Spread Antibodies MOR022714 and MOR022716 were generated by pannings on the N-terminal CXCR2 peptide (aa1-17) Both antibodies recognize cell surface expressed CXCR2 as well as the N-terminal CXCR2 peptides aa1-17 and aa1-48 Affinity maturation resulted in epitope spread to extracellular loop 3 (ECL3) while strong binding to the CXCR2 N-terminus and CXCR2 cell surface recognition were maintained © MorphoSys, September 2013: Discovery on Target 19 Production of Purified CXCR2: Expression Construct Selection Codon-optimized cDNA is synthesized in house (Slonomics) >10 different expression constructs cloned per GPCR − With/without leader sequence − Several N-terminal and C-terminal tags − C-terminally truncated constructs Two mammalian expression hosts compared for each GPCR Microscale expression check of all constructs Selection of most promising GPCR expression construct Large scale transient expression and purification − Refinement of solubilisation buffer (detergents, pH, salt) − Antibody affinity chromatography preferred method for purification Add-on: Stable pool generation © MorphoSys, September 2013: Discovery on Target 20 Expression of CXCR2 Ten different constructs were tested for expression by Western Blot Transient expression in HEK-derived human cell line Purification via anti-Lysozyme Affinity Chromatography 1 2 3 4 5 6 7 8 9 10 FT W E1 E2 E3 E4 E5 E6 kDa 70 55 35 25 15 kDa anti-CXCR2 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. vk_His_hCXCR2 vk_StrepII_hCXCR2_His hCXCR2_His vk_StrepII_hCXCR2 vk_chLys-Flag_hCXCR2 vk_chLys-Flag_hCXCR2_His vk_hCXCR2_eGFP_His hCXCR2_eGFP_His vk_hCXCR2_Flag-chLys_avi hCXCR2_Flag-chLys_avi anti-Lysozyme affinity chromatography 60 50 40 30 chLys-Flag_ hCXCR2_His 25 Final yield of ~0.3-0.5 mg/L for chLys-Flag_hCXCR2_His construct High purity in SDS-PAGE © MorphoSys, September 2013: Discovery on Target 21 Activity Check of Purified CXCR2 Antibody and IL-8 binding to purified CXCR2: CXCR2 and an irrelevant GPCR are immobilized Binding of an anti-CXCR2 antibody or IL-8 is detected using appropriate reagents Anti-CXCR2 antibody binding Ligand binding Specific binding of anti-CXCR2 antibody to the GPCR N-terminus Even more important: Specific binding of the natural ligand IL-8 © MorphoSys, September 2013: Discovery on Target 22 Summary CXCR2 Anti-CXCR2 antibodies can be 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, internalize and mediate ADCC and CDC Several antibodies can antagonize IL-8 signaling as shown by inhibition of β-arrestin recruitment Recombinant CXCR2 can be produced, solubilized and purified in sufficient amounts and quality © MorphoSys, September 2013: Discovery on Target 23 MorphoSys Teams up with Heptares © MorphoSys, September 2013: Discovery on Target 24 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 very difficult GPCRs (instable, low expressing) where stabilization might be needed: Partnership with Heptares © MorphoSys, September 2013: Discovery on Target 25 Summary Generation of specific and functionally active antibodies against GPCRs − Broad range of GPCR-focused 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, September 2013: Discovery on Target Ailanthus spec: Tree of the Gods, Tree of Heaven 26 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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