Antibodies against difficult targets: How to tackle G-protein coupled receptors

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

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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

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„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

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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

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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

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Anti-GPCR Antibodies by Phage Display

© MorphoSys, September 2013: Discovery on Target

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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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

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 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

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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

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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

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MorphoSys Teams up with Heptares

© MorphoSys, September 2013: Discovery on Target

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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

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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!

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