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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
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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
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
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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
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
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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
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
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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
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
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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
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
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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
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
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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
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
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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!
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.