1. Jennifer Könitzer, October 14th 2015
Principal Scientist Immune Modulation & NBE Discovery @ Boehringer
Ingelheim, Biberach, Germany
Using chicken as hosts to derive antagonistic
antibodies against difficult therapeutic targets: A
case study for a GPCR
Web Symposium: How and Why to Use Chickens
for Your Next Antibody Discovery Campaign

2. GPCRs are one of the most important classes for drug
discovery but are difficult targets for antibody development
2
 GPCRs are one of the largest classes of drug-able
targets, but are difficult targets for antibody
development.
 Complex three-dimensional structures, unstable in
absence of membrane lipids, small & poorly antigenic
conserved extracellular loops/domains.
 Challenges in antigen production for immunization
and/or library screening.
 Obtaining functional antagonistic or agonistic Abs (as
opposed to mere binders) is challenging.
 Mogamulizumab (anti-CCR4) is the only GPCR Ab on the
market to date.
 GPCRs are one of the largest classes of drug-able
targets, but are difficult targets for antibody
development.
 Complex three-dimensional structures, unstable in
absence of membrane lipids, small & poorly antigenic
conserved extracellular loops/domains.
 Challenges in antigen production for immunization
and/or library screening.
 Obtaining functional antagonistic or agonistic Abs (as
opposed to mere binders) is challenging.
 Mogamulizumab (anti-CCR4) is the only GPCR Ab on the
market to date.
Boehringer-Ingelheim Project: mAbs for GPCR-X
• Desired activity profile: antagonistic cAMP IC50 <10nM
• Desired affinities in the sub-nanomolar range
• Ideally rodent X-reactive
 This presentation illustrates our experience using chicken as immunization hosts for generating anti-
GPCR-X functional mAbs.
Boehringer-Ingelheim Project: mAbs for GPCR-X
• Desired activity profile: antagonistic cAMP IC50 <10nM
• Desired affinities in the sub-nanomolar range
• Ideally rodent X-reactive
 This presentation illustrates our experience using chicken as immunization hosts for generating anti-
GPCR-X functional mAbs.

3. Using chicken as an alternative antibody production host has resulted in
a much higher number of functional antibodies than classic mouse and
rat hosts
3
Antigen
GPCR-X
ECD-Fc fusion
Recombinant
cell line
DNA
classic mouse &
rat hybridoma
Chicken & GEM
technology
Unique sequences Hybridoma
subclones, not
necessarily unique
 Chicken proved to be a superior host for anti-GPCR-X Abs with both higher overall numbers
and a much higher fraction of antagonistic Abs as opposed to mere binders.
 In addition, almost half of all chicken derived Abs were X-reactive.
 Chicken proved to be a superior host for anti-GPCR-X Abs with both higher overall numbers
and a much higher fraction of antagonistic Abs as opposed to mere binders.
 In addition, almost half of all chicken derived Abs were X-reactive.

4. In primary SPR based screening, affinities for chicken
derived Abs were comparable to mouse and rat Abs
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Human
Mouse
Rat
GPCR-X-ECD
Sensor surfaceY Y Y
Flow
Single Cycle Kinetics
using Cell Culture
Supernatants
 Obtained affinities comparable between hosts.
 Affinities for human-GPCR-X higher than for rodent.
 Obtained affinities comparable between hosts.
 Affinities for human-GPCR-X higher than for rodent.

5. Affinities are not correlated to functional activity but
vary across individual chicken
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cAMPcAMP GPCR-X
Ligand
cAMPcAMP
GPCR-X
Ligand
Y
Y YY
Y
Y Y
Y
AlphaScreen
Agonist
Antagonist
chicken Abs
SPR affinity is not correlated
to antagonistic activity in
cAMP assay.
Individual chicken produce
Abs with distinct affinity
profiles.
SPR affinity is not correlated
to antagonistic activity in
cAMP assay.
Individual chicken produce
Abs with distinct affinity
profiles.

6. A little excursion into chicken antibody structure – longer
HC-CDR3s and stabilizing disulfide bonds
6
Wu et al (2012) J Immunol. 188, 322-333.Wu et al (2012) J Immunol. 188, 322-333.
Chicken
Mouse
Human
 Chicken stabilize longer HC-CDR3 using
disulfide bonds.
 These additional disulfides are an unknown
variable in antibody developability
(humanization, CMC properties &
immunogenicity).
 Chicken stabilize longer HC-CDR3 using
disulfide bonds.
 These additional disulfides are an unknown
variable in antibody developability
(humanization, CMC properties &
immunogenicity).
Naive chicken repertoire

7. The median KD for Cys containing Abs trends lower but
the Cys-free population has the best overall binders
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 Median KD among Cys-Abs is lower, which is particularly pronounced in the antagonistic
Ab population, but absent in the non-antagonistic subset.
 The overall lowest KD Abs are found in the Cys-free population.
 Median KD among Cys-Abs is lower, which is particularly pronounced in the antagonistic
Ab population, but absent in the non-antagonistic subset.
 The overall lowest KD Abs are found in the Cys-free population.

8. The cAMP IC50 in the antagonistic population is not impacted by the
presence or absence of Cys but shows a degree of variation among
individual chicken
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 cAMP IC50 not correlated to Cys content.
 Shows a degree of variation with regards to the
host chicken (less than for SPR KD).
 Only low level of correlation between affinity (SPR
KD) and activity (cAMP IC50).
 Large enough number of Cys free Abs for selection.
 cAMP IC50 not correlated to Cys content.
 Shows a degree of variation with regards to the
host chicken (less than for SPR KD).
 Only low level of correlation between affinity (SPR
KD) and activity (cAMP IC50).
 Large enough number of Cys free Abs for selection.

9. The chicken antibody subset represents greater epitope
diversity than the rodent antibody subset
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Saturating Ab:
• Rodent_reference
Competing_Abs:
• 84 Chicken derived
Abs (supernatants)
• 3 Rodent derived Abs
(purified)
• 1 Reference Ab
(Literature, purified)
• 1 neg control
Signal Ratio: Mean_Plateau_Competing_Ab/Mean_Plateau_Saturating Ab
<1.15 >1.15Competing Abs Non-competing Abs
 Rodent reference Ab, 3 additional rodent Abs and Literature reference Ab define same
epitope on GPCR-X.
 1D binning of 84 chicken Abs reveals additional epitope(s).
 X-binning of chicken Abs (subset, data not shown) defines at least two additional separate
epitopes on GPCR-X that are functional (antagonistic).
 Cys-containing Abs are over-represented among those recognizing distinct epitopes (data
not shown).
 Rodent reference Ab, 3 additional rodent Abs and Literature reference Ab define same
epitope on GPCR-X.
 1D binning of 84 chicken Abs reveals additional epitope(s).
 X-binning of chicken Abs (subset, data not shown) defines at least two additional separate
epitopes on GPCR-X that are functional (antagonistic).
 Cys-containing Abs are over-represented among those recognizing distinct epitopes (data
not shown).
Octet HTX Platform

10. Summary
 For project GPCR-X chicken proved to be the superior host for antagonistic mAb generation.
 A much higher number (172) of unique sequence Abs were obtained than from classic rodent
hybridoma technology (<10).
 A large fraction (75%) were antagonistic on human GPCR-X, about 50% also X-reactive & antagonistic
on rodent GPCR-X.
 Both Cys-containing and Cys-free chicken Abs were obtained.
 The presence of cysteines showed some correlation to affinity, but not overall antagonistic activity.
 A sufficient number of Cys free antibodies showed favorable activity profiles to avoid potential
development liablities of the Cys containing subset.
 The chicken derived antibodies cover a broader epitope space than the rodent derived antibodies.
The cysteine content plays a role in enabling the chicken derived Abs to recognize alternative
epitopes.
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11. Acknowledgements
Boehringer Ingelheim Germany
Robert Augustin
Sebastian Bandholtz
Gerd Luippold
Simon Plyte
Yvonne Roth
Michael Ritter
Nikolai Roosz
Boehringer Ingelheim USA
Qi Pan
Julie Ritchie
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Crystal Biosciences
Bill Harriman
Shelley Izquierdo
Shreya Pramanick
Biberach SiteBiberach Site
Ridgefield, CT SiteRidgefield, CT Site