Antibody immunoperoxidase staining of mouse brain tissue for laboratory class. The focus is on the choroid plexus (a network of blood vessels in the ventricles of the brain that is responsible for producing cerebrospinal fluid). GFAP is helpful for seeing astrocytes and many types of brain tumors. Aquaporin is a water channel protein found on astrocytes surrounding the blood-brain barrier.
Atherosclerosis: New Insights on Foamy Macrophage Induction and PersistenceDr. Marian Laderoute
It is commonly assumed high cholesterol contributes to atherosclerosis which initiates with foamy macrophages. Here it is suggested the induction of foam in human macrophages relates instead to the induction of a newly described foamy virus, human endogenous retrovirus K102 (HERV-K102) in response to high cortisol and/or viruses, other intracellular pathogens, or tumors. HERV-K102 is a newly described human host protection mechanism and the particles are replication competent in vitro and in vivo. In healthy or younger adults, the foamy macrophages lyse on day 7 and release the protective HERV-K102 particles. When immunosenescence is evident (associated with low DHEA and high cortisol) the lysis and release of the HERV-K102 particles is blocked by alpha-fetoprotein (AFP). The dysfunctional and partly activated macrophages promote immunosenescence including atherosclerosis. See Laderoute MP, Discovery Medicine article published December 2015 for more details.
Antibody immunoperoxidase staining of mouse brain tissue for laboratory class. The focus is on the choroid plexus (a network of blood vessels in the ventricles of the brain that is responsible for producing cerebrospinal fluid). GFAP is helpful for seeing astrocytes and many types of brain tumors. Aquaporin is a water channel protein found on astrocytes surrounding the blood-brain barrier.
Atherosclerosis: New Insights on Foamy Macrophage Induction and PersistenceDr. Marian Laderoute
It is commonly assumed high cholesterol contributes to atherosclerosis which initiates with foamy macrophages. Here it is suggested the induction of foam in human macrophages relates instead to the induction of a newly described foamy virus, human endogenous retrovirus K102 (HERV-K102) in response to high cortisol and/or viruses, other intracellular pathogens, or tumors. HERV-K102 is a newly described human host protection mechanism and the particles are replication competent in vitro and in vivo. In healthy or younger adults, the foamy macrophages lyse on day 7 and release the protective HERV-K102 particles. When immunosenescence is evident (associated with low DHEA and high cortisol) the lysis and release of the HERV-K102 particles is blocked by alpha-fetoprotein (AFP). The dysfunctional and partly activated macrophages promote immunosenescence including atherosclerosis. See Laderoute MP, Discovery Medicine article published December 2015 for more details.
In immunology, an antigen is a molecule capable of inducing an immune response in the host organism. High-quality antigens for vaccine development solution are supposed to cover viral antigens, parasitic antigens, bacterial antigens, tumor antigens and so on. https://www.creative-biolabs.com/vaccine/category-antigens-1.htm
• Gene optimization: genes synthesis, vector construction
• Vector construction
• Screening of expression strains
• Small-scale expression
• Protein expression optimization
• Large-scale expression and purification
• Protein folding, modification, renaturation, endotoxin detection and removal
• Protein identification
https://www.creative-biolabs.com/vaccine/virus-like-particles-based-vaccines.htm
Overview of Neuromics A to Z. From high speed filling of transport media for Covid-19 testing to 2 and 3-D Cell Based Assays, defined media and supplements like FBS.
Novel hexavalent GITR agonists stimulate T cells and enhance memory formationThomas Hoeger
Apogenix's novel hexavalent GITR agonists as candidates for immunotherapeutic treatment of cancer – look at the presentation of comprehensive data by Meinolf Thiemann, PhD, at the AACR Annual Meeting in Washington, D.C., on April 4, 2017.
Gamma-irradiated Frozen Cells Validated for GPCR-mediated Kinase SignalingPerkinElmer, Inc.
The use of frozen cells for cell-based screening has become widely accepted within the drug discovery community. Separating cell production from the actual screening campaign not only increases your flexibility but also improves the data consistency as the cellular material can be controlled and validated before running the functional assay.
In immunology, an antigen is a molecule capable of inducing an immune response in the host organism. High-quality antigens for vaccine development solution are supposed to cover viral antigens, parasitic antigens, bacterial antigens, tumor antigens and so on. https://www.creative-biolabs.com/vaccine/category-antigens-1.htm
• Gene optimization: genes synthesis, vector construction
• Vector construction
• Screening of expression strains
• Small-scale expression
• Protein expression optimization
• Large-scale expression and purification
• Protein folding, modification, renaturation, endotoxin detection and removal
• Protein identification
https://www.creative-biolabs.com/vaccine/virus-like-particles-based-vaccines.htm
Overview of Neuromics A to Z. From high speed filling of transport media for Covid-19 testing to 2 and 3-D Cell Based Assays, defined media and supplements like FBS.
Novel hexavalent GITR agonists stimulate T cells and enhance memory formationThomas Hoeger
Apogenix's novel hexavalent GITR agonists as candidates for immunotherapeutic treatment of cancer – look at the presentation of comprehensive data by Meinolf Thiemann, PhD, at the AACR Annual Meeting in Washington, D.C., on April 4, 2017.
Gamma-irradiated Frozen Cells Validated for GPCR-mediated Kinase SignalingPerkinElmer, Inc.
The use of frozen cells for cell-based screening has become widely accepted within the drug discovery community. Separating cell production from the actual screening campaign not only increases your flexibility but also improves the data consistency as the cellular material can be controlled and validated before running the functional assay.
Construction of an A2a GPCR Model using Homology Comparative Protein ModelingROB1323
Experimental procedures such as X-ray diffraction are key in the development of three dimensional structures representing several protein molecules. However, the three dimensional structures of some proteins have yet to be determined. Adenosine G-protein coupled receptor A2a is an example of a protein whose molecular structure does not exist within the protein database. We attempt to find out the three dimensional structure of A2a using the Homology modeling program “Modeller”.
Identify Compounds that Rescue Disease Relevant Mutant Membrane ProteinsDiscoverX Corporation
Learn about diseases caused by protein misfolding and how you can screen for compounds, known as pharmacochaperones, that rescue misfolded proteins and could be used as therapeutics.
B cell sorting platform is capa- 1 Antibody Discovery Based on B Cell Sorting Platform
ble of the high-throughput
screening and generation of
fully human monoclonal
antibodies (mAbs) or natural
mAbs of different species in a
short period.
To achieve better humanization, repeated analysis, construction and test procedures are required. The design of grafted or resurfaced antibodies often involves an iterated approach where sequence designs are generated and tested in binding and/or functional assays. https://www.creative-biolabs.com/antibody-humanization.html
The first and most straightforward method is to screen our human antibody libraries. The second method is to immunize premade “humanized” mice and then raise hybridoma cell lines [or to create an immune humanized antibody library] that produce human antibodies.https://www.creative-biolabs.com/Human-or-Humanized-Antibody-Services.html
Evaluating Ozoralizumab (ATN-203) as a Novel Biotherapeutic Agent for the Tre...jake9606
Poster Presentation which focuses on the background, production and direction of novel Nanobody Technology as a potential biotherapeutic. Technology has been developed by Ablynx (Belgium) and has many benefits particularly in production compared to Antibodies.
Subsequent library screening will fish out the antibody mutants that have high affinity. Two library screening strategies are available. In the first "surface-panning" strategy, decreasing concentrations of antigen is surface immobilized. In the second "solution-sorting" strategy, in which a labeled antigen in solution is used, we have two approaches, selection based on the equilibrium constant (Kd) and selection based on binding kinetics. In the first approach, sub-library phage is incubated with biotinylated antigen at controlled concentrations and bound phages are captured by immobilized NeutrAvidin. Selection based on binding kinetics is also termed off-rate (Koff) selection, in which phage population is allowed to saturate the labeled antigen before a large molar excess of unlabeled antigen is added to the mix for controlled periods of time. This allows the selection of mutant antibodies that have slower off-rates. Since a reduction in Koff usually results in a higher affinity, this selection approach singles out antibody variants with improved Kd.
A computational framework for large-scale analysis of TCRβ immune repertoire ...Thermo Fisher Scientific
TCRβ immune repertoire analysis by next-generation sequencing is emerging as a valuable tool for research studies of the tumor microenvironment and potential immune responses to cancer immunotherapy. Generation of insight from immune repertoire profiling often requires comparative analysis of immune repertoires across research sample cohorts representing immune responses to defined antigens or immunomodulatory agents. Here we describe the development of a computational framework enabling large-scale comparative analysis of immune repertoire data on cloud-based infrastructure.
MIMG 199 P. acnes Poster Final - Lauren and Rachelle
Chicken_mAbs_Webinar
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
4
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
5
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
7
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
8
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
9
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.
10
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
11
Crystal Biosciences
Bill Harriman
Shelley Izquierdo
Shreya Pramanick
Biberach SiteBiberach Site
Ridgefield, CT SiteRidgefield, CT Site