application of label free technology for biologics ......the octet® is a label free platform that...
TRANSCRIPT
Application of Label Free Technology for Biologics Discovery
22 April 2015Pall ForteBio User Meeting
Frances Neal, Scientist 1, ADPE
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Overview
1 Overview of MedImmune
3 Integrating Octet® assays into Biologics Discovery
4 Comparison of assay formats
2 Multiple uses of the Octet®
• Global biologics research and development arm of AstraZeneca
• Approximately 2,500 full-time employees in the US and UK
• Robust pipeline of more than 120 biologics in research and development with more than 30 projects in clinical stage development
• Key therapeutic areas include Respiratory, Inflammation, Autoimmunity; Cardiovascular & Metabolic Disease; Oncology; opportunity-driven in Infection & Vaccines and Neuroscience
MedImmune: A brief overview
3
Rich history, promising future
1993
Enter a research
collaboration that
results in the
discovery of
adalimumab
1995
Collaborate
with HGSI,
later results
in the discovery
of belimumab
1991
CytoGamlaunch
1996
RespiGamlaunch
1999
Acquire U.S. Bioscience(Ethyol)
2003
FluMist
launch
2005
International
Synagis®
launch
2002
Acquire Aviron
Form MedImmuneVentures, Inc
2006
Awarded HHS cell culture contract
First licensing of reverse geneticstechnology
2012
FluMistQuadrivalentapproved
2009
Pandemic
H1N1
vaccine
launched
1991 1993 1997 2001 2005 2009 2013
1998
Synagis®
launch
Expand
cell culture
vaccine
manufacturing
2007
Acquired
by AstraZeneca
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Multiple uses of the Octet® at MedImmune
• Sample quantitation
– For example antibody fragments (scFv), IgG, Fc fusion proteins as unpurified
and purified samples
• Characterisation of tag free proteins
– Beneficial for some commercially available reagents and proteins that are
adversely affected due to labelling
• Inhibition assays such as receptor/ligand and epitope mapping
– Octet benefit: the proteins do not have to be labelled
– HTRF benefits: lower cost and more amenable to higher throughput screening
• Kinetic profiling
• Off rate and affinity ranking
The Octet® is a label free platform that can be used for different applications throughout the biologic therapeutic discovery process.
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Calcitonin Gene Related Peptide
CGRP
Gααααs
CALCRL
RCP
RA
MP
1
Adenylate cyclase
Increase in cAMP
Protein kinase A Vasodilation
IP1
Ca2+ release from intracellular
stores
KeyRAMP1 = Receptor activity modifying protein 1 CALCRL = calcitonin receptor-like receptor RCP = receptor component proteincAMP = Cyclic adenosine monophosphate
CGRP is a 37 amino acid neuropeptide (~3.8kDa) and there are 2 isoforms: CGRPα and CGRPβ
Chronic pain and migraine
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Kinetics profiling and ranking
• To isolate anti-CGRP antibodies an Octet assay was used alongside 3 HTRF based assays to fully characterise potential drug candidates for:
– Species and isoform activity
– Functional activity in a relevant cell
based assay
– Epitope determination
– Kinetics profiling
Octet assays were used as part of a screening cascade to isolate potent functionally active antibodies to CGRP.
Single point screening3512 unpurified scFv
scFv binding HTRF assays (human and mouse/rat CGRPα)
Profiling – 68 purified scFvcAMP neutralisation assays
(human and mouse/rat CGRPα)
Profiling – 11 purified IgGcAMP neutralisation assays
(human and mouse/rat CGRPα, and human, mouse and rat CGRPβ)
Epitope grouping in HTRF assaysKinetics analysis by Octet
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scFv binding assays
• 173 CGRP specific and species cross reactive binders were identified
• 68 scFv that were unique and diverse in sequence across all complementarity determining regions (CDRs) were produced as purified scFv for further characterisation in a cell based assay
Bio
Anti his XL665
SA-K
SA-K = streptavidin europium cryptate
Ex 320nm
Ex 615nm
Ex 665nmFRET
Phage display selections were performed on naïve antibody libraries using immobilised biotinylated CGRP. Selection outputs were screened as single point
unpurified E.coli periplasmic extracts in a simple HTRF binding assay format (3512 samples).
173 (4.9%)
85 (2.4%)
259 (7.4%)
Human CGRPα Mouse/rat CGRPα
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CGRP cell based assay
CGRP signals through its GPCR receptor complex predominantly through adenylate cyclase to increase cAMP levels. An established cAMP assay was
used to test for functional activity in a human neuronal epithelial cell line.
CisBio’s HTRF cAMP assay using SK-N-MC cells
CGRP ligands were active in this assay
CGRP IgGs could inhibit a cAMP response
-15 -14 -13 -12 -11 -10 -9 -8 -7 -6 -50
200
400
600
800
1000
1200
1400human CGRP
human CGRP
mouse/rat CGRP
rat CGRP
mouse CGRP
Irrelevant protein
Forskolin
log10 concentration (M)
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Epitope and kinetics profiling
scFv 5
scFv 6
scFv 4
scFv 1
scFv 2
scFv 3
scFvs
Bio Bio
Streptavidin coated
biosensor
The functionally active IgGs were characterised for epitope in a HTRF assay and for kinetics profiling by Octet which demonstrate two distinct groups of antibodies
Bio
DyLight 650 labelled IgG
SA-K
SA-K = streptavidin europium cryptate
Ex 320nm
Ex 615nm
Ex 665nm
Group 1Clone 2 like epitopeFast on and off rates
Group 2Clone 4 like epitope
Slow on and off rates
FRET
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Lead Isolation Summary
Clone
Geomean IgG IC50 (nM) in cAMP assayEpitope
grouping in HTRF assay
Octet assay(95% CI, n number)
human CGRPα
human CGRPβ
mouse/rat CGRPα
rat CGRPβ
mouse CGRPβ
kd (1/s)
1 Not tested as IgG Clone 2-like 4.4E-02
247 84 59 40 89
Clone 2-like 8.0E-02(31-70, 4) (74-94, 3) (32-97, 4) (26-63, 3) (52-152, 2)
3203 501 249 200 547
Clone 2-like 2.0E-02(82-502, 4) (285-882, 4) (143-432, 3) (77-516, 3) (301-994, 4)
45.9 8.9 145 6.5 158
Clone 4-like 8.4E-04(4.3-8.2, 5) (6.7-11.9, 5) (89-237, 4) (3.4-12.3, 5) (75-333, 4)
54.6 7.1 23 4.4 8.6
Clone 4-like 2.1E-03(3.6-5.9, 4) (6.5-7.7, 4) (16-32, 3) (2.7-7.4, 4) (5.0-15, 4)
639 57 12 40 11
Clone 4-like 3.5E-03(30-50, 4) (52-62, 4) (5.4-26, 4) (27-59, 4) (6.6-19, 4)
A diverse panel of anti CGRP antibodies were isolated
• CGRP clone 2 was chosen to take forward as a potential drug candidate as it showed the most desirable characteristics
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Optimising kinetic assays
• Clone 2 affinity determination was difficult due to scFv dimerisation
• The Octet assay was simply optimised by a series reduction in association time - this allowed the fast monomer to bind but reduced the binding of the slower dimer
• Affinity data highlighted that Clone 2 required affinity maturation
0 100 200 300 400 500 600-1
0
1
2
3
4
Time (secs)
Wavele
ng
th s
hift
(nm
)
Wavele
ngth
shift (n
m)
Although predominantly monomeric, scFv have a natural propensity to dimerisewhich can be clone or batch specific. scFv dimerisation can impede accurate affinity determination but simple assay optimisation can overcome this issue.
5 minute association 40 second association
KD = 500nMKd (1/s) = 2.98E-02
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Effect of scFv dimers & multimers on kinetic profile
Peak 1A
Peak 2
Non fractionated
Peak 5
Peak 3
0 40 80 120 160 200 240 280 320 360-0.5
0.0
0.5
1.0
1.5
Time (secs)
No
rmalis
ed
wavele
ng
th s
hift
5 6 7 8 9 10 11 12
0
20
40
60
Time (mins)
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0n
m a
bs
orb
an
ce
(m
AU
)1A 1B 2 3 54A 4B
scFv monomers, dimers and multimers were manually fractionated using size exclusion chromatography (SEC).
• Off rate profiling using the Octet demonstrated that:
– The off rate for the monomeric peak 5 was very close to non fractionated scFv
– The largest multimerised peak 1A demonstrated avid binding
• Caveat - due to the dynamic nature of the scFv, the composition of the fractions may have changed in between the samples being fractionated and tested for off rate
SEC dataOctet profiling
David Lowne Frances Neal
FractionPeak Time
(min)% Area
A: 5.5 A: 0.2
B: 7.3 B: 0.4
Peak 2 8.1 2.7
Peak 3 8.4 4.5
A: 9.3 A: 5.9
B: 9.9 B: 3.7
Peak 5 10.8 83.4
Peak 4
Peak 1
scFv 2
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Optimisation of anti-CGRP clone 2
Single point screening528 non purified scFv
Octet off rate ranking assay
Profiling – 222 purified scFvcAMP neutralisation assays
(human and mouse/rat CGRPα)
Epitope competition and off rate ranking for a limited panel
Profiling – 58 purified IgGcAMP neutralisation assays
(human and mouse/rat CGRPα, and human, mouse and rat CGRPβ)
Single point screening14960 non purified scFv
HTRF epitope competition assays(human and mouse/rat CGRPα)
An Octet off rate ranking assay using unpurified scFv can be used in combination with HTRF epitope competition assays to isolate affinity improved
scFv.
• Affinity maturation was performed by generation of libraries derived from the parent antibody through a combination of targeted and random mutagenesis of the paratope
• This was followed by selections using phage and ribosome display with increasing stringency
• Typically the antibody off rate is improved during optimisation therefore it was important to evaluate this early in the screening cascade
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Tolerance to unpurified scFv samples
56nM
500nM
167nM
6, 2, 0.7 nM19nM 56nM
500nM
167nM
6, 2, 0.7 nM
19nM
To increase throughput, scFv are expressed in unpurified E.coli periplasmic extracts and screened at a single dilution of unknown concentration. These samples are not well tolerated therefore evaluation of tolerance in the Octet off rate ranking assay
was necessary.
3. Off rates determined from single point testing with unpurified scFv were within 2-3 fold of off rates determined with the gold standard method: titration of purified scFv using full global data fitting
Clone 1kd (1/s) = 7.3E-02
Clone 5kd (1/s) = 1.3E-02
Clo
ne 1
Clo
ne 2
Clo
ne 3
Clo
ne 4
Clo
ne 5
Clo
ne 6
1 10 -4
1 10 -3
1 10 -2
1 10 -1
1 10 0
1. Single point off rate ranking was consistent using unpurified and purified scFv
2. Off rates were consistent between assays using different sample types
kd (1/s) Rank order kd (1/s) Rank order
scFv 1 2.7E-02 5 4.4E-02 5
scFv 2 2.8E-02 6 8.0E-02 6
scFv 3 1.4E-02 4 2.0E-02 4
scFv 4 1.6E-03 1 8.4E-04 1
scFv 5 3.1E-03 2 2.1E-03 2
scFv 6 3.4E-03 3 3.5E-03 3
panel 1
1µM purified scFv25% crude scFv
panel 2
scFv
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Profiling of affinity improved antibodies
• Sequence unique inhibitors in the epitope competition assay were then screened in the scFv off rate ranking assay
• Of the 528 scFv tested 20 had a 2.5 fold slower off rate than clone 2 scFv
scF
v I
C5
0(n
M)
scF
v I
C5
0(n
M)
An Octet off rate ranking assay using unpurified scFv can predict functional activity in a relevant cell based assay.
HTRF epitope competition assay
10,000 fold improvement
Human CGRPα cAMP assay
4,500 fold improvement
Octet unpurified scFv off rate ranking assay
• The off rate improved scFv, and a further 202 scFv of interest from the single point screening, were profiled in the HTRF epitope competition and cAMPassays to monitor inhibition improvements
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Correlation between CGRP assay formats
fold
im
pro
vem
ent in
cA
MP a
ssay
Both HTRF epitope competition and scFv off rate ranking assays can be predictive of functional activity in a relevant cell based assay
Comparison of Octet scFv off rate ranking and cAMP assays
Comparison of epitope competition and cAMP assays
Pearson r = 0.72 62 data points
95% CI 0.57 to 0.8
Assay using Clone 2Pearson r = 0.92
26 data points 95% CI 0.82 to 0.96
Assay using partially optimised clone
Pearson r = 0.55 24 data points
95% CI 0.19 to 0.78
• A comparison of the fold improvements in IC50 values obtained in the assays showed the screening cascade had identified the most relevant scFv
• The activity ranking obtained from both the epitope competition assays and the Octet off rate assay correlated well with the cAMP assay
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Comparison of CGRP assays
HTRF scFvbinding
Octet assaysHTRF epitope competition
cAMP assay
PurposeInitial screen for species cross
reactivity
Kinetics profiling and off rate ranking
Epitope mapping and affinity improvements
Functional activity
Concentration independent
Tolerant to unpurified scFv
Amenable to species cross
reactivity testing
Concentration of human CGRPα
1nM 125nM 0.7 to 2.5nM 0.1nM
Assay format and volume
384 well 10µl
384 well 50 to 100µl*
384 well10µl
384 well20µl
Average cost per well in pence
8p 473p** 6p 15p
A combination of 4 simple assays can be used to fully characterize diverse panels of antibodies and discover potent functionally active antibodies
* Samples can be reused** Biosensors can be regenerated to reduce costs
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Screening in IgG Format
• Express IgGs in a mammalian expression system
– Direct from phage display libraries
• Screening in IgG format desirable for many reasons
– Improved assay tolerance of sample buffer
– Avidity
– Higher expression levels
– Conversion to IgG not required
• Reduced attrition
• Faster to IgG profiling
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Paradigm Shift: Functional Screening of Antibodies
Traditional Approach
Selection
• Reduce naïve library from 1012 to 105-106
Screen
• Crude scFv (E.Coli) in biochemical assay
Purify• Purify bacterially expressed scFv
Screen
• Purified scFv screened in biochemical or cell-based assay
Convert • Lead scFv converted to IgG
Screen• Confirm IgG activity in cell assay
Selection
• Reduce naïve library from 1012 to 105-106
Cloning• Population cloning
Screen
• Crude IgG (mammalian) in cell-based assay
Quantify• IgG quantified (Octet)
Rank
• Rapid ranking of a large panel of IgGs achieved without purification steps
Straight to IgG
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Screening IgG supernatants
% inhib
itio
n
Unpurified IgG supernatants were successfully screened in the cAMP assay and identified potent functionally active IgGs.
• The cAMP assay was tolerant to mammalian expressed IgG supernatants unlike the unpurified bacterial expressed scFv
• 100s of IgG supernatants were quantified by Octet and single point screened in HTRF epitope competition and cAMP assays
• The most interesting supernatants were profiled in the cAMP assays to determine functional potency
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Summary
• The Octet RED 384 provides a platform that can be used for characterisation of key interactions to aid discovery of biologics
– Sample quantitation
– Characterisation of tag free proteins
– Inhibition assays such as receptor/ligand and epitope mapping
– Kinetic profiling
– Off rate and affinity ranking
• Off rate ranking assays can predict functional activity in relevant cell based assays
• Octet assays can be used in conjunction with other assay formats, such as HTRF and functional assays, to isolate potent antibodies to CGRP and the same principle can be applied to other targets
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Acknowledgments
• The Biologic Screening team
• Support Services
– Biologics Expression Team
– Tissue Culture Team
– Bioinformatics
– Laboratory Support Services
• CGRP project team which included:
– Frances Neal
– Joanne Arnold
– Sadhana Podichetty
– David Lowne
– Claire Dobson
– Trevor Wilkinson
– Tristan Vaughan
– Chris Rossant
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