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

5

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)

22

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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