selection of antibodies to signalling networks · application of phage display to high throughput...

Selection of antibodies to signalling networks

Michael Dyson Iontas Ltd

Research and Innovation in Phenotypic screening ELRIG, Telford, 11.3.14

www.iontas.co.uk

• Introduction • Phenotypic screening in

stem cells • Screening for antibodies

for use in IP-MS • Anti-protease antibodies


Affinity

SpecificityEpitope

Iontas Ltd Hopkins Building Department of Biochemistry University of Cambridge

Journey from proteomics project to biotech start-up

!Welcome Trust Sanger Institute, Hinxton

Application of phage display to high throughput antibody generation and characterization. Schofield DJ, et al Genome Biol. 2007;8(11):R254.

IONTAS Ltd www.iontas.co.uk

YOU ARE HERE: BIOMEDTECH CAT CO-FOUNDER DEVELOPS NEW ANTIBODY VENTURE

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CAT CO-FOUNDER DEVELOPS NEWANTIBODY VENTUREThat old CAT magic has a new generation ofantibody technology customers in its spell. ACambridge startup inspired by John McCafferty, co-founder of Cambridge Antibody Technology, iscreating new medical opportunities with its IP.

He has inspired Iontas – a biotechnology businessfocused on the development of novel antibodytherapeutics. It employs its proven expertise in allkey areas of therapeutic antibody discovery anddevelopment.

As well as in-house drug discovery programmes,Iontas is seeking to partner with select companiesto develop new antibody based therapies in theareas of cancer and inflammation.

Iontas uses its proprietary technology to accelerate all aspects of antibody drug discovery from targetselection through to the production and purification of large quantities of fully human antibody pre-clinicalcandidates. Its scientists are developing several novel methods that will revolutionise therapeuticantibody discovery.

Iontas has produced a proprietary high quality human antibody phage display library that has advantagesover existing libraries. For a start it contains over 40 billion antibodies; it also boasts a very highproportion of variable light and heavy inserts and methods to reduce minus insert clones prior toselection. Furthermore, the library is pre-designed for facile conversion to IgG or Fab format.

High throughput time resolved fluorescence assays are employed in lead isolation to rapidly identifybinding hits in primary screening. Lead candidates are then optimised by multi-parallel affinity maturationand secondary screening.

Lead and optimised lead antibody candidates can also be tested by a variety of method to help choosethe optimal pre-clinical candidates. This includes.

CEO McCafferty is a pioneer and inventor of antibody-phage display and co-founder of CambridgeAntibody Technology. After taking CAT to global greatness, a blockbuster drug and acquisition byAstraZeneca, he moved on to fresh challenges. McCafferty established a highly proficient proteomicsgroup at the Wellcome Trust Sanger Institute which developed cutting edge protein expression and highthroughput recombinant antibody isolation.

More recently he has headed a research group within the Department of Biochemistry, University ofCambridge. The focus here was identifying anti-receptor antibodies with therapeutic potential in cancerindications.

Co-founder and group leader Mike Dyson has over 20 years experience in protein and small moleculechemistry, molecular biology and assay development gained during post-doctoral research at theMassachusetts Institute of Technology, Universities of Edinburgh and Cambridge and the biotechnologyindustry.

He has published 27 papers (cited over 800 times, h-index 16), 3 book chapters, edited a book onprotein expression and is inventor on 4 US patent and patent applications. He was previously head ofprotein engineering at Acambis plc, group leader at Sense Proteomic Ltd and was the project leaderresponsible for protein expression within the Atlas Project at Sanger.

McCafferty says Iontas has some wonderful technology within a resurgent antibody segment but that a

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http://www.businessweekly.co.uk/biomedtech-/16537-cat-co-founder-develops-new-antibody-venture

• Founded 1st October 2012 • Self-funded, 10 employees • Focussed on therapeutic

antibody discovery in the areas of cancer and inflammation

• Currently running 4 externally funded projects with UK and USA based biopharmaceutical companies

• Developing in house IP in mammalian display and novel scaffolds to target ion channels and GPCRs

http://www.iontas.co.uk

Select on antigen

Overview of phage antibody selection

ELISA positive clones

Infect phage population into E.coli

population of phage displaying

antibodies

Wash, elute bound phage

Rescue phage

Rescue phage

Sub-clone population screen

Bacterial library of >1 x1010 clones*

*McCafferty Library Schofield et al, (2007) Genome Biology. 8

(11) R254

Iontas Library Improved features

(greater insert ratio, 4 x greater size, built in features to allow facile

conversion to IgG

Recombinant antibody formats

S

CH2

CH3

S SS S

SS

VH

VL

CH1

CL

VHVL

CH1

CL

SS

IgG

VH VL

rCd4(d3)

rCd4(d4)

scFv–rCd4

CH2

CH3

VLVHVL VH

S SS S

scFv-Fc

S S

VH VL

CH1 CL

Fab

VH VL

scFv (expressed in E. coli)

HEK293 or CHO expression

HEK293 or CHO expression

Iontas population

conversion “en masse”

conserving the original VH and

VL pairs

Antibody screening• DELFIA and HTRF assays • Convert to IgG or Fab • In vitro blocking assays • Cell based reporter assays • Affinity measurements

(BIAcore) • Manufacturability (solublity,

expression, stability)

BMG Pherastar

CyBi - Selma



for use in IP-MS • Anti-protease antibodies


Affinity

SpecificityEpitope

4

1

3

2

ligand

Cell membrane

Blocking receptor signalling with antibodies

Bradbury, Sidhu, Duebel, McCafferty (2011) Nature Biotechnology 29 (3) Beyond natural antibodies: the power of in vitro display technologies

e.g. cMet / scatter factor

e.g. Notch / integrins

Functional screening in ES cellsTet prom constitutive prom

Gateway scFv-Fc Gateway puro

Homology arm Homology arm

Transfect ES cells Select PurR

colonies

Anti-FLAG / anti-mouse-IR680

+ doxycycline

scFv-Fc fusion

differentiation

Different outcome in cell expressing blocker

One antibody gene/cell

FGFR signalling

Moosa Mohammadi Lab Tel: (212) 263-7122 Fax: (212) 263-7133 Add: MSB 425 - 431, 550 First Ave, New York, NY 10016

Home Publication Lecture Animation Lab member Collaborator Former member Site map

FGFR Interacts with Intracellular Signaling Molecule

FGF cell surface signaling. The current state of the structural biology of FGFR activation at the cell surface is presented. In the past few years, we haveelucidated the structural basis by which heparin/HS assists FGF and FGFR to form a symmetric 2:2 FGF-FGFR dimer. We have also resolved the crystal structureof the intracellular FGFR1 kinase domain. The structurally unresolved regions of FGFR are drawn as gray lines. The N-terminal and C-terminal lobes of the kinasedomain are colored green and red, respectively. The activation loop (A-loop) in the C-terminal kinase lobe is colored yellow and the side chains of tyrosines 653 and654 are rendered in black. Domain organization of PLCγ-1 and FRS2α, two major intracellular targets, are shown. Receptor dimerization is a universal mechanism in RTK activation. The structural basis for receptor dimerization has been elucidated for several RTKsubfamilies including EGFR, VEGF, TRK. These studies reveal that the mode of receptor dimerization varies greatly among RTK subfamilies and is likely tailored tomeet their individual biological functions. The mechanism by which FGFs transduce their signal across cell membranes has been intensely studied over the past 30years. Recent X-ray crystallographic studies have transformed FGF signaling into is one of the foremost structurally understood ligand-receptor systems amongRTKs (Fig above). The symmetric "two-end" model for FGFR dimerization unifies a wealth of biochemical data, provides a structural basis for FGF-FGFR bindingspecificity and explains how pathogenic mutations in FGFR result in human skeletal disorders. The mode of receptor dimerization observed in the symmetric "two-end" model is highly cooperative and is well-suited to execute the essential roles of FGF signaling in development. This model is based on several synergesticbinding events, including secondary ligand-receptor and direct receptor-receptor interactions, which are facilitated by HS. Importantly, the dimer interface is adjacentto the FGF-D2 portion of the primary interface and therefore binding events at the dimer interface are intimately coupled with binding events at the primary bindingsite. Importantly, this mode of dimerization is capable of sensing and responding to the dynamic changes in extracellular HSPGs that occur during development.Future challenges include structural and biochemical studies of how these HS changes modulate dimer assembly and will provide greater insight into the role ofFGF signaling in physiological and pathophysiological settings.

Created and maintained by Jinghong Ma

Moosa Mohammadi Lab NYU

FGFR activation causes stem cells to be permissive for differentiation

ES colony phenotype variation

ES clones expressing FGF4-blocking antibodies resist differentiation

Selecting antagonistic antibodies that control differentiation through inducible expression in embryonic stem cells. Melidoni AN, M Dyson R, Wormald S, McCafferty J. Proc Natl Acad Sci U S A. 2013 Oct 29;110(44):17802-7.

Selected anti-FGF4 clones maintain Oct4-GFP expression in liquid differentiation in the presence of doxycycline


Exogenous anti-FGF4 antibodies maintain pluripotency

Selection of anti-FGFR1 blocking antibodies by stem cell phenotype screening

B in vitro FGFR1 / FGF4 binding assay


Functional screening in ES cells• Antibody libraries can be clones into ES cells (1

antibody gene per cell) !• Phenotypic selection in semi-solid media with

fluorescent reporter cell-lines !• Antagonistic antibodies selected to the FGF4 signalling

system selected (anti-FGF4 and FGFR1) !• In cell expression reporter system (ICER) will be useful

for: - generation of antibodies for controlling ES cell differentiation outcomes - direct identification of antagonistic or agonistic antibodies to complex signalling events - dissection of signalling pathways

Functional screening in ES cells: What next?

COMMENTARY

Directing stem cell differentiationwith antibodiesMartin Dalziel, Max Crispin, and Raymond A. Dwek1

Department of Biochemistry, Oxford Glycobiology Institute, University of Oxford, Oxford OX13QU, United Kingdom

Stem cells are highly specialized cells en-dowed with unlimited replicative self-renew-ing potential. These cells are capable of eitherlimited multipotent (adult stem cells, ASC) orunlimited pluripotent (embryonic stem cells,ESC) differentiation to somatic cell lineages.

Control of this differentiation process holdsgreat promise in areas such as tissue re-generation and the treatment of chronicdegenerative diseases. The medical exploita-tion of this phenomenon is carried out usingstem cells derived from different sources (e.g.,

ASC/ESC), developmental stages (e.g., mes-enchymal cells), cellular reprogramming (e.g.,induced pluripotent stem cells), or evennonstem cells in a process known as trans-differentiation (Fig. 1). Despite this wide di-versity, all of the differentiation steps thatdrive these cells to the desired somatic celllineage rely on the directed manipulationof endogenous pathways. This process isachieved primarily by direct ligand signalingto cell-surface receptors, or use of distinctcombinations of transcription factors de-rived from these signals. Now in PNAS,Melidoni et al. (1), building upon earlierreports (2, 3), raise the exciting possibility ofusing combinatorial library-derived antibod-ies intimately linked to stem-cell phenotypeas a powerful tool in controlling these differ-entiation events.The use of antibodies provides technical

advantages over traditional techniques thanksto their intrinsic stability and precise speci-ficity. However, an even greater benefit lieswithin the antibody selection method itself.These antibodies are derived from combina-torial libraries that contain a vast repertoireof possible antibody combinations. This di-versity means that as a whole, such librariescontain members that can influence everypossible cell-receptor–signaling event of atarget cell, including potentially unforeseensynergistic phenomena. Coupling antibodyselection to a desirable cell phenotypic traitenables significant opportunities in stem-cell manipulation.The conception and realization of combi-

natorial antibody libraries is arguably themost important advance in immunochemis-try since monoclonal antibodies were discov-ered (4). Combinatorial antibody libraries areone of the most widely used of all biochem-ical libraries. Indeed, they were the first bio-chemically generated libraries and were thesource of the term “combinatorial libraries”(5, 6). Antibodies have now taken centerstage in therapeutics and, in this age ofproteomics, access to an antibody diversity

Fig. 1. Stem-cell differentiation pathways. Stem cells (embryonic and adult) are shown in blue. Somatic cells shownare from top to bottom: epithelial (orange), dendrite (red), granulocyte (purple), and neuron (blue). The approximatepoints at which the different studies discussed are indicated alongside their appropriate reference (numbers in pa-renthesis) with antagonist signaling in red and agonist in green. iPSC, induced pluripotent stem cell; TD, trans-differentiation. *, TD can occur either between nonstem cells or differentiated (progenitor) stem cells to outwith theirestablished differentiation pathway. Although grouped together in this figure, progenitor cells are generally consid-ered to be stem cells that have undergone significant commitment to a specific somatic lineage with concomitantdiminishment of both multipotency and replicative potential.

Author contributions: M.D., M.C., and R.A.D. wrote the paper.

The authors declare no conflict of interest.

See companion article on page 17802.

1To whom correspondence should be addressed. E-mail: [email protected].

17608–17609 | PNAS | October 29, 2013 | vol. 110 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1317614110

Directing stem cell differentiation with antibodies Martin Dalziel, Max Crispin, and Raymond A. Dwek, PNAS, 110, 17608–17609!!Phenotypic directed antibody selection. McCafferty J. Chem Biol. 2014 Feb 20;21(2):170-1.



for use in IP-MS


Affinity

SpecificityEpitope

Cell membrane

Mapping protein interactions using affinity matured antibodies

EGFr

EGF

ras Raf! MEK etcshc

grb2sos1

PP

PP Y313P-S29

gab1shp2AP2A

PTP-N12

MEKK1! JNK etc

By Multipe reaction monitoring (Pawson lab)

Mapping Protein Interactions by Combining Antibody Affinity Maturation and Mass Spectrometry. Dyson MR et al Anal Biochem. 2011 Oct 1;417(1):25-35.

Introducing diversity by chain shuffling

Library size (x 109) Lyn 1.3 Crk 1.8 Zap 70 1.6 PTPN11C 1.3 Vav1 1.7 Nck1 2 Shc1 0.15 Lck1 0.15

• Specificity of V domains determined by “complementarity determining region” (CDRs)

• CDR3 of heavy chain central and most diverse !

• Each heavy chain “meets” limited number of partners in original library !

• Chain shuffling provides many more opportunities to find better partner

Dyson et al, Anal Biochem, 2011, . 2011 Oct 1;417(1):25-35.

Select on biotinylated

antigen

Infect phage population into E.coli

population of phage displaying

antibodies

Overview of stringent antibody selection

Wash, elute bound phage

ELISA positive clones

Chain shuffled library 108-109 clones

Rescue phage

Rescue phage

sub-clone

Anti-SHC1 (poly)

Anti-FLAG

VH VL scFv

SHC1

Screening for what you wantELISA

SHC1

VH VL

Anti-FLAG Eu Eu3+

Affinity capture screen

1 2 3 4 5 6 7 8 9 10 11 12 A 157.5 155.2 144.1 137.1 134.5 132 129.3 129.2 126.6 125.2 125.1 119.8 B 118.3 118.2 116.8 116.4 116 115.1 114.1 114 113.7 113.2 112.4 112.2 C 112.1 111.7 111.5 111.3 110.8 110.8 109.5 109 108.1 107.7 107.3 107.3 D 105 104.7 104.4 104.2 103.6 103.6 103.5 103.4 103.3 103.2 102.8 101.9 E 100.7 100.3 99.95 99.54 99.48 99.08 98.91 98.41 98.03 97.8 96.68 96.49 F 96.06 95.25 95.15 94.2 94.01 93.75 93.68 93.48 93.28 93.25 92.76 92.68 G 92.41 92.39 91.59 91.01 90.52 90.46 89.5 88.98 88.97 88.49 88.47 88.33 H 87.68 87.57 86.98 85.37 85.01 84.95 84.93 84.76 84.75 84.23 83.7 83.58

TRF x 1000

Cell lysatescFv

1 2 3 4 5 6 7 8 9 10 11 12 A 40 36 23 28 26 24 23 26 25 25 23 1826 B 1213 27 1961 27 24 26 23 24 1699 18 24 1396 C 45 30 30 28 29 25 1152 30 22 24 23 1305 D 24 31 1818 30 33 23 23 26 22 29 20 28 E 36 887 26 25 29 552 27 453 21 23 1830 83 F 30 32 31 1459 132 27 23 27 331 24 269 21 G 21 27 862 23 23 27 31 28 26 29 1333 814 H 21 29 26 26 407 317 267 418 26 22 383 42

Anti-rabbit Eu Eu3+

Good correlation between affinity capture screen and immunoprecipitation

CONFIDENTIAL

Mapping protein interactions using affinity matured antibodies

SOS1

ATADDELSFK (m/z=548/2+)

Grb2

PTP-N12

EGFRGab1

Shp2

AP2a

PTB SH2

Y313S29

TPESFVLADMPIR (m/z=738/2+)

ELFDDPSYVNIQNLDK (m/z=955/2+)SHC1

a b c b c

058_E05

072_2B01

0

10

20

30

40

50

60

70

0 0.05 0.1 0.15 0.2 0.25

Dissociation rate (s-1)

% R

etai

ned

Dyson et al (2011) Analytical Biochemistry 417 25–35

Acknowledgement

• Kritika Pershad • John Pavlovik • Brian Kay • Gill Murphy • Chris Tape • Ronny Falk • Anna Melidoni • Kothai Parthaban • John McCafferty

• Structural Genomics Consortium • Human Protein Atlas • Tony Pawson, Karen Colwill

selection of antibodies to signalling networks · application of phage display to high throughput...

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