Blocking(cell(signaling(with( recombinantan:bodies( · 40% ELISA positives elute bound phage Bacteriallibraryof"" >1"x1010clones Schoﬁeld(etal((2007)(Rescue phage Rescue phage No

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Blocking cell signaling with recombinant an:bodies

John McCafferty

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1

3

2

ligand

Cell membrane

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Phage display of an:body fragments

Phage display vector

VL VH

ori

Signal peptide

Gene 3

An:body genes can be fused to gene encoding minor phage coat protein -‐results in func:onal an:body product on surface of phage par:cle

Select on an8gen

Infect phage popula8on into E.coli

popula8on of phage displaying

an8bodies

Overview of phage an:body selec:on

Wash, elute bound phage

ELISA posi8ve clones

An8body library of >1 x1010 bacterial

clones

Rescue phage

Rescue phage

Sub-‐clone popula:on screen

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Controlling Notch signalling with an:bodies

Figs from Gordon et al 2008 Journal of Cell Science 121 (19) 3109-‐3119

Notch receptors (N1-‐N4) and their ligands (Jagged 1,2, dll1, 3, 4) bind promiscuously

Targe:ng Notch nega:ve regulatory domain

Select antibodies

LNR1 LNR2 LNR3 HD1 HD2

ΔS1

rCD4/His10 SP CMV

S2

(LNR= Lin12 Notch repeat)

ßS1

LNR-C LNR-A

LNR-B

ßS2 Ca2+

Ca

Ca2+

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N1_E6 N1_E7 N2_B6 N2_B9 N3_MAB Shc1 293-J1 293T0

0,2

0,4

0,6

0,8

1

1,2293-Notch1 cells, n=2


0,2

0,4

0,6

0,8

1



0,2

0,4

0,6

0,8

1


Rel

ativ

e Lu

cife

rase

sig

nal

An:body mediated blocking of

Notch signalling is specific

Falk et al, Methods (2012) Methods 58 p69-‐78

Inhibi:on of endogenous Notch signalling in neural stem cells

0.01

0.1

1

10

DA

PT

2uM

Con

trol a

b

N1

N2

N1+

N2

Fold

cha

nge

0

2

4

6

8

Control ab DAPT N1+N2+N3

Fold

cha

nge

Reduced expression of direct target gene (Hes5)

Increased neural differen8a8on by Notch inhibi8on (RT-‐PCT of doublecor:n)

Falk et al, Methods (2012) Methods 58 p69-‐78

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Notch blocking an:bodies stabilise closed conforma:on

Wu et al (2010) Therapeu:c an:body targe:ng of individual Notch receptors Nature 464 1050

C-‐Met receptor and cancer

MET928

MET741

MET567

•  Ligand is Hepatocyte growth factor/sca\er factor (SF)

•  Ligand and receptor play a key role in development/:ssue regenera:on

•  Major role in human cancer –  SF causes EMT in epithelial cells –  Over-‐expression in miceàtumours –  Met muta:on or Met over-‐expression enriched

in human tumours

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–  Straussman R et al (2012) Tumour micro-‐environment elicits inate resistance to RAF inhibitors through HGF secre:on

Nature 487 500-‐504

Role of HGF from tumour microenvironment in resistance to targeted

therapies

Iden8fica8on of HGF as the secreted factor

Wilson R et al (2012) Widespread poten:al for growth factor driven resistance to an:-‐cancer kinase inhibitors Nature 487 505-‐509

Select on MET928

Infect phage population into E.coli

population of phage displaying

antibodies

Primary selec:on of Met blocking an:bodies

Wash, elute bound phage 40% ELISA positives

Bacterial library of >1 x1010 clones

Schofield et al (2007)

Rescue phage

Rescue phage

No HGF/SF 1 nM HGF/SFNo HGF/SF 1 nM HGF/SFScatter assay BxPC3 pancreatic carcinoma cells

Chain shuffle

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0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0 30000

10000

3000

1000

300

100 30

10 3 1

0.3

0.1

0.03

0.01 0 0

30000

10000

3000

1000

300

100 30

10 3 1

0.3

0.1

0.03

0.01 0

0.3 nM HGFSF 0 nM HGFSF

Spot intensity

(backgroun

d subtracted

)

7A2 scFv concentra8on (nM)

SKOV3 migra8on to HGFSF: Effect of 8trated scFv clone 7A2 16/10/09; 400V; mean ± standard devia8on of triplicate wells

Iden:fica:on of Met blocking an:bodies

SKOV3 cell migra:on assay

Round 1 100nM 10nM 1nM 100pM

Round 2 1nM 100pM 10pM 1pM

1nM 100pM 10pM 1pM

1nM 100pM* 10pM 1pM

Round 3

7A2 VH

*

Oligo-‐directed mutagenesis of lead clone

11 amino acids Randomised 5 amino acids/oligo (x3 oligos)

Sub-‐clone Pick 1152 clones Sequence/ELISA Select top 146 unique clones BIA-‐Core off-‐rate screen FAb conversion

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Round 1 100nM 10nM 1nM 100pM

Round 2 1nM 100pM 10pM 1pM

1nM 100pM 10pM 1pM

1nM 100pM* 10pM 1pM

Round 3

7A2 VH

*

Oligo-‐directed mutagenesis of lead clone

11 amino acids Randomised 5 amino acids/oligo (x3 oligos)

7A2 (Parent) 12nM scFv 77nM Fab

dattpyYGMdv

LEAD CLONES 0.1-0.3nM scFv 1.2-2.3nM Fab

dattpyWGMxx

Sub-‐clone Pick 1152 clones Sequence/ELISA Select top 146 unique clones BIA-‐Core off-‐rate screen FAb conversion

Role of TACE in tumour microenvironment •  TNFα Conver:ng Enzyme (TACE/ADAM17) is a cri:cal “sheddase” of many

substrates, including EGF family ligands •  TGFa, HB-‐EGF, Aphiregulin, Epiregulin, Epigen, Neuregulin

•  Upregulated in a wide range of cancers including colorectal, hepa:c carcinomas, triple nega:ve breast cancer, ovarian and prostate

•  Expressed by tumour and stromal cells

•  Expression correlates with disease severity and outcome in a number of cancers including gastric cancer, HNSCC and breast cancer

•  Mul:ple RNA knockdown experiments indicate a role for TACE in tumour growth

•  Shedding of EGFR ligands is an acute resistance mechanism in colorectal cancer cells

•  Treatment of CRC with chemotherapies, eg 5-‐FU increases TACE ac:vity •  TACE over-‐expression decreased chemotherapy effect •  TACE abroga:on increases chemotherapy induced apoptosis of CRC

Kyula,Van Schaeybroeck et al. 2010 Chemotherapy-‐induced ac:va:on of ADAM-‐17: A novel mechanism of drug resistance in colorectal cancer Clin Cancer Research 16 3378-‐3389

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Protein model for TACE ectodomain

model based on VAP2B structure Igarashi et al., 2007

Chris Tape

An:-‐TACE an:body D1 inhibits proteoly:c ac:vity

………but doesn’t bind the cataly:c domain

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Chain-‐shuffling iden:fies a VL which directs binding to the cataly:c domain

Tape et al (2011) Cross-‐Domain Inhibi:on of TACE Ectodomain PNAS 108 p5578–5583

Affinity matura:on generates a cross-‐domain inhibitor of TACE

Tape et al (2011) Proc Natl Acad Sci 108 5578-‐5583

KD 26nM

KD 0.46nM (5.2nM on cataly:c domain)

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Phage display selection (FGF4, FGFR1 and FGFR2) Clone antibody gene population into targeting vector

Introduc8on of an8body gene popula8on into Oct4-‐GFP ES reporter cell

ES differen8a8on and an8body expression (in semi-‐solid medium)

Iden8fy modified colonies

Undifferen:ated GFP posi:ve

Differen:ated GFP nega:ve Recover an8body gene

Puromycin selec:on

Melidoni et al Proc. Natl. Acad. Sci ( in press)

ES “In Cell expression and

repor:ng” system (ES-‐ICER)

Introduc8on of an8body gene popula8on into Oct4-‐GFP ES reporter cell

ES differen8a8on and an8body expression (in semi-‐solid medium)

Iden8fy modified colonies

Undifferen:ated GFP posi:ve

Differen:ated GFP nega:ve Recover an8body gene

Puromycin selec:on

-dox

+d

ox (1

ug/

ml)

+dox

(2 u

g/m

l)

Tet-On promoter

TetRVP16 GATEWAY GATEWAY PuroR

ROSA26 Homology

Arm 1

ROSA26 Homology

Arm 2

an:body Fc

pROSA-‐ic

Phage display selection (FGF4, FGFR1 and FGFR2) Clone antibody gene population into targeting vector

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Summary •  Met

–  107_A07 blocks Met and binds IgG1/IgG2 domain –  non-‐HGF binding site, but blocks HGF binding

•  Notch –  blocking by conserving closed conforma:on

•  TACE –  Highly specific blocker –  Chain specific domain recogni:on

FGF4/FGF receptor illustrates: –  poten:al for direct func:onal screening –  Poten:al for controlling stem cell differen:a:on

•  In all cases an:bodies represent highly specific blockers

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c-MET Danielle diCara Ermanno Gherardi Tony Pope

TACE Chris Tape, Gill Murphy

Cambridge Research Institute

Mike Dyson Peter Slavny Kothai Parthiban Tony Pope Aneesh Karatt Vellatt

Notch Ronny Falk

FGF4 and FGF receptor Anna Melidoni, Mike Dyson

Documents

Blocking(cell(signaling(with( recombinantan:bodies( · 40% ELISA positives elute bound phage Bacteriallibraryof"" >1"x1010clones Schoﬁeld(etal((2007)(Rescue phage Rescue phage No