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Antibodies binding the ADAM10 substrate recognitiondomain inhibit Eph function

Lakmali Atapattu1, Nayanendu Saha2, Carmen Llerena1, Mary E. Vail1, Andrew M. Scott3, Dimitar B. Nikolov2,Martin Lackmann1,*,` and Peter W. Janes1,*1Department of Biochemistry and Molecular Biology, Monash University, Victoria 3800, Australia2Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA3Tumour Targeting Program, Ludwig Institute for Cancer Research, Austin Hospital, Heidelberg, Victoria 3084, Australia

*These authors contributed equally to this work`Author for correspondence (martin.lackman@monash.edu)

Accepted 13 October 2012Journal of Cell Science 125, 6084–6093� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.112631

SummaryThe ADAM10 transmembrane metalloprotease cleaves a variety of cell surface proteins that are important in disease, including ligandsfor receptor tyrosine kinases of the erbB and Eph families. ADAM10-mediated cleavage of ephrins, the ligands for Eph receptors, issuggested to control Eph/ephrin-mediated cell-cell adhesion and segregation, important during normal developmental processes, andimplicated in tumour neo-angiogenesis and metastasis. We previously identified a substrate-binding pocket in the ADAM10 C domain

that binds the EphA/ephrin-A complex thereby regulating ephrin cleavage. We have now generated monoclonal antibodies specificallyrecognising this region of ADAM10, which inhibit ephrin cleavage and Eph/ephrin-mediated cell function, including ephrin-inducedEph receptor internalisation, phosphorylation and Eph-mediated cell segregation. Our studies confirm the important role of ADAM10 in

cell-cell interactions mediated by both A- and B-type Eph receptors, and suggest antibodies against the ADAM10 substrate-recognitionpocket as promising therapeutic agents, acting by inhibiting cleavage of ephrins and potentially other ADAM10 substrates.

Key words: ADAM metalloprotease, Eph receptor, Ephrin cleavage, Cell-cell adhesion

IntroductionProteolytic release, or ‘shedding’, of cell surface-bound proteins

acts as an important post-translational switch that regulates

protein function and activity. The ADAM (a disintegrin and

metalloprotease) family of transmembrane proteases are the most

prominent shedding enzymes for membrane-anchored proteins.

ADAMs contain multiple extracellular domains, including a distal

metalloprotease (MP) domain, followed by disintegrin (D)- and

cysteine-rich (C) domains involved in substrate interaction, as well

as transmembrane and variable cytoplasmic sequences (Blobel,

2005). They are important in regulating inflammatory and growth

factor signalling, cell migration, and cell adhesion: in particular,

two closely related, atypical ADAMs, ADAM10 (CD156C,

MADM, Kuzbanian) and 17 [CD156B, TACE (TNFa-convertingenzyme)], shed ligands and/or receptors regulating key cytokine,

chemokine and growth factor signalling pathways important in

disease. These include erbB/EGF receptor family ligands and

receptors, Notch receptors and ligands, TNFa and TNFRI and II,CX3CL1, IL-6R, as well as cadherins and various cellular

adhesion molecules (CAMs), and the amyloid precursor protein

(APP) (Murphy, 2008; Saftig and Reiss, 2011). ADAM10 and 17

are also overexpressed in a variety of cancers (Murphy, 2008;

Saftig and Reiss, 2011; Sanderson et al., 2006). Together this

implies their important involvement in diseases such as

Alzheimer’s, chronic inflammatory and heart diseases, and cancer.

ADAM10 also cleaves ligands for Eph receptors, the largest

family of receptor tyrosine kinases, which together with their

membrane-bound ephrin ligands, control cell migration and

positioning during normal and oncogenic development

(Nievergall et al., 2012; Pasquale, 2010). In this context

ADAM10 association with A-type Eph receptors is promoted by

binding to their ephrin-A ligands on interacting cells (Janes et al.,

2005; Salaita et al., 2010), whereupon ADAM10 cleaves ephrin,

disrupting the Eph–ephrin tether between cells to allow de-

adhesion, or retraction (Hattori et al., 2000; Janes et al., 2005). This

function of ADAM10 is further regulated by kinase activity

(Blobel, 2005; Hattori et al., 2000), which we found to be mediated

through conformational changes in the Eph cytoplasmic domain

(Janes et al., 2009), such that ADAM10 acts as a switch between

cell-cell adhesion and segregation in response to Eph

phosphorylation levels. This switch is thought to be important

for Eph-dependent oncogenesis, where aberrant Eph receptor

expression and/or mutation contributes to tumour development by

promoting neo-angiogenesis, invasion and metastasis (Nievergall

et al., 2012; Pasquale, 2010). Interestingly, while EphB/ephrin-B

cell contacts were reported to be attenuated through protease-

independent trans-endocytosis (Marston et al., 2003; Zimmer et al.,

2003), ADAM10 was also recently found to be required for EphB/

ephrin-B-dependent cell sorting, where EphB2 activation triggers

ADAM10-mediated shedding also of E-cadherin (Solanas et al.,

2011).

Despite considerable efforts to develop ADAM metalloprotease

inhibitors, to date clinical trials based on compounds blocking the

protease catalytic site have failed due to lack of efficacy and

specificity (DasGupta et al., 2009; Moss et al., 2001; Saftig and

Reiss, 2011). To a large extent, this reflects similarity of the MP

6084 Research Article

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active site to matrix metalloproteases (MMPs) (Maskos et al.,

1998), and the mechanism of ADAM substrate specificity, which

does not rely on a typical cleavage signature recognised by the

protease domain, but on non-catalytic interactions between the

substrate and the ADAM C domain (Reddy et al., 2000; Smith

et al., 2002; White, 2003). We have previously used structure/

function studies to identify a substrate-binding pocket within the

ADAM10 C domain, which specifically recognises the Eph/ephrin

complex and thereby specifies cleavage of Eph-bound ephrin

(Janes et al., 2005). We therefore set out to raise monoclonal

antibodies (mAbs) against this region and assess their ability to

block substrate cleavage. We now describe mAbs specific for the

ADAM10 substrate-binding pocket, which inhibit ADAM10-

mediated ephrin cleavage, Eph activity and Eph-dependent cell

behaviour.

ResultsGeneration of monoclonal antibodies recognising

ADAM10 in the context of Eph/ephrin signalling complexes

To generate mAbs that selectively bind the substrate recognition

pocket within the C domain of the native ADAM10 extracellular

domain, we sequentially immunised and boosted mice with

ADAM10/EphA3+ve human embryonic kidney (HEK) 293 cells

and recombinant ADAM10 extracellular domain (ECD)

fragments, respectively. In particular, we used a protein

fragment spanning residues 214–646 of recombinant bovine

ADAM10 ECD (Janes et al., 2005), in keeping with the notion

that the lower homology to mouse sequences within the C domain

(92.7%, compared to 94.8% homology for human; Fig. 1A), may

increase its immunogenicity and bias the mouse immune response

against this domain. Screening of hybridoma cell lines generated

from these mice, using ELISA and Plasmon Resonance analysis to

test binding to recombinant bovine ADAM10 ECD, and

immunoprecipitation of endogenous human ADAM10 from

human embryonic kidney (HEK) 293 cell lysates, yielded three

monoclonal antibodies: 3A8 and 8C7, recognising bovine and

human ADAM10 (Fig. 1B,C), as well as 4G1, binding only to

bovine ADAM10 (Fig. 1B; supplementary material Fig. S1). 8C7

also bound to mouse ADAM10 present in lysates of wild-type

(Wt) mouse embryonic fibroblasts (MEFs), but not in

immunoprecipitates from ADAM2/2 MEFs (Hartmann et al.,2002), indicating its specificity for ADAM10 (Fig. 1D).

We previously reported that association of EphA3 and ADAM10

is increased by ligand-induced receptor clustering (Janes et al.,

2005), a finding since confirmed for other Eph receptors (Salaita

et al., 2010; Solanas et al., 2011). To investigate if our mAbs would

recognise cell surface ADAM10 in the context of associated EphA3,

we analysed EphA3/HEK293 cells expressing abundant endogenous

ADAM10 (above) by confocal microscopy. Staining of the cells

with Alexa647-conjugated 8C7 (Alexa647–8C7), when performed on

ice to prevent endocytosis, was faint (Fig. 2A, top row), consistent

with the reported tightly controlled levels of cell surface ADAM10

(Marcello et al., 2010). Interestingly, incubation with 8C7 at 37 C̊

resulted in prominent punctuate staining and notable internalisation

of stained complexes (Fig. 2A, 60 min, 2nd row), suggesting

antibody-induced clustering and its endocytosis together with

ADAM10-containing protein complexes.

We next sought to test if the 8C7 antibody also recognises

ADAM10 that is associated with EphA3 during activation, by

incubating cells with 8C7 and with ephrin-A5-Fc, which when

cross-linked with anti (a)-human Fc antibodies causes EphA3activation and internalisation (Lawrenson et al., 2002; Vearing

et al., 2005). Again, we added the Alexa647–8C7 mAb and

Alexa488–ephrin-A5-Fc to cells on ice, in order to prevent

endocytosis and internalisation of ADAM10-bound 8C7 prior to

stimulation. Cells that had been fixed immediately (0 min,

ephrin-A5), and those treated for indicated times with a-humanIgG at 37 C̊ to cross-link bound ephrin-A5-Fc, displayed closely

matching 8C7 and ephrin-A5-staining patterns, suggesting that

8C7 effectively binds to ADAM10 that is associated with Eph/

Fig. 1. Specificity of a-ADAM10 monoclonal antibodies. (A) Alignment of mouse, human and bovine ADAM10 cysteine-rich domain sequences (AA 551–

646). In the human and bovine sequences, only residues not homologous to mouse are shown. (B) Comparison of binding of mouse hybridoma (fusion) and

isolated cell clone supernatants to serially diluted, immobilised bovADAM10 ECD by ELISA. Binding of non-immunised mouse serum (control) is shown for

comparison. (C) Binding of endogenous huADAM10 by a-ADAM10 hybridoma clones, or the R&D ADAM10 mAb 1427, was compared by immunoprecipitation

from equivalent HEK293 cell lysates and western blotting with an a-ADAM10 pAb; u, unprocessed; p, processed ADAM10. (D) The specificity of 8C7 for

ADAM10 was tested by immunoprecipitation from lysates of ADAM10 knockout (2/2) and Wt (+/+) mouse embryonic fibroblasts (MEFs), and a-ADAM10

pAb western blot.

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ephrin signalling complexes (Fig. 2B). This is particularly

evident after (cross-linked) ephrin-A5-Fc-mediated EphA3

clustering and endocytosis, revealing pronounced uptake of

8C7 into EphA3-containing endocytic vesicles (Fig. 2B, 15,

60 min).

Unique mAb specificity for the ADAM10 substrate-

recognition module

To assess the epitope specificity of our ADAM10 mAbs, we

tested their ability to recognise isolated human (hu)-ADAM10

protein fragments, corresponding either to the full length ECD

(lacking the Pro domain), or the disintegrin and cysteine-rich

domains, expressed together or separately. Western blot analysis

revealed that both 3A8 and 8C7 recognise recombinant protein

fragments containing the cysteine-rich domain, but not the

isolated disintegrin domain alone (supplementary material Fig.

S2A). In comparison, 8C7 does not bind the isolated D+C

domains from either ADAM17 or ADAM19 (supplementary

material Fig. S2B), confirming its specificity. Importantly,

BIAcore analysis verified binding of the isolated ADAM10 C

domain to 8C7 conjugated to the sensor surface, while in

comparison the R&D a-ADAM10 mAb (clone 1427) recognisesonly the full length ECD, but not the isolated C domain (Table 1).

Interestingly, 8C7 seems to bind with six- to seven-fold higher

affinity to the isolated C domain (,14 nM) as compared to theentire ECD (,93 nM); this is mainly due to a substantiallyhigher association rate, suggesting this interaction might be

conformation dependent, with the epitope partially masked in the

full length molecule.

We next sought to test if our mAbs selectively target the

substrate-binding pocket within the ADAM10 C domain. We

previously identified three glutamic acid residues at positions

573, 578 and 579 which contribute to the negative charge of the

pocket and are essential for efficient binding of the ephrin/Eph

complex, as shown by Glu/Ala substitution at these sites

(ADAM10 3E-A mutant) (Janes et al., 2005). We therefore

compared binding of aADAM10 mAbs to Wt huADAM10–GFPand the ADAM10 3E-A mutant, by immunoprecipitation from

lysates of transfected ADAM102/2 MEFs. When compared to

the R&D a-ADAM10 mAb, which binds equally well to Wt andmutant ADAM10 (supplementary material Fig. S3A), both 8C7

and 3A8 showed significantly attenuated recognition of the

substrate-binding pocket mutant, indicating their specificity for

the Wt form of this region (Fig. 3B). Similarly, Ala substitutions

at residues 617 and 618, which are part of the lip of the binding

pocket (Fig. 3A), also decreased binding of our mAbs, while 8C7

binding to an unrelated, catalytic domain ADAM10 mutant with

a Glu384 to Ala substitution was equivalent to the Wt protein

(supplementary material Fig. S3B).

Fig. 2. Co-staining of cells with ADAM10 mAb 8C7 and ephrin-A5-Fc

reveals colocalisation and co-internalisation with EphA3. (A) EphA3/

HEK293 cells were incubated on ice with Alexa647–8C7 mAb and fixed for

imaging (0 min) or first allowed to warm to 37 C̊ for 60 min. (B) Cells were

labelled with Alexa647–8C7 and with Alexa488–ephrin-A5-Fc and fixed

immediately (0 min) or incubated at 37 C̊ with a-humanFc to cluster ephrin-

A5-Fc for the indicated time periods before fixation. The insets are enlarged

images of the regions within the dotted lines. Cells incubated for 60 min with

Alexa488–ephrin-A5-Fc alone are shown as a control in the bottom panels.

Scale bars: 25 mm.

Table 1. BIAcore affinity measurements of ADAM10 mAb binding to immobilised human ADAM10 ECD or isolated C domain

Analysed protein

8C7 mAb R&D mAb 1427

KD (M) ka (M/s) kd (s21) x2 KD (M) ka (M/s) kd (s

21) x2

ADAM 10 ECD 9.361028 2.36104 2.261023 14.3 2.061028 1.66105 3.361023 31.9ADAM10 C domain 1.461028 4.66105 6.461023 16.1 No binding

The shown affinity (rate) constants are for the interaction between the recombinant soluble ADAM10 extracellular domain proteins binding to BIAcore sensorchip surfaces conjugated with mouse 8C7 or with R&D mAb 1427, determined using a linear kinetic model provided in the BIAcore evaluation software(vers.3.1).

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mAbs against the ADAM10 substrate recognition domain

block ephrin cleavage and Eph activation

These data, suggesting that our mAbs may specifically bind, and

thereby block access to the substrate recognition motif (ephrin/

Eph-binding pocket) of ADAM10, prompted us to test their effect

on ADAM-mediated ephrin cleavage, and its uptake into Eph-

expressing HEK293 cells. For this we monitored cleavage of

ephrin from ephrin-A5-Fc-coated tissue culture plates by

HEK293 cells stably transfected with either EphA3 or with

EphB2, which also binds and is activated by ephrin-A5 (Himanen

et al., 2004). Cells that had been treated with increasing doses of

8C7 were plated onto wells pre-coated with Alexa594-labelled

ephrin-A5-Fc. After 30 min the cells were recovered and

analysed for internalised Alexa594–ephrin-A5 by microscopy

(Fig. 4A). In similar experiments we analysed by flow cytometry

ephrin shedding and uptake by cells treated with 8C7 alone, or

with anti-mouse IgG cross-linked 8C7, with enhanced binding

avidity (Fig. 4B). Both image and flow analysis showed robust

uptake of ephrin-A5 by the cells, and its significant, dose-

dependent inhibition by pre-incubation of cells with 8C7 mAb,

which was most pronounced with cross-linked 8C7.

To test this inhibition of ADAM10 in a physiologically more

relevant model, we assessed ephrin cleavage and uptake in co-

cultures of EphA3/HEK293 cells and GFP–ephrin-A5/HEK293

cells (Janes et al., 2005), by monitoring co-localised GFP–ephrin

within Cell-Tracker-Red-labelled EphA3 cells (Fig. 4C, cont.,

arrows). Pre-incubation of cell cultures with 8C7 significantly

inhibited ephrin uptake. Despite a more modest effect, likely

reflecting the lesser sensitivity of this assay, inhibition by 8C7

was significantly stronger than that achieved by treatment with

the broad MMP/ADAM metalloprotease inhibitors GM6001 or

TAPI1 (Fig. 4C, graph). Inhibition of cleavage was particularly

evident in images in which the GFP-tagged ephrin persisted at

cell-cell junctions, rather than being cleaved and internalised by

the EphA3-expressing cells (Fig. 4C, 8C7, ).

Receptor endocytosis is an important modulator of signalling,

not only in signal attenuation, but importantly by facilitating

cytosolic signalling on endosomes acting as signalling platforms

(Dobrowolski and De Robertis, 2012). We therefore tested the

effect of 8C7 treatment on Eph receptor tyrosine phosphorylation

in co-cultures of EphA3- and ephrin-A5-expressing HEK293 cells.

Pre-incubation of the cells with increasing concentrations of 8C7

caused a reproducible, dose-dependent decrease in EphA3

phosphorylation (Fig. 5), particularly at later time points

(.10 min), consistent with its likely effects on receptorendocytosis. In contrast, 8C7 treatment had no effect on

activation of EphA3 by soluble clustered ephrin-A5-Fc

(Fig. 5C), which causes EphA3 activation and internalisation

independent of ADAM activity, in keeping with 8C7 acting via

inhibition of ephrin cleavage.

a-ADAM10 mAb 8C7 blocks Eph/ephrin-mediated cell

repulsion and cell segregation

High or low levels of Eph kinase activity correlate with cell-cell

segregation or adhesion, respectively (Holmberg et al., 2000; Janes

et al., 2011; Nievergall et al., 2012; Poliakov et al., 2008). Also,

ADAM10-mediated cleavage of ephrin (Hattori et al., 2000; Janes

et al., 2005) and cadherins (Solanas et al., 2011) during Eph/

ephrin-mediated cell-cell interactions suggests essential roles in

severing the bond between cells that allows cell segregation

between the ephrin- and Eph-expressing populations to occur. We

analysed effects of our ADAM10 mAbs specifically on cleavage of

ephrins during Eph/ephrin-mediated cell repulsion, using a

modified stripe assay (Knöll et al., 2007; Walter et al., 1987), in

which Eph-expressing HEK293 cells are plated onto coverslips

pre-coated with stripes of fluorescently labelled ephrin, in our case

Alexa594–ephrinA5-Fc and EphB2/HEK293 cells. As a positive

control for Eph-dependent cell adhesion, we tested cells expressing

a truncated, signalling defective, dominant negative mutant EphB2

(DICD), which cannot segregate in response to ephrin (Janes et al.,2011). EphB2/293 cells were found to actively avoid stripes of

high ephrin density, with only 20% remaining on stripes after

overnight incubation (Fig. 6A, top row). In comparison, EphB2

DICD cells displayed marked adhesion to the stripes (Fig. 6A,bottom row), consistent with the Eph cytoplasmic domain being

required for Eph-mediated cell repulsion (Janes et al., 2011;

Fig. 3. Site-directed mutagenesis of the ADAM10 substrate-binding pocket disrupts mAb binding. (A) Structure of the bovine ADAM10 D and C domains

showing the location of key residues targeted by site-directed mutagenesis. (B) Comparison of aADAM10 mAb binding to Wt and substrate-binding pocket

mutant huADAM10. Alanine substitutions at Glu 573, 578 and 579 (3EA) or at residues 617 and 618 (617AA) were made in huADAM10-GFP, and Wt and

mutant constructs were transfected into ADAM102/2 MEFs (control: untransfected). Binding of a-ADAM10 mAbs was assessed by immunoprecipitation from

equivalent cell lysates, and western blotting with a-ADAM10 pAb (non-relevant lanes removed; the altered molecular mass pattern reflects the GFP-tagged

huADAM10). The graph shows binding of 8C7 and 3A8 relative to the R&D mAb, determined by densitometry (one-way ANOVA; **P,0.01 compared to R&D

sample; n.s., not significant; n53).

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Mellitzer et al., 1999) and validating the assay. Interestingly, pre-

treatment of EphB2/293 cells with increasing concentrations of

8C7 caused significant, dose-dependent inhibition of repulsion

from ephrinA5-Fc stripes, leading to a more random distribution,

similar to the effect of the metalloprotease inhibitor GM6001

(Fig. 6A,B), confirming specific ADAM10 inhibition as effective

in blocking Eph-mediated repulsion. We confirmed that,

underlying this inhibition of cell retraction, 8C7 inhibits EphB2

phosphorylation that is induced with cell-expressed ephrin-A5 to

stimulate its kinase activity: as expected and consistent with its

effects on ephrin-A5/EphB2-mediated repulsion, we observed

marked, dose-dependent inhibition of EphB2 phosphorylation by

8C7 (Fig. 6C).

We also tested effects of 8C7-inhibited ADAM10 activity on the

segregation, or sorting, between Eph- and ephrin-expressing cells,

a process previously shown to be dependent on Eph signalling

Fig. 4. 8C7 a-ADAM mAb blocks ephrin shedding and internalisation. (A) Ephrin internalisation from Alexa594–ephrin-A5-coated tissue culture surfaces.

EphA3/293 cells were pre-incubated with 0, 100 or 400 mg/ml 8C7 for 1 h before plating onto Alexa594–ephrin-A5-Fc-coated tissue culture surfaces. Cells weredetached and plated onto fibronectin-coated glass coverslips for fluorescence microscopy. Representative images from two independent experiments are shown,

and internalised ephrin fluorescence/cell quantified from a minimum of 10 fields of view, using ImageJ software. (B) Cells treated with non-clustered or pre-

clustered (X-lk) 8C7 at the indicated concentrations were plated onto Alexa594–ephrin-A5-Fc as in A and detached cells were analysed by flow cytometry.

Histograms of the different cell populations show the mean and s.e.m. (normalised to the non-treated sample with a value of 1) from three independent

experiments. (C) Ephrin internalisation of GFP–ephrin-A5 cells. GFP–ephrin-A5/293 cells and Cell-Tracker-red-labelled EphA3/293 cells were pre-incubated

with 0, 100, 200 or 400 mg/ml 8C7 mAb, or with metalloprotease inhibitors GM6001 (GM; 50 mM) or TAPI1 (50 mM), then co-cultured for 1 h before fixationand nuclear staining with Hoechst (blue). Images were taken by confocal microscopy; examples are shown from control and treated cultures (8C7, 400 mg/ml).Arrows: internalised GFP–ephrin in red-labelled EphA3 cells, bar ( ) indicates blockade of ephrin cleavage at cell-cell junctions. Ephrin internalisation was

calculated using colocalisation of green (ephrin) with red (Eph/293); values are means 6 s.e.m., n55. *P,0.05, ***P,0.001: significant difference from the

untreated or indicated sample. Scale bars: 20 mm.

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(Poliakov et al., 2008) and on ADAM10 (Solanas et al., 2011). In

co-cultures EphB2/293 cells, co-expressing membrane-targeted

GFP, were found to segregate from ephrin-A5/293 cells, such that

the GFP-labelled EphB2 cells formed tight clusters of increased

fluorescence intensity compared to mono-cultures, as previously

observed when co-cultured with ephrin-B1/293 cells (Janes et al.,

2011; Poliakov et al., 2004). Co-incubation with 8C7 alone, or

cross-linked with anti-mouse antibody, clearly affected this

segregation process, reducing the number of bright segregated

colonies, as determined by image analysis of intensity-thresholded

images (Fig. 7A–C). Inhibition was most pronounced using cross-

linked 8C7, consistent with its greater inhibition of ephrin cleavage

(Fig. 4), and comparable to effects of the less selective inhibitor

TAPI1. In separate experiments, cross-linked 8C7 treatment also

dose-dependently inhibited segregation of EphB2- and ephrin-B1-

293 cells (Fig. 7D), and segregation of U251 glioma and ephrin-A-

expressing HEK293 cells (Fig. 7E), a co-culture model we

previously used as a cancer relevant model of Eph/ephrin-A-

dependent cell sorting (Nievergall et al., 2010).

DiscussionInhibitors of ADAM10 and its closest relative ADAM17 (TACE)

have been actively sought over the last decade. While inhibition of

TNFa shedding by TACE was the initial target for treatment ofinflammatory diseases, including rheumatoid arthritis, the role of

both ADAM10 and 17 in shedding a range of disease relevant

targets has broadened interest in raising ADAM-targeted

therapeutics. This is particularly true in cancer, in which growth

factor/RTK signalling is frequently de-regulated, and where

ADAM-mediated shedding of ligands for the EGF receptor

(erbB, HER) family underlies autocrine feedback and

transactivation of EGFR signalling by stress and G-protein-

coupled receptor signalling (Blobel, 2005; Fischer et al., 2003).

ADAM10- and -17-mediated cleavage of HER2 and HER4,

respectively, also contribute to their signalling and underminereceptor-targeted therapies (Liu et al., 2006; Rio et al., 2000; Sardi

et al., 2006). In addition, ADAM10-mediated shedding regulates

cell-cell interactions important for tumour development and

invasion, in particular via targeting cadherins (Solanas et al.,2011) and ephrins (Hattori et al., 2000), which modulate cell-cell

adhesion and segregation during multiple stages of cancer

development (Nievergall et al., 2012; Pasquale, 2010).

Despite this interest, and the validation of ADAM inhibition in

vitro, no inhibitors targeting the metalloprotease catalytic site

have yet successfully passed phase II clinical trials, due to lack of

specificity, stemming from the structural similarity between theproteolytic cleft of ADAMs and matrix metalloproteases (MMPs)

(DasGupta et al., 2009; Saftig and Reiss, 2011). However, the

Fig. 5. ADAM10 mAb 8C7 inhibits EphA3 phosphorylation in response to

stimulation by cell-bound ephrin. (A) 293/EphA3 cells were pretreated with

0, 10 and 100 mg/ml of 8C7 mAb for 2 h and stimulated for the indicated times.a-EphA3 immunoprecipitates from the cell lysates were analysed by western

blot with a-phosphotyrosine (pY) and a-EphA3 antibodies as indicated. A

representative image from four experiments is shown. (B) EphA3

phosphorylation relative to EphA3 protein levels was calculated from replicate

experiments as described in A, using densitometry analysis. Graph shows

means 6 s.e.m., n54. (C) 8C7 does not inhibit EphA3 phosphorylation induced

by soluble clustered ephrin-A5. EphA3/293 cells, pre-incubated with or without

8C7 (100 mg/ml) for 2 hours, were stimulated for 20 min with pre-clusteredephrin-A5-Fc, or left unstimulated, as indicated. EphA3 immunoprecipitates

from cell lysates were analysed by western blotting as in A.

Fig. 6. ADAM10 mAb 8C7 blocks Eph/ephrin-mediated cell repulsion.

(A) EphB2/HEK293 cells labelled with Cell Tracker Green were pre-treated

with vehicle (Cont), 8C7 (50, 200 or 400 mg/ml), or with GM6001 (GM,50 mM), and plated onto coverslips pre-coated with fibronectin and stripes ofalexa594-labelled ephrin-A5-Fc. As a comparison, cells expressing a

signalling-deficient EphB2 mutant (DICD) were also used. After 18 hours the

cells were imaged by fluorescence microscopy, from which examples are

shown (8C7, 400 mg/ml). Scale bar: 250 mm. (B) The percentage of cellsadhering to ephrin stripes was calculated from ,20 images for eachtreatment; the graph shows the averages 6 s.e.m. from three experiments.

(C) 8C7 inhibits ephrin-A5-induced EphB2 phosphorylation. Effects of 8C7

treatment on activation of EphB2/HEK293 cells by ephrin-A5/HEK293 cells

was assessed as in Fig. 5A, following stimulating for 40 minutes.

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specificity of ADAM function is mediated via enzyme-substrateinteractions outside the protease domain, within the Cys-rich

domain, and we previously elucidated the structure of theADAM10 Cys-rich domain and identified a binding pocket thatconfers selectivity for the EphA/ephrin-A complex formed at

cell-cell junctions (Janes et al., 2005). We thus surmised thatantibodies raised against this pocket might potentially blocksubstrate binding and more specifically inhibit ADAM10-mediated proteolysis.

We now describe a-ADAM10 mAbs, which recognise the Cdomain and appear to specifically target the substrate-bindingpocket, as suggested by loss of binding following mutagenesis of

key exposed residues within this region. Interestingly,considerably higher affinity of these mAbs for the isolated Cdomain compared to the full-length extracellular domain,

principally due to a higher association rate, suggests thebinding epitope may be masked in much of the full-lengthprotein, and thus dependent on the conformation of the ADAM10ECD. Although the structure of the full ADAM10 ECD is yet to

be solved, functional studies of the close relative ADAM17suggest activity-dependent conformation changes (Wang et al.,2009; Willems et al., 2010), and structures of related snake

venom metalloproteases also suggest distinct open and closedconformations that are suggested to regulate substrate access tothe C domain (Guan et al., 2010), a notion which would be

consistent with the binding behaviour of our mAbs to this region.

Our a-ADAM10 antibodies also show specific binding toADAM10 on cells, where staining with 8C7 colocalises with

ephrin-A5-Fc-bound EphA3, consistent with the previousreported interactions of ADAM10 with EphA receptors (Janeset al., 2005; Salaita et al., 2010; Solanas et al., 2011). Moreover,

8C7 is effectively co-internalised following EphA3 receptor

stimulation with soluble ephrin, suggesting a close functionalassociation of ADAM10 and EphA3 in cells and the effective

targeting of this complex by 8C7. Importantly, assays withrecombinant or cell-expressed ephrin show that 8C7 blocks

cleavage and internalisation of ephrin-/Eph complexes, but alsoinhibits receptor phosphorylation and ensuing biological

responses. Thus, repulsion of EphB2-expressing HEK293 cellsfrom stripes of immobilised ephrin-A5-Fc was inhibited, as was

segregation of cells expressing various combinations of A- andB-type Ephs and ephrins. The inhibitory effect of 8C7 on EphB2-

mediated ephrin shedding, cell repulsion and cell-cell segregation

in our assays indicates that in addition to EphA/ephrin-A clusters(Janes et al., 2005; Salaita et al., 2010), ADAM10 also effectively

targets ephrins bound to EphB2: It confirms the recently-reportedinteraction between ADAM10 and EphBs (Solanas et al., 2011),

and demonstrates that 8C7 is effective in blocking the ADAM10-facilitated function of both Eph subtypes.

Interestingly, 8C7 also inhibited Eph receptor phosphorylationin EphA3- and EphB2-expressing HEK293 cells in response to

ephrin-A5/HEK293 cells, but did not affect EphA3 stimulationwith soluble, pre-clustered ephrin-A5-Fc. We argue that rather

than directly affecting ephrin-induced Eph activation, 8C7inhibition of ADAM10 prevents ephrin cleavage and thus

endocytosis of ligated Ephs, resulting in persisting Eph/ephrinclusters at cell-cell junctions (Fig. 4B), highly reminiscent of

those observed in cells overexpressing dominant-negative

ADAM10 (Nievergall et al., 2010). However, the increasedresidency at the cell membrane does not result in a prolonged

activation of EphA3, but in agreement with the lower Ephactivity commonly observed under conditions of Eph-mediated

Fig. 7. 8C7 blocks Eph/ephrin-mediated cell segregation.

(A–C) HEK293 cells expressing EphB2 and membrane-

targeted GFP were co-cultured with ephrin-A5/HEK293 cells

in the presence of 0.4 mg/ml 8C7, with or without crosslinking

with anti-mouse IgG. Wells treated with anti-mouse IgG alone

or with TAPI1 served as negative and positive controls,

respectively. Confluent cultures were analysed by fluorescence

microscopy for GFP, and Hoechst staining of nuclei to show

the total cell population, and representative images are shown

in A. Segregated cell clusters were counted in whole well

images by counting areas of thresholded intensity above a set

size equating to roughly 40–50 cells (Solanas et al., 2011)

(B), and mean numbers (n54) were calculated with standard

errors (C). (D) Segregation assay performed with EphB2–GFP-

and ephrin-B1-expressing HEK293 cells showing key

representative examples and quantification. (E) Segregation

assay performed with Cell-Tracker-Green-labelled EphA3/B2-

expressing U251 glioma cells and HEK293 cells. *P,0.05,

**P,0.01 relative to control (cont).

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cell-cell adhesion (Holmberg et al., 2000; Janes et al., 2011;

Nievergall et al., 2012; Poliakov et al., 2008) leads to an overall

reduced phosphorylation. In agreement, in other studies we

have also noted that inhibition of EphA3 endocytosis by

overexpression of the clathrin coat-associated protein AP180

decreases ligand-induced receptor phosphorylation (C.

Stegmeyer, P.W.J., M.L., unpublished data). The underlying

relationship between reduced EphA3 endocytosis and

phosphorylation is also consistent with the inhibition of EphA3

endocytosis observed upon overexpression of the protein tyrosine

phosphatase (PTP) 1B, which regulates EphA3 function and

trafficking by controlling its phosphorylation and kinase activity

(Nievergall et al., 2010). Reduction of EphA3 phosphorylation by

8C7 was particularly evident at later time points (after 10 min)

coincident with EphA3 endocytosis (Wimmer-Kleikamp et al., 2004)

which would imply it has reduced tyrosine-dependent signalling from

endosomes. This is in keeping with the notion that endosomes, rather

than merely downregulating RTK signalling, compartmentalise and

thus facilitate signals that are prevented at the plasma membrane

(Dobrowolski and De Robertis, 2012; Scita and Di Fiore, 2010). A

prominent mechanism, known to regulate endocytic signalling of

several RTKs including receptors for EGF, PDGF and Insulin, is via

RTK-initiated, localised production of reactive oxygen species

(ROS), which accumulate in endosomes (Ushio-Fukai, 2009), and

transiently inactivate regulatory PTPs to enhance RTK

phosphorylation and signalling activity (Oakley et al., 2009; Rhee,

2006; Tiganis, 2011). While such a mechanism remains to be

confirmed for Ephs, similarities in the regulation of their signalling

activity by regulatory PTPs and endocytosis (Nievergall et al., 2012;

Pasquale, 2010) would suggest a similar concept controlling Eph

signalling.

In conclusion, our novel monoclonal antibodies against

ADAM10, by targeting the substrate recognition pocket,

effectively inhibit its ability to cleave ephrin from cell surfaces

and thus facilitate Eph function. Considering that ADAM10 has a

variety of substrates in addition to ephrins, many of which are

critically implicated in a range of chronic diseases, including

inflammatory, heart and neurodegenerative diseases as well as

cancer, it will be of considerable interest to examine the efficacy

of our antibodies to inhibit shedding of these other ADAM10

substrates in cell and animal-based disease models. Importantly,

the high specificity of our mAbs for ADAM10 will likely provide

a significant advantage over current, less selective, protease-

targeted inhibitors. We expect that highly specific inhibition

of ADAM10 activity in pro-inflammatory and/or oncogenic

signalling pathways will provide a basis for the development of

our antibodies as potential therapeutics.

Materials and MethodsExpression constructs

The sequences encoding the extracellular domain [amino acids (AA) 214–646,lacking the prodomain] from bovine (Howard et al., 1996) and human (OriGene)ADAM10, as well as isolated disintegrin (AA 455–550) and cysteine-rich domains(AA 551–646), or D+C domains combined (AA 455–646), were subcloned as Fcfusions into pcDNA3.1 vector (Invitrogen), including an N-terminal prolactinsignal sequence and a C-terminal thrombin cleavage site followed by the Fcdomain of human IgG, for expression in human embryonic kidney 293 (HEK293)cells. Following initial purification on Protein-A–Sepharose (Amersham), the Fctag was removed by thrombin cleavage, and ADAM10 proteins were furtherpurified to homogeneity by gel filtration chromatography.

Point mutations to human ADAM10-turboGFP (OriGene) were introduced bysite-directed mutagenesis (Quickchange XL, Stratagene). Human ADAM10 3EAand 617AA mutants were generated by introducing Alanines at G573,578,579, or atRH617,618, respectively.

Generation of monoclonal antibodies against ADAM10

Mice were immunised with ADAM10/EphA3-expressing HEK293 cells, followedby subsequent injections with bovine ADAM10 extracellular domain and isolatedD+C fragments. Subsequently, B cells from mouse spleens were fused withmyeloma cells in order to generate hybridomas which were selected formonoclonal antibody production. Hybridoma supernatants from pooled fusionsand from isolated clones were collected and screened for binding to ADAM10using enzyme-linked immunosorbant assay (ELISA), followed by IP/westernblotting, immunofluorescence and surface plasmon resonance (BIAcore) analysis,as described in the text. Antibodies were purified from supernatants on Protein G.

Surface plasmon resonance

Analysis of protein interactions by surface plasmon resonance was carried out on aBIAcore 3000 biosensor (BIAcore) as described previously for other antigenantibody interactions (Lackmann et al., 1996). Monoclonal antibodies wereimmobilized onto BIAcore CM5 sensorchips using NHS chemistry and binding ofADAM10 proteins, diluted between 50 and 0.08 nM into running buffer (10 mMHEPES, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween 20), was performedon sensor-chip surfaces derivatised on parallel channels with non-relevant protein.The binding kinetics was derived from the plasmon resonance sensorgrams aftersubtraction of baseline responses (measured on the control channel) by globalanalysis using the BIA Evaluation software (version 3.02, BIAcore). The surface ofthe chip was regenerated after each injection of sample with 3 M MgCl2, 0.075 MHEPES/NaOH, 25% ethylene glycol, pH 7.2, followed by two washes with runningbuffer.

Cell lines and culture

HEK293 cells stably expressing, membrane-targeted GFP and EphB2, orEphB2DICD, were kindly provided by David Wilkinson and Alexei Poliakov(NIMR, London) and Claus Jorgensen (ICR, London) and were previously described(Janes et al., 2011; Jørgensen et al., 2009; Poliakov et al., 2008). EphA3/HEK293 cellswere also previously described (Lawrenson et al., 2002). Mouse embryonic fibroblasts(MEFs) from Wt or ADAM102/2 mice (Hartmann et al., 2002) were kindly providedby Dr Saftig (Kiel). Cell lines were cultured in DMEM supplemented with 10% fetalcalf serum and transiently transfected using Fugene HD (Roche). Metalloproteaseinhibitors GM6001 and TAPI1 were from Calbiochem.

Immunoprecipitation and western blotting

Cells lysed in buffer containing 1% Triton X-100 and 0.1% SDS (Lawrenson et al.,2002) were immunoprecipitated with antibodies against ADAM10 (R&D systemsmAb 1427, or mAbs 3A8 or 8C7) or turboGFP (OriGene) followed by protein A–Sepharose, or EphA3 (mAb IIIA4 (Lackmann et al., 1996) conjugated toMinileakTM beads). Western blots were performed using rabbit polyclonalantibodies against ADAM10 (Abcam pAb 39177), turboGFP (OriGene) or PTyr(Invitrogen), or a-EphA3 sheep pAb (Nievergall et al., 2010) or a-EphB2 mousemAb (R&D systems). Where indicated cells were stimulated with 1.5 mg/mlephrin-A5-Fc [produced as described previously (Lawrenson et al., 2002)] pre-clustered with 0.75 mg/ml a-human IgG (Jackson Immunoresearch). Densitometricanalysis was with ImageQuant software.

Microscopy

Antibodies or ephrin-A5-Fc proteins were coupled to Alexa dyes (488, 594, 647;Invitrogen) as per the manufacturer’s instructions, and incubated with cells for 1 houror as indicated followed by washing with PBS and fixing with 4% PFA in PBS for30 mins at room temperature. Samples were mounted in Moviol and coverslipped.Confocal microscopy was on a Leica SP5 TCS SP5 II or Nikon C1 confocalmicroscope equipped with 405, 488, 561 and 633 nm lasers. Stripe and segregationassays were imaged on a Leica AF6000LX Live Cell Imaging workstation. Imageswere processed using Leica TCS, ImageJ and Adobe Photoshop and InDesign software.

Ephrin cleavage/internalisation assay

Alexa647–ephrin-A5-Fc was bound to 24-well tissue culture plates (BD Falcon,USA) as follows: to increase binding capacity, plates were pretreated withnitrocellulose dissolved in methanol (Lemmon et al., 1989) under a laminar hood,followed by 100 mg/ml protein A/G (Pierce) treatment for 1 hour at 37 C̊.Unbound protein A/G was removed by two PBS washes. Alexa647–ephrin-A5-Fcwas bound to the protein A/G layer at 5 mg/ml for 1 hour at 37 C̊ followed by twoPBS washes.

293/EphA3 cells and EphB2/GFP 293 cells were treated in suspension with 8C7at the indicated concentrations, with or without pre-cross-linking with a-mouseIgG (Jackson Immunoresearch) at 4:1 ratio for 1.5 hours. They were then added tothe Alexa647–ephrin-A5-Fc coated plates and incubated for 30 min at 37 C̊. Thecells adhered to the bottom of the plate were taken off with two vigorous PBSwashes followed by another wash with 10 ml of PBS. Cells were mounted on toslides for analysis by confocal microscopy and fixed with 2% PFA/PBS for20 min, or fixed in suspension with 2% PFA in FACS buffer (1% FCS, 1 mMEDTA in PBS) for flow cytometry. Flow cytometry analysis was performed using

Function blocking ADAM10 antibodies 6091

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BD LSR II flow cytometer (BD Biosciences) under HeNe laser (18 mW at633 nm). Further analysis was done on FlowJo 7.6.1 software package (Tree StarInc., USA). Only single cells were gated for expression of Alexa647–ephrin-A5-Fc.Histograms were generated for every sample indicating the shift of fluorescence.Mean of the fluorescence distribution was calculated using FlowJo.

For assaying ephrin cleavage from cells, 293/EphA3 cells were stained with CellTracker Red (Invitrogen) as described in the manufacturer’s protocol, allowed toadhere onto eight-well chamber slides (BD falcon), and pretreated with vehicle, 8C7,GM6001 or TAPI1 at the indicated concentrations. 293/ephrinA5 GFP cells werethen added for 40 min at RT followed by fixing with 4% PFA, and nuclear stainingwith DAPI. Cells were mounted and imaged using a Nikon upright C1 confocalmicroscope and 1006 oil objective. Ephrin internalisation was estimated from theMander’s coefficient of colocalisation of green (ephrin) with red (EphA3 cells).

Stripe assay

A silicon matrix (obtained from Prof. Bastmeyer, Karlsruhe Institute ofTechnology, Germany) with channels was placed on a 22622 mm glasscoverslip, and protein A/G (Pierce, USA) solution (20 mg/ml) resuspended inHanks Buffered Salt Solution (HBSS, Invitrogen) was applied to the channels asdescribed (Knöll et al., 2007). Coverslips were then coated with fibronectin(Sigma) at 10 mg/ml in PBS. EphrinA5-Fc labelled with Alexa594 (Invitrogen) at16 mg/ml in HBSS was added to the coverslips for 30 mins at 37 C̊ and washedtwice in HBSS. EphB2/GFP 293 cells treated with or without 8C7 mAb antibodyor GM6001 at the indicated concentrations, or EphB2DICD 293 cells pre-labelledwith Cell Tracker Green, were incubated on the coverslips at 37 C̊ overnightbefore imaging by fluorescence microscopy. To determine the percentage ofmigrated cells in each image the number of cells on the stripes (counted manually)was taken as a percentage of the total number of cells (estimated using the imageanalysis software Cell-Profiler, Broad Institute).

Cell segregation assays

EphB2-expressing HEK293 cells co-expressing membrane-targeted GFP (Poliakovet al., 2008), or U251 cells labelled with Cell Tracker Green (Invitrogen) were co-cultured with ephrin-expressing HEK293 cells in 96-well plates seeded with20,000 cells per well, for 48 h or until confluent. Cells were fixed and nucleistained with Hoechst before imaging on a Leica AF6000LX fluorescencemicroscope, using the tile scanning function to image the entire well. Theimages were then analysed with ImageJ software by thresholding areas offluorescence intensity above that seen in monocultures. Cell clusters greater than aset area corresponding to roughly 40–50 cells (Solanas et al., 2011) were thencounted by particle count of thresholded areas. Each treatment was performed onfour replicate wells, and repeated in three or more experiments.

AcknowledgementsWe thank Momchil Kolev (Sloan-Kettering), Chanley Chheang andJulien Robinson (Monash) for technical assistance, Monash MicroImaging for microscopy assistance, and Kaye Wycherley,Monoclonal Antibody Facility, The Walter and Eliza Hall Instituteof Medical Research, for invaluable help with generating monoclonalantibodies.

FundingThis work was supported by the National Health and MedicalResearch Council of Australia [grant numbers 384242 to M.L.,P.W.J. and D.B.N., 487922 to M.L., and R. D. Wright, and SeniorResearch Fellowships to P.W.J. (334085) and M.L. (384113)].Operational Infrastructure funding from the Victorian Government[to A.M.S.]; National Institutes of Health [grant number NS38486 toD.B.N.]; and the New York State Spinal Cord Injury researchprogram [grant number C-022047 to N.S.].

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.112631/-/DC1

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Function blocking ADAM10 antibodies 6093

http://dx.doi.org/10.1074/jbc.275.19.14608http://dx.doi.org/10.1074/jbc.275.19.14608http://dx.doi.org/10.1074/jbc.275.19.14608http://dx.doi.org/10.1074/jbc.275.19.14608http://dx.doi.org/10.1126/science.1130481http://dx.doi.org/10.1126/science.1130481http://dx.doi.org/10.1126/science.1130481http://dx.doi.org/10.1126/science.1130481http://dx.doi.org/10.1074/jbc.275.14.10379http://dx.doi.org/10.1074/jbc.275.14.10379http://dx.doi.org/10.1074/jbc.275.14.10379http://dx.doi.org/10.1016/j.ejcb.2010.11.005http://dx.doi.org/10.1016/j.ejcb.2010.11.005http://dx.doi.org/10.1126/science.1181729http://dx.doi.org/10.1126/science.1181729http://dx.doi.org/10.1126/science.1181729http://dx.doi.org/10.1080/08977190600634373http://dx.doi.org/10.1080/08977190600634373http://dx.doi.org/10.1080/08977190600634373http://dx.doi.org/10.1016/j.cell.2006.07.037http://dx.doi.org/10.1016/j.cell.2006.07.037http://dx.doi.org/10.1016/j.cell.2006.07.037http://dx.doi.org/10.1038/nature08910http://dx.doi.org/10.1083/jcb.200206023http://dx.doi.org/10.1083/jcb.200206023http://dx.doi.org/10.1083/jcb.200206023http://dx.doi.org/10.1038/ncb2298http://dx.doi.org/10.1038/ncb2298http://dx.doi.org/10.1038/ncb2298http://dx.doi.org/10.1016/j.tips.2010.11.006http://dx.doi.org/10.1016/j.tips.2010.11.006http://dx.doi.org/10.1089/ars.2008.2333http://dx.doi.org/10.1089/ars.2008.2333http://dx.doi.org/10.1158/0008-5472.CAN-05-0758http://dx.doi.org/10.1158/0008-5472.CAN-05-0758http://dx.doi.org/10.1158/0008-5472.CAN-05-0758http://dx.doi.org/10.1158/0008-5472.CAN-05-0758http://dx.doi.org/10.1158/0008-5472.CAN-05-0758http://dx.doi.org/10.4049/jimmunol.0802770http://dx.doi.org/10.4049/jimmunol.0802770http://dx.doi.org/10.4049/jimmunol.0802770http://dx.doi.org/10.1016/j.ceb.2003.08.001http://dx.doi.org/10.1016/j.ceb.2003.08.001http://dx.doi.org/10.1042/BJ20100179http://dx.doi.org/10.1042/BJ20100179http://dx.doi.org/10.1042/BJ20100179http://dx.doi.org/10.1083/jcb.200312001http://dx.doi.org/10.1083/jcb.200312001http://dx.doi.org/10.1083/jcb.200312001http://dx.doi.org/10.1038/ncb1045http://dx.doi.org/10.1038/ncb1045http://dx.doi.org/10.1038/ncb1045

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