development of therapeutic anti-jagged1 antibodies for ......aug 08, 2019 · jagged subfamilies...

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Development of therapeutic anti-JAGGED1 antibodies for cancer therapy

Authors

Massimo Masiero1†, Demin Li1†, Pat Whiteman2, Carol Bentley1, Jenny Greig1, Tasneem

Hassanali1, Sarah Watts1, Stephen Stribbling1, Jenna Yates1, Ellen Bealing1, Ji-Liang Li3,a,

Chandramouli Chillakuri2, Devon Sheppard4, Sébastien Serres5,b, Manuel Sarmiento-Soto5,

James Larkin5, Nicola R. Sibson5, Penny A. Handford2‡, Adrian L. Harris3‡, Alison H.

Banham1‡

Authors’ affiliations

1NDCLS, Radcliffe Department of Medicine, OX3 9DU

2Department of Biochemistry, OX1 3QU

3CRUK Department of Oncology, Weatherall Institute of Molecular Medicine, OX3 9DS

4Sir William Dunn School of Pathology, OX1 3RE

5Cancer Research UK and Medical Research Council Oxford Institute for Radiation

Oncology, Department of Oncology, OX3 7DQ

University of Oxford, Oxford, United Kingdom.

aInstitute of Translational and Stratified Medicine, Faculty of Medicine and Dentistry,

Plymouth University, Plymouth, PL6 8BU, United Kingdom.

bSchool of Life Sciences, University of Nottingham, Nottingham NG7 2UH, United Kingdom.

† ‡ These authors contributed equally to this work

Running title: Anti-JAGGED1 antibodies for cancer therapy

Key words: JAGGED1; cancer therapy; therapeutic antibodies; NOTCH; breast cancer

Corresponding author: Prof. Alison Hilary Banham MA DPhil (Oxon) FRCPath, Professor

of Haemato-oncology, Nuffield Division of Clinical Laboratory Sciences, Radcliffe

Department of Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford,

OX3 9DU, UK.

Email: [email protected] Tel: +44 (0)1865 220246 Fax: +44 (0)1865 228980

Conflict of interest: The authors are inventors (A.H.B., A.L.H., and P.A.H.) and contributors

(M.M., D.L., P.W., C.B., J.G., T.H., S.W., S.S., J.Y., C.C., and D.S.) on a patent application

entitled ‘Antibodies that bind to jagged1’.

Research. on December 11, 2020. © 2019 American Association for Cancermct.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-18-1176

mailto:[email protected]

http://mct.aacrjournals.org/

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Authors’ contributions

Conception and design: P.A.H, A.L.H and A.H.B.

Performed experiments and data acquisition: M.M., D.L., P.W., C.B., J.G., T.H., S.W., S.S.,

J.Y., E.B., J-L.L., C.C., D.S., S.S., M.S-S. and J.L.

Analysis and interpretation of data: M.M., D.L., P.W., S.S., M.S-S., J.L., N.R.S., P.A.H.,

A.L.H. and A.H.B.

Writing, review and/or revision of the manuscript: all authors.

Study supervision: P.A.H., A.L.H. and A.H.B.

Abstract

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology have been

firmly established, making them appealing therapeutic targets for cancer treatment. Here we

report the development and characterization of human/rat-specific JAG1-neutralizing

monoclonal antibodies. Epitope mapping identified their binding to the Notch receptor

interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution

prevented effective binding to the murine Jag1 orthologue. These antibodies were able to

specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells

and to block the heterotypic JAG1-mediated Notch signaling between endothelial and

vascular smooth muscle cells. Functionally, in vitro treatment impaired 3D growth of breast

cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo

testing showed variable effects on human xenograft growth when only tumor-expressed

JAG1 was targeted (mouse models) but a more robust effect when stromal expressed Jag1

was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established

triple receptor negative breast cancer brain metastasis in rats showed a significant reduction

in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial

improvement in blood-brain-barrier function and tumor perfusion. Lastly, JAG1-targeting

antibody treatment did not cause any detectable toxicity, further supporting its clinical

potential for cancer therapy.




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Introduction

Notch signaling is an evolutionary conserved pathway that plays an important role in

different physiologic processes and its dysregulation is involved in pathologic conditions,

including cancer. In mammals, signaling is initiated by the interaction between one of four

Notch receptors (Notch1, 2, 3 and 4) with one of five ligands belonging to the Delta-like or

Jagged subfamilies (Dll1, 3 and 4, and Jagged1 and 2 respectively). Upon ligand binding,

the receptor undergoes a series of proteolytic cleavages that ultimately lead to the release of

its intracellular domain into the cytoplasm and subsequent nuclear translocation, where it

acts as a transcriptional regulator (1, 2). Alterations of the pathway have been described in

many cancer types and affect multiple aspects of tumor biology, these generally confer

oncogenic function but, may also have tumor suppressive activity (1, 3).

Among the Notch ligands, human JAGGED1 (hJAG1) has been closely linked to tumor

biology, with involvement in metastasis formation, cancer stem cell (CSC) number,

angiogenesis, epithelial-to-mesenchymal transition (EMT), cell proliferation, resistance to

therapy and immune function regulation (4). Notably both tumoral and stromal Jagged1 have

been reported to play a role, with the latter implicating endothelial cells (5, 6), osteoblasts (7,

8) and myeloid derived suppressor cells (9). JAGGED1 is expressed in many normal tissues

and Alagille syndrome, caused by inactivating JAG1 mutations, primarily affects the liver,

heart, skeleton, eye, face, kidney and vasculature (10). Due to the multi-functional role

played by Notch signaling in several different tumor types, it is not surprising that a variety of

therapeutic approaches targeting this pathway have been developed, including both small

molecules and neutralizing antibodies. Small molecules are predominantly γ-secretase

inhibitors (GSIs), a class of compounds that inhibit the last proteolytic cleavage step during

Notch receptor activation. These were originally developed for Alzheimer’s treatment (11),

but are under extensive clinical testing for a variety of neoplastic conditions. GSIs are

characterized by their ability to inhibit Notch signaling mediated by any receptor-ligand

combination, but also by their recognition of additional substrates and severe gastrointestinal

toxicity, which currently limit their clinical application (12). A more targeted approach




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involves the use of monoclonal antibodies (mAbs), and this has been employed to neutralize

either individual receptors or ligands. Pre-clinical studies have shown therapeutic potential

for mAbs targeting Notch1 (13, 14), Notch 2 and 3 (13, 15), as well as ligands such as Dll4

(16-18) and more recently hJAG1 (8). Based on a similar targeted rationale, Notch

ectodomain-based decoys have also been developed and these performed positively in pre-

clinical models (19, 20). Clinical testing is underway for some of these approaches (12), but

further work is needed to identify the optimal Notch pathway target, the effective agents and

particularly the right therapeutic setting.

Aggressive triple receptor negative breast cancer (TNBC) represents an important area of

unmet clinical need. Patients present with molecular and clinical heterogeneity, have a high

likelihood of relapse and as yet there are no systemic approved standard of care therapies

beyond classical chemotherapy. Here we show that a therapeutic monoclonal antibody

targeting the Notch receptor binding site on the hJAG1 Delta/Serrate/Lag2 (DSL) domain

can inhibit Notch signaling, target TNBC cancer stem cells and reduce tumor growth in vivo.

Anti-JAG1 immunotherapy offers promise as a future treatment strategy, both in TNBC and

other cancer types.

Materials and Methods

Generation of anti-JAG1 monoclonal antibodies

All in vivo work, in this and in other experiments, was approved by the local ethical review

committee and governed by appropriate UK Home Office establishment, project and

personal licenses and complied with the Guidelines for the Welfare and Use of Animals in

Cancer Research (21). Antibodies were generated by immunization of MF1 mice with the

purified JAG1 DSL-EGF1-3 protein (22) and splenocytes were fused with NS0 cells as

described previously (23). Hybridoma supernatants were screened for the presence of

antibodies that were reactive with the immunogen by ELISA, and positive hybridoma cell

lines were cloned by limiting dilution. For further information on antigen and antibody




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production, ELISA screening, dot blot and Surface Plasmon Resonance analysis, and

antibody humanization see Supplementary Materials and Methods.

Cell lines, culture conditions, treatment of 2D and 3D in vitro models

All cell lines and their growth conditions can be found in the Supplementary table 1. All cell

lines were routinely tested for Mycoplasma using the Plasmo Test Mycoplasma Detection kit

(Invitrogen) every 3 months. All cell lines were used within 15 passages following thawing

(7-8 passages for primary human cells). MDA-MB-231 and MDA-MB-231/BR cells were

authenticated using STR profiling by LGC Standards. For 2D cell treatment, cells were

plated in 6 well plates and 24h later, when cell density was approximately 70-80%, growth

medium was replaced with fresh media containing the treatment. 48h later cells were

harvested for further analysis.

For the HUVEC-HUVSMC co-culture experiments, a first layer of cells was plated on day 0

followed by a second one when the first reached approximately 75% confluency (generally

on day 3). Three co-culture combinations were prepared as follow: HUVEC + HUVSMC (test

sample), HUVEC + HUVEC and HUVSMC + HUVSMC (control samples). HUVEC medium

was used for all co-cultures and treatments were added together with the second cell layer.

48h later, cells were harvested, labeled with anti-CD31 MicroBeads and separated using LS

columns on a MidiMACS separator according to supplier protocol (all from Miltenyi Biotec).

For JAG1- or vector-transduced U87 cells co-culture with the parental line, cells were co-

seeded in a 6 well plate and cultured for 5 weeks. Twice per week cells were split and GFP

positive populations (representing transduced cells) were quantified by FACS.

For luciferase reporter assays, clear bottom 96-well-plate (CELLSTAR) wells were coated

with recombinant Notch ligands in 0.1% BSA-PBS (see Supplementary table 2) overnight

before cell plating (4x104 cells/well; treatments were added at this point). LS174T cells

expressing the luciferase gene under the Notch transcription factor RbPJ were used (24).

Luciferase activity was quantified 24h later by a luminescence assay (Bright-Glo system,

Promega). Each condition was performed in triplicate.




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For 3D spheroids treatment, cells were harvested, plated at the density of 5x103

cells/200µl/well in low-adherence 96 well plates (Corning) and spun at 1800rpm for 10

minutes. Plating medium was normal growth medium (see Supplementary table 1)

supplemented with 2.5% Matrigel (BD Bioscience). One day after plating 100µl of medium

was replaced with fresh media containing a specific mAb at double the final concentration.

Medium was then refreshed every 2 days by replacing half the volume and growth was

monitored by taking a picture of each single spheroid. Ten spheroids per condition were

analyzed in every experiment. Spheroid volume was then calculated with ImageJ software,

assuming perfect sphericity.

All antibodies, including the mouse isotype control IgG1 (R&D Systems), were used at a

concentration of 10µg/ml if not specified, DBZ (Calbiochem) was used at a concentration of

100nM and DMSO (Sigma-Aldrich) was used 1:1000.

RNA extraction, reverse transcription and quantitative PCR

Total RNA from cell cultures was isolated using the RNeasy mini kit (Qiagen) according to

the manufacturer’s instructions. For ex vivo material, xenograft samples were powdered

before RNA extraction. Complementary DNA (cDNA) was synthesized from 0.5-1μg of total

RNA using Superscript III first-strand system (Invitrogen). Quantitative PCR (qPCR) analysis

was performed in triplicate using the SYBR GreenER qPCR SuperMix Universal (Invitrogen)

and Chromo4 fluorescence detector (MJ Research/Bio-Rad). Relative quantification was

done using the ΔΔCt method normalizing to housekeeping gene expression (β2-

Microglobulin and β-Actin for human and rat samples respectively). For primer sequences

see Supplementary table 3.

Mouse xenograft experiments

1x107 tumor cells (U87-vector, U87-JAG1, PC3, MDA-MB-231, and OVCAR3) in 100μl

Matrigel (BD Bioscience) were injected subcutaneously into the flank of BALB/c nu/nu

female mice (Crl:NU-Foxn1nu, Charles River Laboratories). JAG1 blocking mAb at the




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indicated concentration, or an equal volume of PBS were injected on same day

intraperitoneally and then twice every week. Tumor volume was calculated as L x W x H x π

/ 6 (25). For treatment of established OVCAR3 xenografts, tumors were grown to ~100mm3

and grouped into 2 arms of similar size (100mm3) and distribution before twice weekly

treatment with J1-65D (20mg/kg).

Rat subcutaneous xenografts

The subcutaneous MDA-MB-231 xenograft experiment was performed by Charles River

Discovery Services in compliance with the Guide for Care and Use of Laboratory Animals

and accredited by the Association for Assessment and Accreditation of Laboratory Animal

Care International. Briefly, 3 days before cell implantation, Taconic female rnu/rnu rats were

intraperitoneally administered with 150mg/kg cyclophosphamide to favor tumor engraftment.

On day 0, cells were subcutaneously injected (1x107/cells/rat in PBS-50% Matrigel) and J1-

65D antibody treatment (20mg/kg) was then administered intravenously twice per week

starting from day 2 (15 animals/group). Tumor volume and body weight were measured

twice per week. Tumor volume was calculated as L x W2 / 2. Blood sampling was performed

under anesthesia on day 20 and at the time of culling (when one of the groups reached an

average size ≥ 10000mm3).

Rat brain metastasis model

Female nude rats were anaesthetized with 2-3% isoflurane in N2:O2 (70:30), placed in a

stereotactic frame and focally microinjected in the left striatum (+1.2mm and 2.5mm lateral to

Bregma, at a depth of 6.5mm) with 1x104 MDA-MB-231/BR cells in 1µl of sterile PBS using a

75µm-tipped glass microcapillary (Clark Electromedical Instruments).

hJ1-65Dv9 antibody treatment (20mg/kg) was administered intravenously twice weekly

starting from day 18 (6 animals/group) until week 7 post cell injection. At this point animals

were sacrificed and transcardially perfusion-fixed under terminal anesthesia as previously

described (26). The brains were post-fixed, cryoprotected, embedded in tissue-tek (Sakura




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Finetek Europe) and frozen in isopentane at -40˚C. The isotype control antibody used was

human anti-fluorescein IgG1 (Absolute Antibody Ltd). For Magnetic Resonance Imaging

analysis and tumor volume reconstruction see Supplementary Material and Methods.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6 and the tests used are reported

in the figure legends. Results are presented as mean ± SD or mean ± SEM. P ≤ 0.05 was

considered to be statistically significant.

For information on expression constructs, flow cytometry and histologic analysis see

Supplementary Material and Methods (including Supplementary tables 4 and 5 regarding

antibodies used for flow cytometry and immunohistochemistry respectively).

Results

JAG1 antibody production and epitope mapping

To generate antibodies that specifically block hJAG1-induced Notch signaling, the hJAG1

DSL domain and the neighboring 3 EGF domains (amino acids 185-335, Fig.1A) (22) was

used to immunize mice. Hybridomas were generated using classical techniques and

secreted mAbs were tested for reactivity with hJAG1 (Fig.1B). Five mAbs, J1-142B, J1-65D,

J1-156A, J1-183D and J1-187B, bound cell surface hJAG1 on overexpressing cells.

Specificity testing showed no effective binding to other Notch ligands including human

JAGGED2 (hJAG2) and hDLL4 (Fig.1B). Specificity was also confirmed by

immunocytochemical labeling of hJAG1 but not hJAG2 transfected HEK293 cells

(Supplementary Fig.1A). Only J1-142B showed effective binding to the murine Jagged1

(mJag1) orthologue (Fig.1B).

To identify the epitopes recognized by the antibodies, their binding to the original

immunogen (hJAG1 DSL-EGF1-3) was compared to the hJAG1 DSL domain alone. The

murine cross-reactive antibody J1-142B was shown to require the EGF domains to bind




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since, unlike the other four mAbs, it was unable to bind to the DSL domain alone (Fig.1C).

The DSL domain and adjacent EGF1 sequence of human and cynomolgus monkey JAG1

are identical and are also highly conserved in the mouse where only three amino acids (aa)

differ between the orthologues (Fig.1D, aa 190, 228 and 231). To investigate their

contribution to human specific binding by the four JAG1 DSL domain targeting mAbs, these

three amino acids in the hJAG1 DSL-EGF1-3 recombinant protein were individually mutated

to their murine counterpart. Antibody binding to the mutants in a dot blotting assay indicated

that mAb binding was unaffected by Y190H and R231K substitutions, while E228D mutation

completely abolished the binding of J1-65D, J1-156A, J1-183D and J1-187B (Fig.1E).

Thus, in addition to forming part of the epitope for these DSL domain-targeting mAbs, E228

is also the residue that confers their human specificity, and has been shown to contribute to

the JAG1 DSL/Notch1 interface (27). While alanine substitutions of other DSL domain

residues also shown to interact with Notch1 (F199, R201, R203, F207) (Fig.1D) had no

effect on J1-142B and J1-183D mAb binding, they differentially inhibited the binding of the

other three mAbs (Supplementary Fig.1B). The DSL domain-targeting mAbs thus

recognize distinct epitopes that include residues that play a key role in forming the DSL-

Notch1 ligand/receptor binding interface (22, 27).

Surface Plasmon Resonance was performed to quantify the binding affinity of J1-156A, J1-

65D, J1-183D, and J1-187B mAbs towards the immunizing hJAG1 protein. J1-65D and J1-

183D exhibited the highest binding affinity, having dissociation constants (Kd) of 9.7nM and

4.9nM, respectively (Supplementary Fig.1C).

JAG1 antibodies specifically block JAG1-activated Notch signaling

The anti-JAG1 mAbs were assessed for their functional ability to disrupt the hJAG1-Notch1

protein-protein interaction using a FACS-based binding assay (Fig.2A). Three antibodies,

J1-65D, J1-183D, and J1-156A completely blocked the binding of hJAG1-overexpressing

cells to human Notch1 (EGF11-13) coated fluorescent beads, but did not block binding to

cells expressing mJag1.




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The same assay was performed with titrated purified mAbs, to compare the concentrations

required to neutralize the receptor-ligand interaction. With the exception of the murine cross-

reactive mAb J1-142B, the remaining mAbs fully blocked Notch1 binding to hJAG1 at ≤

5µg/ml, with J1-65D and J1-183D blocking at 1µg/ml (Supplementary Table 6). J1-142B

was not evaluated further. Sequences for the variable region of J1-65D, J1-183D, J1-156A

and J1-187B mAbs are provided in Supplementary Table 7.

To verify the mAbs were capable of Notch signaling inhibition, LS174T cells expressing

luciferase under RbPJ/Notch control were stimulated with individual Notch ligands in the

presence of our mAbs versus an IgG control. All four antibodies inhibited Notch signaling

induced by recombinant hJAG1 (Fig.2B) at levels comparable to pan Notch inhibition with

GSI (DBZ). There was no effect on human JAG2- or DLL4-induced Notch signaling, further

confirming ligand specificity of the lead mAbs with greatest functional activity, J1-65D and

J1-183D (Fig.2C and D).

We screened a panel of human tumor cell lines for surface expression of JAG ligands and

Notch receptors, and prioritized five for further mAb testing. These expressed both JAG1

and at least one Notch receptor, and represented different tumor types in which JAG1

expression has been implicated in neoplastic growth (4): MDA-MB-231, HCC1143 (both

TNBC), OVCAR3 (ovarian), PC3 (prostate) and H1993 (lung) (Fig.2E and Supplementary

Fig.2A).

In vitro treatment of these cell lines with either the J1-65D or J1-183D mAb or DBZ showed

a broad spectrum of Notch target gene regulation that did not strongly correlate with the

expression level of any of the individual Notch receptors or ligands (Fig.2F and

Supplementary Fig.2B).

Despite a role in vascular biology (28-30) and both primary endothelial and vascular smooth

muscle cells (HUVEC and HUVSMC respectively) expressing hJAG1, neither substantially

responded to anti-JAG1 mAb treatment (Supplementary Fig.3A-D). As hJAG1 might play a

more important role in heterotypic vascular cell interactions, HUVEC and HUVSMC were co-

cultured in the presence of J1-65D or isotype control antibody and then the two cell types




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were separated based on the exclusive HUVEC CD31 positivity and compared to homotypic

cultures (Supplementary Fig.4A-D). Co-culture induced Notch-target gene expression

(HEY2 and HEYL) in both cell types, while also up-regulating maturation markers in smooth

muscle cells (ACTA2 and SMMHC). Importantly, several changes were significantly inhibited

by J1-65D treatment, confirming signaling between endothelial-smooth muscle cells as a

potential target (Supplementary Fig.4E).

JAG1 antibodies inhibit MDA-MB-231 3D cell growth in vitro

Neither the JAG1 mAbs nor DBZ affected tumor cell line growth/viability in normal 2-

dimensional (2D) culture conditions (Supplementary Fig.5A and B). MDA-MB-231 TNBC

tumor cells, which exhibited high-level JAG1 expression and significant Notch target gene

modulation in 2D culture were grown as 3D spheroids in suspension culture to better mimic

tumor biology. Both JAG1 mAbs and DBZ treatment significantly inhibited MDA-MB-231 3D

spheroid growth (Fig. 3A). This was accompanied by a dramatic inhibition of expression of

the Notch-target HES1 and a reduction in other genes with an important role in breast

cancer growth such as IL6 (31) and CA9 (32) (Fig.3B and C) that was dose-dependent

(Supplementary Fig.5C and D). Interestingly, JAG1 inhibition was as effective as DBZ at

significantly reducing two independent cancer stem cell populations in MDA-MB-231,

defined by CD44+/CD24- and Aldefluor+, respectively (33) (Fig. 3D).

J1-65D inhibits in vivo tumor growth in some mouse xenograft models

Since our lead antibodies were hJAG1 specific, we initially used a cell line model (U87

glioma, which lacked endogenous JAG1) where ectopic hJAG1 expression alone caused

accelerated tumor growth (Fig.4A). This provided a model system to identify the antibody

dose needed to fully neutralize hJAG1 activity in a subcutaneous tumor, identified as the

dose that reverted growth to that of the JAG1-negative parental cell line. In an in vitro co-

culture system, JAG1 overexpressing but not vector-transduced U87 cells, were able to out-




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grow parental cells over time (Fig.4B), and also showed a significant growth advantage in

vivo (Fig.4C).

To evaluate the effect of hJAG1 blockade in this model, J1-65D (which performed best

among the mAbs across our in vitro assays) was administered i.p. twice weekly into tumor-

bearing mice from the time of cell injection. At 10mg/kg J1-65D only partially delayed JAG1-

induced U87 growth (Fig.4D), while 20mg/kg completely abolished the growth promoting

activity of hJAG1 overexpression (Fig.4E).

Having demonstrated effective in vivo hJAG1 blockade, three cell lines with similar hJAG1

levels (Fig.2E) where in vitro experiments showed mAb-mediated modulation of Notch

signaling were tested for antibody activity in vivo. In a preventative setting, where twice

weekly mAb treatment was initiated on the day of tumor inoculation, J1-65D treatment

showed no effect on PC3 xenograft growth, a modest reduction for MDA-MB-231, and

significant inhibition of OVCAR3 growth (Fig.5). J1-65D did not inhibit the growth of

established OVCAR3 tumors (Supplementary Fig.6A and B).

J1-65D reduces MDA-MB-231 tumor growth in rat xenografts

To evaluate both the potential safety and additional benefits of stromal targeting to

therapeutic efficacy we repeated the MDA-MB-231 xenograft experiment in nude rats. Rat

Jag1 (rJag1) lacks the E228D alteration that prevents effective binding of our mAbs to

mJag1 (Supplementary Fig.7A), and these effectively inhibit rJag1-induced Notch signaling

(Supplementary Fig.7B and C), offering the opportunity to test both tumor and host stromal

JAG1 inhibition.

J1-65D treatment (20 mg/kg twice weekly i.v. from day 2 post cell injection) significantly

delayed the growth of MDA-MB-231 subcutaneous xenografts in immunocompromised rats

showing superior efficacy to that observed with only tumor hJAG1 targeting in mice (Fig.6A,

B and Fig.5B). We observed significant reductions in the expression of Notch-target gene

HES1 and the cancer stem cell marker ALH1A1 (34) by tumor cells (Fig.6C). Histological




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analysis showed that J1-65D-treated tumors were less necrotic (Fig.6D), perhaps due to

their reduced growth.

Importantly, aware of the toxicity reported for several anti-Notch therapeutics (12), we

evaluated animal health by monitoring body weight, histology of the intestine and performing

extensive blood testing during and at the end of the experiment. Treated animals showed no

visible signs of toxicity, no altered body weight (Fig.6E) and no effect on intestinal goblet cell

numbers or proliferation (Fig.6F and G). Blood analysis did show some statistically

significant differences between the 2 groups (Supplementary Table 8). These were

generally small and showed opposite trends at the two time points analyzed, probably

primarily reflecting differences in tumor size and disease progression.

J1-65D treatment strongly inhibits breast cancer brain metastasis growth

Metastatic TNBC is generally considered to be an incurable disease, and shows a

predilection for the brain and lung as metastasis sites compared to other breast cancer sub-

types (35). To address this unmet clinical need we moved away from the earlier preventative

s.c. experimental settings and focused on a therapeutic brain metastasis model, with

characterized JAG1 expression (Supplementary Fig.8A) where involvement of the

Notch/JAG1 pathway had been reported (36, 37). MDA-MB-231/BR cells (variant having a

brain tropism) were injected into the striatum of immunocompromised rats. Established

tumors were then treated twice weekly i.v. from day 17/18 with control antibody or a

humanized IgG1 variant of J1-65D (hJ1-65Dv9) having enhanced affinity (13.9pM) for JAG1

(sequence for both heavy and light chains in Supplementary Table 7). At this time point the

tumor is detectable by T2-weighted MRI, and blood-brain-barrier (BBB) breakdown is

imminent (already detectable at day 21, Fig.7D). Tumor growth and vascular function were

assessed weekly by MRI and histologically at the end of the experiment (Fig.7A and B).

Assessment of hyperintense regions evident on T2-weighted MR images suggested a

reduced neoplastic growth (Fig.7B) and histological 3D reconstructions subsequently

confirmed a significant reduction in tumor volume in animals treated with hJ1-65Dv9




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compared to control group (Fig.7C). Gadolinium-enhanced MRI, to assess BBB integrity,

showed that the volume of compromised barrier in control animals progressively increased

over time, as expected. Interestingly, JAG1 inhibition drastically reduced BBB breakdown,

with animals showing no sign of increased signal despite similar volumes of T2

hyperintensity to controls at week 6 by MRI (Fig.7D). Cerebral blood flow (CBF) was also

assessed and showed a progressive reduction in control group tumors over time, whilst

stable flow was observed in hJ1-65Dv9-treated animals and in contralateral normal brain for

both groups (Fig.7E and Supplementary Fig. 8B and C). Unsurprisingly, we found

significant correlations between tumor size and both BBB breakdown (positive correlation)

and tumor perfusion (negative correlation) in control animals. Interestingly, no significant

correlations were observed for tumors treated with hJ1-65Dv9 (Supplementary Fig. 8D),

indicating that the differences observed for BBB breakdown and blood flow cannot be

explained by the reduced tumor growth caused by Jagged1 inhibition.

Discussion

Notch signaling has a well-established role in tumor biology (1), and JAG1 is the ligand with

the broadest demonstrated involvement, with reported roles in several aspects of cancer

growth across a variety of different tumor types (4). Our aim was to generate neutralizing

mAbs to target the receptor-binding region of hJAG1 (22), in order to inhibit this specific

Notch signaling axis, while sparing the others and therefore avoid toxicities often associated

with broader pathway inhibition (12).

Here we report the successful generation and characterization of specific anti-human/rat

JAG1 mAbs, which target the receptor binding site within the DSL domain of the ligand (22).

Selected antibodies were able to inhibit Notch signaling in vitro in a variety of tumor cell lines

proving that JAG1 activates homotypic cell interactions between cancer cells, contrary to

what has been suggested by others (8, 38). Interestingly, the level of inhibition varied, in

terms of the magnitude, and the identity of the genes involved, depending on the cell line.

Generally, pan Notch inhibition with DBZ showed a greater magnitude of gene expression




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changes and the number of affected genes per cell line. JAG1-specific blockade also

significantly inhibited Notch target gene expression in four out of five cell lines proving that

both J1-65D and J1-183D mAbs inhibit endogenous JAG1-mediated Notch signaling.

Interestingly, no individual gene was consistently affected in all lines indicating that JAG1

triggers cell-type specific programs. Intriguingly, JAG1 blockade also up-regulated at least

one Notch target gene in four out of five cell lines, indicating that this ligand may work

contemporarily as both an agonist and antagonist in the same cell type, as previously

reported for endothelial cells (30). This was not predictable based on the cell surface

expression of Notch pathway components, including hJAG1 itself.

Vascular cells proved resistant to mAb treatment in homotypic cell cultures but showed

significant response when treated in co-culture, confirming that hJAG1 is an active ligand

that mediates endothelial-vascular smooth muscle cell interactions (29). As previously

reported by others (14), cell line treatment in 2D did not affect cell viability (including pan

Notch inhibition by GSI treatment) despite the significant effect on gene expression. Growth

was however reduced when cells were grown in 3D, indicating that Notch signaling plays a

more important role in this condition that more resembles in vivo tumor growth. Notch

signaling, including aspects mediated by JAG1, has been reported to regulate the stem cell-

like phenotype in breast cancer (33, 39, 40). Treatment with our mAbs confirmed this by

reducing CSC numbers in MDA-MB-231 cells.

In vivo testing showed that in a U87 model with ectopic hJAG1 expression, our lead

antibodies were extremely effective, being able to completely inhibit hJAG1-induced

enhancement of tumor growth. However, inhibition of endogenous tumor-expressed JAG1

alone (mouse xenograft models in which our mAb cannot inhibit host Jagged1) exhibited

highly variable efficacy, reminiscent of the variation within in vitro gene expression changes

caused by anti-JAG1 antibody treatment. This and the ability to prevent the engraftment of

the ovarian OVCAR3 cell line, but not impair the growth of established OVCAR3 tumors,

suggested that the antibodies should be evaluated in models where the host stroma could

also be targeted, in order to fully assess their therapeutic potential.




16

JAG1 antibody treatment of MDA-MB-231 xenografts (partially responsive in mice)

implanted into nude rats subsequently demonstrated a strong growth inhibitory effect.

Confirming the important roles played by Jag1 in stromal cells (5-8) and indicating that

inhibition of stromal Jag1 is necessary to achieve significant therapeutic benefits in this

model. Importantly, targeting rJag1 in normal tissues did not show any evidence of toxicity,

both at the level of general health and in subsequent histologic/cytological studies. The lack

of gastrointestinal toxicity is consistent with inducible genetic deletion of mJag1 being

dispensable for intestinal stem cells homeostasis (41).

The role of JAG1 in breast cancer has been broadly established (4, 42, 43), and despite

important progress in therapeutic management of the disease, subtypes still show poor

patient outcome due to treatment resistance and metastatic spread (44). TNBC in particular,

shows higher metastatic affinity for the brain, making treatment harder and reducing patient

life expectancy (35). The clinical potential of targeting JAG1 in TNBC bone metastasis has

been demonstrated (8), here we evaluated whether this might also be true for brain

metastasis, a process in which Jagged1-Notch signaling has already been implicated (36,

37, 44). In a model of established brain metastasis, treatment with the hJ1-65Dv9 mAb

significantly reduced neoplastic growth, preserved blood-brain-barrier function and tumor

perfusion. These findings indicate that Jag1 neutralization has a protective effect on the

tumor-associated brain vasculature. Interestingly, this does not seem to be solely a

consequence of reduced tumor burden, since hJ1-65Dv9-treated tumors maintained better

vascular function than size-matched tumors from the control IgG group at earlier time points.

Inhibition of Notch signaling would be expected to negatively affect barrier establishment

and function (45), but the latter could be preserved indirectly by inhibiting Jag1-induced

aberrant angiogenesis (46, 47). Overall, this improvement in tumor vascular function might

indicate a form of vascular normalization, and as such could be exploited to improve the

efficacy of other therapeutic approaches that are normally impaired by poor tumor perfusion,

such as radiotherapy, chemotherapy and immunotherapy (48).

The different therapeutic outcomes in experiments performed in mice and rats are most




17

likely to derive from the targeting of both tumoral and stromal Jagged1 in nude rats.

However, a number of experimental variables, including the route of antibody administration,

cyclophosphamide pre-conditioning and use of the humanized Jagged1 antibody could

contribute to maximizing the therapeutic efficacy of JAG1 neutralizing antibodies. The

ongoing development of a mouse in which the DSL domain has been humanized will enable

this to be tested during future preclinical development.

Further studies are warranted to evaluate the potential for combination therapies involving

our JAG1 antibodies. Tumor vasculature that is immature and poorly covered by pericytes is

sensitive to VEGF-targeting antiangiogenic therapy (49). The discoveries that endothelial

specific genetic Jag1 depletion was associated with poor vessel coverage by VSMC (29)

and that our JAG1 antibodies target heterotypic Notch signaling between endothelial and

vascular smooth muscle cells suggests that anti-JAG1 mAb therapy may benefit patients

who are refractory/relapsed to Bevacizumab. It will be important to identify the most relevant

patient groups and to identify biomarkers of response that encompass the heterogeneity we

have observed in Notch target gene regulation. The recent definition of at least four

molecular subtypes within TNBC (BL1, BL2, M and LAR) has demonstrated that the BL1

subtype responds most effectively to chemotherapy, while BL2 and LAR are thought to be

more likely to present with residual chemo-resistant disease (50). Thus, identifying patients

with particular TNBC subtypes may further stratify those that would benefit most from an

additional therapeutic approach, such as inhibition of Jagged1 signaling. Importantly the

data shown here, in association with supportive findings from other groups (8), clearly

demonstrate the clinical potential of JAG1 neutralizing antibodies for cancer therapy,

including the treatment of metastatic breast cancer.

Acknowledgments and financial support: This work was supported by Cancer Research

UK (CRUK) programme grant A10702 to A.H. Banham, A.L. Harris and P.A. Handford, and

Cancer Research UK grant C5255/A15935 to N.R.Sibson.




18

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Figure legends

Figure 1. Anti-JAG1 monoclonal antibody generation, binding specificity and epitope

mapping.

(A) Domain organization and structure of the N-terminal region of JAG1. The region

comprising the DSL domain and the three neighboring EGF repeats was used as the

immunogen for neutralizing antibody production. (B) Binding specificity of human JAG1

mAbs. The mAbs were used to stain HEK293 cells transfected with hJAG1 or hJAG2 and

B16F10 cells transfected with hDLL4 or mJag1. The expression of hJAG2 was verified using

an anti-JAG2 mAb, and that of hDLL4 and mJag1 by GFP expression from the expression

constructs. (C) The hJAG1 DSL domain alone or DSL-EGF1-3 protein was used to coat

ELISA plates and binding of the anti-JAG1 mAbs was tested by ELISA. (D) Residues in the

hJAG1 DSL domain that are substituted in mJag1 are highlighted in bold. Notably the hJAG1

sequence is also conserved in the cynomolgus monkey (cJAG1), which is widely used in

toxicology studies. Note the proximity of these residues (purple; Y190, E228, R231) to the

residues shown to be important for binding to Notch (blue; F199, R201, R203, R207). (E)

The amino acids at positions 190, 228 and 231 in hJAG1 were each mutated to the mJag1

sequence. These soluble DSL-EGF1-3 recombinant proteins were used in dot blots to

identify the amino acids responsible for preferential mAb binding to the hJAG1 protein.

Figure 2. Anti-JAG1 antibodies inhibit Notch receptor binding and signaling.

(A) Anti-JAG1 mAbs block hJAG1 binding to hNotch1. Soluble Notch1 (EGF11-13) coated

purple fluorescent avidin beads were used to stain hJAG1 or mJag1 overexpressing cells

(HEK293 and B16-F10 respectively) in the presence of JAG1 mAbs. Blocking shifts the bold

line towards the shaded grey control peak on the left. (B-D) Human colon cancer cells

expressing a luciferase reporter gene under Notch/RBPJ control were stimulated with coated

recombinant human JAG1 (rhJAG1), JAG2 (rhJAG2), DLL4 (rhDLL4) or control protein

(IgG2b). Cells were contemporarily treated with different J1-mAbs or controls (mIgG1/DMSO




22

are negative controls while DBZ is a positive control) and luciferase activity was then

analyzed 24h after plating. Bars show the average of 6 technical replicates of representative

experiments. (E) FACS analysis showing surface expressed hJAG1 (J1-65D mAb binding)

on a panel of five human tumor cell lines (J1-183D mAb in Suppl. Fig. 2). (F) qPCR analysis

showing the in vitro effect of J1-mAbs on Notch-target gene expression in the same panel of

cell lines described in (E). Bar graphs show the average ± SD of n = 5 (paired Student’s T-

test; * P < 0.05, ** P < 0.01 and *** P < 0.001). mIgG1 is the isotype-matched negative

control for all mAbs, DBZ is a pan-Notch inhibitor and DMSO is its corresponding vehicle

control.

Figure 3. In vitro effects of JAG1 inhibition on MDA-MB-231 3D growth, gene

expression and cancer stem cells.

(A) Effect of J1-65D and J1-183D on MDA-MB-231 spheroid growth (average ± SD of 10

spheroids/group; nonlinear fit test; one representative experiment shown). (B) qPCR

analysis on the RNA extracted from treated spheroids (average ± SD of n ≥ 5; paired

Student’s T-test). (C) Representative immunohistochemistry images showing reduction in

HES1 protein expression in J1-65D-treated versus isotype control-treated spheroids. (D)

FACS analysis showing reduction of two distinct cancer stem cell subpopulations

(CD44high/CD24low and Aldefluor+) in treated spheroids (average of n ≥ 3; paired Student’s

T-test). mIgG1 is the isotype-matched negative control for both mAbs, DBZ is a pan-Notch

inhibitor and DMSO is the corresponding negative control (vehicle). * P < 0.05, ** P < 0.01,

and *** P < 0.001.

Figure 4. J1-65D mAb prevents hJAG1-induced growth in mouse U87 xenograft

tumors.

(A) FACS analysis showing JAG1 surface expression in U87-vector and U87-JAG1 cells

(control and hJAG1-overexpressing cells respectively). Staining was performed using the J1-

65D mAb. (B) U87 cells overexpressing hJAG1, but not control U87-vector cells, show a




23

growth advantage over parental cells in an in vitro co-culture model. FACS analysis was

used to quantify U87-vector and U87-JAG1 cell number (both GFP+) over parental cells

(GFP-). Dotted lines show the ratio of 50%. (C) U87 cells overexpressing hJAG1 display

accelerated tumor growth in vivo (top) and reduced animal survival (bottom) compared to

control cells (n = 6/group). (D) JAG1-neutralizing mAb J1-65D partially prevents hJAG1-

induced growth acceleration (top) and animal reduced survival (bottom) in U87-JAG1

xenografts at 10 mg/kg (U87-Vector: n = 7, other groups: n = 6). (E) JAG1-neutralizing mAb

J1-65D completely prevents hJAG1-induced growth acceleration (top) and normalizes

animal survival (bottom) in U87-JAG1 xenografts at 20 mg/kg (U87-Vector: n = 7, other

groups: n = 10). All tumor growth graphs show average ± SEM (Student’s T-test). * P < 0.05,

** P < 0.01. NS, no significance.

Figure 5. Differential effects of JAG1 mAb J1-65D treatment on the growth of tumor

xenografts in mice.

J1-65D treatment effect on s.c. tumor growth for: (A) prostate cancer cell line PC3 (n =

5/group), (B) breast cancer cell line MDA-MB-231 (n = 10/group), and (C) ovarian cancer

cell line OVCAR3 (n = 5/group). J1-65D mAb (20 mg/kg) or PBS (control) were administered

twice a week starting from the day of tumor inoculation. For each cell line, average tumor

volume ± SEM. is shown in the top panel, and individual tumor growth curves are shown in

the bottom 2 panels. Student’s T-test. * P < 0.05.

Figure 6. J1-65D treatment reduces MDA-MB-231 subcutaneous tumor growth in rats

without any discernable toxicity.

(A) MDA-MB-231 tumor growth is reduced by J1-65D treatment in a subcutaneous rat

xenograft model (average ± SEM of n = 15/group; nonlinear fit test). Individual tumor growth

is shown in (B) for both groups. (C) qPCR analysis of tumor RNA showed strong

downregulation of the human Notch-target gene HES1 and the stem cell marker ALDH1A1

(n = 4 and 5 for PBS and J1-65D groups respectively; unpaired Student’s T-test). (D)




24

Hematoxylin and eosin staining of the tumors showed important necrosis reduction in J1-

65D-treated animals (n = 5/group). (E) No effect on animal weight was observed during the

course of the treatment (n = 15/group). (F) Histologic analysis of rat intestines showed no

effects of J1-65D on goblet and proliferative cells number (Alcian blue and Ki67 staining

respectively; n = 5/group). (G) All bar graphs represent averages ± SD (unpaired Student’s

T-test). * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 7. Anti-Jagged1 treatment reduces MDA-MB-231-BR tumor growth in a rat brain

metastasis xenograft model.

(A) Scheme of an experiment mimicking the therapeutic treatment of an established breast

cancer brain metastasis with the hJ1-65Dv9 humanized/de-immunized antibody. (B)

Representative T2-weighted MR images of hJ1-65Dv9 and control-treated MDA-MB-231-BR

xenograft tumors (red arrows) at 3- and 7-weeks post cell implantation. (C) Analysis of

hyperintense regions on T2-weighted MR images suggests reduced growth of hJ1-65Dv9-

treated tumors (top panel; average size ± SEM; n = 6 and 5 for hJ1-65Dv9 and control

respectively; nonlinear fit test). Metastasis volume at endpoint (week 7) was assessed

histologically and confirmed reduced growth upon Jagged1 inhibition (bottom panel; average

size ± SD; n = 6/group; unpaired Student’s T-test). (D) BBB leakage/permeability assessed

by gadolinium-based T1-weighted MRI shows reduced BBB breakdown in hJ1-65Dv9-

treated animals (average ± SEM; n = 6 and 5 for hJ1-65Dv9 and control respectively;

nonlinear fit test). (E) MRI-based perfusion analysis shows that Jagged1 inhibition stabilized

tumor perfusion whilst control group tumors showed a progressive reduction in perfusion

over time (average ± SD of tumor values normalized to normal contralateral brain; n =

variable between 2 and 6 per data point; 2way ANOVA). * = P < 0.05; ** = P < 0.01; *** = P

< 0.001.




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hJAG1 TCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNR

mJag1 TCDDHYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPDCNK

cJAG1 TCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNR




Figure. 2

A

m Ig G 1 J 1 -6 5 D J 1 -1 5 6 A J 1 -1 8 3 D J 1 -1 8 7 B D M S O D B Z

0

2

4

6

8

1 0Ig G 2 b rh J A G 1C o a tin g :

Lu

cif

era

se

ac

tiv

ity

(A

.U.)

m Ig G 1 J 1 -6 5 D J 1 -1 8 3 D D M S O D B Z

0

1

2

3

4Ig G 2 b rh J A G 2C o a tin g :

Lu

cif

era

se

ac

tiv

ity

(A

.U.)

B

m Ig G 1 J 1 -6 5 D J 1 -1 8 3 D D M S O D B Z

0

3

6

9

1 2Ig G 2 b rh D L L 4C o a tin g :

Lu

cif

era

se

ac

tiv

ity

(A

.U.)

C

D

E

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

* * *

* *

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

* * * *

* * ** *

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0

* *

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

* *

* *

*

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0

*

HE

S1

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0 * * *

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

*

* *

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

* *

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0

*

HE

S4

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

* * *

* ** *

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

HE

S5

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

**

**

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

***

Re

lati

ve

ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0 *

* *

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

HE

Y1

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

2.5 *

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

**

***

Re

lati

ve

ex

pre

ssio

n

0

1

2

3

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

2.0

2.5

HE

Y2

mIg

G1

J1-6

5D

J1-1

83D

DM

SO

DB

Z

mIg

G1

J1-6

5D

J1-1

83D

DM

SO

DB

Z

mIg

G1

J1-6

5D

J1-1

83D

DM

SO

DB

Z

mIg

G1

J1-6

5D

J1-1

83D

DM

SO

DB

Z

mIg

G1

J1-6

5D

J1-1

83D

DM

SO

DB

Z

F MDA-MB-231 HCC1143 OVCAR3 PC3 H1993

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

100

101

102

103

104

FL3-H

0

20

40

60

80

100

% o

f M

ax

hJA

G1

Control staining No blocking mAb blocking

mJa

g1

J1-142B J1-65D J1-156A J1-183D J1-187B

MDA-MB-231 HCC1143 OVCAR3 PC3 H1993

Control staining JAG1 (J1-65D)




Figure. 3

Time (days)

Vo

lum

e (

mm

3)

0 2 4 6 8 100.0

0.2

0.4

0.6

0.8mIgG1

J1-65D

J1-183D

DMSO

DBZ

mIgG1

DMSO

DBZ

***

J1-65D

J1-183D

mIgG1 J1-65D J1-183D

DMSO DBZ

0

25

50

75**

**

CD

44

+/C

D2

4-cells (

%)

*

A

B

C D

HES1

Re

lati

ve

ex

pre

ssio

n

0.0

0.6

1.2

1.8 ******

***

mIg

G1

J1

-65

D

J1

-18

3D

DM

SO

DB

Z

IL6

Re

lati

ve

ex

pre

ssio

n

0.0

0.6

1.2

1.8 ***

**

mIg

G1

J1

-65

D

J1

-18

3D

DM

SO

DB

Z

CA9

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5*

***

mIg

G1

J1

-65

D

J1

-18

3D

DM

SO

DB

Z

Ald

efl

uo

r+ c

ells (

%)

0

1

2

3

4

5 ****

*

mIg

G1

J1

-65

D

J1

-18

3D

DM

SO

DB

Z

mIgG1

J1-65D

HES1 IHC

spheroid

core

spheroid

core 50 µm




Figure. 4

0 20 40 600

50

100S

urv

ival(%

)

Time (days)

0 20 40 600

50

100

Surv

ival(%

)

Time (days)

0 20 40 600

50

100

Time (days)

Surv

ival(%

)

A

GFP

JA

G1

(J1-6

5D

)

U87-Vector U87-JAG1

100 101 102 103 10410

0

101

102

103

104

100 101 102 103 10410

0

101

102

103

104

C E

0 10 20 30 40 500

200

400

600

800

Time (days)

Tum

our

volu

me (

mm

3)

U87-Vector

U87-JAG1

*

0 10 20 30 400

20

40

60

80

100

0 10 20 30 400

20

40

60

80

100B

Time (day)

U87 + U87-JAG1 U87 + U87-Vector

GF

P+

(%

)

0 10 20 30 40 500

500

1000

1500

2000

D

Time (days)

NS

Tum

our

volu

me (

mm

3)

U87-Vector + PBS

U87-JAG1 + PBS

U87-JAG1 + J1-65D

0 10 20 30 400

500

1000

1500

Tum

our

volu

me (

mm

3)

Time (days)

**

U87-Vector + PBS

U87-JAG1 + PBS

U87-JAG1 + J1-65D




Figure. 5

A

0 10 20 30 400

500

1000

1500

0 10 20 30 400

500

1000

1500

2000

Tu

mou

r vo

lum

e (

mm

3)

PC3

Time (days)

Tum

ou

r vo

lum

e (

mm

3)

Tum

our

volu

me (

mm

3)

0 10 20 30 400

500

1000

1500

2000

Time (days)

0 20 40 600

500

1000

1500MDA-MB-231

B

0 20 40 600

500

1000

1500

2000

0 20 40 600

500

1000

1500

2000

Time (days)

0 20 40 60 800

100

200

300

400

500OVCAR3

C

0 20 40 60 800

200

400

600

800

0 20 40 60 800

200

400

600

800

PBS

J1-65D

*

PBS

J1-65D




Figure. 6

T im e (d a y s )

Tu

mo

ur v

olu

me

(m

m3)

0 7 1 4 2 1 2 8 3 5 4 2 4 9

0

4 0 0 0

8 0 0 0

1 2 0 0 0

1 6 0 0 0

P B S

J 1 -6 5 D

***

Time (days)

0 7 14 21 28 35 42 490

5000

10000

15000

20000

25000

PBS

Tu

mo

ur

vo

lum

e (

mm

3)

T im e (d a y s )

0 7 1 4 2 1 2 8 3 5 4 2 4 9

0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

2 5 0 0 0

J 1 -6 5 D

A

C

B

Re

lati

ve

Ex

pre

ss

ion

0 .0

0 .5

1 .0

1 .5

2 .0 *

H u m a n H E S 1

PBS J1-65D

Re

lati

ve

ex

pre

ssio

n

0.0

0.5

1.0

1.5

*

Human ALDH1A1

PBS J1-65D

% o

f n

ec

ro

sis

0

2 0

4 0

6 0

8 0

1 0 0* * *

N e c ro s is

PBS J1-65D

D

T im e (d a y s )

An

ima

l w

eig

ht

(gr)

0 7 1 4 2 1 2 8 3 5 4 2 4 9

0

5 0

1 0 0

1 5 0

2 0 0

P B S

J 1 -6 5 D

PBS J1-65D

200 µm

Alc

ian

blu

e

Ki6

7

F

E

N.

ce

lls

/ c

ryp

t

0

20

40

60Goblet cells

PBS J1-65D 0

20

40

60

80

100

Ki6

7+ c

ryp

t /

cry

pt

len

gth

(%

) Proliferation

PBS J1-65D

G




Figure. 7

3 4 5 6 70

20

40

60

80

100

Time after tumour injection (weeks)

Vo

lum

e o

f h

yp

eri

nte

nsit

y,

su

rro

gate

fo

r tu

mo

ur

vo

lum

e (

mm

3)

hJ1-65Dv9

Isotype control

***

Time after tumour injection (weeks)

Rela

tive b

loo

d f

low

(tu

mo

ur/

no

rmal c

on

tro

late

ral)

3 4 5 6 70.0

0.5

1.0

1.5

hJ1-65Dv9

Isotype control

**

3 4 5 6 7

0

2 0

4 0

6 0

8 0

1 0 0

T im e a fte r tu m o u r in je c t io n (w e e k s )

Vo

lum

e o

f c

om

pro

mis

ed

blo

od

-bra

in-b

arrie

r (

mm

3)

h J 1 -6 5 D v 9

Is o ty p e c o n tro l

***

Isoty

pe c

trl.

hJ1

-65D

v9

Week 3 Week 7 T

um

ou

r v

olu

me

(m

m3)

0

2

4

6

8 *

Isotype

ctrl.

hJ1-65Dv9

A

C B

D E




Published OnlineFirst August 8, 2019.Mol Cancer Ther Massimo Masiero, Demin Li, Pat Whiteman, et al. cancer therapyDevelopment of therapeutic anti-JAGGED1 antibodies for

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development of therapeutic anti-jagged1 antibodies for ......aug 08, 2019 · jagged subfamilies...

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