Molecular
Cancer
Therapeutics
Large Molecule Therapeutics
Development of Therapeutic Anti-JAGGED1
Antibodies for Cancer Therapy
Massimo Masiero1, Demin Li1, Pat Whiteman2, Carol Bentley1, Jenny Greig1,
Tasneem Hassanali1, Sarah Watts1, Stephen Stribbling1, Jenna Yates1, Ellen Bealing1,
bastien Serres5,
Ji-Liang Li3, Chandramouli Chillakuri2, Devon Sheppard4, Se
5
5
5
Manuel Sarmiento-Soto , James Larkin , Nicola R. Sibson , Penny A. Handford2,
Adrian L. Harris3, and Alison H. Banham1
Abstract
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
1
NDCLS, Radcliffe Department of Medicine, University of Oxford, Oxford, United
Kingdom. 2Department of Biochemistry, University of Oxford, Oxford, United
Kingdom. 3CRUK Department of Oncology, Weatherall Institute of Molecular
Medicine, University of Oxford, Oxford, United Kingdom. 4Sir William Dunn
School of Pathology, University of Oxford, Oxford, United Kingdom. 5Cancer
Research UK and Medical Research Council Oxford Institute for Radiation
Oncology, Department of Oncology, University of Oxford, Oxford, United
Kingdom.
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Current address for J.-L. Li: Institute of Translational and Stratified Medicine,
Faculty of Medicine and Dentistry, Plymouth University, Plymouth PL6 8BU,
United Kingdom; and current address for S. Serres: School of Life Sciences,
University of Nottingham, Nottingham NG7 2UH, United Kingdom.
M. Masiero and D. Li contributed equally to this article.
P.A. Handford, A.L. Harris, and A.H. Banham contributed equally to this article.
Corresponding Author: Alison H. Banham, University of Oxford, Level 4,
Academic Block, John Radcliffe Hospital, Headington, Oxford OX3 9DU, United
Kingdom. Phone: 44-1865-220246; Fax: 44-1865-228980; E-mail:
alison.banham@ndcls.ox.ac.uk
Mol Cancer Ther 2019;18:2030–42
doi: 10.1158/1535-7163.MCT-18-1176
2019 American Association for Cancer Research.
2030 Mol Cancer Ther; 18(11) November 2019
dimensional 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.
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, and 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, 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 multifunctional 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 g-secretase inhibitors (GSI),
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
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The role of Notch signaling and its ligand JAGGED1 (JAG1)
in tumor biology has been firmly established, making them
appealing therapeutic targets for cancer treatment. Here, we
report the development and characterization of human/
rat-specific JAG1-neutralizing mAbs. 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
ortholog. These antibodies were able to specifically inhibit
JAG1-Notch binding in vitro, downregulate Notch signaling in
cancer cells, and block the heterotypic JAG1-mediated Notch
signaling between endothelial and vascular smooth muscle
cells. Functionally, in vitro treatment impaired three-
Anti-JAGGED1 Antibodies for Cancer Therapy
Materials and Methods
Generation of anti-JAG1 mAbs
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 production, ELISA screening, dot blot and Surface
Plasmon Resonance analysis, and antibody humanization, see
Supplementary Materials and Methods.
Cell lines, culture conditions, and treatment of twodimensional and three-dimensional in vitro models
All cell lines and their growth conditions can be found in
Supplementary Table S1. 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 short tandem repeat profiling by LGC Standards. For
two-dimensional (2D) cell treatment, cells were plated in 6-well
plates, and 24 hours later, when cell density was approximately
70% to 80%, growth medium was replaced with fresh media
containing the treatment. Forty-eight hours later, cells were harvested for further analysis.
For the HUVEC-HUVSMC coculture experiments, a first layer of
cells was plated on day 0 followed by a second one when the first
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reached approximately 75% confluency (generally on day 3).
Three coculture combinations were prepared as follow:
HUVEC þ HUVSMC (test sample), HUVEC þ HUVEC, and
HUVSMC þ HUVSMC (control samples). HUVEC medium was
used for all cocultures, and treatments were added together with
the second cell layer. Forty-eight hours 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 cocultured with the
parental line, cells were coseeded 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 S2) overnight before
cell plating (4 104 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 24 hours later by a luminescence assay (Bright-Glo
system, Promega). Each condition was performed in triplicate.
For three-dimensional (3D) spheroids treatment, cells were
harvested, plated at the density of 5 103 cells/200 mL/well in
low-adherence 96-well plates (Corning), and spun at 1,800 rpm
for 10 minutes. Plating medium was normal growth medium (see
Supplementary Table S1) supplemented with 2.5% Matrigel (BD
Bioscience). One day after plating, 100 mL 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 10 mg/mL if not
specified, DBZ (Calbiochem) was used at a concentration of
100 nmol/L, and DMSO (Sigma-Aldrich) was used at 1:1,000.
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 was synthesized from 0.5 to
1 mg of total RNA using Superscript III first-strand system (Invitrogen). 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 DDCt method normalizing to housekeeping gene expression (b2-Microglobulin and b-Actin for human
and rat samples, respectively). For primer sequences, see Supplementary Table S3.
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testing for a variety of neoplastic conditions. GSIs are characterized not only 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 involves the use of monoclonal antibodies (mAbs), and
this has been employed to neutralize either individual receptors
or ligands. Preclinical studies have shown therapeutic potential
for mAbs targeting Notch1 (13, 14), Notch2, and Notch3 (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 preclinical 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-ofcare therapies beyond classical chemotherapy. Here, we show that
a therapeutic mAb targeting the Notch receptor–binding site on
the hJAG1 Delta/Serrate/Lag2 (DSL) domain can inhibit Notch
signaling, target TNBC CSCs, and reduce tumor growth in vivo.
Anti-JAG1 immunotherapy offers promise as a future treatment
strategy, both in TNBC and other cancer types.
Mouse xenograft experiments
Tumor cells, 1 107 (U87-vector, U87-JAG1, PC3, MDA-MB231, and OVCAR3) in 100 mL Matrigel (BD Bioscience), were
injected s.c. into the flank of BALB/c nu/nu female mice (Crl:NUFoxn1nu, Charles River Laboratories). JAG1-blocking mAb at the
indicated concentration or an equal volume of PBS was injected
on same day i.p. and then twice every week. Tumor volume was
calculated as L x W x H x p/6 (25). For treatment of established
Mol Cancer Ther; 18(11) November 2019
2031
Masiero et al.
OVCAR3 xenografts, tumors were grown to approximately 100
mm3 and grouped into 2 arms of similar size (100 mm3) and
distribution before twice weekly treatment with J1-65D
(20 mg/kg).
Rat brain metastasis model
Female nude rats were anaesthetized with 2% to 3% isoflurane
in N2:O2 (70:30), placed in a stereotactic frame, and focally
microinjected in the left striatum (þ1.2 mm and 2.5 mm lateral
to Bregma, at a depth of 6.5 mm) with 1 104 MDA-MB-231/BR
cells in 1 mL of sterile PBS using a 75 mm-tipped glass microcapillary (Clark Electromedical Instruments).
hJ1-65Dv9 antibody treatment (20 mg/kg) was administered
intravenously twice weekly starting from day 18 (6 animals/
group) until week 7 after cell injection. At this point, animals
were sacrificed and transcardially perfusion-fixed under terminal
anesthesia as previously described (26). The brains were postfixed, cryoprotected, embedded in tissue-tek (Sakura Finetek
Europe), and frozen in isopentane at -40 C. The isotype control
antibody used was human anti-fluorescein IgG1 (Absolute Antibody Ltd). For MRI analysis and tumor volume reconstruction,
see Supplementary Materials 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 Materials and Methods
(including Supplementary Tables S4 and S5 regarding antibodies
used for flow cytometry and IHC, 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; ref. 22) were 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
2032 Mol Cancer Ther; 18(11) November 2019
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.
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 crossreactive
mAb J1-142B, the remaining mAbs fully blocked Notch1 binding
to hJAG1 at 5mg/mL, with J1-65D and J1-183D blocking at
1 mg/mL (Supplementary Table S6). 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 S7.
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
Molecular Cancer Therapeutics
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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 150 mg/kg cyclophosphamide to favor tumor
engraftment. On day 0, cells were s.c. injected (1 107/cells/rat in
PBS-50% Matrigel), and J1-65D antibody treatment (20 mg/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 10,000 mm3).
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. S1A). Only J1-142B showed effective binding to the murine
Jagged1 (mJag1) ortholog (Fig. 1B).
To identify the epitopes recognized by the antibodies, their
binding to the original immunogen (hJAG1 DSL-EGF1-3) was
compared with the hJAG1 DSL domain alone. The murine crossreactive antibody J1-142B was shown to require the EGF domains
to bind because, 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 orthologs (Fig. 1D, aa
190, 228, and 231). To investigate their contribution to humanspecific 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, whereas 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). Although 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. S1B). 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
toward the immunizing hJAG1 protein. J1-65D and J1-183D
exhibited the highest binding affinity, having dissociation constants (Kd) of 9.7 and 4.9 nmol/L, respectively (Supplementary
Fig. S1C).
Anti-JAGGED1 Antibodies for Cancer Therapy
B
J1-142B
80
60
60
40
40
40
20
20
20
20
0
4
0
1010010
0
4
0
1010010
0
4
0
1010010
10
3
10
1
2
10
FL4-H
10
3
80
60
40
40
20
20
20
0
10
1
2
10
FL4-H
10
3
0
4
1010010
0
10
1
2
10
FL4-H
10
3
0
10
1
2
10
FL4-H
10
3
60
60
40
20
20
0
0
0
0
4
0
1010010
10
1
2
10
FL4-H
10
3
4
0
1010010
60
10
1
2
10
FL4-H
10
3
60
40
40
40
20
20
20
0
0
0
2
10
FL4-H
10
3
10
4
10
0
10
1
2
10
FL4-H
10
3
10
100
100
80
80
80
% of Max
100
60
60
40
10
10
1
2
10
FL4-H
10
3
10
hJAG2
4
10
1
2
10
FL4-H
10
3
10
4
10
4
10
3
4
10 10010
0
10
1
10
FL4-H
2
10
3
10
4
0
10
1
10
FL4-H
2
10
3
10
4
0
10
1
10
FL4-H
2
10
3
10
4
60
0
10
1
2
10
FL4-H
10
3
4
10 10010
60
40
20
10
0
0
10
1
2
10
FL4-H
10
3
10
4
10
Control
JAG1 mAb
20
0
0
0
40
20
0
10
60
40
20
4
3
10
0
0
10
1
2
10
FL1-H
10
3
10
hDLL4
4
10
0
10
1
2
10
FL1-H
10
3
10
4
mJag1
C-term.
C
DSL
OD450
1.5
DSL-EGF1-3
D
1.0
0.5
E
J1-187B
J1-183D
J1-156A
J1-65D
J1-142B
0.0
J1-142B
J1-65D
J1-156A
190
200
210
220
230
hJAG1TCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNR
mJag1 TCDDHYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPDCNK
cJAG1 TCDDYYYGFGCNKFCRPRDDFFGHYACDQNGNKTCMEGWMGPECNR
J1-183D
J1-187B
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% of Max
1
2
10
FL4-H
60
0
10
10
80
20
0
2
20
40
10
1
80
60
10
FL4-H
40
4
0
1010010
80
% of Max
% of Max
80
10
% of Max
3
1
0
0
60
40
10
10
80
20
2
0
60
80
40
10
FL4-H
4
10 10010
20
4
20
1
3
40
40
10
10
80
1010010
80
% of Max
60
2
10
FL4-H
0
4
1010010
80
0
1
60
40
0
20
10
80
20
% of Max
% of Max
hDLL4
3
60
80
% of Max
10
40
100100
mJag1
2
10
FL4-H
% of Max
% of Max
% of Max
60
80
Positive
control
1
80
0
CRD
10
40
% of Max
2
10
FL4-H
60
% of Max
1
% of Max
10
80
EGF3
60
40
10010
EGF2
60
% of Max
100
80
% of Max
100
80
% of Max
100
80
0
100100
hJAG2
EGF1
J1-187B
J1-183D
100
80
% of Max
DSL domain
J1-156A
J1-65D
100
% of Max
hJAG1
DSL
% of Max
C2 domain
C2
% of Max
N-term.
% of Max
A
R231K
E228D
Y190H
WT
Anti-His
Figure 1.
Anti-JAG1 mAb 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.
www.aacrjournals.org
Mol Cancer Ther; 18(11) November 2019
2033
Masiero et al.
80
80
80
80
40
20
0
1
2
10
FL3-H
10
3
4
1010010
1
2
10
FL3-H
10
3
4
1010010
60
60
20
0
0
10
1
2
10
FL3-H
10
3
4
0
1010010
80
% of Max
80
% of Max
80
10
40
20
0
0
0
10
1
2
10
FL3-H
10
3
4
1010010
80
60
60
40
40
40
40
20
20
20
20
0
0
0
0
0
1
2
10
FL3-H
10
3
10
4
10
0
10
1
2
10
FL3-H
10
3
10
4
10
0
10
10
3
10
4
10
3
10
4
10
0
10
1
2
10
FL3-H
10
3
10
4
10
0
10
1
2
10
FL3-H
10
3
10
IgG2b
1
4
0
mlgG1
12
rhJAG1
8
6
4
2
0
J1-156A
J1-183D
MDA-MB-231
3
1.0
0.5
DBZ
DMSO
J1-183D
mIgG1
DBZ
DMSO
J1-65D
J1-183D
*
2.0
1.5
1.0
0.5
0.0
0.0
mIgG1
0.0
**
1.5
Relative expression
Relative expression
0.5
1.0
0.5
0.0
0.0
2.0
2.0
1.5
1.0
0.5
1.5
1.0
0.5
0.0
2.0
*
**
1.5
1.0
0.5
1.5
1.0
0.5
0.0
0.0
3
2.5
2
1
0
2.0
1.5
1.0
0.5
0.0
DBZ
0.5
0.5
2.5
**
1.0
*
1.5
DMSO
1.0
1.0
Relative expression
1.5
**
**
0.0
*
2.0
1.5
0.0
DBZ
2.0
0.0
2.0
J1-65D
0.5
1.5
0.5
0.0
2.0
Relative expression
1.0
2.0
0.5
0.0
***
0.0
***
1.0
2.0
1.5
0.0
**
**
DMSO
0.5
0.0
1.0
J1-183D
0.5
Relative expression
Relative expression
1.0
0.5
*
1.5
mIgG1
1.0
0.0
1.5
Relative expression
1.5
2.0
1.0
1.5
2.0
2.0
1.5
0.5
Relative expression
0.5
0.0
***
**
**
1.0
Relative expression
1.0
Relative expression
Relative expression
0.5
0.0
2.0
Relative expression
1.5
2.5
1.0
DB Z
H1993
*
1.5
DBZ
2.0
0.0
1.5
0.5
2.0
*
*
Relative expression
0.5
DMSO
H1993
DMSO
0.5
***
1.0
Relative expression
1.0
2.5
1.0
J1-183D
PC3
**
J1-183D
**
*
J1-65D
PC3
Relative expression
**
1.5
0.0
0.0
1.5
mIgG1
OVCAR3
mIgG1
0.5
DBZ
1.5
Relative expression
Relative expression
1.0
Relative expression
Relative expression
Relative expression
Relative expression
rhDLL4
6
Relative expression
HCC1143
2.0
HES5
DBZ
9
OVCAR3
2.0
Relative expression
Relative expression
Relative expression
HES1
HES4
**
***
****
0.0
HEY1
IgG2b
DMSO
JAG1 (J1-65D)
MDA-MB-231
1.5
DMSO
HCC1143
J1-65D
F
J1-187B
J1-65D
J1-65D
Control staining
HEY2
Coating:
J1-183D
0
mIgG1
E
J1-65D
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 toward the shaded gray 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 are negative controls, whereas DBZ is a positive control), and luciferase activity was then analyzed
24 hours 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 Supplementary Fig. S2). 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 ¼ 5 (paired Student t test; , P < 0.05;
, P < 0.01; , P < 0.001; , P < 0.0001). mIgG1 is the isotype-matched negative control for all mAbs, DBZ is a pan-Notch inhibitor, and DMSO is its
corresponding vehicle control.
2034 Mol Cancer Ther; 18(11) November 2019
Molecular Cancer Therapeutics
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Luciferase activity (A.U.)
Coating:
rhJAG2
2
mAb blocking
D
10
Luciferase activity (A.U.)
2
10
FL3-H
2
10
FL3-H
IgG2b
J1-183D
B
1
No blocking
Control staining
1
Coating:
3
mIgG1
10
10
60
20
0
0
80
40
10
4
60
J1-65D
10
40
20
0
0
60
% of Max
40
20
% of Max
60
% of Max
40
60
% of Max
80
% of Max
100
% of Max
100
% of Max
100
60
C
J1-187B
J1-183D
100
10010
mJag1
J1-156A
J1-65D
100
Luciferase activity (A.U.)
J1-142B
% of Max
hJAG1
A
Anti-JAGGED1 Antibodies for Cancer Therapy
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 2D culture conditions (Supplementary Fig. S5A and S5B). 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 (ref. 32; Fig. 3B and
C) that was dose-dependent (Supplementary Fig. S5C and S5D).
Interestingly, JAG1 inhibition was as effective as DBZ at
significantly reducing two independent CSC populations in
MDA-MB-231, defined by CD44þ/CD24 and Aldefluorþ,
respectively (ref. 33; Fig. 3D).
J1-65D inhibits in vivo tumor growth in some mouse xenograft
models
Because 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
www.aacrjournals.org
subcutaneous tumor, identified as the dose that reverted growth
to that of the JAG1-negative parental cell line. In an in vitro
coculture system, JAG1-overexpressing but not vectortransduced U87 cells were able to out-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 10 mg/kg J1-65D only partially
delayed JAG1-induced U87 growth (Fig. 4D), whereas 20 mg/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. S6A and S6B).
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. S7A), and these effectively
inhibit rJag1-induced Notch signaling (Supplementary Fig. S7B
and S7C), 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 after
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 (Figs. 5B and 6A and B). We observed significant
reductions in the expression of Notch-target gene HES1 and the
CSC marker ALH1A1 (34) by tumor cells (Fig. 6C). Histologic
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 antiNotch therapeutics (12), we evaluated animal health by monitoring body weight and 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 two groups
(Supplementary Table S8). These were generally small and
showed opposite trends at the two time points analyzed, probably
primarily reflecting differences in tumor size and disease
progression.
Downloaded from http://aacrjournals.org/mct/article-pdf/18/11/2030/1860290/2030.pdf by guest on 10 October 2022
mAbs versus an IgG control. All four antibodies inhibited Notch
signaling induced by recombinant hJAG1 (Fig. 2B) at levels
comparable with 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. S2A).
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. S2B).
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. S3A–S3D). As hJAG1 might play a more important role in
heterotypic vascular cell interactions, HUVEC and HUVSMC were
cocultured in the presence of J1-65D or isotype control antibody
and then the two cell types were separated based on the exclusive
HUVEC CD31 positivity and compared with homotypic cultures
(Supplementary Fig. S4A–S4D). Coculture induced Notch-target
gene expression (HEY2 and HEYL) in both cell types, while also
upregulating 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. S4E).
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 with other breast cancer subtypes (35).
To address this unmet clinical need, we moved away from the
Mol Cancer Ther; 18(11) November 2019
2035
Masiero et al.
A
mIgG1
J1-65D
J1-183D
DMSO
DBZ
0.6
J1-65D
DMSO
DBZ
J1-183D
J1-65D
J1-183D
**
0.4
mIgG1
mIgG1
DMSO
*
Volume (mm3)
0.8
DBZ
0.2
0.0
0
2
4
6
8
10
Time (days)
HES1
CA9
IL6
1.2
0.6
**
1.0
0.5
m
e ri
bl
Via
Spheroid
core
75
**
DBZ
DMSO
*
25
0
**
**
*
4
3
2
1
0
mIgG1
rim
HES1 IHC
Aldefluor+ cells (%)
le
ab
Vi
Spheroid
core
**
50
5
J1-65D
J1-183D
mIgG1
DBZ
DMSO
J1-183D
D
mIgG1
CD44+/CD24- cells (%)
C
J1-65D
mIgG1
DBZ
DMSO
J1-183D
J1-65D
mIgG1
*
0.0
0.0
50 µm
*
DBZ
0.0
**
J1-65D
0.6
*
DMSO
Relative expression
1.2
**
J1-183D
***
1.5
J1-65D
***
***
1.8
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 t test). C, Representative IHC 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 Aldefluorþ) in
treated spheroids (average of n 3; paired Student 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; , P < 0.001.
2036 Mol Cancer Ther; 18(11) November 2019
Molecular Cancer Therapeutics
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Relative expression
1.8
Relative expression
B
Anti-JAGGED1 Antibodies for Cancer Therapy
4
10
U87-Vector
U87-JAG1
B
3
GFP+ (%)
JAG1
(J1-65D)
10
102
1
10
0
GFP
Tumor volume (mm3)
*
400
200
0
10
40
20
20
30
Time (days)
40
10 20 30 400 10 20 30 40
Time (day)
U87-Vector + PBS
U87-JAG1 + PBS
U87-JAG1 + J1-65D
E
NS
2,000
1,500
1,000
500
0
50
0
10
20
30
40
Time (days)
500
0
0
50
0
20
40
Time (days)
60
10
20
30
Time (days)
40
20
40
Time (days)
60
100
50
0
0
**
1,000
Survival (%)
Survival (%)
50
U87-Vector + PBS
U87-JAG1 + PBS
U87-JAG1 + J1-65D
1,500
100
100
Survival (%)
60
0
20
40
Time (days)
60
50
0
0
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 growth advantage over parental cells in an in vitro coculture model. FACS analysis was used to quantify U87-vector and
U87-JAG1 cell number (both GFPþ) 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 with control cells (n ¼ 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 ¼ 7, other groups: n ¼ 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 ¼ 7, other groups: n ¼ 10). All tumor growth graphs show
average SEM (Student t test). , P < 0.05; , P < 0.01; NS, no significance.
earlier preventative s.c. experimental settings and focused on a
therapeutic brain metastasis model, with characterized JAG1
expression (Supplementary Fig. S8A) 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 days 17 and 18 with control
antibody or a humanized IgG1 variant of J1-65D (hJ1-65Dv9)
having enhanced affinity (13.9 pmol/L) for JAG1 (sequence for
both heavy and light chains in Supplementary Table S7). 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 histologic 3D reconstructions
subsequently confirmed a significant reduction in tumor volume
in animals treated with hJ1-65Dv9 compared with control group
www.aacrjournals.org
(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 was also assessed and showed
a progressive reduction in control group tumors over time,
whereas stable flow was observed in hJ1-65Dv9–treated
animals and in contralateral normal brain for both groups
(Fig. 7E and Supplementary Fig. S8B and S8C). 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. S8D), indicating that
the differences observed for BBB breakdown and blood flow
cannot be explained by the reduced tumor growth caused by
Jagged1 inhibition.
Mol Cancer Ther; 18(11) November 2019
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Tumor volume (mm3)
U87-Vector
U87-JAG1
0
80
0
D
C
600
U87 + U87-JAG1
0
10
800
U87 + U87-Vector
100
Tumor volume (mm3)
A
2037
Masiero et al.
C
PBS
0
0
0
2,000
2,000
800
1,500
1,500
600
1,000
1,000
400
500
500
200
1,500
PC3
500
MDA-MB-231
OVCAR3
J1-65D
400
1,000
1,000
500
500
300
200
*
100
PBS
0
0
0
2,000
2,000
800
1,500
1,500
600
1,000
1,000
400
500
500
200
0
0
0
10
20
30
Time (days)
40
0
20
40
Time (days)
60
J1-65D
0
0
20
40
60
Time (days)
80
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 ¼ 5/group), (B) breast cancer cell line MDA-MB-231 (n ¼ 10/group), and (C) ovarian cancer cell line OVCAR3 (n ¼ 5/group). J1-65D mAb
(20 mg/kg) or PBS (control) was administered twice a week starting from the day of tumor inoculation. For each cell line, average tumor volume SEM is shown
in the top plot, and individual tumor growth curves are shown in the bottom two panels. Student t test. , P < 0.05.
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 receptorbinding 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 changes and the number of affected genes per cell line. JAG1specific 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
2038 Mol Cancer Ther; 18(11) November 2019
affected in all lines indicating that JAG1 triggers cell-type–
specific programs. Intriguingly, JAG1 blockade also upregulated
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
coculture, 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
Molecular Cancer Therapeutics
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Tumor volume (mm3)
Tumor volume (mm3)
B
1,500
Tumor volume (mm3)
A
Anti-JAGGED1 Antibodies for Cancer Therapy
B
16,000
25,000
25,000
PBS
J1-6D5
12,000
***
8,000
4,000
0
0
7
14
21
28
35
42
PBS
Tumor volume (mm3)
Tumor volume (mm3)
A
49
20,000
15,000
15,000
10,000
10,000
5,000
5,000
0
0
0
7
14
21
Time (days)
C
49 0
42
7
14
D
0.5
0.0
*
1.0
0.5
0.0
35
42
49
***
80
60
40
20
0
J1-65D
PBS
J1-65D
28
Necrosis
100
% of necrosis
Relative expression
1.0
21
Time (days)
1.5
1.5
E
PBS
J1-65D
F
PBS
150
J1-65D
100
50
Alcian blue
Animal weight (gr)
200
PBS
J1-65D
0
0
7
14
21
28
35
Time (days)
42
49
200 µm
G
40
20
0
PBS
J1-65D
100
Proliferation
80
Ki67
Goblet cells
Ki67+ crypt/
crypt length (%)
N. cells / crypt
35
60
40
20
0
PBS
J1-65D
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 ¼ 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 ¼ 4 and 5 for PBS and
J1-65D groups, respectively; unpaired Student t test). D, Hematoxylin and eosin staining of the tumors showed important necrosis reduction in J1-65D–treated
animals (n ¼ 5/group). E, No effect on animal weight was observed during the course of the treatment (n ¼ 15/group). F, Histologic analysis of rat intestines
showed no effects of J1-65D on goblet and proliferative cell number (Alcian blue and Ki67 staining, respectively; n ¼ 5/group). G, All bar graphs represent
averages SD (unpaired Student t test). , P < 0.05; , P < 0.01; , P < 0.001.
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
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Relative expression
Human ALDH1A1
*
PBS
60
28
Time (days)
Human HES1
2.0
J1-6D5
20,000
could also be targeted, in order to fully assess their therapeutic
potential.
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
Mol Cancer Ther; 18(11) November 2019
2039
Masiero et al.
C
B
Week 7
100
60
***
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20
0
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Isotype control
80
hJ1-65Dv9
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Time after tumor injection (weeks)
7
Relative blood flow
(tumor/normal contralateral)
Volume of compromised
blood–brain barrier (mm3)
E
100
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*
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Isotype
ctrl.
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6
Time after tumor injection (weeks)
Tumor volume (mm3)
hJ1-65Dv9
Isotype control
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Isotype control
hJ1-65Dv9
**
1.0
0.5
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Time after tumor injection (weeks)
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/deimmunized 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 after cell implantation. C, Analysis of hyperintense
regions on T2-weighted MR images suggests reduced growth of hJ1-65Dv9–treated tumors (top plot; average size SEM; n ¼ 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 plot; average size SD; n ¼ 6/group; unpaired Student 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 ¼ 6 and 5 for hJ1-65Dv9 and control, respectively; nonlinear fit test). E, MRI-based
perfusion analysis shows that Jagged1 inhibition stabilized tumor perfusion, whereas control group tumors showed a progressive reduction in perfusion over
time (average SD of tumor values normalized to normal contralateral brain; n ¼ variable between 2 and 6 per data point; 2-way ANOVA). , P < 0.05;
, P < 0.01; , P < 0.001.
2040 Mol Cancer Ther; 18(11) November 2019
Molecular Cancer Therapeutics
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Isotype ctrl.
Week 3
Volume of hyperintensity,
surrogate for tumor volume (mm3)
A
Anti-JAGGED1 Antibodies for Cancer Therapy
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, whereas BL2 and
LAR are thought to be more likely to present with residual
chemoresistant 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.
Disclosure of Potential Conflicts of Interest
M. Masiero has ownership interest in a patent contributor. D. Li has
ownership interest in patent antibodies that bind to Jagged 1 PCT/GB2014/
050104. P. Whiteman has ownership interest in patent antibodies that bind to
Jagged1. J. Larkin has ownership interest in a patent on methods for arterial spin
labeling. A.H. Banham has ownership interest in a patent for JAG1 mAbs. No
potential conflicts of interest were disclosed by the other authors.
Authors' Contributions
Conception and design: M. Masiero, S. Stribbling, N.R. Sibson, P.A. Handford,
A.L. Harris, A.H. Banham
Development of methodology: D. Li, P. Whiteman, S. Watts, E. Bealing, J.-L. Li,
C. Chillakuri, S. Serres, P.A. Handford, A.L. Harris
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): M. Masiero, D. Li, P. Whiteman, C. Bentley,
J. Greig, T. Hassanali, S. Watts, S. Stribbling, J. Yates, E. Bealing, J.-L. Li,
C. Chillakuri, D. Sheppard, S. Serres, M. Sarmiento-Soto, J. Larkin,
N.R. Sibson, P.A. Handford
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): M. Masiero, D. Li, P. Whiteman, E. Bealing, J.-L. Li,
S. Serres, M. Sarmiento-Soto, J. Larkin, N.R. Sibson, P.A. Handford, A.L. Harris,
A.H. Banham
Writing, review, and/or revision of the manuscript: M. Masiero, D. Li,
P. Whiteman, T. Hassanali, S. Stribbling, J.-L. Li, S. Serres, M. Sarmiento-Soto,
J. Larkin, N.R. Sibson, P.A. Handford, A.L. Harris, A.H. Banham
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): M. Masiero, P. Whiteman, C. Bentley,
T. Hassanali, S. Stribbling, J.-L. Li
Study supervision: N.R. Sibson, A.L. Harris, A.H. Banham
Acknowledgments
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.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Downloaded from http://aacrjournals.org/mct/article-pdf/18/11/2030/1860290/2030.pdf by guest on 10 October 2022
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 cell 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 and
preserved BBB 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,
because 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 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 preconditioning, 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 suggest
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
Received October 15, 2018; revised April 19, 2019; accepted August 2, 2019;
published first August 8, 2019.
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