6, jie lin, jeno gyuris, heidi okamura authors’ affiliation: aveo … · 2015-05-20 · all four...

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23814, an Inhibitory Antibody of Ligand-Mediated Notch1 Activation, Modulates

Angiogenesis and Inhibits Tumor Growth without Gastrointestinal Toxicity

Theresa Proia1, Feng Jiang2, Alisa Bell, Richard Nicoletti, Lingxin Kong, Kelly Kreuter, Laura

Poling3, William M. Winston4, Meghan Flaherty5, Solly Weiler, Samantha Perino, Ronan

O’Hagan6, Jie Lin, Jeno Gyuris, Heidi Okamura

Authors’ Affiliation: AVEO Pharmaceuticals, Inc., 650 E. Kendall Street, Cambridge, MA 02142

Current addresses: 1AstraZeneca, Waltham, MA 02451; 2EMD Serono, Billerica, MA 01821;

3 Mersana Therapeutics, Cambridge, MA 02139; 4Potenza Therapeutics, Cambridge, MA

02138; 5Novartis Institutes for BioMedical Research, Cambridge, MA 01239; 6Merck Research

Laboratories, Boston, MA 02115

Running title: 23814 Notch1 antibody inhibits tumor growth without toxicity

Keywords: Notch1, angiogenesis inhibitor, antibody, tivozanib, toxicity

Corresponding Author: Heidi Okamura, AVEO Pharmaceuticals, Inc., 650 E. Kendall Street,

Cambridge, MA 02142; Phone: 617-299-5897; Fax: 617-995-4995;

E-mail: [email protected]

Conflict of interest: All authors were employees of AVEO Pharmaceuticals when the research

was conducted

Abstract 198

Word count 5371

Tables and figures 6

References 50

on October 16, 2020. © 2015 American Association for Cancer Research. mct.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 May 20, 2015; DOI: 10.1158/1535-7163.MCT-14-1104

http://mct.aacrjournals.org/

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Abstract

Dysregulation of Notch signaling has been implicated in the development of many different

types of cancer. Notch inhibitors are being tested in the clinic, but in most cases gastrointestinal

and other toxicities have limited the dosage and, therefore, the effectiveness of these therapies.

Herein, we describe the generation of a monoclonal antibody against the ligand binding domain

of the Notch1 receptor that specifically blocks ligand-induced activation. This antibody, 23814,

recognizes both human and murine Notch1 with similar affinity, enabling examination of the

effects on both tumor and host tissue in preclinical models. 23814 blocked Notch1 function in

vivo, inhibited functional angiogenesis, and inhibited tumor growth without causing

gastrointestinal toxicity. The lack of toxicity allowed for combination of 23814 and the VEGFR

inhibitor tivozanib, resulting in significant growth inhibition of several VEGFR inhibitor-resistant

tumor models. Analysis of the gene expression profiles of an extensive collection of murine

breast tumors enabled the successful prediction of which tumors were most likely to respond to

the combination of 23814 and tivozanib. Therefore, the use of a specific Notch1 antibody that

does not induce significant toxicity may allow combination treatment with angiogenesis inhibitors

or other targeted agents to achieve enhanced therapeutic benefit.




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Introduction

The Notch signaling pathway regulates many fundamental processes essential for

normal development such as the control of cell differentiation, survival, proliferation, and

angiogenesis [1-3]. Signaling pathways necessary for embryonic development are often found

reactivated during tumorigenesis, and inappropriate activation of Notch signaling has been

implicated in the development of many different types of adult and pediatric cancers [4, 5].

Interestingly, in some tissues, inactivation of Notch signaling may facilitate tumor development,

particularly in the case of squamous cell carcinomas [6-8] and bladder cancer [9]. This

highlights the complexities of Notch signaling, and the importance of understanding the correct

context in which Notch pathway inhibition will be beneficial.

In mammals, there are four Notch receptors (Notch1-4), all with similar functional

domains and structures [10]. The extracellular domains consist of a series of EGF-like repeats

that contain the ligand binding domain (LBD), followed by a negative regulatory region (NRR)

that locks the receptor in the “off” conformation in the absence of ligand [11]. There are five

canonical Notch ligands, including three Delta-like (DLL1, DLL3, DLL4) and two Jagged (Jag1,

Jag2) proteins, which are also membrane-bound. Receptor-ligand activation requires interaction

between neighboring cells, leading to conformational changes that result in sequential cleavage

of the receptor by ADAM and gamma secretase proteases, and release of the Notch

intracellular domain (ICD). The Notch ICD translocates to the nucleus where it complexes with

other transcription factors and coactivators to turn on downstream target genes [10].

Notch1 activation impacts tumorigenesis through multiple mechanisms. Activating

mutations of Notch1 occur in several different types of hematopoietic malignancies, including

>50% of T-cell acute lymphoblastic leukemia (T-ALL) [12] and ~10% of chronic lymphocytic

leukemia (CLL) [13, 14], where Notch1 signaling appears to promote resistance to apoptosis

[15]. Immunohistochemistry shows elevated levels of cleaved Notch1 ICD in several different




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primary human tumor types, suggesting that Notch1 activation is also a common occurrence in

solid tumors [16, 17]. Cancer stem cell maintenance is another area where Notch signaling is

thought to play a key role [18, 19]. Inhibition of Notch1 in mouse models can reduce cancer

stem cell numbers in both hematopoietic and solid tumors, potentially decreasing resistance to

chemotherapy and delaying tumor recurrence [20-22]. Finally, Notch1 activity in the

microenvironment keeps VEGF signaling in check to optimize tumor angiogenesis. Inhibition of

Notch1-DLL4 signaling leads to uncontrolled vascular sprouting, and a significantly greater

number of tumor vessels [23-27]. These vessels, however, are not efficiently perfused [23, 28].

Notch1 inhibition has also been shown to decrease production of endothelial nitric oxide, a

vasodilating agent important for regulating blood flow [29]. Thus, despite increasing vascular

density, Notch1-DLL4 blockade results in impaired vascular function and tumor growth

inhibition, likely due to increased hypoxia.

The multifaceted manner in which Notch1 can facilitate tumor growth underscores the

tremendous potential of targeting this pathway. Gamma-secretase inhibitors (GSI), which inhibit

all four Notch receptors as well as numerous other substrates, have been investigated in the

clinic, where hints of anti-tumor activity have been observed. However, severe gastrointestinal

toxicity due to goblet cell metaplasia, or reduced GSI exposure due to CYP3A4-mediated

deactivation, has forced suboptimal dosing and schedule alterations, limiting their therapeutic

utility [30-33]. Inhibitory antibodies targeting Notch1 have been developed in an effort to limit

toxicity, but gastrointestinal and other toxicities have still been reported in preclinical and clinical

settings [27, 34, 35]. Nevertheless, signs of clinical response have been seen in patients

treated with Notch1 antibody [35], indicating that Notch1 inhibition can be efficacious. Recent

preclinical studies demonstrate that a soluble decoy encompassing a subset of the Notch1

EGF-like repeats can block Notch1 signaling without inducing gut toxicity, suggesting that

inhibition and toxicity can be uncoupled [36].




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Here, we describe the in vitro and in vivo activity of 23814, a human antibody made

against the Notch1 LBD that specifically inhibits ligand-induced activation of the Notch1

receptor. The 23814 antibody possesses cross-reactivity towards human and murine Notch1

with similar affinity, allowing us to examine the effect on both tumor and host tissue. At effective

doses that allow inhibition of Notch1 signaling in both physiological and pathological contexts,

no evidence of gastrointestinal or other toxicity is observed. The lack of toxicity allowed

combination of 23814 with the pan-VEGFR inhibitor tivozanib to achieve significant tumor

growth inhibition in several models that do not respond robustly to tivozanib monotherapy.

These results suggest that treatment with Notch1-specific antibodies may be a viable

therapeutic option in cancer settings resistant to anti-angiogenic treatment.

Materials and Methods

Cell lines and reagents

Karpas 45 was acquired from the German Collection of Microorganisms and Cell

Cultures (DSMZ) cell bank. The HT-1080 cell line was acquired directly from the ATCC and

stored frozen at early passage until use. No additional authentication was performed. Stable

Notch ligand-expressing lines were established by transfecting CHO Flp-In cells (Life

Technologies) with full length Jagged1, Jagged2, DLL1, or DLL4 using Lipofectamine 2000 (Life

Technologies). Notch receptor antibodies for FACS analysis were purchased from BioLegend.

Tumor Xenograft Studies

All mice were treated in accordance with the OLAW Public Health Service Policy on

Human Care and Use of Laboratory Animals and the ILAR Guide for the Care and Use of




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Laboratory Animals. All in vivo studies were conducted following the protocols approved by the

AVEO Pharmaceuticals Institutional Animal Care and Use Committee.

Efficacy studies were performed with 8-10 week old female NCR nude mice. Cells were

resuspended in HBSS containing 50% matrigel and inoculated subcutaneously in the flank of

each mouse. Primary human tumors from surgical resection were obtained through the

Cooperative Human Tissue Network (CHTN). For the Ki-168 patient-derived xenograft (PDX)

study, mice were inoculated with 5x106 cells obtained from a human primary renal clear cell

tumor propagated in vivo and stored frozen prior to use. When tumors approached ~200 mm3,

mice were randomized and received 23814 or control hIgG (Xolair, Novartis AG) at 20 mg/kg

three times weekly, ErbB3 antibody (AV-203) at 20 mg/kg twice weekly, or tivozanib (dissolved

in 0.5% carboxymethylcellulose (CMC)) at 5 mg/kg daily. Mice were inoculated with 3.5x106

HT-1080, or 1x105 BH cells and randomized when tumor size reached 150-200 mm3. Animals

received 23814 or tivozanib as described above, combination treatment with 23814 (20 mg/kg

three times weekly) plus tivozanib (5mg/kg daily), or vehicle control (20 mg/kg hIgG three times

weekly plus daily dosing with 0.5% CMC). Statistical analysis was performed using 1-way

ANOVA and two-tailed t-tests using Graphpad Prism software version 6.

CD31 Staining

Mice were cohorted when tumors approached 250 mm3, and treated with 20 mg/kg

23814 or hIgG antibody on days 1 and 3, and/or dosed daily with 5 mg/kg tivozanib for 3 days.

Tumors were collected on day 4, fixed, processed, and embedded in paraffin. Tumor sections

were immunostained with antibodies to CD31/PECAM1 to detect the vasculature. The average

number of vessels per tumor area (number/μm2) was determined using an Aperio ScanScope

XT (Aperio, Vista, CA) and Microvessel Analysis v1 Parameters Tool image analysis software

(Aperio).




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Thymocyte Depletion

Four week old female mixed background mice (Taconic) were separated into groups of

five, and treated with 20 mg/kg hIgG or 23814 antibody by intraperitoneal injection. Mice were

dosed 2-3 times weekly for 18 days. Thymuses were harvested in 5%FBS/PBS, pushed

through a 100 μm filter, and spun. The cell pellet was resuspended in cold 1X RBC lysis buffer

(eBioscience) for 2 minutes before addition of 5%FBS/PBS. After spinning for 5 minutes at

1000 rpm, the cell pellet was resuspended in 0.5% BSA/PBS. Thymocytes were counted using

the Countess® Automated Cell Counter (Invitrogen).

Alcian Blue Staining

Mice were treated with 23814 or hIgG at 20 mg/kg 3x weekly, or with the GSI

dibenzazepine (DBZ) (Syncom) at 10 µmol/kg daily. All mice were weighed twice weekly. DBZ-

treated animals were taken down after four weeks due to deteriorating health. 23814 and hIgG

arms were treated for eight weeks. For goblet cell detection, sections of small intestine from all

treatment groups were harvested at end of study, fixed in 10% formalin, processed, and stained

using the Alcian Blue pH 2.5 Stain Kit (American Master Tech Scientific) according to the

manufacturer’s instructions.

Immunoassay for 23814 Detection

Serum samples and standards were diluted in fetal bovine serum, and 23814 captured

using recombinant Notch1Fc (R&D Systems) bound to 96-well High Bind plates (Meso Scale

Discovery). Captured 23814 was detected with a SULFO-TAG labeled anti-human F(ab’)2




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(Jackson ImmunoResearch) using electrochemiluminescence with the Meso Scale Discovery

platform.

Luciferase reporter assays

Karpas 45 were infected with lentivirus containing the firefly luciferase gene under

control of the RBP-Jk transcriptional response element (SABiosciences), and selected with

puromycin. Assays were performed by preincubating reporter cells with antibody for 30

minutes, then co-culturing with ligand-expressing CHO Flp-In cells that had been seeded in 96

well plates. 24 hours later, cells were processed using the Bright Glo (Promega) reporter assay

protocol per the manufacturer’s instructions. Lysates were transferred to white–walled 96 well

plates (Greiner Bio-One) and read on a GloMax Luminometer (Promega).

ICD Cleavage and Western Blotting

Recombinant Jag1 mFc and Jag2 mFc were created by fusing cDNA encoding the

extracellular domain of human Jag1 or human Jag2, respectively, to the murine IgG1 Fc

fragment. Constructs were stably transfected into CHOK1SV cells and fusion protein was

purified from the supernatant. 96-well plates were coated with anti-His tag (R&D Sytems) or

anti-mFc (Jackson ImmunoResearch) to capture recombinant DLL4 (R&D Systems), Jag1 mFc,

Jag2 mFc, or mFc (Jackson ImmunoResearch). Karpas 45 were preincubated with 23814 or

hIgG for 30’ before plating onto ligand-coated wells. Cells were harvested after 4 hours, and

lysates analyzed by SDS PAGE and Western blot. Blots were probed with antibody specific for

cleaved Notch1 ICD (Cell Signaling) or β-tubulin (Cell Signaling). Bands were detected using a

ChemiDoc XRS+ imager and ImageLab software (BioRad).




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Expression Analysis

RNA was prepared using Qiashredders and RNeasy miniprep columns (Qiagen), and

cDNA was prepared using High Capacity cDNA reverse transcription kit (Life Technologies).

qPCR was performed on Applied Biosystems HT7900 using TaqMan Gene Expression assays

(DTX1-Hs00269995_m1, HES4-Hs00368353_g1, and NRARP-Hs01104102_s1) and

manufacturer-suggested PCR protocols (Life Technologies). Results were analyzed by the

comparative Ct method (Delta Delta Ct) using β-actin as the loading control, as detailed in

Supplementary Materials and Methods.

Hierarchical Clustering

Unsupervised hierarchical clustering was conducted using Euclidian distance and the

complete linkage method on the BH expression data [37], selecting the top 1,000 most variant

probes as defined by median absolute deviation. The analysis was performed using R version

3.0.2.

Results

Identification and characterization of Notch1-specific antagonistic antibodies

To assess the therapeutic potential of targeting the Notch pathway in cancer, we used

phage display to generate monoclonal antibodies that specifically target EGF-like repeats 11-12,

the LBD of the Notch1 receptor. Candidate clones were screened by ELISA and FACS for

binding to human and murine Notch1, in an effort to identify antibodies that cross-react with the

LBD of both species. To identify antibodies able to inhibit ligand-induced activation, we utilized

a luciferase reporter under the control of an RBP-Jκ transcriptional response element, which

mediates Notch signaling. A stable reporter line was established using Karpas 45, a human T-




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ALL cell line expressing high levels of endogenous Notch1 receptor, and used to screen for

blocking antibodies. This led to the identification of 23814. This antibody was able to block

activation induced by all ligands tested in a dose-dependent manner. Luciferase activity was

inhibited by 23814 when the reporter line was co-cultured with cells expressing Jag1, Jag2,

DLL1, or DLL4 (Fig. 1A). Western blots confirmed that addition of 23814 to Karpas 45 cells

robustly prevented ligand-induced Notch1 ICD cleavage, maintaining Notch1 ICD at background

levels even in the presence of high amounts of ligand (Fig. 1B). Consistent with effective

inhibition of Notch1 signaling, expression of downstream target genes was significantly

decreased in the presence of 23814 (Fig. 1C).

FACS analysis shows that 23814 binds to human and murine Notch1 expressed on the

cell surface, but not to Notch2 or Notch3 (Fig. 1D). Surface plasmon resonance also confirmed

high affinity binding of 23814 to the extracellular domains of human and murine Notch1 (KD =

2.3 nM and 3.7 nM, respectively), but not to those of other Notch family members. These

results demonstrate that 23814 is a neutralizing Notch1-specific antibody that recognizes both

human and murine Notch1 with high affinity.

Treatment of mice with 23814 results in sustained inhibition of Notch1 signaling in vivo

To assess the ability of 23814 to inhibit Notch1 function in vivo, we examined its effect

on thymocyte development, a well-characterized process dependent on Notch1 signaling.

Active Notch1 signaling is required for thymic progenitor cells to commit to the T cell lineage,

and for normal progression through T cell development. Inducible knockout of the Notch1 gene

in neonatal mice has been shown to result in a marked reduction in thymus size, as well as a

five-fold reduction in thymocyte number [38]. Treatment of mice with 23814 resulted in a

dramatic reduction in the number of thymocytes relative to isotype control-treated mice (Fig.

2A). The decrease in thymocyte number was dose dependent and consistent with effective




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sustained inhibition of murine Notch1 activity by 23814. No weight loss or diarrhea was

observed at any of the doses tested (Fig. 2B).

23814 does not induce gut toxicity at efficacious doses

Several Notch inhibitors have entered the clinic, but have limited effectiveness due to the

development of goblet cell metaplasia, an overwhelming conversion of progenitor cells to

secretory goblet cells at the expense of absorptive enterocytes [30, 39]. The ability of 23814 to

recognize murine Notch1 allowed us to investigate its effect on gut toxicity at doses relevant to

the inhibition of Notch1 signaling. To confirm the lack of goblet cell conversion after 23814

treatment, Alcian blue staining was performed on the small intestines of mice treated for 8

weeks with 20 mg/kg of either hIgG or 23814, or for 4 weeks with dibenzazepine (DBZ), a

gamma-secretase inhibitor. In agreement with previously reported results, mice treated with

DBZ developed severe goblet cell metaplasia [30, 40] as indicated by increased Alcian blue

staining of mucin in intestinal crypts (Fig. 2C). In contrast, the small intestines of 23814-treated

mice were histologically similar to those of control mice and did not show increased goblet cell

numbers (Fig. 2C). Moreover, no signs of vascular neoplasm were observed, as has been

reported for some DLL4 and Notch1 inhibitors [34]. 23814 was confirmed to be present at high

concentration in the serum of treated animals at end of study (Fig. 2D). To further investigate

the potential toxicity of Notch1 inhibition, mice were treated twice weekly with 5, 20, or 40 mg/kg

23814 antibody for 4 weeks. Upon termination of the study, all major organs including liver,

heart, spleen, kidney, and thymus were examined for histology. Again, no signs of vascular

neoplasm or vascular proliferation were observed in any of the animals (Supplementary Fig.

S1A-C). Histopathology showed rare instances of single cell necrosis or mitotic figures in the

livers of some 23814-treated animals, but these were not dose-responsive and considered

recoverable. Serum collected at termination showed that clinical pathology analysis was well




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within normal reference range for liver enzymes (Supplementary Table S1), and that 23814

exposure was maintained in a dose-dependent manner (Supplementary Fig. S1D). This

suggests that 23814 treatment for 4 weeks, at doses that achieve functional inhibition of Notch1

signaling in vivo, does not produce severe toxicities.

23814 inhibits tumor growth in kidney PDX model

Expression of DLL4 in endothelial cells is directly induced by VEGF signaling [41].

Therefore, Notch1 activation might be expected in settings where VEGF signaling is high.

Although VEGF is overexpressed in a number of different types of cancer, clear cell renal cell

carcinoma (RCC) is uniquely dependent on VEGF signaling due to the frequency of the von

Hippel-Lindau (VHL) tumor suppressor mutation [42]. To assess the functional activity of 23814

in a tumor model we utilized Ki-168, a PDX model established from a clear cell RCC tumor.

PDX models are thought to retain key characteristics of the original donor tumor and, therefore,

to possess superior potential to predict clinical outcome compared to traditional cell line based

models [43].

Ki-168 PDX tumors were treated with hIgG, 23814, AV-203 (a negative control antibody

against ErbB3), or tivozanib, a potent and selective inhibitor of VEGFR-1, -2 and -3 that has

demonstrated significant activity in the clinic [44]. Blockade of VEGFR signaling due to

tivozanib treatment effectively inhibited Ki-168 tumor growth relative to control. Similarly,

treatment with the 23814 Notch1 antibody resulted in potent tumor growth inhibition of Ki-168. In

contrast, inhibition of ErbB3 signaling with AV-203 had no effect in this model (Fig. 3). These

results show that monotherapy treatment with 23814 is able to inhibit growth of a patient-derived

RCC tumor, a type of tumor especially sensitive to inhibitors of angiogenesis.




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Treatment with 23814 alters tumor vasculature and inhibits tumor growth in models that

do not respond well to VEGFR inhibitors

It has previously been demonstrated that inhibition of the Notch pathway through

blockade of DLL4 can negatively impact tumor growth in preclinical models that are resistant to

anti-VEGF therapy [23, 24, 26, 28, 45]. To see if specific inhibition of the Notch1 receptor can

achieve similar results, we utilized the highly vascular HT-1080 xenograft model, which has

been widely used to study tumor angiogenesis. HT-1080 tumors were treated with 23814, either

alone or in combination with tivozanib. Treatment with either 23814 or tivozanib monotherapy

resulted in partial tumor inhibition relative to control. Combination treatment using 23814 and

tivozanib together resulted in significantly greater efficacy than either drug alone (p<0.001)

(Fig.4A).

To determine whether 23814 was affecting tumor angiogenesis, immunohistochemistry

was performed on HT-1080 tumors. Established tumors that had been treated for three days

with either 23814, tivozanib, or a combination of the two were collected and stained with anti-

CD31 to examine vessel density. Quantification of CD31 staining demonstrates that treatment

with 23814 markedly increased tumor vascular density (Fig. 4B), consistent with specific

inhibition of Notch1-DLL4 signaling [23-25, 27]. As expected, inhibition of VEGF signaling with

tivozanib resulted in greatly decreased tumor vasculature. Combination treatment with 23814

and tivozanib resulted in reduced tumor vessel density relative to vehicle control, similar to that

observed with tivozanib alone (Fig. 4B).

To determine whether 23814 could inhibit tumor growth in other VEGF inhibitor-resistant

models, we examined a collection of breast cancer tumors derived from chimeric mice

engineered to inducibly express mutated HER2 (V659E) [37, 46]. More than 100 individual

primary tumors from these breast Her2 (BH) mice have been propagated, characterized, and

shown to have diverse characteristics across the collection by histopathology, microarray

analysis, and variation in response to drug treatment due to their chimeric nature [37].




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Accordingly, tumors from BH models differ widely in their response to tivozanib monotherapy,

ranging from complete regression to varying degrees of resistance (Fig. 5A, Supplementary Fig.

S2, and data not shown). Several different BH tumor models that did not robustly respond to

tivozanib monotherapy were chosen to examine whether increased efficacy could be achieved

through blockade of Notch1 signaling using 23814. While most of the models tested had a

moderate response to either 23814 or tivozanib alone, all but one model exhibited significantly

increased tumor inhibition when treated with a combination of tivozanib and 23814 (Fig. 5A,

Supplementary Fig. S2, and data not shown). To confirm that 23814 and tivozanib were

effectively inhibiting the Notch and VEGFR signaling pathways, respectively, expression of

downstream genes was examined using TaqMan assays. Analysis of the treated tumors

demonstrated that 23814 inhibited Notch1 signaling, as canonical Notch targets such as Hey1

and Hes1 were downregulated (Fig. 5B). Tivozanib treatment decreased Flt4 (VEGFR3)

expression as expected, but 23814 significantly increased Flt4 expression (Fig. 5B), in

accordance with findings suggesting that increased VEGFR3 signaling is responsible for the

deregulated angiogenesis observed upon Notch1 inhibition [47]. Hypoxia-regulated gene

expression also increased upon treatment with 23814, tivozanib, and combination therapy,

consistent with inhibition of angiogenesis (Fig. 5C). CD31 staining demonstrated a marked

increase in tumor vessels relative to vehicle control in 23814-treated tumors consistent with

Notch1 blockade, while tivozanib and combination treatment greatly decreased vessel number

(Fig. 5D). Interestingly, significant increases in vessel sprouting were also observed in some

BH tumors treated with 23814 monotherapy whose growth was not robustly inhibited

(Supplementary Fig. S2), suggesting that aberrant vessel density is a marker of Notch1

inhibition, but is not predictive of potent tumor growth inhibition. Although addition of 23814 to

tivozanib treatment did not decrease the number of tumor vessels relative to tivozanib alone

(Fig. 5D), the combination of the two inhibitors did significantly increase the amount of tumor

necrosis observed in BH224 (Supplementary Fig. S3).




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Hierarchical clustering predicts response to 23814 and tivozanib combination treatment

As not all BH tumors are equally inhibited by 23814 and tivozanib, it would be desirable

to be able to predict which tumors would best respond to combination treatment. Since BH224

exhibited robust tumor inhibition in response to combination therapy (Fig. 5A),

unsupervised, hierarchical clustering was used to determine which BH tumor models were most

biologically similar to BH224. BH224 clustered in a distinct subgroup that did not include any of

the other BH tumors tested in initial studies with 23814 (Fig. 6A). To see if tumors in this

subgroup share a dependence on Notch1/VEGF signaling, two of the closest neighbors to

BH224, BH270 and BH228, were assessed for response to combination treatment with 23814

and tivozanib. Both BH270 and BH228 tumor models responded strongly to combination

therapy, and exhibited greater sustained inhibition with 23814/tivozanib treatment than

previously tested BH models that clustered in separate subgroups (Fig. 6B). These results

suggest that there is an underlying biology in the tumors clustering within the BH224 subgroup

that predisposes them to dependence on Notch1/VEGF signaling, making them especially

susceptible to inhibitors of these pathways.

Discussion

Here we report the identification of a Notch1-specific antibody that exhibits anti-tumor

efficacy and modulates tumor angiogenesis, without causing gastrointestinal or other toxicities

previously reported for Notch pathway inhibitors. Several pan-Notch inhibitors have entered the

clinic, but a major obstacle in their progress has been the induction of dose-limiting toxicity due

to intestinal goblet cell metaplasia. Studies in conditional knockout mice have suggested that

goblet cell metaplasia requires inhibition of both Notch1 and Notch2 receptors [48]. Therefore,

much effort has gone into the development of antibodies that specifically target various epitopes




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of Notch1 [20, 21, 27, 49, 50], with the expectation that this would eliminate gut toxicity.

Surprisingly, however, it was reported that treatment of mice with antibodies that target Notch1

through binding to the negative regulatory region (NRR) can induce some level of goblet cell

metaplasia [27]. One possible explanation for the difference in toxicity observed between 23814

and the previous report is the differing mechanisms of action. While 23814 binds to the Notch1

LBD and prevents association of the receptor with its activating ligands, the NRR antibody

allows ligand binding but keeps the receptor locked in an “off” state. It is possible that Notch1

receptors bound to an NRR antibody are able to bind and sequester ligands in this inactive

conformation, thereby affecting signaling of other Notch family receptors. Other Notch1-specific

antibodies detailed in the literature either do not cross-react with murine Notch1 or are not

characterized in in vivo experiments, so it is unknown whether induction of goblet cell

metaplasia is a feature of anti-Notch1 NRR antibodies in general, or a specific property of

certain antibodies. Another possible explanation for the lack of toxicity for 23814 versus other

Notch1 antibodies is simply that there is a difference in potency or antibody kinetics that leads to

a larger therapeutic window. Perhaps for inhibitors of targets such as Notch, which regulate

stem cell and normal tissue homeostasis, the key is not to treat at the maximum tolerated dose,

but instead to treat at the minimum efficacious dose.

Our data suggests that Notch1 inhibitors can be effective in settings where anti-

angiogenesis therapies are indicated. We have demonstrated potent tumor inhibition in a

patient-derived primary RCC model using 23814 monotherapy, as well as in combination with

tivozanib in several models that exhibit partial resistance to VEGFR inhibitor alone. Although

monotherapy treatment of the BH tumors with 23814 resulted in only modest tumor inhibition,

23814 clearly potentiated the anti-tumor activity of tivozanib when used in combination.

Addition of 23814 to tivozanib treatment resulted in significantly greater tumor necrosis, as well

as increased induction of hypoxia genes, compared to tivozanib monotherapy (Fig. 5C and

Supplementary Fig. S3). This is consistent with an anti-angiogenic mechanism of inhibition, and




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is similar to what has been observed when DLL4 and VEGF inhibitors are combined [28], further

suggesting that inhibition of the Notch1 and VEGF pathways is not redundant [23, 24, 45]. Use

of a Notch1 antibody may have the added advantage of simultaneously blocking oncogenic

activation of Notch1 signaling not only through DLL4 in the stroma, but also through other

ligands such as Jag1 or Jag2 in the tumor itself, possibly impacting pro-survival signals in

addition to angiogenesis.

Undoubtedly, there are many mechanisms leading to VEGF inhibitor resistance, and

discovering which tumors are likely to respond to Notch1 inhibition is essential for success in the

clinic. Our BH tumors, derived from chimeric primary mouse models, demonstrate a wide range

of responses to combined tivozanib 23814 therapy, likely reflecting what would be observed in

the patient population. It is well-established that the expression level of Notch1 receptor itself is

not predictive of response to Notch inhibitors in the clinic, and response to combination therapy

is unlikely to depend on Notch signaling alone. Therefore, instead of limiting our scope to

differences in canonical Notch pathway genes, we used unsupervised hierarchical clustering in

an attempt to predict a priori which tumor models would respond to tivozanib/23814 treatment.

The two models selected for testing from the same subgroup as the best responder exhibited

robust tumor inhibition in response to tivozanib/23814 combination treatment that significantly

exceeded response to either therapy alone. This underscores the ability to enrich for

responders based on the unique combination of biological pathways that are activated in

subgroups of tumors.

Our findings demonstrate that the 23814 Notch1 antibody exhibits anti-tumor efficacy

that is not redundant with VEGF pathway inhibition, and that angiogenesis inhibitors with

different mechanisms of action can be combined to achieve improved outcome. The lack of

toxicity may allow combination of 23814 with VEGF inhibitors or other therapies at efficacious

doses, and suggests that evaluation of specific Notch1 inhibitors as a treatment option for VEGF

inhibitor-resistant tumors is warranted.




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Acknowledgements

The authors gratefully acknowledge Hamid Tissire, Sara Haserlat, and Sandra Abbott for

their contributions to antibody screening and engineering, Lynette Cook for 23814 purification,

and Tom Magee for toxicology assistance.




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25

Figure Legends

Figure 1. In vitro characterization of 23814 activity. A, Notch1 luciferase reporter line co-

cultured with stable lines expressing Jag1, Jag2, DLL1 and DLL4 ligands in the presence of

23814 or control hIgG antibodies. Results represented as percent inhibition of luciferase activity.

B, Karpas 45 plated on wells coated with recombinant Jag1, Jag2, DLL4 or Fc (no ligand) in the

presence of 23814 or hIgG control (10 ug/ml). Lane 1, no ligand + hIgG; lane 2, ligand + hIgG;

lane 3, ligand + 23814 antibody. Western blots were probed with antibody specific for cleaved

Notch1 ICD (Cell Signaling). Equal amounts of lysate transferred to a separate blot were

probed with β-tubulin antibody as a loading control. C, Karpas 45 co-cultured with ligand-

expressing CHO lines in the presence of 23814 or hIgG. Total RNA was isolated after 24 hours

and gene expression assessed by TaqMan assay (* p<0.05 by t-test). D, FACS analysis of

23814 binding to CHO cells expressing human or murine Notch1, human Notch2, or human

Notch3.

Figure 2. 23814 inhibits Notch1 signaling in vivo and does not cause gut toxicity. A, 23814

treatment of mice (n = 5 animals/group) results in thymocyte depletion (***p<0.001; NS, not

significant). B, at doses where 23814 effectively inhibits Notch1 signaling in vivo, no weight loss

occurs. C, mice were treated with hIgG, 23814 (8 weeks), or the gamma secretase inhibitor

DBZ (4 weeks). Alcian blue staining of small intestines shows significant goblet cell conversion

in DBZ-treated mice, while 23814-treated mice looked similar to control animals. D, 23814 in

serum was captured with recombinant Notch1Fc and detected with SULFO-TAG labeled anti-

human F(ab’)2. A1-A5, mice treated with hIgG; B1-B5, mice treated with 23814.




26

Figure 3. 23814 inhibits growth of a renal clear cell PDX tumor. NCR nude mice (n = 10)

received either hIgG control, anti-ErbB3 (AV-203), 23814, or tivozanib. 23814 and tivozanib

monotherapy significantly inhibited tumor growth relative to control (*** p<0.001).

Figure 4. 23814 treatment increases tumor vasculature. A, HT-1080 tumor model (n = 10) was

treated with control hIgG, 23814, tivozanib, or a combination of 23814 plus tivozanib.

Combination treatment results in significantly greater tumor growth inhibition than either

tivozanib or 23814 alone (*** p<0.001). B, CD31 staining and microvessel density quantification

of HT-1080 tumors from mice (n = 3) treated for 3 days with hIgG, tivozanib, and/or 23814 (***

p<0.001, ** p<0.01).

Figure 5. Combined inhibition of Notch1 and VEGFR pathways enhances anti-tumor efficacy in

models partially resistant to VEGFR inhibitor monotherapy. A, BH tumor models (n = 10) exhibit

a range of responses to treatment with 23814, tivozanib, or a combination of the two (***

p<0.001, ** p<0.01, NS= not significant). For B, C, and D, analysis was performed on BH224

tumors harvested after 3 days of treatment. B, TaqMan analysis of Notch1 target gene

expression. Results normalized to the mean expression level of vehicle-treated tumors (n = 3).

*p<0.05. C, TaqMan analysis shows increased expression of the hypoxia-regulated genes ADM

(adrenomedullin), NDRG1 (N-myc downstream-regulated 1) and VEGFA, although statistical

significance was not reached. D, CD31 staining of treated tumors.

Figure 6. Unsupervised hierarchical clustering identifies tumors likely to respond to

23814/tivozanib combination therapy. A, cluster analysis demonstrates that BH tumors




27

separate into distinct subgroups. BH413 and BH226 (labeled in blue), representing a non-

responder and partial responder, respectively, cluster in different subgroups from BH224, while

BH228 and BH270 (labeled in green) are biologically similar. B, BH228 and BH270 tumor

growth inhibition is significantly increased by combination treatment with 23814/tivozanib

relative to tivozanib monotherapy (**p<0.01, *p<0.05).




Figure 1

D C

Jag2

DLL1

Jag1

DLL4

B

hNotch1 CHO Control CHO

hNotch1

mNotch1 CHO Control CHO

mNotch1 hNotch2


hNotch3


A

0

0.2

0.4

0.6

0.8

1

1.2

DTX1 HES4 NRARP

Rela

tive

Exp

ressio

n

hIgG 23814

* * *

30

40

50

60

80

100

220

30

40

50

60

80

100

220

30

40

50

60

80

100

220

b-Tubulin 50 50 50

Notch1 ICD

1 2 3

DLL4 1 2 3

Jag2 1 2 3

Jag1




Figure 2

B A

hIgG 23814 DBZ

C

0

100

200

300

20 mpk 20 mpk 10 mpk 2 mpk

hIgG 23814 Notch1 mAb

Tota

l T

hym

ocyte

Num

ber

(x10

6)

NS

*** ***

Days

Bo

dy

we

igh

t (g

)

0 5 10 15 200

5

10

15

20

25

hIgG (20mpk)

23814 (20mpk)

23814 (10mpk)

23814 (2mpk)

0.00

200.00

400.00

600.00

800.00

A1 A2 A3 A4 A5 B1 B2 B3 B4 B5

hIgG 23814

An

tib

od

y C

on

c [u

g/m

l]

a-Notch1 Ab at end of study

D




Figure 3

***

Days

***




Figure 4

B

A

tivozanib Vehicle 23814 23814+tivozanib

Days

Tu

mo

r vo

lum

e (

mm

3)

0 5 10 15 200

500

1000

1500

2000

Control

Tivozanib

23814

23814 + Tivozanib

0.00E+00

1.00E-04

2.00E-04

3.00E-04

4.00E-04

5.00E-04

6.00E-04

7.00E-04

Vehicle Tivo 23814 Combo

Nu

mb

er V

esse

ls/m

m2

Microvessel Density

***

** ***

***




Figure 5

B

A

0 5 10 15 20 250

500

1000

1500

2000

2500VehicleTivozanib23814Tivozanib+23814

days

tum

or v

olu

me

(m

m3

)

BH224

**

0 5 10 15 200

500

1000

1500

2000

2500

VehicleTivozanib23814Tivozanib+23814

daystu

mo

r v

olu

me

(m

m3

)

BH226

***

0 5 10 15 200

500

1000

1500

2000

2500VehicleTivozanib23814Tivozanib+23814

days

tum

or v

olu

me

(m

m3

)

BH413

NS

C

0

0.5

1

1.5

2

HEY1 HES1 FLT4

Re

lati

ve

Ex

pre

ss

ion

Vehicle

Tivozanib

23814

23814+Tivo

0

1

2

3

4

ADM NDRG1 VEGFA

Re

lati

ve E

xpre

ssio

n

Vehicle

Tivozanib

23814

23814+Tivo

D Vehicle 23814

Tivozanib Tivo+23814

*

* *




Figure 6

B

A

0 5 10 15 200

500

1000

1500

2000

2500


days

tum

or

vo

lum

e (

mm

3)

0 5 10 15 200

500

1000

1500

2000

2500


days

tum

or

vo

lum

e (

mm

3)

BH270 BH228

* **




Published OnlineFirst May 20, 2015.Mol Cancer Ther Theresa Proia, Feng Jiang, Alisa Bell, et al. without Gastrointestinal ToxicityActivation, Modulates Angiogenesis and Inhibits Tumor Growth 23814, an Inhibitory Antibody of Ligand-Mediated Notch1

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