targeting tissue factor for immunotherapy of triple ... · human igg1 fc. we named this construct...
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Targeting Tissue Factor for Immunotherapy of Triple-Negative Breast Cancer Using a
Second-Generation ICON
Short title: TF as immunotherapy target in triple-negative breast cancer
Zhiwei Hu1*, Rulong Shen2, Amanda Campbell3, Elizabeth McMichael3, Lianbo Yu4,
Bhuvaneswari Ramaswamy5, Cheryl A. London6, Tian Xu7 and William E. Carson III1
1Department of Surgery Division of Surgical Oncology, The Ohio State University
Wexner Medical Center and The OSU James Comprehensive Cancer Center, Columbus,
OH 43210; 2Department of Pathology, The Ohio State University Wexner Medical
Center, Columbus, OH 43210. 3Biomedical Sciences Graduate Program, The Ohio State
University, Columbus, OH 43210. 4Center for Biostatistics, The Ohio State University,
Columbus, OH 43210. 5Department of Medical Oncology, The Ohio State University
Wexner Medical Center, Columbus, OH 43210. 6Department of Veterinary Biosciences,
College of Veterinary Medicine, The Ohio State University, Columbus, OH, USA; 7Department of Genetics, Yale University School of Medicine, New Haven, CT 06520
Corresponding author: Zhiwei Hu, The Ohio State University Wexner Medical Center,
410 W 12th Avenue, CCC308, Columbus, OH, USA. Email: [email protected];
Phone: 614-685-4606; Fax: 614-688-4028
Conflict of Interest: Z.H. is co-inventor of U.S. patents on the original neovascular-
targeted immunoconjugates (ICON) and is the inventor of U.S. patent application on the
second-generation ICON (L-ICON1). Other authors declare no potential conflicts of
interest.
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Abstract
Triple-negative breast cancer (TNBC) is a leading cause of breast cancer death and is
often associated with BRCA1 and BRCA2 mutation. Due to the lack of validated target
molecules, no targeted therapy for TNBC is approved. Tissue factor (TF) is a common
yet specific surface target receptor for cancer cells, tumor vascular endothelial cells and
cancer stem cells in several types of solid cancers including breast cancer. Here we report
evidence supporting the idea that TF is a surface target in TNBC. We used in vitro cancer
lines and in vivo tumor xenografts in mice, all with BRCA1 or BRCA2 mutations,
derived from patients’ tumors. We showed that TF is over-expressed on TNBC cells and
tumor neovasculature in 50% to 85% of TNBC patients (n = 161) and in TNBC cell line–
derived xenografts (CDX) and patient-derived xenografts (PDX) from mice, but was not
detected in adjacent normal breast tissue. We then describe the development of a second-
generation TF-targeting immunoconjugate (called L-ICON1, for lighter or light chain
ICON) with improved efficacy and safety profiles compared to the original ICON. We
showed that L-ICON1 kills TNBC cells in vitro via antibody-dependent cell-mediated
cytotoxicity and can be used to treat human and murine TNBC CDX as well as PDX in
vivo in orthotopic mouse models. Thus TF could be a useful target for the development
of immunotherapeutics for TNBC patients, with or without BRCA1 and BRCA2
mutations.
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Introduction
Breast cancer is the most common newly diagnosed cancer and second leading cause of
cancer death among women in the United States in 2016 (1). Breast cancer deaths are
largely the result of recurrent and metastatic disease that typically does not respond well
to current treatments (2). Triple-negative breast cancer (TNBC), so-called because the
tumor cells lack expression of three targetable proteins, the estrogen receptor, the
progesterone receptor, and the epidermal growth factor receptor HER2, is difficult to treat
malignancies and is usually considered incurable (3) (4-10). TNBC comprises
approximately 15% of globally diagnosed breast cancer (4,11,12). At present, there are
no molecularly targeted therapies approved for TNBC.
TNBC is often associated with mutations in breast cancer predisposition genes BRCA1
and BRCA2 (13). The identification of BRCA1 (14) and BRCA2 (13) in the mid-1990s
has led to a better understanding of the molecular pathogenesis of hereditary breast
cancer. BRCA1 and BRCA2 are human genes encoding tumor suppressor proteins that
ensure the stability of DNA. When either of these genes is mutated, or altered, such that
its protein product either is not made or does not function correctly, DNA damage may
not be repaired properly. As a result, BRCA1 and BRCA2 mutations are associated with
an increased risk for female breast and ovarian cancer. Approximately 20% of TNBC
patients harbor a mutation in either the BRCA1 or BRCA2 genes (15). Thus,
identification of biomarkers and oncotargets for BRCA1- and 2-associated hereditary
breast cancer and development of corresponding targeted immunotherapy could provide
efficacious treatment regimens for TNBC, with or without BRCA1 and BRCA2
mutation, and reduce the mortality associated with the malignancy.
To identify a therapeutic target in TNBC, we focused on tissue factor (TF), based on
previous findings in breast cancer patients (16) and human breast tumor xenografts from
animal studies (17-20). TF is a 47kDa membrane-bound cell surface receptor that
initiates the extrinsic coagulation cascade pathway upon injury to vessel wall integrity
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(21-23) and modulates angiogenesis (24-27). It is highly expressed in many types of
solid cancers (28,29), including non-TNBC breast cancer. In breast cancer, TF expression
can be detected on both the breast cancer cells and the tumor vascular endothelial cells in
breast cancer patients (16) and in a mouse model of human breast cancer (17), but not in
adjacent normal breast tissues (16). We reported that TF is an angiogenic-specific
receptor in VEGF-stimulated angiogenic vascular endothelial models (30) and is also
could be a target for cancer stem cells (CSCs) in several types of solid cancer (31),
including breast cancer. Other studies have also connected TF with TNBC (18,19).
However, it is still not known whether TF is expressed in TNBC cells and tumor
neovasculature, and if so, to what extent. If TF is indeed highly and selectively expressed
in TNBC, it could serve as a target for immunotherapy of TNBC.
TF expression has been detected previously on other types of solid cancers, leukemia,
and sarcomas (28,29), including melanoma (32,33), prostate cancer (34), and head and
neck cancer (35), suggesting that it might be a useful therapeutic target. Pursuing that
approach, we developed the TF-targeting immuno-conjugate agent named ICON, which
consists of full-length factor VII peptide (406 amino-acid residues, aa) fused to the Fc
region of IgG1 (32-35), in which fVII, the TF-targeting domain, binds TF with specificity
(30) and high affinity [pico-molar dissociation constant (Kd)] (36), and the IgG1 Fc binds
complement and Fc receptors (CD16) on immune cells such as natural killer (NK) cells.
After binding to the Fc domain, NK cells and the complement cascade kill ICON-bound
target cancer cells via antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC), respectively (35). Intralesional ICON
therapy of experimental murine and human melanoma (32,33), prostate (34), and head
and neck tumors (35) with an ICON adenoviral vector leads to growth inhibition of
tumors with minimal effects on normal tissues (33). However, ICON is large (210 kDa)
(35). In addition, hypercoagulation is documented in virtually all cancer types (37), albeit
at different rates, and is a second leading cause of death in cancer patients (38).It is
partially the result of TF produced by and released from cancer cells (39,40). To
eliminate the coagulation activity of fVII in the ICON, a coagulation-active lysine reside
at position 341 (K341A) was mutated to an alanine in fVII peptide by site-directed
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mutagenesis (34). With this mutation (K341A), the pro-coagulant effects of ICON were
reduced but not eliminated (34). In the current work we developed a strategy to eliminate
the pro-coagulant effects of ICON, reduce its molecular mass, and retain its binding
activity to TF so that ICON could be more effective for treatment of cancer patients when
administered systemically.
To identify TF as a surface target in TNBC, we investigated TF expression on TNBC
cancer cells and tumor vascular endothelial cells in TNBC cell-derived xenografts (CDX)
and patient-derived xenografts (PDX) from mice as well as in a total of 161 TNBC tumor
tissues from patients with TNBC, including 14 cases of paraffin-embedded whole tumor
tissues and 147 cases of TNBC tumors on tissue microarray (TMA) slides. Because
TNBC is associated with BRCA1 and BRCA2 mutations (41), we also investigated TF
expression on human TNBC cell lines and in CDX and PDX tumors with or without
BRCA1 or BRCA2 mutations.
To develop a TF-targeting ICON with improved safety for targeted immunotherapy of
TNBC, we modified ICON by removing the heavy chain (153-406 aa) of fVII. The
resulting construct contains the non-coagulating fVII light chain (1-152 aa) fused to
human IgG1 Fc. We named this construct light chain ICON or lighter ICON (L-ICON1).
We compared molecular weights, binding activities, coagulation activities of L-ICON1
with ICON in vitro and compared the in vivo efficacy of L-ICON1 and ICON for
immunotherapy of TNBC in orthotopic CDX and PDX mouse models.
Materials and Methods
Cell lines and reagents. Chinese hamster ovary cells (CHO-K1) were purchased from
American Type Culture Collection (ATCC, Manassas, VA) in 2004 and were grown in
F12K complete growth medium (ATCC) supplemented with 10% heat-inactivated fetal
bovine serum (HI-FBS) (Sigma) and 1 × penicillin and streptomycin (Invitrogen). Human
TNBC cell lines MDA-MB-231 (BRCA1-wt, BRCA2-mutant), BT20 (BRCA1-wt,
BRCA2-wt) and MDA-MB-468 (BRCA1-wt), non-TNBC lines MCF7/MDR and
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MCF7/MDR/TF (BRCA1-wt, BRCA2-mutant), murine breast cancer cells 4T1 and
EMT6 were purchased from ATCC in 2001-2009 except for murine TNBC line 4T1
(kind gift from Dr. William Carson at The Ohio State University, 2013) and human non-
TNBC lines MCF7 and MCF7/MDR (kind gift of Dr. Zping Lin at Yale University,
2008). The cancer cells were grown in DMEM or RPMI1640 complete growth medium
supplemented with 10% HI-FBS and 1 × penicillin and streptomycin (Invitrogen).
MCF7/MDR line is a low TF-expressing MCF7 line with multi-drug resistance
(MCF7/MDR) (17) and MCF7/MDR/TF is a high-TF expressing MCF7/MDR
(MCF7/MDR/TF) derived from MCF7/MDR by being infected with retroviral vector
encoding TF (kind gift of Dr. Michael Bromberg) (42). MDA-MB-231 and 4T1 lines
were transfected using xtremeGene HP transfection reagent (Roche, 3 μl transfection
reagent: 1 μg plasmid DNA) (Roche) with plasmid vectors (1 μg ACT PBase and 1 μg
PB Transposon, constructed and kindly provided by Dr. Tian Xu’s laboratory at Yale
University) encoding luciferase (Luc) and green fluorescent protein (GFP). The
transfected cell lines were selected and maintained for stable expression of Luc and GFP
by supplementing growth media with blasticidin (Invitrogen) (at a final concentration of
10 μg/ml for selection and 5 μg/ml for maintaining the cell culture). Following the
manufacturer’s instructions, ADCC effector cells, a Jurkat-based cell line transfected
with CD16 and N-FAT/Luciferase, were purchased from Promega (Cat. No. G7102) in
2014 and were grown in RPMI 1640 complete growth medium supplemented with 100
μg/ml G418, 250 μg/ml hygromycin, 10% HI-FBS, 1 × nonessential amino acids, 1 ×
sodium pyruvate and 1 × penicillin and streptomycin (Invitrogen). 293AD cell line was
purchased from Cell Biolabs (Cat. No. AD-100, 2014) and was grown in DMEM (high
glucose) supplemented with 10% H-FBS, 0.1 mM MEM Non-Essential Amino Acids, 2
mM L-glutamine and 1 × penicillin and streptomycin (Invitrogen). All cell cultures were
maintained mycoplasma free by routinely supplementing with Plasmocin (Invivogen,
Cat. No. Ant-mpt, 25 μg/ml for treatment and 5 μg/ml for maintenance) or Ciprofloxacin
HCl (Enzo, Cat. No. 380-288-G025, 10 μg/ml) in cell culture medium. Cultures were
tested annually for mycoplasma contamination. The latest date they were tested by using
Mycoalert Mycoplasma Detection Kit (Lonza, Cat. No. LT07-118) was June 26, 2017.
Authenticity of cancer cell lines was determined by validating TF expression by flow
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cytometry, Western blots and/or cell ELISA, as described below. Thawed cancer cells
were maintained in culture for one month. CHO-K1 producer cells for L-ICON1 protein
production were maintained for 2 months.
Immunohistochemistry (IHC) for staining TF expression. The use of patients’ tissues
with non-identifiable information was reviewed and approved by the Ohio State
University (OSU) Cancer Institutional Review Board (IRB) and was conducted following
the OSU Human Research Protection Program in accordance with the ethical principles
and guidelines summarized in the Belmont Report
(https://web.archive.org/web/20111017133845/http://www.hhs.gov/ohrp/archive/docume
nts/19790418.pdf). Fourteen TNBC tumor tissues, confirmed by pathology diagnosis to
lack detectable estrogen receptor, progesterone receptor and epidermal growth factor
receptor Her2, were obtained from The Cooperative Human Tissue Network of The Ohio
State University (OSU). Generation of OSU TNBC tissue microarrays was previously
described (43). Tissue microarray (TMA) slides with 147 cases of TNBC tumor tissues
and 147 cases of matched normal breast tissues (usually adjacent to the tumor tissues)
were obtained from OSU Pathology. For each patient, TMA slides had two cores of
tumor tissues and two cores of matched normal tissue adjacent tumor. Some samples
were lost as samples detached from the slides during IHC staining. Some tumor tissues
that did not contain tumor cells were excluded. TF expression was stained with a mouse
monoclonal antibody (HTF1, kind gift of Dr. William Konigsberg at Yale University)
and/or with a commercial goat anti-human TF (Sekisui Diagnostics, REF 4501), both of
which we showed bind to human TF, the same antigen/receptor that is recognized by
ICONs and fVII peptides (30). The detailed procedures have been similarly described in
our previous publications (17,44).
The IHC procedures for staining TF and CD31 in TNBC TMA slides were optimized and
performed by the OSU Pathology Core Facility. Paraffin-embedded 4- m TMA slides of
patients’ TNBC tumors were placed on a 60 C oven for 1 hour, cooled, deparaffinized
and rehydrated through xylene and graded ethanol solutions to water. All slides were
quenched for 5 minutes in a 3% hydrogen peroxide aqueous solution to block for
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endogenous peroxidase. Antigen retrieval was performed in a 1× solution of Target
Retrieval Solution pH6.0 (Dako) by Heat-Induced Epitope Retrieval for 25 min at 96oC
using a vegetable steamer (Black & Decker). Slide were stained with the Intellipath
Autostainer Immunostaining System using mouse monoclonal anti-human TF antibody
(HTF1, 1:100, 1mg/ml stock) for staining TF or using mouse monoclonal anti-CD31
(1:80, Culture supernatant) (Dako, Cat. M0823, Clone JC70A) for staining endothelial
cells at room temperature for 30 min, followed by MACH3 Mouse HRP-Polymer
Detection (Biocare Medical) for 20 min at room temperature and then by 5 min
incubation with liquid DAB+ Chromogen (Dako). TF staining was verified by using a
commercial polyclonal antibody against human TF (Sekisui Diagnostics, REF 4501). The
slides were counterstained in Richard Allen hematoxylin (Thermo Fisher Scientific),
dehydrated through graded ethanol solutions, cleared with xylene, and coverslipped.
The IHC procedure for CDX and PDX tumors was performed by the Comparative
Pathology and Mouse Phenotyping (CPMPSR) Laboratory of the OSU Veterinary
School. Human TNBC CDX (MDA-MB-231) and PDX (JAX TM00089, breast tumor
markers: TNBC ER-/PR-/HER2-, BRCA1 V757fs) slides were derived from formalin-
fixed and paraffin-embedded control tumors from previously published mouse studies
(20,45) or from an untreated PDX mouse in this study. Paraffin-embedded 4- m tissue
slides were de-paraffinized, rehydrated, pretreated with Antigen Retrieval Solution and
then 3% hydrogen peroxide, blocked with serum free protein and stained with 5 μg/ml of
goat anti-human TF antibody (with cross reaction with murine, rat and rabbit TF, Sekisui
Diagnostics, REF 4501, CT, USA) or anti-CD31 antibody (1:80, Dako, Cat. M0823,
Clone JC70A) diluted in Dako antibody diluent with background reducing agents.
Finally, the sections were incubated using 2 μg/ml of Vector biotinylated rabbit anti-goat
IgG secondary Ab followed by Vector TRU ABC Elite complex for anti-TF Ab or
MACH3 Mouse HRP-Polymer Detection (Biocare Medical) for anti-CD31 Ab. Positive
staining for VECs and TF was visualized by Nova Red substrate. Cell nuclei were
counterstained by hematoxylin. Human MDA-MB-231 CDX and PDX slides were also
stained with hematoxylin and eosin (H&E) by routine methods for visualization of tumor
pathology.
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After IHC staining, two investigators, including a board-certified anatomic pathologist,
independently reviewed and scored the stained slides and representative views were
photographed. The Scores for TF expression were graded as follows: negative (-),
moderately positive (+), positive (++), strongly positive (+++) and very strongly positive
(++++). Positive percentages included all cases graded from moderately positive through
very strongly positive.
Construction of plasmid DNA vectors encoding murine and human L-ICON1.
Construction of an expression plasmid vector pcDNA3.1(+) encoding cDNA for the wild
type human fVII fused to human IgG1 Fc has been described (32,34). Plasmid vectors
pcDNA3.1(+) encoding mouse and human L-ICON1 were similarly constructed using
recombinant DNA techniques. Briefly, the cDNA for the first 152 amino acids of mouse
and human fVII light chain were PCR amplified from the plasmid vectors encoding their
parental ICONs. We used primers as follows. The cDNAs of human and mouse fVII light
chain were subcloned in frame into pcDNA3.1(+) containing human IgG1 Fc between
EcoRI and BamHI. The cDNA sequences of ICON and L-ICON1 were verified by
Sanger-based DNA sequencing at the Genomic Shared Resources at Yale University and
at The Ohio State University. The cDNA sequence of L-ICON1 is deposited with
accession no. KX760097 at GenBank. To make recombinant L-ICON1 proteins, the
plasmid vectors encoding mouse and human L-ICON1 cDNAs were transfected into
CHO-K1 cells (ATCC) using xtremeGene HP transfection reagent (Roche). The stably
transfected L-ICON1 producer CHO-K1 cells were selected under 1 mg/ml G418 (Gibco)
and were cultured in suspension at a start density of 2.5 × 105 cells/ml in CHO serum free
medium (SFM4CHO, Hyclone) supplemented with 1 μg/ml vitamin K1 (Sigma). The
SFM was collected twice a week and L-ICON1 protein was purified from the
supernatants of SFM by a 5 ml HiTrap rProtein A FF affinity column (GE Healthcare)
following the manufacturer’s instructions, as described (35). Molecular weights of L-
ICON1 and ICON proteins were analyzed by 8-16% SDS-PAGE non-reducing gel (Bio-
Rad Laboratories). The concentration of L-ICON1 protein was determined by Protein
Assay Reagent (Bio-Rad Laboratories) using bovine serum albumin standards (Pierce).
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Construction and production of adenoviral vectors encoding L-ICON1. The
adenoviral system we used to make adenoviral vector encoding L-ICON1 was RAPAD
CMV Adenoviral Expression System (Cell Biolabs, Cat. No. VPK-252). Briefly, the
cDNA of L-ICON1 was subcloned into the pacAd5 CMVK-NpA shuttle vector between
EcoRI and Not I. Pac I-digested shuttle vector pacAd5 CMV-L-ICON1 and the Ad
backbone DNA (pacAd5 9.2-100) were separated linearized with Pac I and co-transfected
of the Pac I-digested shuttle vector encoding L-ICON1 and the backbone DNA (at a ratio
of 4μg:1μg) into adenoviral packaging cell line 293AD (Cell BioLabs) using xtremeGene
HP transfection reagent (Roche) following the manufacturer’s instructions. A blank
control vector (AdBlank) was similarly produced using the pacAd5 CMVK-NpA vector
without encoding a transgene and padAd5 9.2-100 backbone DNA. The adenoviral
vectors were amplified by infecting 293AD cells (Cell Biolabs) and purified by CsCl
(Sigma) ultracentrifugation, as described (32-35). The titer of infectious viral particles
was determined by measuring Optical density (OD) of 260 nm of 20-fold diluted viral
particles in 0.1% sodium dodecyl sulfate (SDS) and calculated using the following
formula: 1 OD260 nm = 1 × 1012 viral particles per ml, as described (34). OD260nm was
assessed on Spectrophotometer (Beckman, Model DU650). To verify the production of
L-ICON1 protein by AdL-ICON1 vectors, MDA-MB-231 cancer cells in 6-well plate
were infected with CsCl-purified AdL-ICON1 (GenBank KX760097), AdhICON (human
ICON, hfVII/hIgG1Fc; GenBank AF272774), AdmICON (mouse ICON,
mfVII/hIgG1Fc; GenBank AF272773) and AdBlank (as negative control) vectors at an
MOI of 3000:1 by incubating viral vectors with cancer cells in serum containing growth
medium at 37°C and 5% CO2 for 2 hrs. Next morning the cells were washed once with 1
× DPBS and grown in SFM4CHO supplemented with 1 μg/ml Vitamin K1 (Sigma) for 4
days. The supernatants of SFM from viral vector-infected cancer cells were assayed in
MDA-MB-231 cancer cell ELISA above for determining the concentration and binding
activity of L-ICON1 and ICONs. The L-ICON1 and ICON proteins in SFM were further
confirmed by immunoprecipitation-Western blot (IP-WB), as described below.
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Factor VII coagulation activity assay. Following the manufacturer’s instruction, the
retained coagulation activity of fVII light chain peptide in the L-ICON1 protein was
determined by Factor VII Human Chromogenic Activity Kit (Abcam), which was
developed to determine human fVII coagulation activity, specifically the ability of fVII to
bind and form a complex with TF and then to activate factor X (fX) to fXa. The
amidolytic activity of the TF/FVIIa complex was quantitated by the amount of fXa
produced using an FXa substrate that releases a yellow para-nitroaniline (pNA)
choromophore. The change in absorbance of the pNA at 405nm (A405nm) is directly
proportional to the FVII enzymatic (coagulation) activity.
Briefly, L-ICON1 protein at 1.25-10 nM was added to a 96-well strip assay plate, in
which a polyclonal antibody against human FVII was pre-coated. After two hours
incubation at 37°C, the strip plate was washed 5 times with wash buffer then the freshly
prepared assay mix, which contains recombinant human TF and FX, was added and
incubated at 37°C for 30 min followed by adding FXa substrate. The absorbance at 405
nm was read every 5 minutes for 30 minutes on a microplate reader (SpectraMax i3,
Molecular Devices). The chromogenic activity curve of human FVII standard was
generated by serially diluting a stock solution of 0.2 IU/ml (70 ng/ml) 1:2 to produce 0.1,
0.05, 0.025 and 0.0125 IU/ml. The positive control for the assay was an active form of
FVII (FVIIa) (America Diagnostica) and the negative control was an active site inhibited
FVIIa (FFR-FVIIa, America Diagnostica). For comparison with its parental ICONs,
active site mutated ICON (hfVIIK341A-hIgG1Fc, or ICON K341A) and wild type ICON
(ICON WT without active site mutation) were also tested in parallel with L-ICON1. All
of these proteins were tested at 10 nM, a physiological concentration of FVII in
circulation, with 1:2 serial dilutions to 5, 2.5 and 1.25 nM.
Flow cytometry and cancer cell ELISA for binding activity assays. The binding
activity of L-ICON1 was assayed by flow cytometry and enzyme-linked immunosorbent
assay (ELISA), as previously described (12,32,34,35). Briefly, the flow cytometry
procedure involves incubation of non-enzymatic dissociated cancer cells with 10 μg/ml
L-ICON1 or ICON proteins for their binding activity, or mouse monoclonal anti-Human
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TF antibody (anti-HTF1, kind gift of Dr. William Konigsberg of Yale University) for TF
expression followed by 1:50 diluted FITC conjugated anti-human IgG or anti-mouse IgG
conjugate (Sigma), respectively. Controls were incubated with normal human IgG or
mouse IgG as isotype controls in replacement of L-ICON1 or HTF1 antibody. For cancer
cell ELISA, cancer cells (MDA-MB-231, BT20, MCF7/MDR/TF, 4T1 and EMT6) were
seeded at 1 × 104 cells per well in 96-well flat microplates. When the cells reached about
95% confluence, they were washed once with warm PBS, fixed in 4% paraformaldehyde
at room temperature for 20 min, blocked with 1% BSA in calcium containing buffer (1x
TBS/Ca, 10 mM Tris HCl pH8.0, 150 mM NaCl, 10 mM CaCl2) and then incubated with
L-ICON1, ICON and an isotype control (human IgG, Sigma) proteins in 1 × TBS/Ca
buffer followed by incubation with HRP-conjugated anti-human IgG1Fc (human specific,
1:10,000) (Sigma) for detection of human IgG1 Fc portion of ICON and L-ICON1
proteins. After final wash, OPD was added as substrate and the absorbance at 490nm
(A490nm) was read on SpectraMax i3 microplate reader (Molecular Devices).
Sandwich ELISA assays for measuring the concentration of L-ICON1 in cell culture
medium and mouse plasma samples. L-ICON1 concentration was determined by paired
factor VII antibody Sandwich ELISA (Cedarlane Laboratory) following the
manufacturer’s instructions with modifications as follows. Anti-human fVII capture
antibody (1:200) was coated in EIA 96-well strip plates, was blocked with 1% BSA then
incubated with cell culture supernatants in serum free medium (SFM4CHO) or 1:5
diluted mouse plasma samples in PBS followed by HRP-conjugated anti-human IgG1Fc
(human specific, 1:10,000) for detection of full-length peptides of ICON and L-ICON1,
as described above. The standard proteins in Sandwich ELISA were HiTrap rProtein A
affinity purified-ICON and L-ICON1 proteins, as described above and earlier (35).
ADCC effector assay. The effect of L-ICON1-mediated ADCC to TNBC cells was
determined using an ADCC effector assay (Promega) (46) following the manufacturer’s
instruction with a modification to the assay medium by using DMEM medium, instead of
RPMI 1640. DMEM medium contains a higher calcium concentration (1.8 mM) than
RMPI 1640 (0.42 mM) per the information on calcium concentrations in cell culture
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medium on the Sigma-Aldrich website (Calcium is required for ICON and fVII binding
to its receptor TF) (32). Briefly, human TNBC (MDA-MB-231 and BT20) and non-
TNBC (MCF7/MDR/TF) cells (1 × 104 cells per well in 100 μl growth medium) were
seeded in duplicates into the 60 inner wells and grown overnight in 96-well white assay
plate (Corning) at 37 C and 5% CO2. Then, 95 μl of growth medium was removed from
each well. Pre-warmed 25μl ADCC effector assay medium, (DMEM supplemented with
0.5% super low IgG fetal bovine serum (HyClone)), was added to 60 inner wells whereas
75 μl ADCC effector assay medium was added to 36 outer wells. Then 25 μl of ICON
protein at serial dilutions of 3 × or 5 × concentrations was added in duplicate wells to the
cancer cells, whereas human IgG (Sigma) was used as an IgG isotype control. After 15
min incubation at 37 C, 25 μl ADCC effector cells (Promega) (a T cell-derived leukemia
cell line Jurkat transfected with Fc receptor CD16 and NFAT-Luciferase under the
control of CD16 binding followed by NFAT activation) were added (2.5 × 105 cells per
well) to achieve a ratio of effector to target (E:T) at 25:1 and incubated for 6 or 19 hours
at 37 C and 5% CO2. At the end of 6-hr or 19-hr incubation, the plate and Bio-Glo assay
reagent (Promega) were equilibrated at room temperature for 15 min and then equal
volume (75 μl) of Bio-Glo reagents was added to 60 inner wells and two outer wells with
medium only as background control. Raw bioluminescence (RLU) was read on
SpectraMax i3 (Molecular Devices).
Generation of orthotopic mouse models of TNBC cell-derived xenografts (CDX) and
patient-derived xenografts (PDX). The animal study protocol was reviewed and
approved by the Institutional Animal Care and Use Committee of The Ohio State
University (OSU IACUC). To generate orthotopic mouse models of tumor line-derived
xenografts, 5 × 105 human TNBC MDA-MB-231/Luc+GFP cells in 50 μl of 1:1 mixed
PBS/Matrigel (BD Biosciences) or 5 × 105 murine TNBC 4T1/Luc+GFP cells in 50 μl
PBS were injected into the fourth left mammary gland fat pad in 4-6 weeks-old, female
CB-17 SCID (Taconic Farms) or Balb/c mice (Charles River Laboratories), respectively.
Both CB-17 SCID and Balb/c mice have intact and functional NK cells that are crucial to
mediate ICON and antibody therapy (35). To generate an orthotopic TNBC PDX model,
we purchased a TNBC PDX donor NSG mouse (NOD SCID gamma, no mature B, T, NK
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cells and no complement) with BRCA1 mutation from Jackson Laboratory (JAX
TM00089, breast tumor markers: TNBC ER-/PR-/HER2-, BRCA1 V757fs). Surgeries
were performed in accordance with the OSU IACUC Rodent Surgery Policy. Mice were
anesthetized with isoflurane. For the donor NSG mouse (JAX, TM00089), an incision
was made over the flank of tumor to extract the tumor tissue, which was washed once in
RPMI medium supplemented with 1 x penicillin and streptomycin and was cut in sterile
PBS into ~3 millimeters (mm) pieces in diameter for implantation into the recipient mice.
CB-17 SCID mice (Taconic Farms) were used as recipient mice. For recipient mice, an
incision was made over the fourth right mammary gland, for direct implantation of one
piece of ~3 mm PDX tumor tissues from the donor tumor tissue into it. One drop of tissue
adhesive (Vetbond) and/or 1 wound clip (Sigma) was used to close the incision site in
routine fashion. Animals were monitored for recovery from anesthesia before returning to
routine husbandry. Animals were checked daily until wounds were healed, and analgesics
(buprenorphine, s.c. 0.1 mg/kg in 100 l sterile PBS) were administered to provide pain
relief for up to 72 hours after surgery.
Adenoviral gene therapy using AdL-ICON1 in vivo in mouse models of human and
murine TNBC CDX and PDX. When tumor size reached 150-200 mm3, the mice were
randomized into control, L-ICON1-treated or ICON-treated groups. The control mice
were intratumorally (i.t.) injected with 1 or 4 × 1010 VP of AdBlank and the treated mice
were injected with 1 or 4 × 1010 VP of AdL-ICON1 or AdICON vectors weekly for
orthotopic human TNBC CDX and PDX or every 4 days for orthotopic 4T1 CDX, as
specified in figure legends. Therapeutic efficacy was determined by measuring tumor
width (W) and length (L) with calipers in millimeters (mm) and calculating tumor
volume (mm3) using the formula (W)2 × L/2 (mm3), as described (34,35). The efficacy
was also monitored using small animal imaging for bioluminescence and fluorescence
(IVIS, Caliper) before the first treatment, during treatment and at the end of experiment.
At the time of sacrifice, i.e., two days after last intra-lesional injection of AdL-ICON1 or
AdBlank, mouse plasma samples were prepared from the L-ICON1 treated and control
SCID mice bearing orthotopic TNBC CDX tumors and were assayed for L-ICON1
protein concentration by ELISA, as described above.
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In vivo mouse models and Bioluminescence imaging of breast cancer xenografts. To
image tumor cells that stably express Luc and GFP, the mice were i.p. injected with 100
μl of Luciferin solution. Ten minutes after injection of luciferin, the mice were imaged
for quantitatively measuring tumor bioluminescence intensity under anesthesia using
inhalation of isoflorane under IVIS Bioluminescence Imaging System (Caliper). The
results from IVIS imaging were presented as Raw Bioluminescence (RLU).
Statistical analysis. The data in vitro and in vivo are presented as mean SEM (or mean
SD as specified) and analyzed by ANOVA and t-test for statistical significance using
Prism software (GraphPad) and SAS 9.4 software. Fisher’s exact test was used to test
IHC score percentage difference between whole tumor tissues and TMA tissues. For
analyses of statistical significance, duplicate or triplicate wells in each group were used
for in vitro assays in tissue culture plates and 5 mice per group were used in vivo in
animal studies. Statistical significance is presented as *: P < 0.05; **: P < 0.01; ***: P
< 0.001; ****: P < 0.0001 and “ns” stands for no (statistical) significance.
Results
TF is expressed by TNBC cancer cells and tumor vascular endothelial cells
To examine TF expression in TNBC with wild type (wt) and mutated BRCA1 and
BRCA2, we first determined TF expression on TNBC lines. Supplementary Fig. S1
showed that TF was expressed by TNBC cell lines, either with wt or mutated BRCA1 and
BRCA2 (47,48), including MDA-MB-231 (BRCA1-wt, BRCA2-mutant)
(Supplementary Fig. S1a), BT20 (BRCA1-wt, BRCA2-wt) (Supplementary Fig. S1b)
and MDA-MB-468 (BRCA1-wt) (Supplementary Fig. S1c). To verify the binding
specificity for TF of the monoclonal anti-human TF (HTF1) used in the flow cytometry,
we infected a non-TNBC human breast cancer MCF7/MDR line (Supplementary Fig.
S1d), which was derived from MCF7 line (BRCA1-wt, BRCA2-mutant) with multi-drug
resistance (MDR) and both are known for low TF expression (17), with a retroviral
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vector encoding human TF (42). We showed that the resulting MCF7/MDR/TF line
expressed TF as detected by anti-TF (Supplementary Fig. S1e).
To translate the findings from TNBC cell lines to preclinical mouse models of TNBC, we
examined TF expression in human TNBC CDX and PDX tumor tissues from severe-
combined immunodeficiency (SCID) mice. With immunohistochemistry, TF expression
was detected on the TNBC cells (Supplementary Fig. S2a) in TNBC CDX tissues, but
not in adjacent normal tissues (Supplementary Fig. S2a). In addition, TF expression was
also detected on the TNBC cells (Supplementary Fig. S2c) and tumor vascular
endothelial cells (VECs) (Supplementary Fig. S2c) in TNBC PDX tumors, whose
vascular origin was confirmed by positive staining for endothelial marker CD31
(Supplementary Fig. S2d). Isotype Ab control (Supplementary Fig. S2b) and 2nd Ab
alone (Supplementary Fig. S2e) were used as negative controls. Mitosis was observed in
PDX tumor tissues (Supplementary Fig. S2c-S2f).
To translate the findings from mouse models to TNBC patients, we then examined TF
expression in primary TNBC tumor tissues from 14 patients. By immunohistochemistry,
12 of 14 (85.7%) TNBC tumor tissues expressed TF on the surface of TNBC cells (Fig.
1a and Supplementary Fig. S2c). Nine patients who expressed TF on tumor cells also
expressed TF on the surface of tumor VECs (64.3% of 14 cases) (Fig. 1b). The
endothelial origin of tumor VECs was verified through expression of endothelial marker
CD31 (Supplementary Fig. S3a). Normal breast gland cells and normal VECs in
adjacent normal breast tissues or adenosis (a benign inflammation with enlarged milk-
producing glands but not a cancer) were negative for TF expression (Fig. 1c). Mouse IgG
isotype was used as a negative control (Fig. 1d).
In light of these results, and to determine statistical significance, we investigated TF
expression in 147 additional cases of TNBC tumors and 147 matched normal breast
tissues from TNBC patients via tissue-microarray (TMA). Of TNBC tumor samples, 71
of 147 (48.3%) were positive for TF expression on the surface of TNBC cancer cells
(Fig. 2a, 2b and 2d) and 11 of 147 (7.5%) were positive for TF on tumor VECs
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(Supplementary Fig. S3b and S3c), whereas normal breast tissues (n = 147) were
negative for TF expression (Fig. 2a, 2c and Supplementary Fig. S3d). There was a
significant difference of IHC score percentage of TF expression on TNBC cancer cells
between whole tumor tissues and TMA tissues (P = 0.0002474, Fisher’s exact test) (Fig. 2d).
Modification in L-ICON1eliminated its pro-coagulant effects
To target TF, an oncotarget for TNBC, we developed a second-generation ICON by
modifying the first-generation TF-targeting ICON (Fig. 3a). Following the tradition of
making mouse and human ICONs (32,34) so that the mouse version can be tested in
mouse studies and the human version can be used in future clinical trials, mouse and
human L-ICON1s were constructed (Supplementary Fig. S4a). DNA sequencing
analysis confirmed that both the mouse (mL-ICON1) and human L-ICON1 (hL-ICON1
or L-ICON1) (GenBank accession no. KX760097) have the same sequence of human
IgG1 Fc as those in mouse and human ICONs (GenBank accession no. AF272773 and
AF272774) (i.e., a 16-amino acid residue hinge region followed by constant heavy chain
domains 2 and 3) (Supplementary Fig. S4a). SDS-PAGE non-reducing gel analysis
showed that the molecular weight of Protein A-affinity purified L-ICON1 protein was
approximately 100 kDa (Fig. 3b). Thus L-ICON1 is less than half the molecular weight
of ICON (210 kDa) (Fig. 3b).
To verify that the coagulation activity of fVII light chain in L-ICON1 was eliminated, the
coagulation activity of L-ICON1 was measured and compared to those of fVII
coagulation active site-mutated ICON (i.e., ICON K341A), wild type-fVII ICON (i.e.,
ICON WT), activated form of fVII (FVIIa) (as a positive control) and active site inhibited
FVIIa (FFR-FVIIa) (as a negative control). For this assessment, L-ICON1 was used at 10
nM, a physiological concentration of zymogen fVII in blood circulation. Compared to the
coagulation activity (100%) of FVIIa, L-ICON1 at the same concentration (10 nM) had
undetectable coagulation activity (-6.061 ± 0.000%), comparable to FFR-FVIIa (~4%) (P
= 0.0026 for L-ICON1 vs. FFR-FVIIa), whereas the first-generation ICON (K341A) and
the wild-type ICON (WT) exhibited approximately 5% and 58% coagulation activity (P =
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0.0104 for L-ICON1 vs. ICON K341A and P = 0.0179 for L-ICON1 vs. ICON WT),
respectively. (Fig. 3c, Supplementary Fig. S5 and Supplementary Table S1).
ICON and L-ICON1 similarly bound TNBC and non-TNBC cancer cells
To compare L-ICON1 and ICON binding activity on cancer cells, we used flow
cytometry and cancer cell ELISA assays. L-ICON1 and ICON bound similarly to TNBC
MDA-MB-231 cells in flow cytometry assay (Fig. 3d). By cancer cell ELISA, L-ICON1
showed an increased activity to MDA-MB-231 cells compared to ICON (P < 0.01 to
0.0001, L-ICON1 vs. ICON) (Fig. 3e). The results showed that the modifications in L-
ICON1 did not reduce its binding activity for TF-expressing TNBC. Murine and human
L-ICON1s could bind both the human (MDA-MD-231) (Supplementary Fig. S4b) and
the murine cancer cells (4T1 and EMT6) (Supplementary Fig. S4c and S4d). However,
hL-ICON1 had significantly stronger binding activity to those cancer cells than mL-
ICON1 (P < 0.0001) (Supplementary Fig. S4b-S4d). Thus, hL-ICON1 (L-ICON1 for
simplicity) was used in vitro and in vivo experiments thereafter.
L-ICON1 induced antibody-dependent cell-mediated cytotoxicity (ADCC)
To elucidate the mechanism of action of L-ICON1, we performed an ADCC effector
assay (Fig. 4). L-ICON1 binds to two TNBC lines (MDA-MB-231 and BT20) and one
non-TNBC chemoresistant line (MCF7/MDR/TF as a TF-positive control)
(Supplementary Fig. S1e and Fig. 4a). Furthermore, L-ICON1 could induce ADCC to
those TNBC lines and non-TNBC line (Fig. 4b), whereas the human IgG isotype
negative control had no binding (Fig. 4a) and had no ADCC effect (Fig. 4b) on those
breast cancer cells. The results demonstrate that L-ICON1 can mediate ADCC effect on
killing human TNBC and non-TNBC cells.
L-ICON1 is more effective than ICON in vivo for treatment of TNBC xenografts
To compare the efficacy of L-ICON1 and ICON, we constructed adenoviral vectors
encoding L-ICON1 (AdL-ICON1) and verified that AdL-ICON1 vectors could produce
L-ICON1 protein after infecting MDA-MB-231 cells in vitro, and that cells so treated
could secrete L-ICON1 protein into the cell culture medium. Cancer cell ELISAs showed
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that the concentrations (mean ± SD) of L-ICON1, murine ICON (mICON, murine fVII
K341A fused to human IgG1Fc) and human ICON (hICON, human fVII K341A fused to
human IgG1Fc) were 15842.0 ± 234.4 ng/ml, 9028.4 ± 1388.0 ng/ml and 9611.7 ±
1111.1 ng/ml in serum-free culture media 4 days after infection (Supplementary Table
S2) (P < 0.001 for AdL-ICON1 vs. AdmICON and AdhICON; no statistical significance
for AdmICON vs. AdhICON by ordinary one-way ANOVA), whereas AdBlank did not
produce any L-ICON1 or ICON protein (0.0 ± 0.0 ng/ml).
After having verified the production of L-ICON1 and ICON proteins by AdL-ICON1 and
AdICON vectors in vitro, we compared the in vivo effects of ICON and L-ICON1, as
intratumoral injection of adenoviral vectors encoding L-ICON1 or ICON, for
immunotherapy of human TNBC xenografts in an orthotopic mouse model using MDA-
MB-231/Luc+GFP cell line. The results demonstrated that L-ICON1 showed a
significantly increased therapeutic effect (80% reduction as compared to control tumor on
day 21) in vivo than ICON treatment (60% reduction as compared to control tumor on
day 21) (P = 0.0042) (Fig. 5a). Furthermore, the results in Fig. 5 showed that L-ICON1
inhibited orthotopic TNBC growth in mice (11% smaller on day 21 than its starting
volume on day 0) (P < 0.0001), as measured by tumor size (83.6% reduction as
compared to control tumor on day 23) (Fig. 5a) and by in vivo bioluminescence (83.6%
reduction as compared to control tumor on day 23) (Fig. 5b-5d), compared to control
adenoviral vector (AdBlank). Tumor weights also showed a significant difference ex vivo
(Fig. 5e).
Body weight was assessed as an indicator of safety, per previously recorded models
(17,44). There was no difference in whole mouse body weights between L-ICON1 and
control groups (Fig. 5f). At the time of sacrifice, which was two days after the last
injection of AdL-ICON1 (4 × 1010 VP per injection), the L-ICON1 protein was detected
in mouse plasma from the AdL-ICON1-treated mice at a concentration of 794.8 ± 156.4
ng/ml (~8 nM; ~200 ng/ml L-ICON1 protein produced/1010 VP intra-lesionally injected),
whereas there was no L-ICON1 protein was detected in the plasma samples from control
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vector-injected mice (Supplementary Table S2. P < 0.0001 for AdL-ICON1 vs.
AdBlank).
L-ICON1 is effective and safe for treatment of murine TNBC
To examine the efficacy of L-ICON1 for murine TNBC in immunocompetent mice, we
transfected the murine breast cancer line 4T1 for stably expressing GFP and luciferase
(Supplementary Fig. S6). 4T1 cells serve as a model of murine TNBC (49) for
generating an orthotopic mouse model in syngeneic Balb/c mice. L-ICON1 showed a
decrease (65.8% reduction as compared to the control tumor on day 10 after initiation of
treatment) in tumor size in this murine TNBC model (P < 0.001) (Fig. 6a). The average
tumor volume was 192 mm3 at the time of initiation of treatment (day 0) in the AdL-
ICON1 treated mice, which was significantly bigger (68.4% bigger) than the average
tumor volume (114 mm3) in the control group at the same time (P < 0.05) (Fig. 6a). Mice
were humanely sacrificed when evidence of extreme sickness was present, per IACUC
protocol. On day 10 post-treatment, only one of five L-ICON1-treated mice had to be
sacrificed (Fig. 6a), compared with five of five (100%) control mice by the same time-
point (P = 0.0203 for AdL-ICON1 vs. AdBlank control) (Fig. 6b). Under in vivo
bioluminescence imaging, no metastasis was observed in both L-ICON1 and control
groups at the time of sacrifice of these animals (day 10 post-treatment, i.e., day 18 after
4T1 cell implantation).
L-ICON1 treated TNBC patient-derived xenografts in an orthotopic mouse model
To translate L-ICON1 therapy for TNBC to the clinic, we tested its efficacy in an
orthotopic TNBC PDX (with BRCA1 mutation) model in CB-17 SCID mice. The results
demonstrated that L-ICON1 inhibited orthotopic PDX growth in mice as measured by
tumor size (P < 0.0001, 93.7% reduction as compared to control tumor on day 43) (Fig.
7a). PDX tumors in control mice grew quickly; these mice had to be sacrificed on day 43
after the first i.t. injection of control vectors. Tumor weights also showed a significant
difference ex vivo (P = 0.0044, L-ICON1-treated tumors on day 58 vs. control tumors on
day 43) (Fig. 7b).
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Discussion
TNBC is not only a significant clinical malignancy lacking molecularly defined surface
targets, but also is a molecularly heterogeneous disease associated with BRCA1 and
BRCA2 mutations. To identify TF as a surface target in TNBC, we investigated TF
expression on human TNBC lines with or without BRCA1 and BRCA2 mutations. To
translate the findings from in vitro to in vivo and ultimately to the clinic, here we also
investigated TF expression on the TNBC cancer cells and tumor vascular endothelial
cells in human TNBC CDX and PDX models from mice and in TNBC patients’ tumors.
PDX models are considered a better cancer xenograft model than the standard CDX
model for evaluating anticancer drug response; results from PDX models better predict
the clinical outcome of anticancer agents (50). The percentage (48%) of TF-positive
TNBC on TMA slides (n = 147) was lower than that (85%) found by examination of
regular paraffin-embedded whole TNBC tumor tissues (n = 14). This was possibly due to
the fact that tumors are heterogeneous and that only small tissue samples are taken for
making TMA slides, which is one of the limitations of TMA technique (51). In future
studies, we would suggest that multiple specimens from each tumor should be used for
IHC screening for enrollment of patients into clinical trials of L-ICON1 therapy. TF is
also expressed by cancer stem cells isolated from TNBC cell lines, from TNBC tumor
xenografts and from breast cancer patients (30). Taken together, our findings in this study
along with other groups’ findings (18,19) suggest that TF might be a useful therapeutic
target for treatment of TNBC, prompting us to improve upon the TF-targeting ICON that
we had previously developed (32-35). To develop a therapy for TNBC that uses TF as a target, we constructed an improved
second-generation ICON named L-ICON1, for light chain ICON or lighter ICON. When
Dr. Garen and I designed the first generation ICON as a TF-targeting agent, we attempted
to eliminate the coagulation activity of fVII in ICON by introducing a mutation at the
amino acid residue Lysine 341 (32), because Ruf and colleagues identified fVII surface
residues as key to TF binding and protease catalytic function (52). The pro-coagulant
effects of ICON-encoded fVII were thereby reduced, but not eliminated, as measured by
prothrombin time assay (34) and by fVII chromogenic assay in this study. In addition, the
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large MW of ICON (210 kDa), which is even bigger than an IgG1 antibody, may limit
the ability of ICON to penetrate tumor tissues when administered as recombinant protein
via intravenous injection. These findings prompted us to improve ICON by eliminating
its coagulation activity to avoid potential coagulation disorders in cancer patients when
administered systemically and reducing its size to improve penetration of ICON into
tumor tissues. To accomplish these objectives, we constructed a second-generation ICON
consisting of only the light chain (1-152aa) of fVII fused to IgG1 Fc. At 100 kDa, L-
ICON1 was less than half the size of ICON (210 kDa). The MW of L-ICON1 observed
by gel electrophoresis is consistent with the MW calculated from its predicted protein
sequence as derived from cDNA sequence (GenBank KX760097). That is, the L-ICON1
protein is predicted to contain 425 amino acids (47.56 kDa for monomer and approximate
95 kDa as homodimer). The first 38 amino acids are the signal peptide, and the following
387 residues are the mature peptide. The 387-aa mature peptide would be predicted to be
43.32 kDa as a monomer and 87 kDa as a homodimer, without consideration of the mass
of glycosylated side chains.
L-ICON1 has several advantages over ICON. First, the MW of L-ICON1 is reduced more
than 50% compared to ICON. Second, L-ICON1 has no detectable coagulation activity,
whereas the coagulation activities in ICON (K341A) and ICON (WT) are about 5% and
58% of that for FVIIa, respectively. L-ICON1 may therefore be a safer product than
ICON. Third, human L-ICON1 binds both mouse and human breast cancer cells, whereas
human ICON bound strongly to a human melanoma line, but weakly to a mouse
melanoma line (34). These features of L-ICON1 will allow us to test human L-ICON1 in
preclinical mouse models of human and murine cancers and translate the findings directly
to clinical trials. Lastly, the current study showed that L-ICON1 was more effective than
ICON in treating TNBC tumor xenografts in the orthotopic mouse model and in vivo
studies demonstrated that L-ICON1 immunotherapy via intralesional injections of AdL-
ICON1 was effective and safe for the treatment of human TNBC (MDA-MB-231 with
BRCA2 mutation) and murine TNBC (4T1) (49) in orthotopic CDX mouse models. L-
ICON1 could arrest the PDX tumor growth of TNBC with BRCA1-mutation in an
orthotopic mouse model.
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ICON is efficacious and safe in preclinical animal (mouse, rat, pig and non-human
primate) models of cancer (32-35), age-related macular degeneration (AMD) (53,54) and
endometriosis (55,56). ICON is being tested in Phase 1 and 2 clinical trials in patients
with AMD (wet form) and ocular melanoma. Indeed, as in angiogenic endothelial cells in
cancers, TF is aberrantly expressed by the angiogenic VECs in vitro (30) and in vivo in
nonmalignant diseases that are angiogenesis dependent, such as AMD (53) and
endometriosis (55). As an improved neovascular-targeting agent, L-ICON1 might have
therapeutic potential to treat pathological angiogenesis-dependent malignancy and non-
malignant diseases.
To target TF as part of cancer immunotherapy, in addition to the ICON and L-ICON
molecules that are designed to bind to TF by using its natural ligand fVII, either full-
length peptide with mutated pro-coagulation active site (K341A) or light-chain peptide
lacking pro-coagulation activity, several humanized monoclonal antibodies (TF-HuMab)
and/or antibody-drug conjugates (TF-ADC) are also being studied in preclinical and
clinical studies (19,57). ICON and L-ICON have several advantages compared to anti-
TF and TF-ADC: (1) The Kd for fVII binding to TF is up to 10-12 M (58), in contrast to
anti-TF that has a Kd in the range of 10-8 to 10-9 M for TF (59). (2) ICON and L-ICON
are produced by recombinant DNA technology, allowing these TF-targeting protein
agents to be made from human sources for clinical trials without need for humanization
process that is required for monoclonal antibodies (57). (3) Because ADC is made by
covalently conjugating drugs to antibodies, most ADCs exist as heterogeneous mixtures
and require site-specific conjugation technologies (60). Nevertheless, these studies of TF-
targeting Ab and ADC support the idea that TF-targeted therapies have the potential to
treat a range of solid cancers (28,29).
We report that TF is a useful target in treatment of TNBC models and immunotherapy
with TF-targeting L-ICON1 showed better efficacy and safety than the original ICON.
Immunotherapy targeted at TF may warrant further investigation for TNBC patients. If
such immunotherapy proves effective and safe in clinical trials, such treatment regimens
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might reduce the mortality associated with TNBC. TF is highly expressed on cancer cells
on non-TNBC breast cancer as well as in many other solid cancers, acute myeloid
leukemia, acute lymphocytic leukemia, and sarcomas (28,29): it is expressed in 80%-
100% of breast , 40%-80% of lung, 84% of ovarian, 95% of primary melanoma, 100% of
metastatic melanoma, 53%-90% of pancreatic, 57%-100% of colorectal, 63%-100% of
hepatocellular carcinoma, 60%-78% of primary and metastatic prostate, and 47%-75% of
glioma tumors. Thus, TF-targeting immunotherapy may have the potential to treat a
variety of TF-positive solid cancers, leukemias, and sarcomas (28,29).
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Acknowledgments: We thank Dr. Beth Schachter, Dr. Stephen M. Smith, Mr. Aaron
Conley and Dr. Thomas Mace for critical reading and editing, Dr. William Konigsberg at
Yale University and Dr. Michael Bromberg formerly with Yale University (currently
with Temple University) for kind gifts of monoclonal antibody HTF1 against TF and
retroviral vector-encoding TF and control vectors. We thank Longzhu Piao for technical
assistance. Data and materials availability: The cDNA sequence of L-ICON1 is
deposited at GenBank with accession no. KX760097. Funding: This work was supported
by the Dr. Ralph and Marian Falk Medical Research Trust Awards Programs. The project
described was also partly supported by Award Number UL1TR001070 from the National
Center for Advancing Translational Sciences through a Phase 1 L-Pilot Award and a
voucher award from the OSU Center for Clinical and Translational Science and by a Seed
Award from the Translational Therapeutics Program of OSU Comprehensive Cancer
Center. The plasmids for Luc+GFP were generated by Dr. Tian Xu lab at Yale University
with the support from the HHMI.
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FIGURE LEGENDS
Figure 1. TF is an oncotarget and a surface marker for the TNBC cancer cells and
tumor vascular endothelial cells. a. TF expression on TNBC cancer cells (TF positive
staining in brown, arrows); b. TF expression on the TNBC vascular endothelial cells (TF
positive staining in brown, arrowheads); c. No TF expression in normal breast gland
tissues with adenosis (TF negative); d. IHC staining with a mouse IgG isotype as a
negative control. Original magnification: 400 × in a-c and 100 × in d. TF expression
shown above was stained with a mouse monoclonal antibody (HTF1) and was
independently verified with goat anti-human TF (Sekisui Diagnostics).
Figure 2. TF expression on TNBC cells, but not on normal breast tissue, on Tissue
Microarray (TMA) slides. a. TF expression on TMA with two cores from tumor tissues
(upper row, TF positive cells stain brown) and two cores from normal tissues (bottom
row, no TF expression) from the same TNBC patient (40 ×). b. TF expression on the
TNBC cells (brown staining) (400 ×). c. No TF expression in normal breast tissues (400
×). d. Immunohistochemical staining for TF expression on TNBC cells in TNBC whole
tumor tissues and TMA tissues. TF expression shown above was stained with a mouse
monoclonal antibody (HTF1) and was independently verified with goat anti-human TF
(Sekisui Diagnostics). * The Scores for TF expression were graded as follows: negative (-
), moderately positive (+), positive (++), strongly positive (+++) and very strongly
positive (++++). Positive percentages included all cases graded from moderately positive
through very strongly positive. ** Fisher’s exact test was used to test IHC score
percentage difference between whole tumor tissues and TMA tissues, there is a
significant difference with P = 0.0002474.
Figure 3. L-ICON1 has improvements over the first-generation ICON. a. Diagrams
of L-ICON1 and ICON. fVII light chain: 1-152 aa and heavy chain: 153-406 aa; IgG1
Fc: Hinge region followed by two constant regions (CH2 and CH3). –S-S-: Disulfide
bonds in the hinge region to form homodimer of ICON and L-ICON1. b. SDS-PAGE
analysis shows a 50% reduction in molecular weight of L-ICON1 as compared with
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36
ICON. c. Depletion of coagulation activity of L-ICON1 as compared to those in ICON
(K341A) and ICON (WT). FVIIa: Active form of fVII (American Diagnostica) as a
positive coagulation control; FVIIa-FFR: Active site inhibited FVIIa (American
Diagnostica) as coagulation-inactive control. All fVII proteins were at FVII physiological
plasma concentration (10nM). Assays were in duplicate wells and repeated once with
consistent observations. Representative results (mean ± SEM) from two independent
experiments. d. Binding activity of L-ICON1 and ICON to MDA-MB-231 cells by flow
cytometry analysis. 2nd Ab-FITC alone or unstained cells were controls. e. Binding
activity of L-ICON1 and ICON to MDA-MB-231 cancer cells using ELISA analysis.
Human IgG was an isotype control. Representative results (mean ± SEM) in d & e from
two or three independent experiments. NS (not significant), *, **, *** and **** for
statistical significance: P values >0.05, <0.05, <0.01, <0.001 and <0.0001 by ANOVA
model, respectively.
Figure 4. L-ICON1 can mediate ADCC for TNBC and non-TNBC cancer cells. a. L-
ICON1 binding to TNBC cancer lines (MD-MB-231 and BT20) and non-TNBC line
(MCF7/MDR/TF). hIgG: Human IgG isotype control. b. L-ICON1 mediates ADCC
effector assay for TNBC and non-TNBC cancer lines. a & b: P values were analyzed by
ANOVA model. Representative results (mean ± SEM) from three independent
experiments.
Figure 5. L-ICON1 is more effective than ICON for the treatment of human TNBC
(MDA-MB-231/Luc+GFP) in an orthotopic CDX model in CB-17 SCID mice. a.
Therapeutic efficacy of L-ICON1 and ICON was compared in vivo by measuring tumor
volume using calipers (n = 5 in each group). b. Therapeutic efficacy of L-ICON1 for
TNBC was further verified by in vivo bioluminescence imaging (AdBlank and AdL-
ICON1 were the same group of mice as shown in panel a, n = 5 in each group). Arrows
in a & b indicate the dates when adenoviral vectors were intratumorally (i.t.) injected. c-
d. Photos of TNBC-bearing control mice (c) and treated mice (d) before (day 0) and after
treatment (Tx) (day 23). e. Tumor weights of control mice and L-ICON1-treated mice (P
= 0.0017). f. Whole body weights indicate that L-ICON1 has no toxicity to host mice (P
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37
= 0.2190). Tumor weights (e) and body weights (f) were measured when mice were
sacrificed on day 23. Ad: adenoviral vectors for control (AdBlank) and L-ICON1 (AdL-
ICON1). Data are presented as mean ± SEM from one experiment. P values were
analyzed by 2-way ANOVA (GraphPad). P values were analyzed by linear mixed model
with random subject effects and unequal variances over days using t-test.
Figure 6. L-ICON1 is effective for the treatment of murine TNBC (4T1/Luc+GFP)
in an orthotopic CDX model. Tumor growth curves (a) and mouse survival curves (b)
after intra-tumoral injection of AdL-ICON1 or AbBlank control vectors for murine breast
cancer 4T1 in an orthotopic model in Balb/c mice (n = 5 in each group). Representative
results (mean ± SEM) from two independent experiments. P values were analyzed by
linear mixed model with random subject effects (a) or by Kaplan-Meier analysis (b).
Figure 7. L-ICON1 is effective for the treatment of patient’s TNBC in an orthotopic
PDX mouse model in CB-17 SCID mice. a. Therapeutic efficacy of L-ICON1 was
determined in vivo by measuring tumor volume using calipers as compared to a control
vector (AdBlank)-treated PDX (n = 5 in each group). b. Tumor weights of control mice
and L-ICON1-treated mice (P = 0.0044). Tumor weights from the control mice were
measured when the control mice had to be sacrificed on day 43, whereas L-ICON1-
treated tumors were measured at the end of experiment on day 58. Intratumoral injections
of adenoviral vectors for control (AdBlank) and L-ICON1 (AdL-ICON1) were done on
days 0, 4, 7, 14, 21, 28, 35 and 42. The L-ICON1 treated mice were additionally i.t.
injected on days 49 and 56. Data are presented as Mean ± SEM from one experiment. P
values were analyzed by 2way ANOVA (a) and by t-test (b) (GraphPad).
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Published OnlineFirst April 5, 2018.Cancer Immunol Res Zhiwei Hu, Rulong Shen, Amanda Campbell, et al. Breast Cancer Using a Second-Generation ICONTargeting Tissue Factor for Immunotherapy of Triple-Negative
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