Targeting Tissue Factor for Immunotherapy of Triple ... · human IgG1 Fc. We named this construct...

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.

on July 2, 2020. © 2018 American Association for Cancer Research. cancerimmunolres.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 April 5, 2018; DOI: 10.1158/2326-6066.CIR-17-0343

http://cancerimmunolres.aacrjournals.org/

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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.




15

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




16

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




17

(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 =




18

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




19

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




20

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).




21

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




22

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.




23

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




24

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).




25

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.




26

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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




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




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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