targeting tissue factor for immunotherapy of triple ... · research article targeting tissue factor...

Research Article

Targeting Tissue Factor for Immunotherapyof Triple-Negative Breast Cancer Using aSecond-Generation ICONZhiwei Hu1, Rulong Shen2, Amanda Campbell3, Elizabeth McMichael3, Lianbo Yu4,Bhuvaneswari Ramaswamy5, Cheryl A. London6, Tian Xu7, and William E. Carson III1

Abstract

Triple-negative breast cancer (TNBC) is a leading cause of breastcancer death and is often associated with BRCA1 and BRCA2mutation.Due to the lackof validated targetmolecules, no targetedtherapy for TNBC is approved. Tissue factor (TF) is a common yetspecific surface target receptor for cancer cells, tumor vascularendothelial cells, and cancer stem cells in several types of solidcancers, including breast cancer. Here, we report evidence support-ing the idea that TF is a surface target in TNBC. We used in vitrocancer lines and in vivo tumor xenografts inmice, all withBRCA1orBRCA2mutations, derived from patients' tumors. We showed thatTF is overexpressed on TNBC cells and tumor neovasculature in50% to 85% of TNBC patients (n ¼ 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 lightchain ICON) with improved efficacy and safety profiles comparedwith the original ICON. We showed that L-ICON1 kills TNBCcells in vitro via antibody-dependent cell-mediated cytotoxicityand can be used to treat human and murine TNBC CDX as wellas PDX in vivo in orthotopic mouse models. Thus, TF could be auseful target for the development of immunotherapeutics forTNBC patients, with or without BRCA1 and BRCA2 mutations.Cancer Immunol Res; 6(6); 1–14. �2018 AACR.

IntroductionBreast cancer is themost common newly diagnosed cancer and

second leading cause of cancer death amongwomen in theUnitedStates in 2016 (1). Breast cancer deaths are largely the result ofrecurrent and metastatic disease that typically does not respondwell to current treatments (2). Triple-negative breast cancer(TNBC), so-called because the tumor cells lack expression of threetargetable proteins, the estrogen receptor, the progesterone recep-tor, and the epidermal growth factor receptor HER2, is difficult totreat malignancies and is usually considered incurable (3–10).TNBC comprises approximately 15%of globally diagnosed breastcancer (4, 11, 12). At present, there are no molecularly targetedtherapies approved for TNBC.

TNBC is often associated with mutations in breast cancerpredisposition genes BRCA1 and BRCA2 (13). The identifica-tion of BRCA1 (14) and BRCA2 (13) in the mid-1990s has ledto a better understanding of the molecular pathogenesis ofhereditary breast cancer. BRCA1 and BRCA2 are human genesencoding tumor suppressor proteins that ensure the stability ofDNA. When either of these genes is mutated, or altered, suchthat its protein product either is not made or does not functioncorrectly, DNA damage may not be repaired properly. As aresult, BRCA1 and BRCA2 mutations are associated with anincreased risk for female breast and ovarian cancer. Approxi-mately 20% of TNBC patients harbor a mutation in either theBRCA1 or BRCA2 genes (15). Thus, identification of biomarkersand oncotargets for BRCA1- and 2-associated hereditary breastcancer and development of corresponding targeted immuno-therapy could provide efficacious treatment regimens for TNBC,with or without BRCA1 and BRCA2 mutation, and reduce themortality associated with the malignancy.

To identify a therapeutic target in TNBC, we focused on tissuefactor (TF), based on previous findings in breast cancer patients(16) and human breast tumor xenografts from animal studies(17–20). TF is a 47-kDa membrane-bound cell surface receptorthat initiates the extrinsic coagulation cascade pathway uponinjury to vessel wall integrity (21–23) and modulates angio-genesis (24–27). It is highly expressed in many types of solidcancers (28, 29), including non-TNBC. In breast cancer, TFexpression can be detected on both the breast cancer cells andthe tumor vascular endothelial cells (VECs) in breast cancerpatients (16) and in a mouse model of human breast cancer(17), but not in adjacent normal breast tissues (16). Wereported that TF is an angiogenic-specific receptor in VEGF-stimulated angiogenic vascular endothelial models (30) and is

1Department of Surgery Division of Surgical Oncology, TheOhio State UniversityWexner Medical Center and The OSU James Comprehensive Cancer Center,Columbus, Ohio. 2Department of Pathology, The Ohio State University WexnerMedical Center, Columbus, Ohio. 3Biomedical Sciences Graduate Program, TheOhio State University, Columbus, Ohio. 4Center for Biostatistics, The Ohio StateUniversity, Columbus, Ohio. 5Department of Medical Oncology, The Ohio StateUniversity Wexner Medical Center, Columbus, Ohio. 6Department of VeterinaryBiosciences, College of Veterinary Medicine, The Ohio State University, Colum-bus, Ohio. 7Department of Genetics, Yale University School of Medicine, NewHaven, Connecticut.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Zhiwei Hu, The Ohio State University Wexner MedicalCenter, 420 West 12th Avenue, Columbus, OH 43210. Phone: 614-685-4606;Fax: 614-688-4028; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0343

�2018 American Association for Cancer Research.

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also could be a target for cancer stem cells (CSC) in severaltypes of solid cancer (31), including breast cancer. Otherstudies have also connected TF with TNBC (18, 19). However,it is still not known whether TF is expressed in TNBC cells andtumor neovasculature, and if so, to what extent. If TF is indeedhighly and selectively expressed in TNBC, it could serve as atarget for immunotherapy of TNBC.

TF expression has been detected previously on other types ofsolid cancers, leukemia, and sarcomas (28, 29), including mel-anoma (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-targetingimmuno-conjugate agent named ICON, which consists of full-length factor VII peptide (406 amino-acid residues, aa) fused tothe Fc region of IgG1 (32–35), in which fVII, the TF-targetingdomain, binds TF with specificity (30) and high affinity [pico-molar dissociation constant (Kd); ref. 36], and the IgG1 Fc bindscomplement and Fc receptors (CD16) on immune cells such asnatural killer (NK) cells. After binding to the Fc domain, NKcells and the complement cascade kill ICON-bound targetcancer cells via antibody-dependent cell-mediated cytotoxicity(ADCC) and complement-dependent cytotoxicity (CDC),respectively (35). Intralesional ICON therapy of experimentalmurine and human melanoma (32, 33), prostate (34), andhead and neck tumors (35) with an ICON adenoviral vectorleads to growth inhibition of tumors with minimal effects onnormal tissues (33). However, ICON is large (210 kDa; ref. 35).In addition, hypercoagulation is documented in virtually allcancer types (37), albeit at different rates, and is a secondleading cause of death in cancer patients (38). It is partiallythe result of TF produced by and released from cancer cells(39, 40). To eliminate the coagulation activity of fVII in theICON, a coagulation-active lysine reside at position 341(K341A) was mutated to an alanine in fVII peptide by site-directed mutagenesis (34). With this mutation (K341A), theprocoagulant effects of ICON were reduced but not eliminated(34). In the current work, we developed a strategy to eliminatethe procoagulant effects of ICON, reduce its molecular mass,and retain its binding activity to TF so that ICON could be moreeffective for treatment of cancer patients when administeredsystemically.

To identify TF as a surface target in TNBC, we investigated TFexpression on TNBC cells and tumor VECs in TNBC cell–derivedxenografts (CDX) and patient-derived xenografts (PDX) frommice as well as in a total of 161 TNBC tumor tissues from patientswith TNBC, including 14 cases of paraffin-embedded wholetumor tissues and 147 cases of TNBC tumors on tissuemicroarray(TMA) slides. Because TNBC is associatedwithBRCA1 andBRCA2mutations (41), we also investigated TF expression on humanTNBC cell lines and in CDX and PDX tumors with or withoutBRCA1 or BRCA2 mutations.

To develop a TF-targeting ICON with improved safety fortargeted immunotherapy of TNBC, we modified ICON byremoving the heavy chain (153–406 aa) of fVII. The resultingconstruct contains the noncoagulating fVII light chain (1–152aa) fused to human IgG1 Fc. We named this construct lightchain ICON or lighter ICON (L-ICON1). We compared molec-ular weights, binding activities, coagulation activities ofL-ICON1 with ICON in vitro and compared the in vivo efficacyof L-ICON1 and ICON for immunotherapy of TNBC inorthotopic CDX and PDX mouse models.

Materials and MethodsCell lines and reagents

Chinese hamster ovary cells (CHO-K1) were purchased fromthe ATCC in 2004 and were grown in F12K complete growthmedium (ATCC) supplemented with 10% heat-inactivated fetalbovine serum (HI-FBS; Sigma) and 1 � penicillin and strepto-mycin (Invitrogen). Human TNBC cell lines MDA-MB-231(BRCA1-wt, BRCA2-mutant), BT20 (BRCA1-wt, BRCA2-wt), andMDA-MB-468 (BRCA1-wt), non-TNBC lines MCF7/MDR andMCF7/MDR/TF (BRCA1-wt, BRCA2-mutant), murine breast can-cer cells 4T1 and EMT6 were purchased from ATCC in 2001 to2009 except formurine TNBC line 4T1 [kind gift fromDr.WilliamCarson at The Ohio State University (OSU), 2013] and humannon-TNBC linesMCF7andMCF7/MDR(kindgift ofDr. Zping Linat YaleUniversity, 2008). The cancer cellswere grown inDMEMorRPMI1640 complete growth medium supplemented with10% HI-FBS and 1 � penicillin and streptomycin (Invitrogen).MCF7/MDR line is a low TF-expressingMCF7 line withmultidrugresistance (MCF7/MDR; ref. 17) and MCF7/MDR/TF is a high TF-expressing MCF7/MDR (MCF7/MDR/TF) derived fromMCF7/MDR by being infected with retroviral vector encoding TF(kind gift of Dr. Michael Bromberg; ref. 42). MDA-MB-231 and4T1 lines were transfected using xtremeGene HP transfectionreagent (Roche, 3 mL transfection reagent: 1 mg plasmid DNA;Roche) with plasmid vectors (1 mg ACT PBase and 1 mg PBTransposon, constructed and kindly provided by Dr. Tian Xu'slaboratory at Yale University) encoding luciferase (Luc) and greenfluorescent protein (GFP). The transfected cell lines were selectedand maintained for stable expression of Luc and GFP by supple-menting growth media with blasticidin (Invitrogen; at a finalconcentration of 10 mg/mL for selection and 5 mg/mL for main-taining the cell culture). Following the manufacturer's instruc-tions, ADCC effector cells, a Jurkat-based cell line transfected withCD16 andN-FAT/Luciferase, were purchased from Promega (Cat.No. G7102) in 2014 and were grown in RPMI 1640 completegrowth medium supplemented with 100 mg/mL G418, 250 mg/mL hygromycin, 10% HI-FBS, 1 � nonessential amino acids, 1 �sodium pyruvate and 1 � penicillin and streptomycin (Invitro-gen). The 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, 0.1mmol/LMEMNon-EssentialAmino Acids, 2 mmol/L L-glutamine and 1 � penicillin andstreptomycin (Invitrogen). All cell cultures were maintainedmycoplasma free by routinely supplementing with Plasmocin(Invivogen, Cat. No. Ant-mpt, 25 mg/mL for treatment and5 mg/mL for maintenance) or Ciprofloxacin HCl (Enzo, Cat.No. 380-288-G025, 10 mg/mL) in cell culture medium. Cultureswere tested annually for mycoplasma contamination. The latestdate they were tested by using Mycoalert Mycoplasma DetectionKit (Lonza, Cat. No. LT07-118)was June 26, 2017. Authenticity ofcancer cell lines was determined by validating TF expression byflow cytometry, Western blots, and/or cell ELISA, as describedbelow. Thawed cancer cells were maintained in culture for 1month. CHO-K1 producer cells for L-ICON1 protein productionwere maintained for 2 months.

IHC for staining TF expressionThe use of patients' tissues with nonidentifiable information

was reviewed and approved by OSU Cancer Institutional ReviewBoard and was conducted following the OSU Human Research

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Protection Program in accordance with the ethical principles andguidelines summarized in the Belmont Report (https://web.archive.org/web/20111017133845/http://www.hhs.gov/ohrp/archive/documents/19790418.pdf). Fourteen TNBC tumor tis-sues, confirmed by pathology diagnosis to lack detectable estro-gen receptor, progesterone receptor and epidermal growth factorreceptor Her2, were obtained from The Cooperative HumanTissue Network of OSU. Generation of OSU TNBC TMAs waspreviously described (43). TMA slides with 147 cases of TNBCtumor tissues and 147 cases of matched normal breast tissues(usually adjacent to the tumor tissues) were obtained from OSUPathology. For each patient, TMA slides had two cores of tumortissues and two cores of matched normal tissue adjacent tumor.Some samples were lost as samples detached from the slidesduring IHC staining. Some tumor tissues that did not containtumor cells were excluded. TF expression was stained with amouse monoclonal antibody (HTF1, kind gift of Dr. WilliamKonigsberg at Yale University) and/or with a commercial goatanti-human TF (Sekisui Diagnostics, REF 4501), both of whichwe showed bind to human TF, the same antigen/receptor thatis recognized by ICONs and fVII peptides (30). The detailedprocedures have been similarly described in our previouspublications (17, 44).

The IHC procedures for staining TF and CD31 in TNBC TMAslides were optimized and performed by theOSU Pathology CoreFacility. Paraffin-embedded 4-mm TMA slides of patients' TNBCtumors were placed on a 60�C oven for 1 hour, cooled, depar-affinized, and rehydrated through xylene and graded ethanolsolutions to water. All slides were quenched for 5 minutes in a3%hydrogen peroxide aqueous solution to block for endogenousperoxidase. Antigen retrieval was performed in a 1� solutionof Target Retrieval Solution pH6.0 (Dako) by Heat-InducedEpitope Retrieval for 25 minutes at 96�C using a vegetablesteamer (Black & Decker). Slides were stained with the Intelli-path Autostainer Immunostaining System using mouse mono-clonal anti-human TF antibody (HTF1, 1:100, 1 mg/mL stock)for staining TF or using mouse monoclonal anti-CD31 (1:80,culture supernatant; Dako, Cat. M0823, Clone JC70A) forstaining endothelial cells at room temperature for 30 minutes,followed by MACH3 Mouse HRP-Polymer Detection (BiocareMedical) for 20 minutes at room temperature and then by 5-minute incubation with liquid DABþ Chromogen (Dako). TFstaining was verified by using a commercial polyclonal anti-body against human TF (Sekisui Diagnostics, REF 4501). Theslides were counterstained in Richard Allen hematoxylin(Thermo Fisher Scientific), dehydrated through graded ethanolsolutions, cleared with xylene, and coverslipped.

The IHCprocedure for CDXand PDX tumorswas performedbythe 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 fromformalin-fixed and paraffin-embedded control tumors from pre-viously published mouse studies (20, 45) or from an untreatedPDX mouse in this study. Paraffin-embedded 4-mm tissue slideswere deparaffinized, rehydrated, pretreatedwithAntigenRetrievalSolution and then 3% hydrogen peroxide, blocked with serum-free protein and stained with 5 mg/mL of goat anti-human TFantibody (with cross reaction with murine, rat and rabbit TF,Sekisui Diagnostics, REF 4501) or anti-CD31 antibody (1:80,Dako, Cat. M0823, Clone JC70A) diluted in Dako antibody

diluent with background reducing agents. Finally, the sectionswere incubated using 2 mg/mL of Vector biotinylated rabbit anti-goat IgG secondary Ab followed by Vector TRUABCElite complexfor anti-TF Ab or MACH3 Mouse HRP-Polymer Detection(Biocare Medical) for anti-CD31 Ab. Positive staining for VECsand TF was visualized by Nova Red substrate. Cell nuclei werecounterstained by hematoxylin. Human MDA-MB-231 CDX andPDX slides were also stained with hematoxylin and eosin (H&E)by routine methods for visualization of tumor pathology.

After IHC staining, two investigators, including a board-certi-fied anatomic pathologist, independently reviewed and scoredthe stained slides, and representative views were photographed.The scores for TF expression were graded as follows: negative (�),moderately positive (þ), positive (þþ), strongly positive (þþþ),and very strongly positive (þþþþ). Positive percentages includedall cases graded from moderately positive through very stronglypositive.

Construction of plasmid DNA vectors encoding murine andhuman L-ICON1

Construction of an expression plasmid vector pcDNA3.1(þ)encoding cDNA for the wild-type (WT) human fVII fused tohuman IgG1 Fc has been described (32, 34). Plasmid vectorspcDNA3.1(þ) encoding mouse and human L-ICON1 were sim-ilarly constructed using recombinantDNA techniques. Briefly, thecDNA for the first 152 amino acids ofmouse and human fVII lightchain were PCR amplified from the plasmid vectors encodingtheir parental ICONs. We used primers as follows. The cDNAs ofhuman and mouse fVII light chain were subcloned in frame intopcDNA3.1(þ) containing human IgG1 Fc between EcoRI andBamHI. The cDNA sequences of ICON and L-ICON1were verifiedby Sanger-based DNA sequencing at the Genomic SharedResources at Yale University and at OSU. The cDNA sequence ofL-ICON1 is deposited with accession no. KX760097 at GenBank.To make recombinant L-ICON1 proteins, the plasmid vectorsencoding mouse and human L-ICON1 cDNAs were transfectedinto CHO-K1 cells (ATCC) using xtremeGene HP transfectionreagent (Roche). The stably transfected L-ICON1 producerCHO-K1 cells were selected under 1 mg/mL G418 (Gibco) andwere cultured in suspension at a start density of 2.5� 105 cells/mLin CHO serum-free medium (SFM4CHO, Hyclone) supplemen-ted with 1 mg/mL vitamin K1 (Sigma). The SFM was collectedtwice a week, and L-ICON1 protein was purified from the super-natants of SFM by a 5 mL HiTrap rProtein A FF affinity column(GE Healthcare) following the manufacturer's instructions, asdescribed (35). Molecular weights of L-ICON1 and ICON pro-teins were analyzed by 8% to 16% SDS-PAGE nonreducing gel(Bio-Rad Laboratories). The concentration of L-ICON1 proteinwas determined by Protein Assay Reagent (Bio-Rad Laboratories)using bovine serum albumin standards (Pierce).

Construction and production of adenoviral vectorsencoding L-ICON1

The adenoviral system we used to make adenoviral vectorencoding L-ICON1 was RAPAD CMV Adenoviral ExpressionSystem (Cell Biolabs, Cat. No. VPK-252). Briefly, the cDNA ofL-ICON1 was subcloned into the pacAd5 CMVK-NpA shuttlevector between EcoRI and NotI. Pac I–digested shuttle vectorpacAd5 CMV-L-ICON1 and the Ad backbone DNA (pacAd59.2-100) were separated by Agarose gel electrophoresis, recoveredfrom the gel slices, and cotransfected of the Pac I–digested shuttle

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vector encoding L-ICON1 and the backbone DNA (at a ratio of 4mg:1 mg) into adenoviral packaging cell line 293AD (Cell BioLabs)using xtremeGene HP transfection reagent (Roche) following themanufacturer's instructions. Ablank control vector (AdBlank)wassimilarly produced using the pacAd5 CMVK-NpA vector withoutencoding a transgene and padAd5 9.2-100 backbone DNA. Theadenoviral vectors were amplified by infecting 293AD cells (CellBiolabs) and purified by CsCl (Sigma) ultracentrifugation, asdescribed (32–35). The titer of infectious viral particles wasdetermined by measuring Optical density (OD) of 260 nm of20-fold diluted viral particles in 0.1% sodium dodecyl sulfate(SDS) and calculated using the following formula: 1 OD260 nm¼ 1 � 1012 viral particles per mL, as described (34). OD260 nmwas assessed on Spectrophotometer (Beckman, Model DU650).To verify the production of L-ICON1 protein by AdL-ICON1vectors, MDA-MB-231 cancer cells in 6-well plate were infectedwithCsCl-purifiedAdL-ICON1 (GenBankKX760097), AdhICON(human ICON, hfVII/hIgG1Fc; GenBank AF272774), AdmICON(mouse ICON, mfVII/hIgG1Fc; GenBank AF272773), andAdBlank (as negative control) vectors at an MOI of 3,000:1 byincubating viral vectors with cancer cells in serum containinggrowth medium at 37�C and 5% CO2 for 2 hours. Next morning,the cells were washed once with 1 � DPBS and grown inSFM4CHO supplemented with 1 mg/mL Vitamin K1 (Sigma) for4 days. The supernatants of SFM from viral vector-infected cancercells were assayed in MDA-MB-231 cancer cell ELISA above fordetermining the concentration and binding activity of L-ICON1and ICONs. The L-ICON1 and ICONproteins in SFMwere furtherconfirmed by immunoprecipitation-Western blot (IP-WB), asdescribed below.

Factor VII coagulation activity assayFollowing the manufacturer's instruction, the retained coag-

ulation activity of fVII light chain peptide in the L-ICON1protein was determined by Factor VII Human ChromogenicActivity Kit (Abcam), which was developed to determinehuman fVII coagulation activity, specifically the ability of fVIIto bind and form a complex with TF and then to activate factorX (fX) to fXa. The amidolytic activity of the TF/FVIIa complexwas quantitated by the amount of fXa produced using an FXasubstrate that releases a yellow para-nitroaniline (pNA) chro-mophore. The change in absorbance of the pNA at 405 nm(A405 nm) is directly proportional to the FVII enzymatic(coagulation) activity.

Briefly, L-ICON1 protein at 1.25 to 10 nmol/L was added to a96-well strip assay plate, in which a polyclonal antibody againsthuman FVII was precoated. After 2-hour incubation at 37�C, thestrip plate was washed 5 times with wash buffer and then thefreshly prepared assay mix, which contains recombinant humanTF and FX, was added and incubated at 37�C for 30 minutesfollowed by adding FXa substrate. The absorbance at 405 nmwasread every 5 minutes for 30 minutes on a microplate reader(SpectraMax i3, Molecular Devices). The chromogenic activitycurve of human FVII standard was generated by serially diluting astock 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 wasan active form of FVII (FVIIa; America Diagnostica) and thenegative control was an active site inhibited FVIIa (FFR-FVIIa,America Diagnostica). For comparison with its parental ICONs,active-sitemutated ICON (hfVIIK341A-hIgG1Fc or ICONK341A)and ICON WT (without active-site mutation) were also tested in

parallel with L-ICON1. All of these proteins were tested at 10nmol/L, a physiological concentration of FVII in circulation, with1:2 serial dilutions to 5, 2.5, and 1.25 nmol/L.

Flow cytometry and cancer cell ELISA for binding activity assaysThe binding activity of L-ICON1was assayed by flow cytometry

and enzyme-linked immunosorbent assay (ELISA), as previouslydescribed (12, 32, 34, 35). Briefly, the flow cytometry procedureinvolves incubationof nonenzymatic dissociated cancer cellswith10 mg/mL L-ICON1 or ICONproteins for their binding activity, ormouse monoclonal anti-Human TF antibody (anti-HTF1, kindgift of Dr. William Konigsberg of Yale University) for TF expres-sion followed by 1:50 diluted FITC conjugated anti-human IgGoranti-mouse IgG conjugate (Sigma), respectively. Controls wereincubated with normal human IgG or mouse IgG as isotypecontrols in replacement of L-ICON1orHTF1 antibody. For cancercell ELISA, cancer cells (MDA-MB-231, BT20, MCF7/MDR/TF,4T1, and EMT6) were seeded at 1 � 104 cells per well in 96-wellflat microplates. When the cells reached about 95% confluence,they were washed once with warm PBS, fixed in 4% paraformal-dehyde at room temperature for 20 minutes, blocked with 1%BSA in calcium containing buffer (1� TBS/Ca, 10 mmol/LTris–HCl pH8.0, 150mmol/L NaCl, 10 mmol/L CaCl2) and thenincubated with L-ICON1, ICON, and an isotype control (humanIgG, Sigma) proteins in 1� TBS/Ca buffer followed by incubationwith HRP-conjugated anti-human IgG1Fc (human specific,1:10,000; Sigma) for detection of human IgG1 Fc portion ofICON and L-ICON1 proteins. After final wash, OPD was addedas substrate and the absorbance at 490nm(A490nm)was readonSpectraMax i3 microplate reader (Molecular Devices).

Sandwich ELISA assays for measuring the concentration ofL-ICON1 in cell culture medium and mouse plasma samples

L-ICON1 concentration was determined by paired factor VIIantibody Sandwich ELISA (Cedarlane Laboratories) following themanufacturer's instructions with modifications as follows. Anti-human fVII capture antibody (1:200) was coated in EIA 96-wellstrip plates, was blocked with 1% BSA, and then incubated withcell culture supernatants in serum-free medium (SFM4CHO) or1:5 diluted mouse plasma samples in PBS followed by HRP-conjugated anti-human IgG1Fc (human specific, 1:10,000) fordetection of full-length peptides of ICON and L-ICON1, asdescribed above. The standard proteins in Sandwich ELISA wereHiTrap rProtein A affinity purified-ICON and L-ICON1 proteins,as described above and earlier (35).

ADCC effector assayThe effect of L-ICON1–mediated ADCC to TNBC cells was

determined using an ADCC effector assay (Promega; ref. 46)following the manufacturer's instruction with a modification tothe assay medium by using DMEM medium, instead of RPMI1640. DMEM medium contains a higher calcium concentration(1.8 mmol/L) than RMPI 1640 (0.42 mmol/L) per the informa-tion on calcium concentrations in cell culture medium on theSigma-Aldrich website (calcium is required for ICON and fVIIbinding to its receptor TF; ref. 32). Briefly, human TNBC (MDA-MB-231 and BT20) and non-TNBC (MCF7/MDR/TF) cells (1 �104 cells per well in 100 mL growth medium) were seeded induplicate into the 60 inner wells and grown overnight in 96-wellwhite assay plate (Corning) at 37�C and 5% CO2. Then, 95 mL ofgrowth medium was removed from each well. Prewarmed 25 mL

Hu et al.





ADCC effector assay medium (DMEM supplemented with 0.5%super low IgG fetal bovine serum (HyClone) was added to 60inner wells, whereas 75 mL ADCC effector assay medium wasadded to 36 outer wells. Then, 25 mL of ICON protein at serialdilutions of 3 � or 5 � concentrations was added in duplicatewells to the cancer cells, whereas human IgG (Sigma) was used asan IgG isotype control. After 15-minute incubation at 37�C, 25mL ADCC effector cells (Promega; a T cell–derived leukemia cellline Jurkat transfected with Fc receptor CD16 and NFAT-Lucif-erase under the control of CD16 binding followed by NFATactivation) were added (2.5 � 105 cells per well) to achieve aratio of effector to target (E:T) at 25:1 and incubated for 6 or19 hours at 37�C and 5% CO2. At the end of 6-hour or 19-hourincubation, the plate and Bio-Glo assay reagent (Promega)were equilibrated at room temperature for 15 minutes andthen equal volume (75 mL) of Bio-Glo reagents was added to 60inner wells and two outer wells with medium only as back-ground control. Raw bioluminescence (RLU) was read onSpectraMax i3 (Molecular Devices).

Generation of orthotopic mouse models of TNBCCDXs and PDXs

The animal study protocol was reviewed and approved by theInstitutional Animal Care and Use Committee of OSU. Togenerate orthotopic mouse models of tumor line–derivedxenografts, 5 � 105 human TNBC MDA-MB-231/LucþGFP cellsin 50 mL of 1:1 mixed PBS/Matrigel (BD Biosciences) or 5 � 105

murine TNBC 4T1/LucþGFP cells in 50 mL PBS were injectedinto the fourth left mammary gland fat pad in 4 to 6-week-old,female CB-17 SCID (Taconic Farms) or Balb/c mice (TheCharles River Laboratory), respectively. Both CB-17 SCID andBalb/c mice have intact and functional NK cells that are crucialto mediate ICON and antibody therapy (35). To generate an

orthotopic TNBC PDX model, we purchased a TNBC PDXdonor NSG mouse (NOD SCID gamma, no mature B, T, NKcells and no complement) with BRCA1 mutation from TheJackson Laboratory (JAX TM00089, breast tumor markers:TNBC ER�/PR�/HER2�, BRCA1 V757fs). Surgeries were per-formed in accordance with the OSU IACUC Rodent SurgeryPolicy. Mice were anesthetized with isoflurane. For the donorNSG mouse (JAX, TM00089), an incision was made over theflank of tumor to extract the tumor tissue, which was washedonce in RPMI medium supplemented with 1� penicillin andstreptomycin and was cut in sterile PBS into �3-mm pieces indiameter for implantation into the recipient mice. CB-17 SCIDmice (Taconic Farms) were used as recipient mice. For recipientmice, an incision was made over the fourth right mammarygland, for direct implantation of one piece of �3-mm PDXtumor tissues from the donor tumor tissue into it. One drop oftissue adhesive (Vetbond) and/or 1 wound clip (Sigma) wasused to close the incision site in routine fashion. Animals weremonitored for recovery from anesthesia before returning toroutine husbandry. Animals were checked daily until woundswere healed, and analgesics (buprenorphine, s.c. 0.1 mg/kg in100 mL sterile PBS) were administered to provide pain relief forup to 72 hours after surgery.

Adenoviral gene therapy using AdL-ICON1 in vivo in mousemodels of human and murine TNBC CDX and PDX

When tumor size reached 150 to 200 mm3, the mice wererandomized into control, L-ICON1–treated or ICON-treatedgroups. The control mice were intratumorally (i.t.) injectedwith 1 or 4 � 1010 VP of AdBlank and the treated mice wereinjected with 1 or 4 � 1010 VP of AdL-ICON1 or AdICONvectors weekly for orthotopic human TNBC CDX and PDX orevery 4 days for orthotopic 4T1 CDX, as specified in figure

Figure 1.

TF is an oncotarget and a surfacemarker for the TNBC cancer cells andtumorVECs.A,TFexpressiononTNBCcancer cells (TF-positive staining inbrown, arrows); B, TF expression onthe TNBC VECs (TF-positive stainingin brown, arrowheads); C, No TFexpression in normal breast glandtissues with adenosis (TF negative);D, IHC staining with a mouse IgGisotype as a negative control. Originalmagnification: 400 � in A–C and100 � in D. TF expression shownabove was stained with a mousemonoclonal antibody (HTF1) and wasindependently verified with goat anti-human TF (Sekisui Diagnostics).






legends. Therapeutic efficacy was determined by measuringtumor 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 alsomonitored using small animal imaging for bioluminescenceand fluorescence (IVIS, Caliper) before the first treatment,during treatment, and at the end of experiment. At the timeof sacrifice, i.e., 2 days after last intralesional injection of AdL-ICON1 or AdBlank, mouse plasma samples were prepared fromthe L-ICON1–treated and control SCID mice bearing orthoto-pic TNBC CDX tumors and were assayed for L-ICON1 proteinconcentration by ELISA, as described above.

In vivo mouse models and bioluminescence imaging of breastcancer xenografts

To image tumor cells that stably express Luc and GFP, the micewere i.p. injected with 100 mL of luciferin solution. Ten minutesafter injection of luciferin, themicewere imaged for quantitativelymeasuring tumor bioluminescence intensity under anesthesiausing inhalation of isoflorane under IVIS Bioluminescence Imag-ing System (Caliper). The results from IVIS imaging were pre-sented as RLU.

Statistical analysisThe 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) andSAS 9.4 software. Fisher exact test was used to test IHC scorepercentage difference between whole tumor tissues and TMAtissues. For analyses of statistical significance, duplicate ortriplicate wells in each group were used for in vitro assays intissue culture plates and 5 mice per group were used in vivo inanimal studies. Statistical significance is presented as �, P < 0.05;��, P < 0.01; ��, P < 0.001; ��, P < 0.0001 and "ns" stands forno (statistical) significance.

ResultsTF is expressed by TNBC cancer cells and tumor VECs

To examine TF expression in TNBC with WT and mutatedBRCA1 and BRCA2, we first determined TF expression on TNBClines. Supplementary Fig. S1 showed that TF was expressed byTNBC 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; Supple-mentary Fig. S1B), andMDA-MB-468 (BRCA1-wt; SupplementaryFig. S1C). To verify the binding specificity for TF of the mono-clonal anti-human TF (HTF1) used in the flow cytometry, weinfected a non-TNBC human breast cancer MCF7/MDR line(Supplementary Fig. S1D), which was derived from the MCF7line (BRCA1-WT, BRCA2-mutant) with multidrug resistance(MDR) and both are known for low TF expression (17), with a

Figure 2.

TF expression on TNBC cells, but noton normal breast tissue, on TMA slides.A, TF expression on TMA with twocores from tumor tissues (top row,TF-positive cells stain brown)and two cores from normal tissues(bottom row, no TF expression) fromthe same TNBC patient (40�).B, TF expression on the TNBC cells(brown staining; 400 �). C, No TFexpression in normal breast tissues(400�). D, Immunohistochemicalstaining for TF expression on TNBCcells in TNBC whole tumor tissues andTMA tissues. TF expression shownabove was stained with a mousemonoclonal antibody (HTF1) and wasindependently verified with goat anti-human TF (Sekisui Diagnostics). � , Thescores for TF expression were gradedas follows: negative (�), moderatelypositive (þ), positive (þþ), stronglypositive (þþþ), and very stronglypositive (þþþþ). Positivepercentages included all cases gradedfrom moderately positive throughvery strongly positive. �� , Fisher exacttest was used to test IHC scorepercentage difference betweenwhole tumor tissues and TMA tissues;there is a significant differencewith P ¼ 0.0002474.

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retroviral vector encoding human TF (42). We showed that theresulting MCF7/MDR/TF line expressed TF as detected by anti-TF(Supplementary Fig. S1E).

To translate the findings from TNBC cell lines to preclinicalmouse models of TNBC, we examined TF expression in humanTNBCCDX and PDX tumor tissues from severe-combined immu-nodeficiency (SCID) mice. With IHC, TF expression was detectedon the TNBC cells (Supplementary Fig. S2A) in TNBCCDX tissues,but not in adjacent normal tissues (Supplementary Fig. S2A). Inaddition, TF expression was also detected on the TNBC cells(Supplementary Fig. S2C) and tumor VECs (Supplementary Fig.S2C) in TNBC PDX tumors, whose vascular origin was confirmedbypositive staining for endothelialmarkerCD31 (SupplementaryFig. S2D). Isotype Ab control (Supplementary Fig. S2B) andsecond Ab alone (Supplementary Fig. S2E) were used as negativecontrols. Mitosis was observed in PDX tumor tissues (Supple-mentary Fig. S2C–S2F).

To translate the findings frommousemodels to TNBC patients,we then examined TF expression in primary TNBC tumor tissuesfrom 14 patients. By IHC, 12 of 14 (85.7%) TNBC tumor tissuesexpressed TF on the surface of TNBC cells (Fig. 1A; SupplementaryFig. S2C). Nine patients who expressed TF on tumor cells alsoexpressed TF on the surface of tumor VECs (64.3%of 14 cases; Fig.1B). The endothelial origin of tumor VECs was verified throughexpression of endothelial marker CD31 (Supplementary Fig.S3A). Normal breast gland cells and normal VECs in adjacentnormal breast tissues or adenosis (a benign inflammation withenlarged milk-producing glands but not a cancer) were negativefor TF expression (Fig. 1C). Mouse IgG isotype was used as anegative control (Fig. 1D).

In light of these results, and to determine statistical signif-icance, we investigated TF expression in 147 additional cases ofTNBC tumors and 147 matched normal breast tissues fromTNBC patients via TMA. Of TNBC tumor samples, 71 of 147

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 ICON. C, Depletion of coagulation activity of L-ICON1 ascompared with those in ICON (K341A) and ICON (WT). FVIIa: Active form of fVII (American Diagnostica) as a positive coagulation control; FVIIa-FFR: Active-siteinhibited FVIIa (American Diagnostica) as coagulation-inactive control. All fVII proteins were at FVII physiological plasma concentration (10 nmol/L). Assayswere in duplicate wells and repeated once with consistent observations. Representative results (mean � SEM) from two independent experiments. D, Bindingactivity of L-ICON1 and ICON to MDA-MB-231 cells by flow cytometry analysis. Second Ab-FITC alone or unstained cells were controls. E, Binding activity ofL-ICON1 and ICON to MDA-MB-231 cancer cells using ELISA analysis. Human IgG was an isotype control. Representative results (mean � SEM) in D andE from two or three independent experiments. NS (not significant), � , �� , �� , and �� indicate statistical significance: P values >0.05, <0.05, <0.01, <0.001,and <0.0001 by ANOVA model, respectively.






(48.3%) were positive for TF expression on the surface ofTNBC cancer cells (Fig. 2A, B and D) and 11 of 147 (7.5%)were positive for TF on tumor VECs (Supplementary Fig. S3Band S3C), whereas normal breast tissues (n ¼ 147) werenegative for TF expression (Fig. 2A and C; SupplementaryFig. S3D). There was a significant difference of IHC scorepercentage of TF expression on TNBC cancer cells betweenwhole tumor tissues and TMA tissues (P ¼ 0.0002474, Fisherexact test; Fig. 2D).

Modification in L-ICON1eliminated its procoagulant effectsTo target TF, an oncotarget for TNBC, we developed a

second-generation ICON by modifying the first-generationTF-targeting ICON (Fig. 3A). Following the tradition of makingmouse and human ICONs (32, 34) so that the mouse versioncan be tested in mouse studies and the human version canbe used in future clinical trials, mouse and human L-ICON1swere constructed (Supplementary Fig. S4A). DNA sequencinganalysis confirmed that both the mouse (mL-ICON1) andhuman L-ICON1 (hL-ICON1 or L-ICON1; GenBank accessionno. KX760097) have the same sequence of human IgG1 Fc asthose in mouse and human ICONs (GenBank accession nos.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 nonreducing gel analysisshowed that the molecular weight of Protein A-affinity purifiedL-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 inL-ICON1 was eliminated, the coagulation activity of L-ICON1was measured and compared with those of fVII coagulationactive-site mutated ICON (i.e., ICON K341A), WT-fVII ICON(i.e., ICON WT), activated form of fVII (FVIIa; as a positivecontrol), and active-site inhibited FVIIa (FFR-FVIIa; as a nega-tive control). For this assessment, L-ICON1 was used at10 nmol/L, a physiological concentration of zymogen fVII inblood circulation. Compared with the coagulation activity(100%) of FVIIa, L-ICON1 at the same concentration (10nmol/L) had undetectable coagulation activity (�6.061 �0.000%), comparable with FFR-FVIIa (�4%; P ¼ 0.0026 forL-ICON1 vs. FFR-FVIIa), whereas the first-generation ICON(K341A) and the WT ICON (WT) exhibited approximately 5%and 58% coagulation activity (P¼ 0.0104 for L-ICON1 vs. ICONK341A and P ¼ 0.0179 for L-ICON1 vs. ICON WT), respectively(Fig. 3C; Supplementary Fig. S5; Supplementary Table S1).

Figure 4.

L-ICON1 can mediate ADCC for TNBCand non-TNBC cancer cells.A, L-ICON1binding to TNBC cancer lines(MD-MB-231 and BT20) andnon-TNBC line (MCF7/MDR/TF). hIgG:Human IgG isotype control.B, L-ICON1mediates ADCC effector assay forTNBC and non-TNBC cancer lines. Aand B: P values were analyzed by theANOVAmodel. Representative results(mean � SEM) from threeindependent experiments.

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ICON and L-ICON1 similarly bound TNBC and non-TNBCcancer cells

To compare L-ICON1 and ICON binding activity on cancercells, we used flow cytometry and cancer cell ELISA assays.L-ICON1 and ICON bound similarly to TNBC MDA-MB-231cells in flow cytometry assay (Fig. 3D). By cancer cell ELISA,L-ICON1 showed an increased activity to MDA-MB-231 cellscompared with ICON (P < 0.01 to 0.0001, L-ICON1 vs.ICON; Fig. 3E). The results showed that the modifications in L-ICON1did not reduce its binding activity for TF-expressing TNBC.Murine andhumanL-ICON1s couldbindboth thehuman (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 cancercells thanmL-ICON1 (P<0.0001 ; Supplementary Fig. S4B–S4D).Thus, hL-ICON1 (L-ICON1 for simplicity) was used in vitro andin vivo experiments thereafter.

L-ICON1 induced ADCCTo elucidate the mechanism of action of L-ICON1, we

performed an ADCC effector assay (Fig. 4). L-ICON1 bindsto 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; Fig. 4A). Furthermore,L-ICON1 could induce ADCC to those TNBC lines and non-TNBC line (Fig. 4B), whereas the human IgG isotype–negativecontrol had no binding (Fig. 4A) and had no ADCC effect(Fig. 4B) on those breast cancer cells. The results demonstratethat L-ICON1 can mediate ADCC effect on killing humanTNBC and non-TNBC cells.

L-ICON1 is more effective than ICON in vivo for treatment ofTNBC xenografts

To compare the efficacy of L-ICON1 and ICON, we con-structed adenoviral vectors encoding L-ICON1 (AdL-ICON1)and verified that AdL-ICON1 vectors could produce L-ICON1protein after infecting MDA-MB-231 cells in vitro, and that cellsso treated could secrete L-ICON1 protein into the cell culturemedium. Cancer cell ELISAs showed that the concentrations(mean � SD) of L-ICON1, murine ICON (mICON, murinefVII K341A fused to human IgG1Fc), and human ICON(hICON, human fVII K341A fused to human IgG1Fc) were15,842.0 � 234.4 ng/mL, 90,28.4 � 1,388.0 ng/mL and9,611.7 � 1,111.1 ng/mL in serum-free culture media 4 daysafter infection (Supplementary Table S2; P < 0.001 forAdL-ICON1 vs. AdmICON and AdhICON; no statistical

Figure 5.

L-ICON1 ismore effective than ICON forthe treatment of human TNBC(MDA-MB-231/Luc þ GFP) in anorthotopic CDX model in CB-17 SCIDmice. A, Therapeutic efficacy ofL-ICON1 and ICON was comparedin vivo by measuring tumor volumeusing calipers (n ¼ 5 in each group).B, Therapeutic efficacy of L-ICON1 forTNBC was further verified by in vivobioluminescence imaging (AdBlankand AdL-ICON1 were the same groupof mice as shown in A, n ¼ 5 in eachgroup). Arrows in A and B indicate thedates when adenoviral vectors wereintratumorally (i.t.) injected. C and D,Photos of TNBC-bearing control mice(C) and treated mice (D) before (day0) and after treatment (Tx; day 23).E, Tumor weights of control mice andL-ICON1–treated mice (P ¼ 0.0017).F, Whole body weights indicate thatL-ICON1 has no toxicity to host mice(P ¼ 0.2190). Tumor weights (E) andbodyweights (F)weremeasuredwhenmice were sacrificed on day 23. Ad:adenoviral vectors for control(AdBlank) and L-ICON1 (AdL-ICON1).Data are presented as mean � SEMfrom one experiment. P values wereanalyzed by two-way ANOVA(GraphPad).P valueswere analyzedbythe linear mixed model with randomsubject effects and unequal variancesover days using the t test.






significance for AdmICON vs. AdhICON by ordinary one-wayANOVA), whereas AdBlank did not produce any L-ICON1 orICON protein (0.0 � 0.0 ng/mL).

After having verified the production of L-ICON1 and ICONproteins by AdL-ICON1 and AdICON vectors in vitro, we com-pared the in vivo effects of ICON and L-ICON1, as intratumoralinjection of adenoviral vectors encoding L-ICON1 or ICON, forimmunotherapy of human TNBC xenografts in an orthotopicmousemodel usingMDA-MB-231/LucþGFP cell line. The resultsdemonstrated that L-ICON1 showed a significantly increasedtherapeutic effect (80% reduction as compared with controltumor on day 21) in vivo than ICON treatment (60% reductionas compared with control tumor on day 21; P¼ 0.0042; Fig. 5A).Furthermore, the results in Fig. 5 showed that L-ICON1 inhibitedorthotopic TNBC growth inmice (11% smaller on day 21 than itsstarting volume on day 0; P < 0.0001), as measured by tumorsize (83.6% reduction as compared with control tumor on day23; Fig. 5A) and by in vivo bioluminescence (83.6% reduction ascompared with control tumor on day 23; Fig. 5B–D), comparedwith control adenoviral vector (AdBlank). Tumor weights alsoshowed a significant difference ex vivo (Fig. 5E).

Body weight was assessed as an indicator of safety, perpreviously recorded models (17, 44). There was no differencein whole mouse body weights between L-ICON1 and controlgroups (Fig. 5F). At the time of sacrifice, which was 2 days afterthe last injection of AdL-ICON1 (4� 1010 VP per injection), theL-ICON1 protein was detected in mouse plasma from theAdL-ICON1–treated mice at a concentration of 794.8 �156.4 ng/mL (�8 nmol/L; �200 ng/mL L-ICON1 proteinproduced/1010 VP intralesionally injected), whereas noL-ICON1 protein was detected in the plasma samples fromcontrol vector–injected mice (Supplementary Table S2;P < 0.0001 for AdL-ICON1 vs. AdBlank).

L-ICON1 is effective and safe for treatment of murine TNBCTo examine the efficacy of L-ICON1 for murine TNBC in

immunocompetentmice, we transfected themurine breast cancerline 4T1 for stably expressing GFP and luciferase (SupplementaryFig. S6). 4T1 cells serve as a model of murine TNBC (49) forgenerating an orthotopic mouse model in syngeneic Balb/c mice.L-ICON1 showed a decrease (65.8% reduction as compared withthe control tumoronday10after initiationof treatment) in tumorsize in this murine TNBCmodel (P < 0.001; Fig. 6A). The averagetumor volume was 192mm3 at the time of initiation of treatment(day 0) in the AdL-ICON1–treated mice, which was significantlybigger (68.4%bigger) than the average tumor volume (114mm3)in the control group at the same time (P < 0.05; Fig. 6A). Micewere humanely sacrificed when evidence of extreme sicknesswas present, per IACUC protocol. On day 10 after treatment,only 1 of 5 L-ICON1–treated mice had to be sacrificed (Fig. 6A),compared with 5 of 5 (100%) control mice by the same timepoint (P¼ 0.0203 for AdL-ICON1 vs. AdBlank control; Fig. 6B).Under in vivo bioluminescence imaging, no metastasis wasobserved in both L-ICON1 and control groups at the time ofsacrifice of these animals (day 10 after treatment, i.e., day 18after 4T1 cell implantation).

L-ICON1 treated TNBC PDXs in an orthotopic mouse modelTo 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 measuredby tumor size (P < 0.0001, 93.7% reduction as compared withcontrol tumor on day 43; Fig. 7A). PDX tumors in control micegrew quickly; these mice had to be sacrificed on day 43 after thefirst i.t. injection of control vectors. Tumor weights also showed asignificant difference ex vivo (P ¼ 0.0044, L-ICON1–treatedtumors on day 58 vs. control tumors on day 43; Fig. 7B).

DiscussionTNBC not only is a significant clinical malignancy lacking

molecularly defined surface targets, but also is a molecularlyheterogeneous disease associated with BRCA1 and BRCA2 muta-tions. To identify TF as a surface target in TNBC,we investigated TFexpression on human TNBC lines with or without BRCA1 andBRCA2mutations. To translate the findings from in vitro to in vivoand ultimately to the clinic, here we also investigated TF expres-sion on the TNBC cancer cells and tumor VECs in human TNBCCDX and PDX models from mice and in TNBC patients' tumors.PDX models are considered a better cancer xenograft model thanthe standard CDXmodel for evaluating anticancer drug response;

Figure 6.

L-ICON1 is effective for the treatment of murine TNBC (4T1/Luc þ GFP) in anorthotopic CDX model. Tumor growth curves (A) and mouse survival curves(B) after intratumoral injection of AdL-ICON1 or AbBlank control vectors formurine breast cancer 4T1 in an orthotopic model in Balb/c mice (n ¼ 5 in eachgroup). Representative results (mean � SEM) from two independentexperiments. P values were analyzed by the linear mixed model with randomsubject effects (A) or by Kaplan–Meier analysis (B).

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results from PDX models better predict the clinical outcome ofanticancer agents (50). Thepercentage (48%)of TF-positive TNBCon TMA slides (n ¼ 147) was lower than that (85%) found byexamination of regular paraffin-embedded whole TNBC tumortissues (n¼ 14). This was possibly due to the fact that tumors areheterogeneous and that only small tissue samples are taken formaking TMA slides, which is one of the limitations of the TMAtechnique (51). In future studies, we would suggest that multiplespecimens from each tumor should be used for IHC screening forenrollment of patients into clinical trials of L-ICON1 therapy. TF isalso 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 othergroups' findings (18, 19) suggest that TF might be a usefultherapeutic target for treatment of TNBC, prompting us toimprove upon the TF-targeting ICON that we had previouslydeveloped (32–35).

To develop a therapy for TNBC that uses TF as a target, weconstructed an improved second-generation ICON namedL-ICON1, for light chain ICON or lighter ICON. When designingthe first-generation ICON as a TF-targeting agent, Alan Garen andZhiwei Hu at Yale University attempted to eliminate the coagu-lation activity of fVII in ICON by introducing a mutation at theamino acid residue Lysine 341 (32), because Ruf and colleaguesidentified fVII surface residues as key to TF binding and proteasecatalytic function (52). The procoagulant effects of ICON-encoded fVII were thereby reduced, but not eliminated, as mea-sured by prothrombin time assay (34) and by fVII chromogenicassay in this study. In addition, the largeMWof ICON (210 kDa),which is even bigger than an IgG1 antibody, may limit the abilityof ICON to penetrate tumor tissues when administered as recom-binant protein via intravenous injection. These findingsprompted us to improve ICON by eliminating its coagulationactivity to avoid potential coagulation disorders in cancer patientswhen administered systemically and reducing its size to improve

penetration of ICON into tumor tissues. To accomplish theseobjectives, we constructed a second-generation ICON consistingof only the light chain (1–152aa) of fVII fused to IgG1 Fc. At 100kDa, L-ICON1was less than half the size of ICON (210 kDa). TheMWof L-ICON1observed by gel electrophoresis is consistentwiththe MW calculated from its predicted protein sequence as derivedfromcDNAsequence (GenBankKX760097). That is, the L-ICON1protein is predicted to contain 425 amino acids (47.56 kDa formonomer and approximate 95 kDa as homodimer). The first 38amino acids are the signal peptide, and the following 387 residuesare the mature peptide. The 387-aa mature peptide would bepredicted to be 43.32 kDa as a monomer and 87 kDa as ahomodimer, without consideration of the mass of glycosylatedside chains.

L-ICON1 has several advantages over ICON. First, the MWof L-ICON1 is reduced more than 50% compared with ICON.Second, L-ICON1 has no detectable coagulation activity, where-as the coagulation activities in ICON (K341A) and ICON (WT)are about 5% and 58% of that for FVIIa, respectively. L-ICON1may 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 melanomaline, but weakly to a mouse melanoma line (34). These featuresof L-ICON1 will allow us to test human L-ICON1 in preclinicalmouse models of human and murine cancers and translate thefindings directly to clinical trials. Lastly, the current studyshowed that L-ICON1 was more effective than ICON in treat-ing TNBC tumor xenografts in the orthotopic mouse model,and in vivo studies demonstrated that L-ICON1 immunother-apy via intralesional injections of AdL-ICON1 was effectiveand safe for the treatment of human TNBC (MDA-MB-231with BRCA2 mutation) and murine TNBC (4T1; ref. 49) inorthotopic CDX mouse models. L-ICON1 could arrest the PDXtumor growth of TNBC with BRCA1 mutation in an orthotopicmouse model.

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 determinedin vivo by measuring tumor volume using calipers as compared with a control vector (AdBlank)-treated PDX (n ¼ 5 in each group). B, Tumor weights of controlmice and L-ICON1–treated mice (P ¼ 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 tumorsweremeasured 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 presentedas mean � SEM from one experiment. P values were analyzed by two-way ANOVA (A) and by t test (B; GraphPad).






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; refs. 53, 54) and endome-triosis (55, 56). ICON is being tested in phase I and II clinical trialsin patients with AMD (wet form) and ocular melanoma. Indeed,as in angiogenic endothelial cells in cancers, TF is aberrantlyexpressed by the angiogenic VECs in vitro (30) and in vivo innonmalignant diseases that are angiogenesis dependent, such asAMD (53) and endometriosis (55). As an improved neovascular-targeting agent, L-ICON1might have therapeutic potential to treatpathologic angiogenesis-dependent malignancy and nonmalig-nant diseases.

To target TF as part of cancer immunotherapy, in addition tothe ICON and L-ICON molecules that are designed to bind toTF by using its natural ligand fVII, either full-length peptidewith mutated procoagulation active site (K341A) or light-chainpeptide lacking procoagulation activity, several humanizedmonoclonal antibodies (TF-HuMab) and/or antibody–drugconjugates (TF-ADC) are also being studied in preclinical andclinical studies (19, 57). ICON and L-ICON have severaladvantages compared with anti-TF and TF-ADC: (i) the Kd forfVII binding to TF is up to 10�12 M (58), in contrast to anti-TFthat has a Kd in the range of 10�8 to 10�9 M for TF (59); (ii)ICON and L-ICON are produced by recombinant DNA tech-nology, allowing these TF-targeting protein agents to be madefrom human sources for clinical trials without need for human-ization process that is required for monoclonal antibodies (57);(iii) because ADC is made by covalently conjugating drugs toantibodies, most ADCs exist as heterogeneous mixtures andrequire site-specific conjugation technologies (60). Neverthe-less, these studies of TF-targeting Ab and ADC support the ideathat TF-targeted therapies have the potential to treat a range ofsolid cancers (28, 29).

We report that TF is a useful target in treatment of TNBCmodels, and immunotherapy with TF-targeting L-ICON1 showedbetter efficacy and safety than the original ICON. Immunotherapytargeted at TF may warrant further investigation for TNBCpatients. If such immunotherapy proves effective and safe inclinical trials, such treatment regimensmight reduce themortalityassociated with TNBC. TF is highly expressed on cancer cells onnon-TNBC breast cancer as well as in many other solid cancers,acute myeloid leukemia, acute lymphocytic leukemia, and sarco-mas (28, 29): it is expressed in 80% to 100% of breast, 40% to80% of lung, 84% of ovarian, 95% of primary melanoma, 100%ofmetastaticmelanoma, 53% to 90%of pancreatic, 57% to 100%of colorectal, 63% to 100% of hepatocellular carcinoma, 60% to

78% of primary and metastatic prostate, and 47% to 75% ofglioma tumors. Thus, TF-targeting immunotherapy may have thepotential to treat a variety of TF-positive solid cancers, leukemias,and sarcomas (28, 29).

Disclosure of Potential Conflicts of InterestZ. Hu is co-inventor of U.S. patents on the original neovascular-targeted

immunoconjugates (ICON) and is the inventor of U.S. patent application onthe second-generation ICON (L-ICON1). No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: Z. Hu, C.A. London, W.E. Carson IIIDevelopment of methodology: Z. HuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. Hu, R. Shen, A. Campbell, E. McMichaelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Hu, R. Shen, A. Campbell, E. McMichael, L. Yu,B. RamaswamyWriting, review, and/or revision of themanuscript: Z. Hu, A. Campbell, L. Yu,B. Ramaswamy, C.A. London, W.E. Carson IIIAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. HuStudy supervision: Z. HuOther (provided plasmid DNAs): T. Xu

AcknowledgmentsThis work was supported by the Dr. Ralph andMarian FalkMedical Research

Trust Awards Programs. The project described was also partly supported byAward Number UL1TR001070 from the National Center for Advancing Trans-lational Sciences through a Phase I L-Pilot Award, a voucher award from theOSU Center for Clinical and Translational Science, and a Seed Award fromthe Translational Therapeutics Program of OSU Comprehensive CancerCenter. The plasmids for Luc þ GFP were generated by Dr. Tian Xu lab atYale University with the support from the HHMI. Z. Hu conceived of theideas and initiated the project during his tenure at Yale University and com-pleted the work during his employment at OSU.

We thank Dr. Beth Schachter, Dr. Stephen M. Smith, Mr. Aaron Conley, andDr. Thomas Mace for critical reading and editing; Dr. William Konigsberg atYale University and Dr. Michael Bromberg formerly with Yale University(currently with Temple University) for kind gifts of monoclonal antibody HTF1against TF and retroviral vector-encoding TF and control vectors. We thankLongzhu Piao for technical assistance.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 30, 2017; revised January 17, 2018; accepted March 27, 2018;published first April 5, 2018.

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Published OnlineFirst April 5, 2018.Cancer Immunol Res Zhiwei Hu, Rulong Shen, Amanda Campbell, et al. ICONTriple-Negative Breast Cancer Using a Second-Generation Targeting Tissue Factor for Immunotherapy of

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