Epithelial Junction Opener JO-1 Improves Monoclonal ...Therapeutics, Targets, and Chemical Biology Epithelial Junction Opener JO-1 Improves Monoclonal Antibody Therapy of Cancer InesBeyer1,RuanvanRensburg1,RobertStrauss3,ZongYiLi1,HongjieWang1,JonasPersson1,RomaYumul1,

Therapeutics, Targets, and Chemical Biology

Epithelial Junction Opener JO-1 Improves MonoclonalAntibody Therapy of Cancer

InesBeyer1, RuanvanRensburg1, Robert Strauss3, ZongYi Li1, HongjieWang1, JonasPersson1, RomaYumul1,Qinghua Feng2, Hui Song1, Jiri Bartek3,4, Pascal Fender5, and Andr�e Lieber1,2

AbstractThe efficacy of monoclonal antibodies (mAb) used to treat solid tumors is limited by intercellular junctions

which tightly link epithelial tumor cells to each another. In this study, we define a small, recombinant adenovirusserotype 3-derived protein, termed junction opener 1 (JO-1), which binds to the epithelial junction proteindesmoglein 2 (DSG2). In mouse xenograft models employing Her2/neu- and EGFR-positive human cancer celllines, JO-1 mediated cleavage of DSG2 dimers and activated intracellular signaling pathways which reducedE-cadherin expression in tight junctions. Notably, JO-1-triggered changes allowed for increased intratumoralpenetration of the anti-Her2/neumAb trastuzumab (Herceptin) and improved access to its target receptor, Her2/neu, which is partly trapped in tight junctions. This effect translated directly into increased therapeutic efficacy oftrastuzumab in mouse xenograft models using breast, gastric, and ovarian cancer cells that were Her2/neu-positive. Furthermore, combining JO-1 with the EGFR-targeting mAb cetuximab (Erbitux) greatly improvedtherapeutic outcomes in a metastatic model of EGFR-positive lung cancer. A combination of JO-1 with anapproach that triggered transient degradation of tumor stroma proteins elicited eradication of tumors. Takentogether, our findings offer preclinical proof of concept to employ JO-1 in combination withmAb therapy. CancerRes; 71(22); 7080–90. �2011 AACR.

Introduction

Trastuzumab (Herceptin) and cetuximab (Erbitux) arehumanized monoclonal antibodies (mAb) used for the therapyof Her2/neu- and EGFR-positive cancers, respectively. Themechanisms of trastuzumab and cetuximab action include theactivation of antibody-dependent or complement-dependentcytotoxicity, and interference with tyrosine kinase receptorsignaling (1). A unifying aspect among these mechanisms isthat tumor cell growth inhibition is dependent on thebindingofthese mAbs to their corresponding receptors, that is, Her2/neuand EGFR. Therefore, molecules that prevent access and bind-ing to the receptor, either by physically inhibiting intratumoraltransport from blood vessels to malignant cells or masking ofreceptors, are predicted to block trastuzumab and cetuximab

activity (2). Several studies showed that the expression orupregulation of epithelial proteins correlated with increasedresistance to trastuzumab (3) and cetuximab (4) therapy.Epithelial cells maintain several intercellular junctions (tightjunctions, adherens junctions, gap junctions, anddesmosomes),a feature which is often conserved in epithelial cancers in situand in cancer cell lines (5). Epithelial junctions are composed ofadhesive dimers consisting of cadherinmolecules derived from2 neighboring cells (6). Desmoglein 2 (DSG2), an epithelialcatherin, is overexpressed in a series of epithelial malignancies,including breast cancer (ref. 7; Supplementary Fig. S1), ovariancancer (ref. 7; Supplementary Fig. S1), lung cancer (7), gastriccancer (8), squamous cell carcinomas (9), melanoma (10),metastatic prostate cancer (11), and bladder cancer (12).

Recently, we showed that a group of human adenoviruses(Ads; Ad serotype 3, 7, 11, and 14) use DSG2 as a primaryattachment receptor for the infection of cells (7). Importantly,in epithelial cells, Ad3 binding to DSG2 triggered activation ofsignaling pathways resulting in the transient opening of epi-thelial junctions (7). The opening of the epithelial junctionswas also achieved with recombinant subviral particles, such asAd3 penton-dodecahedra (PtDd; Fig. 1A). We subsequentlygenerated a minimal Ad3-derived DSG2 ligand formed by 2fiber knob domains (13). This protein, with a molecular weightof approximately 50 kDa, is produced in E. coli and can be easilypurified. In a series of functional studies, we showed that thisprotein efficiently triggers the opening of junction. In thefollowing study, we therefore refer to this protein as junctionopener-1 (JO-1).

Authors' Affiliations: 1Division of Medical Genetics; 2Department ofPathology,University ofWashington, Seattle,Washington; 3DanishCancerSociety, Center for Genotoxic Stress Research, Copenhagen, Denmark;4Institute of Molecular and Translational Medicine, Palacky University,Olomouc, Czech Republic; and 5Unit of Virus Host Cell Interactions,Grenoble, France

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Andr�e Lieber, University of Washington, Box357720, Seattle, WA 98195. Phone: 206-221-3973; Fax: 206-685-8675;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-2009

�2011 American Association for Cancer Research.

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Figure 1. Transient opening of epithelial junctions by JO-1. A, structure of Ad3 viral particles. Left: complete, infectious Ad3 particle. The capsid proteinsfiber and penton base are shown in green and blue, respectively. The trimeric fiber knob is shown in red. Middle: Ad3 PtDd formed by spontaneousassembly of 12 recombinant pentons (fiberþ penton base). Right: dimeric Ad3 fiber (JO-1). B, schematic structure of JO-1 containing anN-terminal His-tag, a dimerization domain (K-coil ref. 32), a flexible linker, one fiber shaft motif, and the homotrimeric Ad3 fiber knob domain. C, left: simplified structureof epithelial junctions with tight junctions, desmosomes, and adherens junctions. Confocal immunofluorescence microscopy of T84 cells. Shown arestacked XZ images. Cells were treated with JO-1 (5 mg/mL) for 1 hour on ice. After removal of JO-1, cells were incubated at 37�C and analyzed 0, 30, and60 minutes later. Top: DSG2 (green) seems at the apical site of baso-lateral junctions marked by claudin 7 (red). Middle: within 30 minutes after addingJO-1, claudin 7 staining increases and DSG2 staining becomes visible along the upper part of the lateral membrane (yellow signals). Bottom: by 60minutes, lateral junctions resemble those of time point "0 minute." The scale bar is 40 mm. D, transmission electron microscopy of junctional areas ofpolarized colon cancer T84 cells. Cells were either treated with PBS (left) or JO-1 (right) for 1 hour on ice, washed, and then incubated for 1 hour at 37�C.At this time, the electron-dense dye ruthenium red (33) was added together with the fixative. The scale bar is 1 mm. E, 14C-PEG-4,000 diffusion throughmonolayers of T84 cells at different time points after adding JO-1 or anti-DSG2 antibody 6D8 (directed against ECD3/4). F, effect of various DSG2ligands on the TEER of polarized T84 epithelial cells. Cells were treated as described in (E).

Cotherapeutic for Anticancer mAbs

www.aacrjournals.org Cancer Res; 71(22) November 15, 2011 7081




In this study, we have partially delineated the in vivomechanism of JO-1–mediated junction opening. Furthermore,we show that JO-1 treatment greatly increases the permeationof mAbs in tumors and significantly enhances the efficacy oftrastuzumab and cetuximab therapy in a series of xenografttumor models.

Material and Methods

ProteinsJO-1 (also known as Ad3-K/S/Kn) is produced in E. coli as

described previously (13). Recombinant Ad3 PtDd proteincomplexes were produced in insect cells and purified asdescribed elsewhere (14).

Cell linesBT474-M1 is a tumorigenic subclone of BT474 (ATTC,

HTB-20) that was generously provided by Mien-Chie Hung(Department of Molecular and Cellular Oncology, Universityof Texas MD Anderson Cancer Center, Houston) in 2009 (15).BT474-M1 and HCC1954 cells (ATTC, CRL-2338) werecultured in RPMI-1640 with 10% FBS, 1% Pen/Strep andL-Glutamine. A549 (ATCC, CCL-185) and T84 (ATCC,CCL-248) were cultured in DMEM/F:12 with 10% FBS, 1%Pen/Strep and L-Glutamine. To achieve cell polarization, 1.4� 105 T84 cells were cultured in collagen-coated 6.5 mmTranswell inserts (0.4 mm pore size; Costar TranswellClears) for a period of 14 to 20 days until transepithelialresistance was stable (7). Cell lines from the ATTC wereobtained in December 2010. All cell lines have been passagedfor fewer than 6 months. Cell surface expression of Her2/neu (BT474-M1, HCC1954) and/or EGFR1 (A549, T84)was confirmed by immunofluorescence analysis in January2011.

Immunofluorescence analyses were done as describedrecently (7).

Western blots were done as described recently (7).

Transepithelial electrical resistance and PEGpermeability assays

A total of 5 � 105 T84 cells were seeded on 12 mmtranswell inserts (PET membrane, with 0.4 mm pore size)and cultured for 20 days. Culture medium was changedevery 2 to 3 days. The cells were exposed to DSG2 ligands(20 mg/mL) in adhesion medium (DMEM, 1% FBS, 2 mmol/LMgCl2, 20 mmol/L HEPES) for 15 minutes at room temper-ature and transepithelial electrical resistance (TEER) wasmeasured and calculated as described elsewhere (16). Forpermeability assays 15 minutes after adding the DSG2ligands, 1 mCi of [14C] polyethylene glycol-4000 (PEG-4000; Perkin Elmer) was added to the inner chamber. Medi-um aliquots were harvested from the inner and outerchambers and measured by a scintillation counter. Perme-ability was calculated as described elsewhere (17).

Transmission electron microscopyTransmission electron microscopy (TEM) was done as

described previously (13).

Hematopoietic stem cell–based relaxin expressionThe protocol has been described elsewhere (18). Briefly,

transplant recipients were 6- to 10 weeks old, female CB17severe combined immunodeficient (SCID)-beige mice, suble-thally irradiated with 350 cGy immediately before tail veininjection with 6 � 105 lentivirus vector—transduced bonemarrow cells from 5-FU—treated mice. After engraftment ofcells in the recipients' bone marrow was confirmed, a total of4 � 106 HCC1954 were injected into the mammary fat pad.The lentivirus vector expressing relaxin under the control ofdoxycycline (Dox) has been described previously (18).

Human IgG (Herceptin) ELISAA polyclonal goat anti-human IgG antibody (G-101-C-ABS,

R&D Systems) was used as a capture antibody. Tissues werelysed as for Western blots. Purified human IgG served as astandard. Bindingwas detectedwith amousemonoclonal anti-human IgG1 Fc antibody (MAB 110, R&D Systems), followed byan anti-mouse IgG-HRP conjugate.

Animal studiesBreast cancer xenografts were established by injecting 4 �

106 cancer cells into the mammary fat pad of CB17 SCID-beigemice. Trastuzumab was injected intraperitoneally (i.p.) ata dose of 10 mg/kg. PtDd or JO-1 was given i.v. at a dose of2 mg/kg. Tumor volumes were measured as described previ-ously (19). Mice were sacrificed when the tumor volumereached 1,000 mm3 or ulcerated. Lung cancer xenografts wereestablished by injecting 4� 106 A549 s.c. into the right flank ofCB17 SCID-beigemice. Cetuximabwas injected at 10mg/kg i.p.For the disseminated lung tumor model, mice were intrave-nously injectedwith 2� 106 A549 cells. Animals were sacrificedwhen the first mouse of the control group wasmoribund. Indiaink (15% in PBS) was injected intratracheally prior to theremoval of the lungs.

Statistical analysisAll results are expressed as mean � SD. Student t test or 2-

wayANOVA formultiple testing, were appliedwhen applicable.A value P < 0.05 was considered significant.

Results

JO-1 triggers opening of epithelial junctionsAs the large size of Ad3 or PtDd particles can affect their

egress fromblood vessels and tissue penetration, we attemptedto generate smaller Ad3-derived DSG2 ligands that are func-tionally active as epithelial junction openers. We thereforedesigned JO-1 (aka Ad3-K/S/Kn; ref. 13), a small, self-dimeriz-ing Ad3 fiber derivative (Figs. 1A and B; ref. 13). JO-1 has amolecular weight of approximately 50 kDa and is produced inE. coli prior to purification by affinity chromatography. Incontrast, PtDd have to be produced in insect cells and havea molecular weight of 4,860 kDa and a diameter of approxi-mately 50 nm.

The functional activity of JO-1 was tested on polarized coloncancer T84 cells. Incubation of T84 cells with JO-1 triggeredremodeling of epithelial junctions, as shown by confocal

Beyer et al.

Cancer Res; 71(22) November 15, 2011 Cancer Research7082




microscopy for claudin 7 and DSG2 (Fig. 1C). Opening of thetight junctions, which are localized apical to the desmosomaland adherence junctions, is illustrated by electron microscopy(Fig. 1D). Microphotographs of untreated epithelial cells showintact tight junctions as judged by the exclusion of the apicallyapplied electrone-dense dye ruthenium red from basolateralspace. Incubation of epithelial cells with JO-1 for 1 hourresulted in the disassembly of tight junctions and leakage ofruthenium red into the basolateral space (Fig. 1D, right panel).Exposure of polarized epithelial cells to JO-1 also increased thetransepithelial permeability, as shown by transflux of 14C-PEG-4000with amolecular weight of 4,000 Da (Fig. 1E). Importantly,monoclonal antibodies against different regions of the extra-cellular domain of DSG2 did not significantly increase transe-pithelial permeability. We speculate that the ligation of severalDSG2 molecules is required to trigger the opening of the

junctions. Finally, transient opening of junction was confirmedby measuring the TEER in polarized epithelial cells (Fig. 1F).Notably, JO-1 had no significant effect on the TEER whenstudies were done in subconfluent cell cultures where maturejunction had not yet formed (i.e., when TEER was notconstant).

JO-1 triggers intracellular signaling and increasespenetration of mAb in epithelial tumors in vivo

An orthotopic breast cancer xenograft model (HCC1954)was used to study the effect of JO-1 on epithelial junctions invivo. HCC1954 xenograft tumors resembled the histology ofbreast cancer in humans (20), that is, tumorswere vascularizedand contained nests of epithelial cells glued together byepithelial junctions and surrounded by extracellular matrixes(Supplementary Fig. S2). JO-1 was injected intravenously into

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Figure 2. Analysis of mechanism of JO-1 action in tumors in an orthotopic (HCC1954) breast cancer model. When tumors reached a volume of approximately200mm3, JO-1 (2mg/kg in 200mLPBS)was injected intravenously. Tumorswere harvested either 1 or 12 hours after JO-1 injection. Controlmice received 200mLPBSand tumorswere collected 1hour later. A, kinetics of JO-1 accumulation in tumors. Left: immunofluorescence analysis of tumor sectionsusing anti-Histag antibodies (for visualization of JO-1). The scale bar is 20 mm. Right: Western blot analysis of tumor tissue using Ad3-fiber knob specific antibodies (7). B,analysis ofDSG2 in tumors. Left: immunofluorescence analysis of tumor sections usingDSG2antibodies (mAb6D8against extracellular domain 3/4 ofDSG2).The inserts show a higher magnification. Right: the same anti-DSG2 antibody was used for Western blot analysis of tumor tissue. C, intracelluar signaling invivo. Left: Western blot analysis of tumor tissue for E-cadherin and phosphorylated E-cadherin, Erk 1/2, phosphorylated Erk1/2, claudin 7, and vimentin.Antibodies against g-tubulinwere used to assess sample loading ("loading control"). Right: immunofluorescenceanalysis using antibodies against E-cadherinand phosphorylated Erk1/2.






mice with preestablished tumors. JO-1 could be detected in thetumors by immunofluorescence microscopy as early as 1 hourpostinjection. JO-1 accumulated in the tumors as is indicatedby the increased immunofluorescence at 12 hours postinjec-tion (Fig. 2A, left 3 panels). This is also confirmed by Westernblot analysis of tumor lysates (Fig. 2A, right panel). Analysis ofDSG2 on tumor sections by immunofluorescence microscopyin PBS-treated animals showed membrane localized signals(Fig. 2B, left panel). One hour subsequent to JO-1 injection,DSG2 molecules were mostly found in the cytoplasm of thetumor cells (second panel). By 12 hoursmembrane localizationof DSG2 seemed to be partly restored (third panel). Westernblot analysis using anti-DSG2 antibodies against the extracel-lular domain of DSG2 revealed smaller fragments of the DSG2(80 and 45 kDa) at the 1 hour time point (Fig. 2B, right panel).These fragments represent the extracellular domains (ECD)and proteolytic cleavage products of the ECD. Proteolyticcleavage of DSG2 to stable fragments in normal epithelialtissue and cancer has been reported before (21–23).

Recently, it was found in in vitro studies that Ad3 binding toDSG2 of epithelial cells triggered intracellular signaling includ-ing pathways that are involved in epithelial-to-mesenchymaltransition (EMT; 7). Among the feature that characterize EMTare decreased expression of epithelial markers and activationof Erk1/2 (MAPK; 5). In our studies with xenograft tumors, wefound less nonphosphorylated and phosphorylated forms ofE-cadherin in tumors 12 hours after intravenous injection ofJO-1 (Fig. 2C, left panel). Preceding the changes in E-cadherin,was a transient increase in phosphorylated Erk1/2 [Fig. 2C,

compare pErk1/2 PBS vs. JO-1 (1 hour)]. The decrease in E-cadherin and an increase in signals for phosphorylated Erk1/2upon JO-1 injection were also observed by immunofluorescencemicroscopy (Fig. 2C, right panels). These studies indicate thatJO-1 triggers transient activation of Erk1/2 pathways in vivo.

Next, we tested whether JO-1-triggered opening of epithelialjunctions in tumors would increase the penetration ofmAbs inxenograft tumors. Trastuzumab, a humanized IgG1 mAb, wasinjected intraperitoneally at a dose of 10 mg/kg into HCC1954tumor-bearing mice (24). In tumor sections and Western blotanalyses, trastuzumabwas detectable 1 hour postinjection andat higher levels 12 hours after injection (Fig. 3A and B).Quantitative analysis of human IgG1 in tumor lysates by ELISAshowed approximately 6 fold higher levels inmice that receivedJO-1 injectionþ trastuzumab (12 hours time point) comparedwith mice that received trastuzumab alone (Fig. 3C).

In conclusion, intravenous injection of JO-1 1 hour prior tothe administration of trastuzumab, significantly increased theamount of trastuzumab in the tumors, indicating either betteregress from blood vessels, better intratumoral penetration,and/or longer intratumoral half-life.

mAb targets are trapped in epithelial junctionsIn breast cancer xenograft sections and in cultured breast

cancer cells, we found costaining of Her2/neu and the adherensjunction protein claudin 7 (Fig. 4A, Supplementary Fig. S3).Confocalmicroscopy of breast cancer BT474 cells confirmed thetrapping of Her2/neu in lateral junctions. Incubation of theHer2/neu positive breast cancer cell lines BT474 (Fig. 4) or

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Figure 3. JO-1 improves penetrationof trastuzumab in HCC1954 breastcancer tumors in situ. Tumor-bearing mice were intravenouslyinjected with PBS or JO-1 (2 mg/kg)followed by trastuzumab 1 hourlater. Tumors were harvested 1 or12 hours after trastuzumabinjection. A, sections were stainedfor human IgG(i.e., trastuzumab). Positive stainingseems green. The scale bar is 20mm. B, Western blot analysis forhuman IgG (trastuzumab) in tumors.Heavy (HC) and light (LC) Ig chainsare indicated by arrows. C, ELISAfor human IgG1 in tumor lysates.Total protein concentration in alllysates was adjusted to 5 mg/mL.Shown is the ratio of human IgG1concentrations in tumors of PBS-treated mice versus mice thatreceived JO-1 and/or trastuzumabtreatment. n ¼ 3; �, P < 0.05.

Beyer et al.





HCC1954s (Supplementary Fig. S3) with JO-1 changed thecomposition of the lateral epithelial junctions within 1 hour.As a result of this, Her2/neu staining at the cell surface becamemore intense, while it faded in areas distal of the cell surface.This suggests that JO-1 mediated junction opening triggered atranslocation of Her2/neu from lateral membranes to the cellsurface. Being trapped in epithelial junctions also seems to be aproblem for EGFR as costaining for EGFR and the tight junctionproteinE-cadherin suggests (Fig. 4B, SupplementaryFig. S3B). Inour studies with cetuximab, we focused on a lung cancer model(A549 cells), asmost colon cancer cell lines havemutations in K-ras, which confers resistance to cetuximab (25). Similar to whatwe observed for Her2/neu, incubation of A549 cells with JO-1resulted in a translocation of EGFR to the cell surface.Release of mAb receptors from trapping is supported by the

enhanced killing of cancer cells by trastuzumab and cetux-imab. In vitro killing of BT474 breast cancer and A549 lungcancer cells by trastuzumab and cetuximab, respectively, wasinefficient (Fig. 4C andD). Pretreatment of these cells with JO-1significantly increased in vitro cytotoxicity of both antibodiesin the corresponding cell lines, although the effect of JO-1 wasrelatively modest.

JO-1 improves trastuzumab therapy in vivoJO-1's potential enhancement of trastuzumab therapy was

first tested in an orthotopic breast cancer model based onHer2/neu positive BT474-M1 cells. JO-1 injection alone had nosignificant effect on tumor growth (Fig. 5A). BT474-M1 tumorsinitially responded well to trastuzumab, however, preinjectionof JO-1 significantly enhanced the therapeutic efficacy oftrastuzumab (Fig. 5A). The enhancing effect of JO-1 pretreat-ment becomes more apparent when treated mice were fol-lowed long term, that, for 136 days. Although 60% of theanimals that received trastuzumab monotherapy relapsedaround day 100, none of the animals treated with JO-1þtrastuzumab showed tumor regrowth (data not shown).

A second breast cancer model involved HCC1954 cells.Tumors derived from these cells are more resistant to tras-tuzmab (Fig. 5B). As seen in the BT474-M1 model, JO-1pretreatment significantly improved trastuzumab therapy andstalled tumor growth. On the basis of our previous study withPtDd (7), we chose a time interval of 10 hours between JO-1 andtrastuzumab injections. This regimen is supported by thekinetics of JO-1 accumulation in tumors and the kinetics ofE-cadherin decrease (see Fig. 2A and C). On the other hand,

Figure 4. JO-1 increases mAbkilling of cells in which the targetreceptors are trapped in epithelialjunctions. A, confocal microscopyof Her2/neu (green) and DSG2 (red)staining on polarized BT474 cellcultures (XY and XZ images). Cellstreated with PBS are shown in theleft panel. Middle and right: Cellswere treated with JO-1 (20 mg/mL)for 1 hour on ice. After removal ofJO-1, cells were incubated at 37�Cand analyzed 1 and 16 hours later.XY images show the cell surface(left) and a section 2 mm below thecell surface. The scale bar is 40 mm.B, confocal microscopy of EGFR(red) and the tight junction proteinE-cadherin (green) on polarizedA549 lung cancer cells. C, JO-1enhances killing of Her2/neu-positive breast cancer cells bytrastuzumab. BT474 cells wereincubated with JO-1 (5 mg/mL) orPBS. Trastuzumab (15 mg/mL) wasadded 1 hour later. Cell viability wasmeasured after 3 hours by WST-1assays as described earlier (34).Viability of PBS-treated cells wastaken as 100%. n ¼ 5, (D) JO-1enhances cetuximab killing ofEGFR-positive A549 cells.n ¼ 5; �, P < 0.05.

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events that seem to be linked to junction opening, that is, DSG2cleavage or Erk1/2 activation, occur already within 1 hour afterJO-1 injections. We therefore investigated how simultaneousJO-1/trastuzumab injection and injection of trastuzumab 1hour after JO-1 application influenced the therapeutic out-come (Fig. 5C). In this study, no significant difference wasfound when compared with the treatment approach usedinitially (trastuzumab 10 hours after JO-1). We speculate thatthis is due to the relative slow accumulation of the protein inthe tumors. To further consolidate the clinical relevance of JO-1as a cotherapeutic for trastuzumab, we conducted efficacystudies in Her2/neu-positive gastric cancer (NCI-N87) andovarian cancer (SKOV3-1ip) models (Supplementary Fig. S4).Similar to the breast cancer model, we found costaining ofHer2/neu and claudin 7 in NCI-N87 cultures and xenograftedtumors, suggesting trapping of Her2/neu in epithelial junc-tions. Pretreatment of NCI-N87 tumor-bearing mice with JO-1significantly improved trastuzumab therapy as reflected bydelayed tumor growth (Supplementary Fig. S4A). To establishthe ovarian cancer model, SKOV3-1ip, cells were injectedintraperitoneally and survival was monitored after treatment(Supplementary Fig. S4B). Although all mice treated withtrastuzumab alone had reached the endpoint by day 80, 80%of the animals that received the combination treatment JO-1plus trastuzumab were still alive at this time.

JO-1 improves cetuximab therapy in vivoCetuximab treatment of mice with preestablished subcuta-

neousA549 tumors did not result in a significant delay of tumorgrowthwhen comparedwith treatmentwith PBS (Fig. 6A). JO-1was injected intravenously or intraperitoneally followed bycetuximab 12 hours later. Both treatment approaches had asignificant therapeutic effect and resulted in a decrease oftumor volumes. An additional combination of intravenouslyinjected JO-1 with an intratumoral application of the junctionopener did not further increase the therapeutic efficacy. Asseen in the breast cancer model, JO-1 treatment alone did notexert a significant antitumor effect. JO-1 pretreatmentenhanced cetuximab therapy to a similar degree as seen withPtDd (Fig. 6B).

Next, the cotherapy approachwas tested in ametastatic lungcancer model. In this model, mice became morbid within 37days of tumor cell transplantation with predominant tumorlocalization to the lung (Fig. 6C, "PBS" group). Treatment ofmice was started at day 10. All animals were sacrificed at day40. Although lungmetastases were clearly visible in the controlgroup, JO-1 group, and the cetuximab-treated animals, 80% ofthe lungs in the JO-1þcetuximab-treated animals were free oftumor when inspected macroscopically. Microscopy of lungsections showed that in PBS-treated animals, tumor cellsalmost completely replaced normal lung tissue and also filled

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Figure 5. JO-1 improvestrastuzumab therapy in Her2/neupositive breast cancer models. A,BT474-M1 breast cancer model:When tumors reached a volume ofapproximately 100 mm3, micereceived an intravenous injection ofJO-1 or PBS, followed by anintraperitoneal injection oftrastuzumab or PBS 10 hours later.A second treatment cycle wasstarted at day 36 (marked byarrows). Shown is the increase intumor volume (compared topretreatment levels at day 29).n ¼ 5. �, P < 0.05. B, HCC1954breast cancer model: mice weretreated as in (A). Mice received thefirst treatment at day 12. Treatmentwas repeated at day 18. n ¼ 5.�, P < 0.05. C, mice bearingHCC1954 breast cancer tumorswere injectedwith amixture of JO-1and trastuzumab, JO-1 followed bytrastuzumab 1 hour later and, JO-1followed by trastuzumab 10 hourlater. Injections were repeatedweekly. n ¼ 5. All JO-1 cotherapiesare significantly more effective thantrastuzumab alone. There is notsignificant difference between thedifferent cotherapy regimens.

Beyer et al.





the broncioli (Fig. 6C, right panels). Although cetuximab-treated animals had considerable, infiltrating tumor growth,themajority of JO-1þ cetuximab-injected animals showed onlymicrometastases.

Combined tumor stroma protein degradation andjunction openingExtracellular matrix proteins forming the tumor stroma

tightly surround nests of malignant breast and colon cancercells (26). We have recently shown that transient degrada-

tion of tumor stroma proteins by intratumoral expression ofthe peptide hormone relaxin significantly enhanced trastu-zumab therapy (24). Here, we utilized the HCC1954 model totest whether additional transient tumor stroma proteindegradation, would further increase the effect of JO-1 ontrastuzumab therapy (Fig. 7A). To deliver the relaxin geneto the tumor, we employed an approach based on hemato-poietic stem cells (HSC; ref. 26). The approach involved theex vivo transduction of bone marrow derived HSCs withlentivirus vectors expressing relaxin under the control of a

Figure 6. JO-1 improvescetuximab therapy in lung cancerxenograftmodels. A, subcutaneousA549 tumor model: JO-1 wasinjected at day 12 and 15intravenously (2 mg/kg) orintraperitoneally (4 mg/kg) followedby an intraperitoneal injection ofcetuximab or PBS 10 hours later.One group received 1 mg/kg of JO-1 intravenously and 1 mg/kgintratumorally. n ¼ 5. All JO-1cotherapies are significantly moreeffective than trastuzumab alone.The difference between JO-1injection routes was not significant.B, mice received an intravenousinjection of 2 mg/kg PtDd followedby an intraperitoneal injection ofcetuximab (10 mg/kg) or PBS 10hours. A second treatment cyclewas started at day 14 (marked byarrows). n ¼ 5. Cetuximab versusPtDd plus cetuximab P < 0.001. C,metastatic A549 lung cancermodel: 10 days after intravenousinjection of A549 cells, micereceived an intravenous injection of2 mg/kg JO-1 or PBS, followed byan intraperitoneal injection ofcetuximab (10 mg/kg) or PBS 10hours later. The treatment wasrepeated every 3 days until day 38.n ¼ 10. Left: lungs from individualmice stained with India ink. Healthytissue seems black. Tumor tissuestains white. Right: representativesections of lungs stained with H&E.

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Dox-inducible transcription cassette, and the transplanta-tion of these cells into myelo-conditioned recipients, wherethey engraft in the bone marrow and provide a long-termsource of genetically modified cells that will home intotumors. This study showed that relaxin expression alonesignificantly delayed tumor growth and increased trastuzu-mab therapy (Fig. 7B). The combination of relaxin expres-sion and JO-1 treatment stopped tumor growth. Tumors didnot regrow when treatment was terminated, in contrast togroups that received either relaxinþtrastuzmab or JO-1þtrastuzumab therapy. Histologic analyses of residualmasses in the JO-1/relaxin/trastuzumab group at the endof the observation period, showed only connective tissue. Incontrast, explanted tumors from the other groups containedtumor cells, which could be cultured in vitro upon proteasedigestion of tumors. Notably, no adverse side effects wereobserved in mice that received the triple combination (JO-1/relaxin/trastuzumab) treatment.

Our data underscore that physical obstacles in tumors areinvolved in mediating resistance to trastuzumab therapy.

Discussion

JO-1 as new cotherapeuticThe epithelial phenotype of cancer, that is, intercellular

junctions, creates obstacles to mAb therapy. The small

recombinant protein JO-1 increased the penetration oftrastuzumab in the tumor and allowed for better access tomAb target receptors, which, in turn, facilitated mAb ther-apy in a series of xenograft models involving human epi-thelial tumor cells. Potentially, the combination of JO-1 withtrastuzumab and cetuximab might allow for the reductionof the effective dose of these mAbs, thereby reducing criticalside effects, that is, trastuzumab-associated cardiotoxityand acne-like rashes that often occur during cetuximabtherapy.

Mechanisms of actionOur data suggest that JO-1 triggers junction opening in

epithelial tumors through several, potentially connected,mechanisms: (i) cleavage of the DSG2 ECD, and disruption ofDSG2 dimers between neighboring cells; (ii) intracellular sig-naling that leads to a transient decrease of E-cadherin andpotentially other junction proteins; and (iii) changes in themembrane distribution ofHer2/neu. JO-1 treatment resulted intransient phosphorylation of Her2/neu in tumors (Supplemen-tay Fig. S5). However, trastuzumab treatment alone also trig-gered Her2/neu phosphorylation, a phenomenon that has beenobserved before (27), and JO-1 plus trastuzumab cotherapy didnot further increase the levels of phosphorylated Her2/neu.Thismakes it unlikely that JO-1 enhances trastuzumab therapythrough its effect on the biology of Her2/neu.

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Figure 7. Combination therapy ofJO-1 and relaxin in the HCC1854breast cancer model. A, schematicillustration of the experiment.Lethally irradiated mice receivedeither mock transduced or LV-EF1a/Rlx transduced Lin� bonemarrow cells. Six weeks later, afterengraftment of HSCs, mice wereinjected into the mammary fat padwith 4�106HCC1954cells. Relaxin(Rlx) expression was activated byDox 7 days later. Mice were thengiven weekly treatment of PBS,PBS/trastuzumab or JO-1/trastuzumab and tumor volumeswere measured. B, tumor volumesof individual mice. n ¼ 5.(trastuzumab vs. JO-1 þtrastuzumab, P < 0.001; Rlxþ JO-1þ trastuzumab vs. Rlx þtrastuzumab, P < 0.001; Rlx þ PBSvs. PBS, P < 0.001; Rlx þ PBS vs.Rlx þJO-1 þ PBS, P < 0.001; RlxþPBS vs. Rlx þ JO-1 þtrastuzumab, P < 0.001).

Beyer et al.





Side effects on normal epithelial tissuesBecause the mouse orthologue of DSG2 is not recognized by

Ad3 or JO-1 (7), we generated transgenic mice containing thehumanDSG2 locus. The expression pattern and level of humanDSG2 in these animals were similar to those found in humans.Furthermore, we showed that JO-1 binding to human DSG2 intransgenic mouse epithelial cells triggered junction opening toa degree similar to data observed in human cells. In preliminarystudies with DSG2-transgenic mice we did not find critical sideeffects of intravenous JO-1 injection (2 mg/kg; 28). We spec-ulate that DSG2 in normal epithelial cells is not readilyaccessible to intravenously applied JO-1. On the other hand,greater leakage of tumor-associated blood vessels and the lackof strict cell polarization might make epithelial tumors moreresponsive to JO-1. Lack of toxicity after intravenous injectionof JO-1 ligands is also underscored by studies with adeno-viruses containing Ad3 fibers (29).

JO-1 immunogenicityAs JO-1 is a viral protein, adaptive immune responses might

develop in humans, particularly after repeated injection. Thismight, however, not be a problem clinically because bothtrastuzumab and cetuximab are used in combination withimmunosuppressive chemotherapeutic drugs.

Potential risk to enhance tumor invasion andmetastasisIn agreement with other studies (8, 9), we found a higher

DSG2 expression in malignant tissues than in the surroundingnormal epithelial tissue. There are, however, also studiesreporting a reduction in the amounts of DSG2 in invasivepancreatic or gastric cancer (23, 30). The latter, and the findingthat JO-1 triggers EMT-like signaling, raises the question of

whether JO-1would facilitatemetastasis. Notably, in allmodelsused in this study, we did not see stimulation of tumor growthor macroscopic/microscopic signs of metastasis in animalstreated with JO-1 alone. Tumor invasion and metastasisrequires more than transient activation of EMT pathways.Detachment from epithelial cancers and migration of tumorcells is only possible after long-term crosstalk between malig-nant cells and the tumor microenvironment, resulting inchanges in the tumor stroma and phenotypic reprogrammingof epithelial cells into mesenchymal cells (31).

In summary, the epithelial junction opener JO-1 has thepotential to improve mAb therapies of cancer both in terms ofefficacy and safety, that is, by allowing lower therapeutic mAbsdoses. This study also sheds light on the mechanisms of Ad3infection of epithelial cells.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

The work was supported by NIH grants R01 CA080192, R01 HLA078836 (A.Lieber), the Pacific Ovarian Cancer Research Consortium/Specialized Programof Research Excellence in Ovarian Cancer Grant P50 CA83636, FHCRC BreastCancer Research Program Pilot Project Found, the Danish Cancer Society, theDanish National Research Foundation (R. Strauss and J. Bartek), and theEuropean Commission (grant CZ.1.05/2.1.00/01.0030; J. Bartek). I. Beyer is arecipient of a postdoctoral fellowship award from "Deutsche Krebshilfe"(108988).

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 16, 2011; revised September 13, 2011; accepted September 28,2011; published OnlineFirst October 11, 2011.

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Correction

Correction: Epithelial Junction Opener JO-1Improves Monoclonal Antibody Therapy ofCancer

In this article (Cancer Res 2011;71:7080–90), which was published in the November15, 2011, issue of Cancer Research (1), there is an error in Fig. 3A. The panel"trastuzumab (1 h p.i.)" was inadvertently duplicated and appeared as panel"trastuzumab (12 h p.i.)." The correct version of Fig. 3A appears below. Furthermore,in the legend of Fig. 3A, "Positive staining seems green" should read "Positivestaining appears in light gray." The authors regret this error.

Reference1. Beyer I, van Rensburg R, Strauss R, Li Z, Wang H, Persson J, et al. Epithelial junction opener

JO-1 improves monoclonal antibody therapy of cancer. Cancer Res 2011;71:7080–90.

Published OnlineFirst March 19, 2014.doi: 10.1158/0008-5472.CAN-14-0557�2014 American Association for Cancer Research.

Trastuzumab(1 h p.i.)

JO-1 + trastuzumab(1 h p.i.)PBS

Trastuzumab(12 h p.i.)

JO-1 + trastuzumab(12 h p.i.)A

Figure 3A.

CancerResearch

www.aacrjournals.org 2131

2011;71:7080-7090. Published OnlineFirst October 11, 2011.Cancer Res Ines Beyer, Ruan van Rensburg, Robert Strauss, et al. Antibody Therapy of CancerEpithelial Junction Opener JO-1 Improves Monoclonal

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Epithelial Junction Opener JO-1 Improves Monoclonal ...Therapeutics, Targets, and Chemical Biology Epithelial Junction Opener JO-1 Improves Monoclonal Antibody Therapy of Cancer InesBeyer1,RuanvanRensburg1,RobertStrauss3,ZongYiLi1,HongjieWang1,JonasPersson1,RomaYumul1,