Targeting Tumor Neoangiogenesis via Targeted Adenoviral ...Evaluated vector for cancer gene therapy...

MOLECULAR CANCER THERAPEUTICS | MODELS AND TECHNOLOGIES

Targeting Tumor Neoangiogenesis via TargetedAdenoviral Vector to Achieve Effective Cancer GeneTherapy for Disseminated Neoplastic Disease A C

Myungeun Lee1, Zhi Hong Lu1, Jie Li1, Elena A. Kashentseva1, Igor P. Dmitriev1, Samir A. Mendonca1, andDavid T. Curiel1,2

ABSTRACT◥

The application of cancer gene therapy has heretofore beenrestricted to local, or locoregional, neoplastic disease contexts.This is owing to the lack of gene transfer vectors, which embodythe requisite target cell selectivity in vivo required for metastaticdisease applications. To this end, we have explored novel vectorengineering paradigms to adapt adenovirus for this purpose.Our novel strategy exploits three distinct targeting modalitiesthat operate in functional synergy. Transcriptional targeting isachieved via the hROBO4 promoter, which restricts transgeneexpression to proliferative vascular endothelium. Viral binding ismodified by incorporation of an RGD4C peptide in the HI loopof the fiber knob for recognition of cellular integrins. Liversequestration is mitigated by ablation of factor X binding to the

major capsid protein hexon by a serotype swap approach. Thecombination of these technologies into the context of a single-vector agent represents a highly original approach. Studies ina murine model of disseminated cancer validated the in vivotarget cell selectivity of our vector agent. Of note, clear gains intherapeutic index accrued these vector modifications. Whereasthere is universal recognition of the value of vector targeting, veryfew reports have validated its direct utility in the context ofcancer gene therapy. In this regard, our article validates the directgains that may accrue these methods in the stringent deliverycontext of disseminated neoplastic disease. Efforts to improvevector targeting thus represent a critical direction to fully realizethe promise of cancer gene therapy.

IntroductionA wide range of strategies have been developed to apply gene

therapy to the context of neoplastic disease (1–4). The central goalembodied in these molecular interventions is the achievement of animproved therapeutic index compared with conventional cancer ther-apies. Heretofore, such in vivo cancer gene therapies have been appliedfor local, or locoregional, neoplastic disease. This is owing to the factthat currently available gene transfer vectors lack the in vivo target cellselectivity mandated for the clinical context of disseminated disease.

In this regard, there has been a field-wide recognition of the need forgene transfer vectors, which embody the capacity for target cellselectivity (5). Indeed, an NIH report on gene therapy highlightedthis goal as the highest mandate for the field. For cancer, such a vector-targeting capacity thus represents the sine qua non for practicaladvancement of these promising strategies to the problematic clinicalsetting of metastatic disease. Given this consideration, the paucity ofreports of the successful application of vector targeting for cancer genetherapy is noteworthy.

To this end, we have endeavored modification of adenoviral vectors(Ad) to address this key gene delivery mandate. On the basis of the

unique molecular promiscuity of the parent virus, we hypothesizedthat targeting might be achieved by exploiting multiple biologic axes.Furthermore, we sought to combine such distinct targeting strategiesto realize functional synergy vis-�a-vis the achievement of target cellselectivity. On this basis, the requirement for in vivo selectivity, in thecontext of disseminated neoplastic disease, might be achieved.

Materials and MethodsAdenovirus production

The replication incompetent E1-deleted Ad5 vectors used for thestudy were prepared using a two-plasmid cloning method. Untargetedor triple-targeted Ad5 encoding the GFP reporter gene or the HSVtktherapeutic gene were produced in accordance with the standardtechniques (6). Briefly, adenoviral genome–including plasmids weredigested with PacI for releasing the recombinant viral genomes, andtransfected into HEK293 cells. Rescued viruses were serially amplified,and then purified by centrifugation on CsCl gradients according to thestandard protocols. For in vitro and in vivo study, viruses were dialyzedagainst PBS containing 10%glycerol, and stored at�80�C.The titers ofphysical viral particles were determined by methods described byMaizel and colleagues (7).

In vitro validation of adenovirusHUVEC (human primary endothelial), bEnd-3 (mouse primary

endothelial), and NIH/3T3 (mouse embryonic fibroblast) cells wereobtained from the ATCC and maintained for assays according to themanufacturer's instructions. Total expression levels of HSVtk proteinsin whole-cell lysate were determined with anti-tk antibody (kindlyprovided by Dr. William Summers, Yale University, New Haven, CT)by Western blot analysis in accordance with the standard protocols.For HSVtk/ganciclovir-killing activity assay, cells were infected withvirus encoding either GFP or HSVtk gene, ganciclovir (Selleckchem)prodrugwas administered to cells via serial diluted drug concentration.

1Division of Cancer Biology, Department of Radiation Oncology, School ofMedicine, Washington University in St. Louis, St. Louis, Missouri. 2BiologicTherapeutics Center, Department of Radiation Oncology, School of Medicine,Washington University in St. Louis, St. Louis, Missouri.

Corresponding Author: David T. Curiel, Washington University School ofMedicine, Department of Radiation Oncology, 660 S. Euclid Ave., Campus Box8224, St. Louis, MO 63108. Phone: 314-747-5443; Fax: 314-362-9790; E-mail:[email protected]

Mol Cancer Ther 2020;19:966–71

doi: 10.1158/1535-7163.MCT-19-0768

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To measure the cellular ATP contents (Promega) as a marker for cellviability, assay plates were read in amicroplate luminometer (Bertholddetection system) and cell viability was analyzed. Dose–responseanalysis curves were plotted by Graph Pad Prism v7.0c software.

Murine xenograft modelsTriple immunodeficient NOD/SCID/IL2Rg (NSG) mice were

injected subcutaneously with 1 � 106 786-0 renal carcinoma cells(mCherry expressed cell line). Two weeks later, the mice were intra-venously injectedwith 1� 1011 viral particles of untargetedAd5.CMV.GFP or triple-targeted Ad-GFP viruses. To perform histopathologicanalysis in tumor or organs, mice were sacrificed under anesthesia(Avertin, Sigma-Aldrich) at 3 days post-virus injection. The tumor-bearing tissues were harvested, followed by postfixed in 4% parafor-maldehyde for 2 hours at room temperature, cryopreserved in 30%sucrose for 16 hours at 4�C, and cryo-embedded in NEG50 (ThermoFisher Scientific) over 2-methylbutane/liquid nitrogen. Histopatho-logic images were captured by epifluorescence microscopy. To per-form histopathologic analysis in SPC6 subcutaneous tumor, all assayprocedures were conducted in accordance with the same protocols.

Tissue harvest and immunofluorescence stainingMice were administrated with 1 � 1011 viral particles of triple-

targeted adenoviral vectors via tail vein injection. Three days post-virus infection, mice were anesthetized and tissues (liver, lung, pan-creas, spleen, kidney, small bowel, heart, muscle, and brain) harvestedfor immunofluorescence staining. For frozen sections, organ sliceswere cryo-preserved in 30% sucrose in PBS at 4�C overnight, embed-ded in NEG50 Mounting Medium (Thermo Fisher Scientific), andthen frozen in a liquid nitrogen prechilled 2-methylbutane containingbucket. Sectioning of frozen organs was carried out using the CryoJaneTaping System (Leica Biosystems Inc). All frozen section slides weresubject to immunofluorescence staining analysis in accordance withthe standard techniques (8). Primary antibodies used in this studyincluded hamster anti-CD31 (EMD Millipore), rat anti-endomucin(eBioscience), rat anti-PDGFRb (eBioscience), rabbit anti-HSVtk(Dr. Summers's laboratory), and Alexa Fluor 488 or 594-conjugatedsecondary antibodies (Jackson ImmunoResearch Laboratories).

Animal studies for therapeutic index gainsTriple-immunodeficient NSG mice allow for modeling of all met-

astatic sites as published previously (8, 9). For systemic metastases,mice were injected intracardially with 5 � 105 786-0 renal carcinomacells (Luciferase gene expressed). Tumor growth was monitored andquantified using bioluminescence imaging (BLI) analysis. Three weeks

after tumor implantation,micewere intravenously injected via tail veinwith 1� 1011 viral particles of each virus. During the following 7 days,mice received daily intraperitoneal injection of ganciclovir (50 mg/kg/7 days) according to the different treatment groups [(n ¼ 8 mice/group): untargeted Ad5-GFP, untargeted Ad5-HSVtk, and triple-targeted Ad-HSVtk plus ganciclovir]. Mice were weighed daily togauge health and survival during the assays. For study of tumorprogression, final ROI values were monitored and compared(increased fold ¼ last ROl value/initial ROl value) in the survivalmice. Survival data were plotted on a Kaplan–Meier curve in all animalgroups by Graph Pad Prism v7.0c software. The studied was termi-nated at 8 weeks (56 days posttumor implantation) according to theapproved protocols and pursuant to NIH guidelines for the care anduse of laboratory animals’ standards. All animal experiments werereviewed and approved by the Institutional Animal Care and UseCommittee of the Washington University in St. Louis, School ofMedicine (protocol #.20160292, St. Louis, MO).

ResultsGenerated triple-targeting vector for gene therapy

As the main limit to the systemic employment of adenoviral vectorsis liver sequestration (10), we first sought to address this issue. Ourstudies, and those of others, had linked this phenomenon to vectorcapsid association with serum factor X (11, 12). On this basis, weemployed a strategy of capsid chimerism whereby the major capsidprotein hexon of the serotype 5 vector base was substituted with acognate from the alternate human adenovirus serotype 3. We hadpreviously shown that this modification substantially mitigatedsequestration in the reticuloendothelial system in the context ofsystemic vector administration (12, 13). This liver “untargeting”method was then combined with modifications designed to direct theexpression of delivered transgenes to tumor vascular endothelium.Specific vector particle binding was enhanced for the target cells by theincorporation into the adenoviral vectors capsid fiber knob of theRGD4C peptide (14, 15). This ligand exhibits preferentiality forintegrins of avb3 and aVb3 class overexpressed in angiogenic endo-thelium. This approach was also combined with a transcriptionaltargeting strategy to restrict transgene expression target cells. In thiscase, the promoter of the roundabout guidance receptor 4 (ROBO4)gene, which is selectively inductive for proliferative endothelium, wasemployed. We hypothesized that the combination of the liver untar-geting and vector-targetingmethods (Fig. 1) would provide functionalsynergy with respect to the achievement of the in vivo selectivitymandated for disseminated neoplastic disease cancer gene therapy.

Figure 1.

Schema of “triple-targeted” genetic modifications ofadenovirus to accomplish in vivo targeting of tumorendothelium. Liver untargeting has previously beenaccomplished via genetic modification of hexondomains that can associate with serum factor X.Hexon ectodomain hypervariable regions (HVR7) ofthe vector were replaced with base human adeno-virus serotype 5 with the corresponding domainsfrom human adenovirus serotype 3 (H5/H3). In addi-tion, for transcriptional targeting, the 50 upstreamregion of the therapeutic gene for roundabout 4(ROBO4; endothelial-specific promoter) was config-ured within the deleted adenovirus E1A/B site. Theintegrin targeting peptide RGD4C (CDCRGDCFC)was incorporated into HI loop of adenoviral vectorsfiber knob for transductional targeting.

Triple-Targeted Adenoviral Vector for Cancer Gene Therapy

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Figure 2.

In vitro validation of adenoviral vectors. A, Assessment of inclusion of the HSVtk gene into E1 region from purified viral genomes (i, Ad5; ii, Ad5.CMV.HSVtk; and iii,triple-targeted Ad-HSVtk) by PCR using HSVtk-specific primers. Viral genomes are shown with the black arrow, and amplified HSVtk gene from each of the viralgenomes is shownwith the red arrow. B, Validation of HSVtk gene delivery and expression via adenoviral vectors in HUVEC cells usingWestern blotting analysis. C,Evaluation of cell killing efficiency by HSVtk/ganciclovir (GCV) treatment on endothelial cells. Each of HUVEC (human) and bEnd-3 (mouse) were infected with eachvirus encoding eitherGFPorHSVtk and then treatedwith ganciclovir prodrug (serial diluted drug concentration shown in x-axis). Results are presented as the relativepercentage of cell viability measured by cellular ATP contents. D, Assessment of transduction efficacy in vitro. Each of NIH/3T3 (mouse embryonic fibroblast) andbEnd-3 (mouse endothelial cell) were infectedwith triple-targetedAd5-GFPwith two different MOI (500 and 1,000). Blue, nuclei (stainedwith DAPI) and green, GFPsignal through adenoviral vectors.

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Evaluated vector for cancer gene therapySuch a triple-targeted adenoviral vectors was thus constructed

to achieve expression of the HSVtk therapeutic gene selectivelywithin proliferative tumor endothelium in vitro. This gene has beenapplied historically for molecular chemotherapy cancer gene therapyapproaches (16, 17). Its employment here thus served to reconcile ourfindings with the extensive literature relating to the use of HSVtk forcancer gene therapy. After rescue and upscale, the virion was subjectedto genomic analysis (Fig. 2A). This study confirmed that the control

untargeted adenovirus, and the triple-targeted adenovirus, both con-tained the cassette with the HSVtk therapeutic gene. Infection ofhuman endothelial cells (HUVEC), with follow-on Western blotanalysis, confirmed expression of HSVtk in cells infected via bothvectors (Fig. 2B). Next, analysis of the specificity of expression wascarried out utilizing an in vitro assay whereby HSVtk expressionsensitized cells to the cytotoxic effects of ganciclovir. Target cells werehuman endothelial (HUVEC) or murine endothelial cells (bEnd-3). Inthis analysis, it could be seen that the target cells were sensitive to

Figure 3.

Evaluation of vector targeting via systemicdelivery inmurine xenograftmodels (triple immunodeficientNSGmice).A,Comparison analysis of targeting capacitywithGFP reporter maker by histopathologic methods. Experimental procedures for this study are described in Materials and Methods. The cryo sections of the liver (top)and tumor (bottom) were obtained from each of the Ad5 or triple-targeted Ad5-GFP–injected mice at 3 days later. All assays were conducted and compared inparallel conditions. Blue, nuclei (stained with Hoechst 33258); red, mouse CD31/endomucin in the liver; or mCherry signal from the tumor and green, GFP signalthrough adenoviral vectors. B, In vivo confirmation of triple-targeted Ad5-GFP transduction on tumor endothelium. The cryo section of the tumor (human 786-0subcutaneous) was obtained from triple-targeted Ad5-GFP–infected mice (same as A) and was subject to immunofluorescence staining analysis with specificantibodies (anti-Cd31/anti-endomucineor anti-PDGFPb) as an endotheliummarker. In the tumor, endotheliumand triple-targetedAd5-GFPare colocalized inyellow.C, In vivo validation of ectopic gene delivery and transduction with our triple-targeted Ad5-GFP. NSG mice were intravenously injected with 1 � 1011 viral particles(each of Ad5-GFP or triple-targeted Ad5-GFP) and then harvested of all tissues (liver, lung, spleen, kidney, heart, small bowel, pancreas, brain, and muscle) forhistopathologic assayby immunofluorescence staining. Blue, nuclei (stainedwithHoechst 33258); red,mouseCD31/endomucin.D,Evaluation of targeting specificityof our triple-targeted Ad5-GFP in the context of SGH SHPC6 subcutaneous tumor (SHPC6: Syrian hamster pancreatic carcinoma). Experimental procedures for thisstudywere in accordance as inA. Blue, nuclei (stainedwith Hoechst 33258); red, mouse CD31/endomucin in the tumor; and green, GFP signal through triple-targetedadenoviral vectors. Tumor endothelium and triple-targeted Ad5-GFP are colocalized in yellow (arrow).






vector-mediated cytotoxicity exclusively after infection with the triple-targeted adenoviral vectors encoding HSVtk (Fig. 2C), under ganci-clovir treatment. In addition, regarding to the transduction efficacy, weconfirmed with NIH/3TC cells (mouse embryonic fibroblast) includ-ing demonstration of the target cells specificity (Fig. 2D). These studiesthus validated the endothelial selectivity of our vector targetingschema.

Advanced vector targeting to tumor in vivoWe next sought to validate the in vivo selectivity of our vector

methods in a systemic delivery context. For this analysis, we derivedversions of the control and triple-targeted adenovirus, whichencoded the GFP reporter gene. Immunodeficient NSG micewere xenografted with subcutaneous nodules of the human renalcarcinoma cell line 786-0 (8). Animals were then treated with 1 �1011 particles of the control and targeted adenoviral vectorswith analysis of harvested tumor and organs at 3 days later. Asthe major site for ectopic adenoviral vectors localization is the liver,analysis was limited to this site and target tumor. In these studies itcould be seen that untargeted adenoviral vectors was largely

sequestered in the liver, with no detectable reporter noted withinvascular areas of the tumor (Fig. 3A). In marked contrast, the triple-targeted adenoviral vectors avoided ectopic localization withinthe liver and spleen. Of note, high levels of reporter gene wereseen within the harvested subcutaneous tumor nodules via second-ary staining analysis that confirmed the identity of gene modifiedcells as tumor endothelium (Fig 3B). Our findings confirm thatthe hexon modification of our triple-targeted adenoviral vectorsachieves the desired goal of liver untargeting, without the accrual ofmajor new sites of ectopic gene delivery (Fig. 3C). Furthermore, asan even more stringent model (xenografted with subcutaneousnodules of the Syrian golden hamster pancreatic carcinoma cellline, SHPC6), the targeting specificity of our triple-targeted ade-novirus in the context of other tumors could be seen (Fig. 3D).Thus, our triple-targeting strategy achieves highly specific in vivoselectivity.

Improved vector to achieve the therapeutic index gains in vivoFinally, we endeavored evaluation of the therapeutic index

gains, which accrue synergistic targeting. The therapy experiment

Figure 4.

Assessment of the therapeutic index gains in metastatic murine models. A, The immunodeficient NSG mice were intracardiacally injected with 5 � 105 786-0 renalcarcinoma cells (Luciferase/mCherry gene expressed cell line). Three weeks after tumor implantation, NSG mice were intravenously injected with 1 � 1011 viralparticles of viruses (n¼ 8mice/each of groupwith Ad5.CMV.GFP, Ad5.CMV.HSVtk, and triple-targetedAd-HSVtk) and treatedwith ganciclovir (50mg/kg/7 days byintraperitoneal injection). Mice wereweighed daily to gauge health and survival, upon a 20%ormore decrease in initial bodyweight micewere considered nonviableand sacrificed. The comparative analysis of therapeutic index gains was assessed between the groups by the tumor progression in B and survival time in C. Thestatistical significance of differences between data determined as a P value (��, P < 0.05, by Graph Pad Prism v7.0c software). Tumor progression was detectedthrough ROI value (final ROI value divided by initial ROl) by BLI analysis. The final ROI valueswere only determined from the three survivingmice at the conclusion ofthe study (56 days post–tumor implantation). All mice from Ad5.CMV.HSVtk virus–injected group died 3 weeks later due to hepatotoxicity as shown in A.

Mol Cancer Ther; 19(3) March 2020 MOLECULAR CANCER THERAPEUTICS970

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with targeted adenovirus (vs. nontargeted adenovirus) was employedin amurinemodel ofmetastatic disease. The triple-targeting specificitymitigated the vector-associated toxicity known to be associated withthe systemic application of untargeted adenoviral vectors encodingHSVtk (ref. 18; Fig. 4A). In parallel with that, of note, it could be seenthat extended survival time accrued to the treatment group thatreceived only the triple-targeted adenoviral vectors encoding HSVtkwith significant differences (��,P< 0.005;Fig. 4C). Our demonstrationof antitumor therapy was accomplished in a context more stringentthan that required only for local control; however, it could be seen asthe potential for improvement of the therapeutic effect (Fig. 4B). Also,it could be seen the persistence of the vector in the metastatic tumor aswell. These findings demonstrated directly the improved therapeuticindex, which accrued the employment of vector targeting for thisestablished cancer gene therapy approach.

DiscussionOur study highlights the direct benefits that may derive from the

application of vector-targeting methods to the context of cancer genetherapy. In this regard, in vivo cancer gene therapies have beenrestricted to local diseases contexts owing to the limits of currentlyavailable gene transfer vectors. Thus, the problematic clinical contextof metastatic cancer has not been addressable to this point via genetherapy methods. Further in this regard, the universal recognition ofthe potential value of vector targeting has not been translated to thecontext of cancer gene therapy studies demonstrating therapeuticgains. The advent of our novel vector-targeting technology nowprovides adequate selectivity in vivo to test this hypothesis of field-wide significance. Our early finding reported here thus provide therationale for future studies designed to apply cancer gene therapy tothis problematic clinical context most warranties novel interventions.

Disclosure of Potential Conflicts of InterestD.T. Curiel is a paid consultant for Samyang Biopharmaceuticals and is an

advisory board member (unpaid consultant) for Precision Virologics, Inc., UnleashImmunoOncolytics, Inc., DNAtri, Inc., and Altimmune, Inc. No potential conflicts ofinterest were disclosed by the other authors.

Authors’ ContributionsConception and design: M. Lee, Z.H. Lu, I.P. Dmitriev, D.T. CurielDevelopment of methodology: Z.H. Lu, E.A. Kashentseva, I.P. Dmitriev,S.A. MendoncaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): M. Lee, Z.H. Lu, E.A. KashentsevaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Lee, J. LiWriting, review, and/or revision of the manuscript:M. Lee, Z.H. Lu, I.P. Dmitriev,D.T. CurielAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): M. Lee, E.A. Kashentseva, I.P. DmitrievStudy supervision: Z.H. Lu, I.P. Dmitriev

AcknowledgmentsWe thank Dr. Arbeit for providing critical precedent research. We also appreciate

Dr. Summers's laboratory for providing HSVtk antibody essential to this study. Wealso thank Dr. Toth's laboratory for providing SHPC6 (Syrian Hamster PancreaticCarcinoma) cells essential to this study. We especially thank Amanda Baker Wilms-meyer for extensive advice on the article and the scientific graphics. This study wasfunded by the NIH (RO1's CA211096, to principle investigator D.T. Curiel) researchgrants.

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

Received August 7, 2019; revised October 24, 2019; accepted December 31, 2019;published first January 6, 2020.

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2020;19:966-971. Published OnlineFirst January 6, 2020.Mol Cancer Ther Myungeun Lee, Zhi Hong Lu, Jie Li, et al. Neoplastic Diseaseto Achieve Effective Cancer Gene Therapy for Disseminated Targeting Tumor Neoangiogenesis via Targeted Adenoviral Vector

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