Ad5 -A20: A Tropism-Modiﬁed, avb6 Integrin-Selective ... · wild-type Ad5 for tumor therapy. We have generated a novel virotherapy vector, Ad5 NULL-A20, with altered, tumor-selective

Translational Cancer Mechanisms and Therapy

Ad5NULL-A20: A Tropism-Modified, avb6Integrin-Selective Oncolytic Adenovirus forEpithelial Ovarian Cancer TherapiesHanni Uusi-Kerttula1, James A. Davies1, Jill M. Thompson2,Phonphimon Wongthida2, Laura Evgin2, Kevin G. Shim2, Angela Bradshaw3,Alexander T. Baker1, Pierre J. Rizkallah4, Rachel Jones5, Louise Hanna6,Emma Hudson6, Richard G. Vile2, John D. Chester1,6, and Alan L. Parker1

Abstract

Purpose: Virotherapies are maturing in the clinical setting.Adenoviruses (Ad) are excellent vectors for the manipulabilityand tolerance of transgenes. Poor tumor selectivity, off-targetsequestration, and immune inactivation hamper clinicalefficacy. We sought to completely redesign Ad5 into a refined,tumor-selective virotherapy targeted to avb6 integrin, whichis expressed in a range of aggressively transformed epithelialcancers but nondetectable in healthy tissues.

Experimental Design: Ad5NULL-A20 harbors mutations ineach major capsid protein to preclude uptake via all nativepathways. Tumor-tropism via avb6 targeting was achieved bygenetic insertion of A20 peptide (NAVPNLRGDLQVLAQK-VART) within the fiber knob protein. The vector's selectivityin vitro and in vivo was assessed.

Results: The tropism-ablating triple mutation completelyblocked all native cell entry pathways of Ad5NULL-A20 viacoxsackie and adenovirus receptor (CAR), avb3/5 integrins,and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and

selectively transduced avb6þ cell lines and primary clinicalascites-derived EOC ex vivo, including in the presence ofpreexisting anti-Ad5 immunity. In vivo biodistribution ofAd5NULL-A20 following systemic delivery in non–tumor-bearing mice was significantly reduced in all off-targetorgans, including a remarkable 107-fold reduced genomeaccumulation in the liver compared with Ad5. Tumor up-take, transgene expression, and efficacy were confirmed in aperitoneal SKOV3 xenograft model of human EOC, whereoncolytic Ad5NULL-A20–treated animals demonstrated sig-nificantly improved survival compared with those treatedwith oncolytic Ad5.

Conclusions: Oncolytic Ad5NULL-A20 virotherapies rep-resent an excellent vector for local and systemic targetingof avb6-overexpressing cancers and exciting platformsfor tumor-selective overexpression of therapeutic anticancermodalities, including immune checkpoint inhibitors.Clin Cancer Res; 24(17); 4215–24. �2018 AACR.

IntroductionOvarian cancer remains the deadliest gynecologic cancer with

global 5-year survival rates below 50% (1). The early stages ofthe disease are commonly asymptomatic, with the result thatmost patients have advanced, incurable disease, at presenta-tion. Ovarian cancer metastasizes with large volumes of malig-nant, intraperitoneal ovarian ascites (OAS) providing a protu-

morigenic microenvironment (2). Chemoresistance rapidlydevelops during treatment, requiring alternative regimens.Epithelial ovarian cancer (EOC) is the most common (90%)ovarian cancer type (3). One-third of patients with EOC havecells expressing an epithelial cancer-specific marker, avb6 integ-rin (4). Upregulation of avb6 expression in cancer has beenlinked to aggressive transformation, metastasis, and poor prog-nosis (5–8). avb6 is absent in healthy epithelium (5, 9) butwidely overexpressed in plethora of cancers, including ovarian,lung, skin, esophageal, cervical, and head and neck cancers (4),thus making it a promising target for therapeutic vectors. avb6is an activator of TGFb1 signaling that promotes metastasis byenhancing angiogenesis, immune cell suppression, and epithe-lial-to-mesenchymal transition (reviewed in ref. 10).

Cancer virotherapy is undergoing renewed interest, includ-ing recent regulatory approval for clinical use of herpessimplex type 1–based talimogene laherparepvec (T-VEC), thefirst oncolytic immunotherapy approved for advanced mela-noma (11). Very recently, oncolytic viruses were shown tosensitize difficult-to-treat tumors, including triple-negativebreast cancer (TNBC; ref. 12) and glioblastoma (13) to subse-quent immunotherapies with immune checkpoint inhibitors.This highlights the potential of virotherapies for combination

1Division of Cancer and Genetics, Cardiff University, Cardiff, United Kingdom.2Department of Molecular Medicine, Mayo Clinic, Rochester, Minnesota. 3BHFGlasgow Cardiovascular Research Centre, Glasgow, United Kingdom. 4Divisionof Infection and Immunity, Cardiff University, Cardiff, United Kingdom. 5SouthWest Wales Cancer Institute, Singleton Hospital, Swansea, United Kingdom.6Velindre Cancer Centre, Cardiff, United Kingdom.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Alan L. Parker, Division of Cancer and Genetics, HenryWellcome Building, Cardiff University School of Medicine, Heath Park, CardiffCF14 4XN, United Kingdom. Phone: 44-2922510231; Fax: 44-2920687006;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-18-1089

�2018 American Association for Cancer Research.

ClinicalCancerResearch

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studies in the clinical setting, and the scope for generating avector capable of systemically targeting tumors following intra-venous introduction. Adenovirus serotype 5 (Ad5) has beencommonly deployed in clinical trials of cancer and gene ther-apies (14), due to ease of genetic manipulation and capacity forlarge transgenes (15). However, this serotype has suboptimalfeatures that hamper its wider clinical use. As a commonrespiratory virus with high seroprevalence rates (16), efficientneutralization of vector by neutralizing antibodies (nAb) limitsefficacy. Other limitations include significant and rapid off-target sequestration to spleen and liver via complexing of thevirion with human coagulation factor 10 (FX; ref. 17) andpotentially other coagulation factors (reviewed extensively inref. 18), "bridging" the complex to heparan sulphate proteo-glycans (HSPG), abundant on hepatocytes (19). In vitro, Ad5enters host cells via coxsackie and adenovirus receptor (CAR;ref. 20) that is ubiquitous within tight junctions on polarizedepithelial cells (reviewed in ref. 21) but commonly down-regulated in progressive cancers (22–26), limiting use ofwild-type Ad5 for tumor therapy.

We have generated a novel virotherapy vector, Ad5NULL-A20,with altered, tumor-selective tropism. We ablated all nativetropisms of Ad5 by mutating key residues in the three maincapsid proteins (hexon, fiber, and penton) and retargeted theresulting vector, Ad5NULL, to the tumor-selective integrin avb6through incorporation of an avb6-binding peptide (A20,NAVPNLRGDLQVLAQKVART) within the fiber knob domain HIloop, generating the novel vector Ad5NULL-A20. A20 peptide wasoriginally derived from foot-and-mouth disease virus (FMDV)capsid protein VP1, and has high affinity for its native receptor,avb6 integrin (27, 28). We have investigated potential clinicalutility of an oncolytic variant of Ad5NULL-A20 (D24/T1) forintraperitoneal treatment of ovarian cancer by investigatingits biodistribution, tumor-selective oncolytic capabilities andavoidance of immune neutralization using in vitro and in vivomodels of human EOC.

Materials and MethodsAdenovirus vectors, cell lines, and clinical ascites

All vectors generated in this study included a luciferase (Luc)reporter gene. Genetic modifications were carried out by AdZhomologous recombineering methods (29) as described pre-viously (30). Viruses were produced in T-REx-293 or HEK293-b6 cells (for A20-modified viruses) and purified as describedpreviously (30, 31). A triply detargeted vector genome,Ad5NULL, was generated by introducing mutations in key genesencoding of each of the major capsid proteins to precludecellular uptake by all known native Ad5 pathways. Ablationof binding to CAR was achieved via the KO1mutation in the ABloop of the L5 fiber knob gene; ablation of binding to co-agulation factor 10 (FX) via a mutation in hypervariable region7 of the L3 hexon gene; and ablation of avb3/5 integrin bindingvia RGD-to-RGE mutation in the L2 penton base gene. avb6retargeting was achieved by insertion of sequences encodingpeptide A20 (NAVPNLRGDLQVLAQKVART) into the fiberknob HI loop (between residues G546 and D547) of Ad5NULL,generating Ad5NULL-A20. Replication-deficient variants ofAd5NULL-A20 carry a complete E1/E3 deletion. Oncolytic var-iants have a 24-base pair deletion (dl922–947) in the retino-blastoma protein (pRB) binding domain of E1A (D24; ref. 32)and a single adenine insertion at position 445 within theendoplasmic reticulum (ER) retention domain of E3/19K(T1 mutation; ref. 33). Additional details of genetic modifica-tions are provided in Supplementary Methods.

Homology modeling was performed using the previouslypublished Ad5 fiber knob structure (PDB ID: 1KNB; ref. 34) andFMDV O PanAsia VP1 protein in complex with anb6 (PDB ID:5NEM; ref. 35). The peptide sequence forming the inter-action with anb6 (NVRGDLQVLAQKVART) was edited to con-form with the A20 peptide sequence used in this study(NAVPNLRGDLQVLAQKVART), docked to the Ad5 fiber knobstructure in the HI loop and the KO1 mutation added usingWinCoot (36) and PyMol 2.0 (37). The crude Ad5NULL-A20structure was aligned with the existing 5NEM structure and thecomplex energy minimized using the YASARA algorithm (38).Binding energy calculations were performed using PISA (39),and surface charge was calculated using APBS tool in PyMol2.0 (37).

avb6-high/CARþ SKOV3-b6 cell line was generated in-houseby retroviral transfection of SKOV3 cells (that natively express theav subunit; ref. 40) with integrin beta6 pBABE puro plasmid toexpress the b6 subunit. Primary EOC cells from ascites wereobtained through Wales Cancer Bank under existing ethicalpermissions (WCB 14/004). Cells were processed and subcul-tured as described previously (30, 31), and tested regularly forMycoplasma infection by commercially available PCR-basedmethods.

In vitro and in vivo studiesCell surface receptor expression was assessed by flow cyto-

metry (30). The presence of anti Ad5 antibodies in OAS andserum was determined by ELISA as previously reported (41).Antigen specificity of the antibodies was assessed by Westernblot. Transduction efficiency was assessed by standard lucifer-ase assays, described previously (30, 31). Animal experimentswere approved by Institutional Care and Use Committee(IACUC) and performed at Mayo Clinic, Rochester, MN.Animals were age and sex matched. Animal handling and

Translational Relevance

Virotherapies are emerging as clinically important anti-cancer agents, demonstrating synergy with immune check-point inhibitors in several recent, high-profile studies.Because these agents have not evolved to be intrinsicallytumor selective, therapeutic index could be furtherenhanced by a thorough redesign of the virus capsid toimprove tumor selectivity following intravascular delivery.To this end, we have systematically refined the adenovirusserotype 5 (Ad5) capsid to genetically preclude uptake viaall known native cellular entry pathways, to generate a basaland more biocompatible vector, Ad5NULL. To empower thisvector with tumor selectivity, we further engineered theAd5NULL capsid to present a high-affinity avb6 integrin-binding oligopeptide, A20. The resultant virotherapy,Ad5NULL-A20 demonstrates exquisite tumor selectivity bothin vitro and in vivo, with basal "off-target" uptake. Ad5NULL-A20 thus represents a powerful platform for targeted in situoverexpression of immunomodulatory modalities for futuretranslational applications.

Uusi-Kerttula et al.

Clin Cancer Res; 24(17) September 1, 2018 Clinical Cancer Research4216



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injections were performed by a veterinary technologist. In vivoexperiments are further described in detail in SupplementaryMethods.

Statistical analysesFigures and statistical analyses were generated using GraphPad

Prism v6.03. In vitro and ex vivo assays were analyzed by two-tailed unpaired t tests or one-way ANOVA with the Dunnettmultiple-comparisons post hoc test. In vivo data were normal-ized and analyzed by one-way ANOVA or Kruskal–Wallis testwith Sidak or Dunn multiple-comparisons post hoc test, respec-tively. Overall survival (%) following oncolytic treatment isshown as a Kaplan–Meier survival curve; survival proportionswere analyzed by the Gehan–Breslow–Wilcoxon test.

ResultsWe generated and produced to very high viral titers replication-

defective and oncolytic variants of a novel Ad5NULL-A20 vector(Fig. 1A) with three detargeting mutations and an A20 peptideinsertion that retargets the vector toavb6 integrin-expressing cells(Fig. 1B). Additionally, we generated replication deficient andoncolytic versions of Ad5.A20, which harbors the avb6 targeting-peptide A20 insertion, in the absence of any detargeting modi-fication. The multiple genetic manipulations did not have asignificant impact on viral titer (Fig. 1A).

We generated a homology model of the Ad5NULL-A20 fiberknob protein in complex with the anb6 dimer (Fig. 1C). The A20peptide (dark blue) occupies space spanning both an (green) and

b6 (purple) subunits. The predicted Ad5NULL-A20 interactingresidues of the A20 peptide (dark blue) and the native knobstructure (cyan) against the approximated charge surface of theanb6 (red is negative, blue is positive; Fig. 1D). The anb6 hasmostly negative surface potential in this region (1D), comple-mentary to the predominantly positive charge of theAd5NULL-A20interface (Supplementary Fig. SI A). The adjacent CD loop of thenative Ad5 fiber knob contributes two polar residue interactionsfrom Lys-442 and Gly-443 (1D), binding to an additional threean residues (Supplementary Fig. SI B). The binding energy of theanb6-Ad5NULL-A20 fiber knob complex is calculated to be –24.3Kcal/mol, suggesting an exceptionally stable interface (Supple-mentary Fig. SI C), providing confidence that our avb6 targetingstrategy was feasible.

The transduction efficiency of replication-deficient vectors wasassessed in cell lines expressing variable levels of CAR and avb6integrin. The detargeting mutation triplet of Ad5NULL-A20completely abolished entry via CAR in CHO-CAR cells (CARþ),while Ad5 transduced these cells at expectedly high efficiency(Fig. 2A). The HVR7mutation abolished Ad5 vector transductionvia FX pathway (Fig. 2B; ref. 42). As expected, FX significantlyincreased transduction of Ad5 into CHO-K1 cells as comparedwith FX-free culture conditions (Fig. 2B, left). Conversely, addi-tion of human FX in culture medium had no effect on thetransduction efficiency of the FX binding-ablated Ad5.HVR7control vector in these cells (Fig. 2B; right). Furthermore, theenhanced transduction seen for Ad5 was reversed by the additionof a 3:1 molar excess of Gla-domain interacting protein, antico-agulant X-bp, which binds and inactivates FX in the medium

Figure 1.

Generated vectors. A, Viral titers andexpected tropisms of Ad5 and triplydetargeted, avb6 integrin retargetedvector, Ad5NULL-A20;B,Vectormap ofthe oncolytic Ad5NULL-A20; C,Homology modeling of the Ad5 fiberknob with A20 peptide(NAVPNLRGDLQVLAQKVART; darkblue) within the HI loop of fiber knobdomain (Ad5.A20; in light blue) incomplex with an (green) and b6(magenta) integrin subunits shows apotential mechanism for the Ad5NULL-A20 interface. D, Residues in both anand b6 subunits form hydrogen bonds(red dashes), stabilizing a chargedinterface (anb6, negative; A20,positive). Residues in Ad5s CD loopform further polar interactions. CAR,coxsackie and adenovirus receptor;FX, coagulation factor 10; HVR7,hypervariable region 7 (42); KO1,CAR-binding mutation in fiber knobAB loop (53); Luc, luciferasetransgene; repl. def., replication-defective; vp, viral particle.

Ad5NULL-A20: An Exquisitely Tumor-Selective Virotherapy

www.aacrjournals.org Clin Cancer Res; 24(17) September 1, 2018 4217




(ref. 19; Fig. 2B, left). On the contrary, FX depletion did not affectthe transduction of Ad5.HVR7 vector (Fig. 2B, right).

We confirmed avb6 integrin as the primary entry receptorfor the triply detargeted, integrin retargeted Ad5NULL-A20 vector(Fig. 3). Ad5NULL-A20 transduced avb6þ/CAR� BT-20 breast can-cer cells with 305-fold higher efficiency (Fig. 3A; P¼ 0.0270) andprimary, patient-derived EOC004 cells (avb6þ/CAR�) at 69-foldincreased efficiency (Fig. 3B; P ¼ 0.0090) relative to Ad5. Com-petition assays using a function-blocking anti-avb6 antibody

(10D5) significantly inhibited cell transduction by Ad5NULL-A20 vector in SKOV3-b6 cells (avb6þ/CARþ; Fig. 3C; P ¼0.0010), confirming the vector's selectivity for avb6 integrin.

We next evaluated the ability of the Ad5NULL-A20 vector toretain its infectivity in the highly neutralizing environmentpresented by OAS. To this end, freshly isolated clinical OASsamples from 20 patients with ovarian cancer were screenedfor the presence of anti-Ad5 antibodies by direct ELISA. Thetiters of anti-Ad5 abs in malignant OAS were scrutinized againstthe serum anti-Ad5 antibody titer of a healthy adult malevolunteer (Fig. 4A). Equal proportions of patients were foundto have lower and higher antibody titers than the control serum(Fig. 4A, black dashed line). Ascites from patient 001 (OAS001)were chosen for subsequent neutralization assays due to itssimilar antibody titer with the control serum. Antibodies inOAS001 and control serum appeared specific for the viral fiberprotein, whilst the most abundant capsid protein—hexon—was recognized only at very low levels in Western blot usingdenatured whole viral particles (Fig. 4B). The neutralizing effectof OAS001 on transduction efficiency of Ad5NULL-A20 wasassessed in avb6þ/CAR� EOC004 primary cells. Ad5NULL-A20 showed up to 902-fold higher transduction efficiency inprimary human EOC cultures relative to Ad5 at OAS concen-trations of 2.5%, 5%, and 10%, whilst Ad5 was not capable oftransducing these cells at detectable levels (Fig. 4C).

We next evaluated biodistribution of virus infection in im-munocompetent, non–tumor-bearing mice. Mice were injectedintravenously with replication-defective vectors to assess in vivotropism (Fig. 5A), in particular the effect of the three detarget-ing mutations on biodistribution of virus infection. As expect-ed and as previously documented, the Ad5 vector showedintense localization in the area of liver and spleen, whileluminescence by the Ad5NULL-A20 vector was completely un-detectable at the 72-hour time point (Fig. 5B). Animals inoc-ulated with Ad5 vector had significantly higher whole-bodyluminescence than the control animals (P < 0.0001) or theAd5NULL-A20 vector (P < 0.0001; Fig. 5C). The liver, spleen,lungs, ovaries, and heart were resected postmortem and quan-tified for ex vivo luminescence (for luminescence heatmaps,see Supplementary Fig. SIIA–C). The livers of Ad5-challengedanimals emitted significantly more luminescence than thePBS control or Ad5NULL-A20 groups (both P < 0.0001;Fig. 5D). Similarly, Ad5NULL-A20 had significantly decreasedtransgene expression in the spleen, lungs, ovaries and heart,relative to Ad5 (Fig. 5E–H; P < 0.0001 for all). For fold changes

Figure 2.

Ablation of native receptor tropisms. A,Binding of replication-deficient Ad5 andAd5NULL-A20 vectors to coxsackie andadenovirus receptor (CAR). Ratio of viraltransgene expression from Ad5NULL-A20relative to Ad5 is indicated above bars. B,Binding of replication-deficient Ad5 andHVR7-mutated Ad5 variant (42) tocoagulation factor 10 (FX) was assessed inluciferase assays by infecting CHO-K1 cells inthe presence of human FX with (þ) orwithout (�) anticoagulant X-bp. HVR7, FX-bindingmutation. Statistical significance: ns,P > 0.05; �� , P < 0.01.

Figure 3.

In vitro assessment of avb6 integrin retargeting. Transduction efficiency ofreplication-deficient wild-type (Ad5) and triply detargeted, integrin retargeted(Ad5NULL-A20) vectors in (A) avb6þ BT-20 breast cancer cells and (B) avb6þ

primary EOC cells from patient 004. C, Competition inhibition of avb6 integrin-mediated cell entry in SKOV3-b6 cells. The highest 10% avb6-expressingSKOV3-b6 cells were sorted by FACS, subcultured, and infected. IgG, normalmouse IgG control; 10D5, anti-avb6 function-blocking antibody. Ratio of viraltransgene expression is indicated above bars. Statistical significance: ns,P > 0.05; � , P < 0.05; �� , P < 0.01; �� , P < 0.001; �� , P < 0.0001.






in luminescence intensity in each off-target organ, see Supple-mentary Fig. SIID.

Confirmation that the modifications in Ad5NULL-A20 result-ed in reduced sequestration of virus in multiple normaltissues was performed via quantitation of viral load by qPCR.Genome copy-number of the Ad5NULL-A20 vector was 10million times lower in the liver relative to the Ad5 (Fig. 6A;P < 0.0001). Similarly, Ad5NULL-A20 genome copy-number wasover 700-fold lower in the spleen compared with Ad5 (Fig. 6B;P < 0.0001). In addition, the Ad5NULL-A20 vector showedimproved off-target profiles in all organs relative to Ad5, withviral load 105, 104, and 103 lower in the lungs, heart, andovaries, respectively (Fig. 6C–E). Successful detargeting of theliver being due to our genetic modifications of Ad5 is support-ed by immunohistochemical staining of liver sections,which showed high expression levels of CAR, whilst avb6 was

undetectable (Supplementary Fig. SIIIA). Confirmation of thedetargeting effects of genetic modifications in Ad5NULL-A20is provided by the observation that liver sections from miceshowed positive staining for Ad capsid proteins in the Ad5group, but not in livers of mice that had been challenged withthe Ad5NULL-A20 vector (Supplementary Fig. SIIIB).

To evaluate efficacy in a human EOC model in vivo, SKOV3human ovarian cancer xenografts were established in immuno-compromised NOD/SCID mice. Animals developed large solidtumors at the cell injection site and at various sites within theperitoneal cavity within 14 days after intraperitoneal implanta-tion of SKOV3 cells (for tumor localization and take rate; seeSupplementary Fig. SIV) and by day 49, tumors were spreadthroughout the peritoneal cavity with accumulation of largevolumes of ascites. Based on these observations, we performedvirotherapy efficacy studies by delivering three intraperitoneal

Figure 4.

The effect of malignant OAS on vector transduction ex vivo. A, Quantification of anti-Ad5 antibodies in 20 clinical OAS samples and control serum from ahealthy male volunteer (solid black line) by ELISA. Horizontal lines indicate 50% and 100% binding of anti-Ad5 abs in the control serum. B, Antigenspecificity of anti-Ad5 antibodies in ascites and serum by Western blot, using denatured whole virus particles. C, Vector transduction efficiency of replication-defective (Ad5) and Ad5NULL-A20 vectors, in the absence and presence of varying dilutions of ascites from an ovarian cancer patient 004 in primary ex vivoculture of EOC cells from patient 004. Cells were preincubated with ascending concentrations of ascites and infected.






doses of oncolytic variants of Ad5, Ad5.A20, and Ad5NULL-A20vectors on days 14, 16, and 18 after implantation of SKOV3 cells.

IVIS imaging at 48 hours after first virotherapy treatmentdose (day 16) showed widespread luminescence throughoutthe abdominal region in animals with SKOV3 xenografts andtreated with the oncolytic Ad5 vector, with highest intensity inthe liver/spleen region (Fig. 7B). This distribution was main-tained, but at lower intensity, until 5 days later, day 21 (Fig.7B). In contrast, the oncolytic Ad5NULL-A20 vector, however,showed selective tumor localization, with significantly reducedoverall luminescence relative to Ad5, consistent with successfuldetargeting of non-tumor tissues. The distribution of infectionmediated by the oncolytic Ad5.A20 vector was intermediatebetween the Ad5 and Ad5NULL-A20. Quantitation of total bodyluminescence showed uptake of the Ad5NULL-A20 vector to besignificantly lower than Ad5 both on day 16 (Fig. 7C; P < 0.05

and 0.05;� , P < 0.05; �� , P < 0.01; �� , P < 0.001;�� , P < 0.0001.






known native tropisms and retargeted to an overexpressed, prog-nostic cancer marker—avb6 integrin (43). Integrin avb6 is apromising target for therapeutic cancer applications due to itsoverexpression in aggressively transformed cancers (4). A20 pep-tide is a feasible tool for a variety of clinical applications andhas been used for imaging diagnostics in an avb6þ pancreatictumor model (44) and in a humanized single-chain Fv antibodyB6-2 (45). avb6 is emerging as a promising target for a range ofadvances therapies, including those based on chimeric antigenreceptor (CAR) T-cell immunotherapies (reviewed in ref. 46),where efficacy in the avb6 expressing SKOV3 cell lines hasbeen demonstrated. Furthermore, the avb6-blocking antibody264RAD showed promising in vivo efficacy in HER2þ/avb6þ

breast cancers in combination with the monoclonal antibodytrastuzumab (47) and is being developed for phase I clinicaltrials. avb6 therefore represents a highly appealing target forcancer treatment across a range of technologies and therapeuticapplications.

In silico evaluation of the Ad5NULL-A20 interface with anb6by homology modeling (Fig. 1; Supplementary Fig. SI) predictsthe Ad5NULL-A20 fiber knob domain to form a low entropyinterface with anb6. A20 possesses the putative RGD integrininteracting motif (48), but specificity to the b6 subunit isderived from the helical motif C-terminal of RGD. It is furtherstabilized by electrostatic interactions across the interface and

polar bonds between an and the Ad5 CD loop. Each fibertrimer possesses three copies of the A20 peptide, with 12trimeric fibers per adenovirus capsid, thus Ad5NULL-A20 pos-sesses 36 potential anb6 interaction sites per viral particle.While not all these sites will be utilized in a single cellularinteraction, it is extremely likely that the virus benefits from apotent avidity effect when interacting with a cell possessingmultiple anb6 copies.

In the present study, we presented the Ad5NULL-A20 as ahighly selective vector platform. A replication-defective form ofAd5NULL-A20 vector successfully detargeted viral uptake by cellsvia native viral uptake pathways (Fig. 2), instead selectivelyretargeting avb6þ cells, in vitro and ex vivo (Fig. 3). Although theefficacy-limiting interactions that occur in the systemic deliveryof adenoviral vectors can, theoretically, be bypassed by intra-cavity administration of the vector via the i.p. route, in practicethis approach presents challenges because wild-type Ad5 issequestered by preexisting anti-Ad5 immunity in the form ofneutralizing antibodies (nAb) in ascitic fluid (41, 49, 50). Wetherefore assessed the transduction efficiency of Ad5NULL-A20in the presence of freshly isolated clinical OAS from patientswith ovarian cancer with confirmed high levels of anti-Ad5nAbs (Fig. 4A). Unlike the Ad5 vector, Ad5NULL-A20 retained itsability to transduce avb6þ cells, even at relatively high OASconcentrations (Fig. 4C).

Figure 6.

Viral genome copy-number in off-target organs at 72 hours followingsystemic delivery. Adenovirus genomecopy-number from tissues excisedfrom animals in Fig. 5: (A) liver, (B)spleen, (C) lungs, (D) ovaries, and(E) heart, as determined by qPCR forthe hexon gene, following systemicvector delivery. Error bars, SEM;n ¼ 5/group; ns, P > 0.05; � , P < 0.05;�� , P < 0.01; �� , P < 0.001;�� , P < 0.0001. Numbers belowgraphs indicate fold decrease ofthe Ad5NULL-A20 group relative tothe Ad5 group.






Clinical efficacy of therapeutic Ad5 vectors with unmodifiedcapsids is also significantly limited by off-target tissue sequestra-tion, particularly in the liver. We demonstrate that Ad5NULL-A20significantly altered the biodistributionof theAd5 vector in vivobyreducing the sequestration in remarkable magnitudes. In tumor-free mice, replication-deficient Ad5NULL-A20 demonstrated sig-nificantly reduced viral transgene expression the liver, spleen, andlungs compared with the parental Ad5 (Fig. 5), and lower viralgenome copy-number in all off-target organs relative to the Ad5vector (Fig. 6).

To test the efficacy of an oncolytic form of our detargeted/retargeted Ad5NULL-A20 vector, we established an orthotopic i.p.

xenograft model of human EOC SKOV3 in immunocompro-misedmice. Themore localized biodistributionof virally encodedtransgene expression of oncolytic Ad5NULL-A20 following intra-peritoneal administration was consistent with reduced off-targetsequestration and/or tumor-selective virus uptake (Fig. 7B–E).This was supported by the superior survival of animals treatedwith Ad5NULL-A20 relative to Ad5 in a SKOV3 xenograft model(Fig. 7E), although extended survival (compared with unmodi-fiedAd5)was not observed inmice treatedwith the oncolytic Ad5.A20 variant. This observation highlights that efficacy in vivodepends upon both the combination of complete ablation of allnative means of cellular uptake via hCAR, avb3/5 integrins and

Figure 7.

Oncolytic efficacy study:intraperitoneal delivery of oncolyticvectors in ovarian cancer xenograftmodel. A, Study schedule.Intraperitoneal xenografts of humanovarian cancer SKOV3 cells wereimplanted into immune-compromisedmice (n¼ 5/group), then animalsweretreated with 3 doses of intravenousoncolytic Ad5, avb6 integrinretargeted Ad5.A20 or triplydetargeted, avb6 integrin retargetedAd5NULL-A20, on days 14, 16, and 18. B,Luminescence heatmap images andquantitation of total bodyluminescence were determined at 48hours after the first treatment (C; day16), and at 7 days after the firsttreatment (D; day 21). E, Overallsurvival of animals inoculated withSKOV3 xenografts (avb6-low/CARþ)and then treated with virus, as above,shown as a Kaplan–Meyer survivalcurve until the final study endpoint of101 days. i.p., intraperitoneal; IVIS, invivo imaging system; vp, viral particle;� , P < 0.05; �� , P < 0.01; �� , P < 0.001.






FX, coupled with an efficient and selective retargeting mechanismto tumor-associated ligands, such as the avb6: A20 receptor:ligand interaction. This observation likely explains previous stud-ies (51, 52), which described no improved efficacy (comparedwith oncolytic Ad5) of virotherapies targeted to avb6 integrin,because the vectors used in those studies lacked modifications inat least two of the three native infectious pathways (the hexon: FXand penton base: avb3/5 interactions). Additional studies will beneeded to fully evaluateavb6þ cancer retargeting in vivo, as well asto dissect the fate in tissues and immunologic responses to theAd5NULL-A20 vector.

Local, i.p. Ad5NULL-A20 administration presents a promisingtreatment option for advanced, chemotherapy-resistant, avb6þ

ovarian cancer. Here, we describe a novel vector that can befurther manipulated for various clinical applications, with thescope of selective targeting to avb6 integrin-expressing cellsand minimal off-target effects that limit current Ad5-basedtherapies. Ad5NULL-A20 vector provides an agile and versatileplatform that could ultimately be modified for precisionvirotherapy applications by various innovative approaches,potentially providing a platform for the local, tumor-selectiveoverexpression of additional, virally encoding therapeuticmodalities, such as immunotherapies.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: R. Jones, R.G. Vile, A.L. ParkerDevelopment of methodology: H. Uusi-Kerttula, J.A. Davies, P. Wongthida,K.G. Shim, A.T. Baker, A.L. ParkerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H. Uusi-Kerttula, J.A. Davies, J.M. Thompson,

P. Wongthida, L. Evgin, K.G. Shim, A. Bradshaw, A.T. Baker, R. Jones, L. Hanna,E. Hudson, R.G. VileAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):H.Uusi-Kerttula, J.A.Davies, A. Bradshaw, A.T. Baker,P.J. Rizkallah, R.G. Vile, J.D. Chester, A.L. ParkerWriting, review, and/or revision of the manuscript: H. Uusi-Kerttula,J.A. Davies, P.Wongthida, A.T. Baker, R. Jones, L. Hanna, E. Hudson, J.D. Chester,A.L. ParkerStudy supervision: A.L. Parker

AcknowledgmentsWe are most grateful to patients at Velindre Cancer Centre, Cardiff, UK, who

donated ascites samples. We would like to thank Mrs. Dawn Roberts fortechnical assistance, Dr. Richard Stanton for his expertise in AdZ recombineer-ing, Dr. Alexander Greenshields-Watson for his assistance with homologymodeling and structural biology, the team who maintain the YASARA energyminimization server, Dr. EdwardWang for his guidance in immunohistochem-istry, Dr. Lisa Spary and clinicians at Velindre Cancer Centre for access to clinicalascites samples, Mr. William Matchett for his help with the IVIS 200 software,and Prof. Gavin Wilkinson, Dr. Michael Barry, and Dr. John Marshall forinsightful discussions.

H. Uusi-Kerttula was supported by a Cancer Research Wales PhD stu-dentship to A.L. Parker, J.A. Davies is supported by a Cancer ResearchUK Biotherapeutics Drug Discovery Project Award to A.L. Parker (projectreference C52915/A23946). A.T. Baker is supported by a Tenovus CancerCare PhD studentship to A.L. Parker (project reference PhD2015/L13).A.L. Parker is funded by Higher Education Funding Council for Wales.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received April 8, 2018; revised May 11, 2018; accepted May 15, 2018;published first May 24, 2018.

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2018;24:4215-4224. Published OnlineFirst May 24, 2018.Clin Cancer Res Hanni Uusi-Kerttula, James A. Davies, Jill M. Thompson, et al. Oncolytic Adenovirus for Epithelial Ovarian Cancer Therapies

6 Integrin-Selectiveβvα-A20: A Tropism-Modified, NULLAd5

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Ad5 -A20: A Tropism-Modiﬁed, avb6 Integrin-Selective ... · wild-type Ad5 for tumor therapy. We have generated a novel virotherapy vector, Ad5 NULL-A20, with altered, tumor-selective