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Translational Science

An Oncolytic Virus Expressing a T-cell EngagerSimultaneously Targets Cancer andImmunosuppressive Stromal CellsJoshua D. Freedman1, Margaret R. Duffy1, Janet Lei-Rossmann1, Alice Muntzer2,Eleanor M. Scott1, Joachim Hagel1, Leticia Campo1, Richard J. Bryant3, Clare Verrill3,4,Adam Lambert3, Paul Miller5, Brian R. Champion2, Leonard W. Seymour1, andKerry D. Fisher1

Abstract

Effective immunotherapy of stromal-rich tumors requiressimultaneous targeting of cancer cells and immunosuppres-sive elements of the microenvironment. Here, we modifiedthe oncolytic group B adenovirus enadenotucirev to expressa stroma-targeted bispecific T-cell engager (BiTE). This BiTEbound fibroblast activation protein on cancer-associatedfibroblasts (CAF) and CD3e on T cells, leading to potentT-cell activation and fibroblast death. Treatment of freshclinical biopsies, including malignant ascites and solidprostate cancer tissue, with FAP-BiTE–encoding virusinduced activation of tumor-infiltrating PD1þ T cells to killCAFs. In ascites, this led to depletion of CAF-associatedimmunosuppressive factors, upregulation of proinflamma-tory cytokines, and increased gene expression of markers

of antigen presentation, T-cell function, and trafficking.M2-like ascites macrophages exhibited a proinflammatoryrepolarization, indicating spectrum-wide alteration of thetumor microenvironment. With this approach, we haveactively killed both cancer cells and tumor fibroblasts,reversing CAF-mediated immunosuppression and yieldinga potent single-agent therapeutic that is ready for clinicalassessment.

Significance: An engineered oncolytic adenovirus thatencodes a bispecific antibody combines direct virolysis withendogenous T-cell activation to attack stromal fibroblasts,providing a multimodal treatment strategy within a singletherapeutic agent. Cancer Res; 78(24); 6852–65. �2018 AACR.

IntroductionCancer-associated fibroblasts (CAF) facilitate invasion (1),

coordinate angiogenesis (2), and maintain an immunosuppres-sive microenvironment in solid carcinomas (3). Their immuno-modulatory functions include production of indoleamine2,3-dioxygenase (IDO) and regulatory cytokines such as VEGF,FGF, IL10, and TGFb (4–8). Notably secreted TGFb can accumu-late in the stromal matrix, exerting a powerful immunosuppres-sive effect on newly infiltrating na€�ve immune cells (9, 10), while

CAF-produced CXCL12 can block entry of CD8þ cells into thetumor and attract regulatory T cells, inhibiting effector T-cellproliferation (11, 12).

CAFs are pivotal to tumor immunology, making it difficult toenvisage cancer immunotherapy achieving its full potential with-out addressing their deleterious effects. CAF depletion can reverselocal immune suppression and improve tumor immunotherapy.Genetic-based CAF depletion in an autochthonous pancreaticcancermodel uncovered the ability of anti-PD-L1 to inhibit tumorgrowth and improve survival (13). While such an approach has astrong therapeutic rationale, implementation can be difficult dueto the lack of unique target antigens on theCAF surface, withmostof their known surfacemarkers alsopresent onnormalfibroblasts.

One promising target antigen is fibroblast activation protein(FAP), which is upregulated on CAFs across a broad range of solidmalignancies (14) but also found on normal fibroblasts in con-nective tissue in the muscle, gall bladder, bladder, and bonemarrow stromal cells (BMSC; ref. 15). Eliminationof FAP-positivecells with mAbs or FAP-targeted CAR-T cells demonstrated thepotential to reverse tumor-associated immune suppression, par-ticularlywhen combinedwith immunotherapeutic strategies suchas cancer vaccines (16, 17). However, FAP expression on extra-tumoral cells is concerning, with previous FAP-targeting preclin-ical studies showing extensive bonemarrow toxicity and cachexiathat would caution against clinical development of systemic FAP-targeted treatments (15, 18).

Bispecific T-cell engagers (BiTE) show powerful targeted killingof cancer cells, but can also be deployed against stromal targets

1Department of Oncology, University of Oxford, Oxford, United Kingdom.2PsiOxus Therapeutics Ltd., Abingdon, United Kingdom. 3Nuffield Departmentof Surgical Sciences, University of Oxford, Oxford, United Kingdom. 4OxfordNIHR Biomedical Research Centre, University of Oxford, Oxford, UnitedKingdom. 5Churchill Hospital, Oxford University Hospital NHS Trust, Oxford,United Kingdom.

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

Current address for J. Hagel: Experimental Medicine Division, Nuffield Depart-ment of Medicine, University of Oxford, Oxford, United Kingdom.

Corresponding Author: Leonard W. Seymour, University of Oxford, Old RoadCampus Research Building, Old Road Campus, Headington, Oxford, OX3 7DQ,United Kingdom. Phone: 4418-6561-7040; Fax: 4418-6561-7028;E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-1750

�2018 American Association for Cancer Research.

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such asCAFs. BiTEs crosslink T cells (viaCD3e) to antigen-positivetarget cells, independent of HLA presentation, and can activateany T-cell to engage with and destroy adjacent target cells (19).Moreover, BiTE-mediated T-cell activation can overcome ele-ments of tumor-associated immunosuppression that limit phys-iologic immune responses, leading to reactivation and prolifer-ation of exhausted tumor-specific T cells (20–22). BiTEs targetedto a CAFmarker such as FAP could be a potent strategy to activateintratumoral T cells to attack anddepleteCAFs.However, systemicdelivery of the BiTE would likely mediate significant toxicity byactivating circulating T cells to attack normal fibroblasts andBMSCs. Accordingly, this potentially powerful approach is frus-trated by challenges of site-specific delivery.

With their ability to encode and specifically express biologics indisseminated tumors, oncolytic viruses (OV) are an ideal solu-tion. One promising candidate is enadenotucirev (EnAd), whichhas demonstrated good blood stability and systemic bioavail-ability in several early-phase clinical trials (23–25). An encodedFAP-specific BiTE would be produced and secreted only uponvirus infection of tumor cells, allowing it to access tumor-infil-trating lymphocytes (TIL). This approach has been validatedusing BiTEs to target T-cell cytotoxicity to tumor cell antigens(21, 26–28). However, a virus-encoded BiTE that activates T cellsto kill tumor stromal fibroblasts would provide a "multimodal"therapeutic agent that simultaneously targets two distinct celltypeswithin the tumor. Alongside directOV-mediated cytolysis oftumor cells, which is often proinflammatory (29), secretion ofFAP-specific BiTEs should activate TILs to attack anddeplete CAFs,acting to reverse CAF-induced immune suppression. Thisapproach combines direct cytotoxicity, immune stimulation, andreversal of local immunosuppression, thereby transforming animmunologically inactive "cold" tumor intoone that is "hot," thatis, with greater immune infiltration, yielding an integrated andmore effective immunotherapeutic response.

Materials and MethodsCell lines

DLD, SKOV3, A549, HEK293A (ATCC), and normal humandermal fibroblast (NHDF; Lonza) cells were cultured in DMEM(Sigma-Aldrich). Chinese Hamster Ovary (CHO, ATCC), normalhuman bronchial epithelial (NHBE) cells (Lonza) were culturedin RPMI1640 (Sigma-Aldrich). All cells were authenticated byshort tandem repeat profiling (CRUKCambridge Institute, UnitedKingdom) and routinely tested each month for Mycoplasma(MycoAlert Mycoplasma Detection Kit, Lonza). Cell lines werepassaged no more than ten passages after thawing before use inexperiments. Growthmediumwas supplemented with 10% (v/v)FBS (Thermo Fisher Scientific). Cells were incubated at 37�C and5% CO2. A FAP-expressing stable CHO cell line was generatedusing the FAP gene sequence (ID: 1149, NCBI) as describedpreviously (21).

BiTE engineering and productionA FAP-targeted BiTE was produced by joining the DNA

encoding two single-chain antibody fragments (scFvs) recog-nizing human FAP and CD3e with a sequence encoding aflexible glycine-serine (GS) linker. An N-terminal immuno-globulin signal sequence for mammalian secretion andC-terminal decahistidine tag for detection were added.DNA sequences were synthesized and inserted into a CMV

promoter–driven expression vector (pSF-CMV-Amp; OxfordGenetics) by standard cloning techniques. Recombinant BiTEprotein was produced by transfecting HEK293A cells with poly-ethylenimine [PEI, linear, MW 25000, Polysciences; DNA:PEIratio of 1:2 (w/w)]. Cells maintained in serum-free DMEM.Supernatants were harvested, concentrated 50-fold using10,000 MWCO Amicon Ultra-15 Filter Units (Millipore), andstored at �80�C. BiTE protein concentration was determined bydot blot using decahistidine-tagged cathepsin D (BioLegend) as astandard. Specific binding of the FAP BiTE to recombinant FAPprotein was confirmed by ELISA (data not shown).

Generation of BiTE-expressing enadenotucirevModified EnAd were produced by direct insertion of the BiTE

cassette into the parental EnAd cloning plasmid pEnAd2.4using Gibson assembly (30, 31). Additional viruses with FAPBiTE expression linked to red fluorescent protein (RFP) via aP2A site were also generated. Plasmid DNA was linearized byrestriction digest with AscI (New England Biolabs) and trans-fected into HEK293A cells for virus production in DMEM (2%FBS). Upon extensive plaque formation, cells were harvested,and virus released by three freeze–thaw cycles. Single cloneswere selected by serial dilution and amplified by serial infec-tion, followed by double CsCl banding to produce concentrat-ed virus stocks. Stocks were titered by the Quant-iT PicogreendsDNA assay (Thermo Fisher Scientific) and infectious dosedetermined by serial titration on A549 cells.

Processing and culture of human PBMCs and clinical biopsysamples

PBMCs were isolated from leukocyte cones (NHS Blood andTransplant, UK) by density-gradient centrifugation. CD3þ cellswere extracted by depleting non-CD3 cells using the Pan T-cellIsolation Kit (Miltenyi Biotec). For CD4þ and CD8þ cells,CD4þ Microbeads were used (Miltenyi Biotec). Primary humanmalignant ascites samples and human prostate tissue sampleswere obtained from the Churchill Hospital (Oxford UniversityHospitals NHS Foundation Trust) following written informedpatient consent and approval by the institutional review boardand research ethics committee of the Oxford Centre forHistopathology Research (Reference 09/H0606/5þ5) in accor-dance to the UK Human Tissue Act 2004 and the Declaration ofHelsinki. For ascites, samples were immediately processed withcells and fluid separated by centrifugation (300 � g), with thecellular fractions treated with red blood cell lysis buffer(Qiagen). For ex vivo T-cell activation and cytotoxicity, cellswere used immediately, or adherent cells were expanded byserial passage. For human prostate tissue specimens, tissue wastransported in RPMI and stored on ice until slicing within twohours of surgery. Tissue cores were embedded in UltraPure lowmelting-point agarose (4% w/v, Thermo Fisher Scientific), and300-mm tissue slices were prepared using a vibratome (Leica VT1200S, Leica Microsystems). Each ex vivo tissue slice was trans-ferred to a 0.6 cm2 PTFE insert (Millipore) in 24-well platescontaining 1 mL of cultivation media for prostate tissue (Sup-plementary Material). After overnight culture, the media werereplaced, and tissue slices were treated with BiTE or recombi-nant virus. On day 0, 4, and 7 postinfection, 30% of thesupernatant was collected, frozen, and replaced. On day 7,slices were fixed in paraformaldehyde (4%) and embedded inparaffin for IHC.

Oncolytic Adenovirus Expressing FAP BiTE

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In vitro and ex vivo coculture experimentsFor in vitro coculture studies, PBMCs were seeded with the

appropriate target cells (E:T ratio, 5:1) in flat-bottom 96-wellplates in 100-mLmedium. Target cell lines were prepared with celldissociation buffer to preserve cell surface antigens. For ex vivoexperiments, unpurified total cells from bone marrow or asciteswere seeded in culture medium or fluid from the same exudatesample, respectively. To assess T-cell activation by virus-infectedcells or BiTE-containing supernatants (300 ng/mL), cocultureswere treated with 100-mL supernatant or infected with 100 vp/cellin 100-mL medium. Where appropriate, CD3/CD28 Dynabeads(Thermo Fisher Scientific) were included as positive controls forT-cell activation. T cells were harvested by pooling the culturemedia and a subsequent PBS wash. If adherent cells are alsorequired, cell dissociation buffer was used to detach from platesurface and cells were pooled with nonadherent cells.

Characterization of human T-cell activationT-cell activation was measured by staining for surface expres-

sion of activation markers (CD69, CD25) and analyzed by flowcytometry. To study T-cell proliferation, T cells were labeled with5 mmol/L carboxyfluorescein succinimidyl ester (CFSE) dye(Thermo Fisher Scientific) prior to culturing with target cells.After five days, T cells were harvested and analyzed byflow cytometry. As a surrogate for proliferation in mixed cellpopulations (e.g., whole ascites samples), total T-cell number perwellwas determinedusing precision counting beads (BioLegend).To measure T-cell degranulation, the externalization of CD107awas assessed by adding a CD107a antibody directly to the well atthe start of the experiment. After 1-hour incubation, GolgiStop(6mg/mL,BDBiosciences)was added, followedbyflowcytometryanalysis after an additional five hours. IL2 and IFNg quantitiesweremeasured using theHuman IL-2 ELISAMAX kit (BioLegend)or Human IFNg ELISA MAX Kit (BioLegend). A flow cytometricmultiplex bead immunoassay was performed using LEGENDplexTh Kit (BioLegend).

Target cell cytotoxicity assayTo assess target cell cytotoxicity by free BiTE or virus, release of

LDH into the supernatant (CytoTox 96 Non-Radioactive Cyto-toxicity Assay; Promega) or MTS viability assay (CellTiter 96 CellProliferationAssay, Promega)wereused. Todetermine viability ofspecific cell types, total cells were harvested by cell dissociationbuffer, and residual number of viable target cells measured byflow cytometry using an amine-reactive fluorescence live–deadstain. For observation of cell viability in real-time, xCELLigencetechnology (Acea Biosciences) was used. TGFb and VEGF quan-tities were measured using TGF beta-1 Human/Mouse ELISA Kit(Thermo Fisher Scientific) and LEGENDplex Growth Factor Kit(BioLegend), respectively.

Flow cytometryTo classify different cellular populations, antibodies specific for

CD11b (ICRF44), EpCAM (9C4), FAP (427819, R&D Systems,USA), CD3 (HIT3a), CD4 (OKT4), CD8a (HIT8) were used. Toanalyze T-cell populations, the following antigens were used:CD69 (FN50), CD25 (BC96), IFNg (4S.B3), CD107a (H4A3),PD1 (H4A3). To analyze macrophage populations, cells weretreated with Fc receptor block (Miltenyi Biotec) and stained withCD163 (GHI/61), CD206 (15-2), CD64 (10.1), and CD86(IT2.2). The appropriate isotype control antibody was used in

each case. All antibodies were acquired from BioLegend unlessstated otherwise. Analysis was performed on a FACSCalibur flowcytometer (BD Biosciences) and data processed with FlowJov10.0.7r2 software (TreeStar Inc.).

IHCAutomated staining was carried out with the Leica BOND-

MAX autostainer (Leica Microsystems). Antigen retrieval wasperformed at 100�C using Epitope Retrieval Solution 2 (LeicaBiosystems), followed by incubation with antibodies for CD8(Agilent Technologies), CD25 (Atlas Antibodies), EpCAM(BioLegend), FAP (R&D Systems) or adenoviral hexon (Milli-pore). Detection was performed using the BOND PolymerRefine Detection System (Leica Biosystems). Alternatively,for the FAP primary antibody only, anti-sheep HRP-DABStaining Kit (R&D Systems) was used. Sections were incubatedwith hematoxylin and imaged (Aperio CS2 slice scanner,Leica Microsystems).

Quantitative PCRAdenovirus genomes were measured by qPCR using primers

and probe against hexon (primers: 50-TACATGCACATCG-CCGGA-30/50-C GGGCGAACTGCACCA-30, probe: 50-FAM-CCGGACTCAGGTACTCCGAAGCATCCT-TAMRA-30). At the spe-cific timepoint, total cell and supernatants were harvested, andDNA extracted (PureLink Genomic DNAMini Kit; Thermo FisherScientific). In brief, primers and probe were mixed withDNA samples and added to QPCRBIO Probe Mix Hi-Rox (PCRBiosystems) Master Mix. To measure levels of FAP mRNA inascites, reverse transcription qPCR was performed. The total cellfraction was harvested after 72 hours of treatment, RNA wasextracted (RNAqueous-Micro Total RNA Isolation Kit; ThermoFisher Scientific), and cDNA prepared (Superscript III First-StrandSynthesis SuperMix; Thermo Fisher Scientific). FAP expressionwas quantified using FAP-specific primers (50-TCAGTGTGAG-TG CTCTCATTGTAT-30/50-GCTGTGCTTGCCTTATTGGT-30) and2xqPCRBIO SyGreen Blue Mix Hi-ROX Master Mix (PCR Bio-systems). Expression of the 18S gene was also measured as anormalization control (50-GCCCGAAGCGTTTACTTTGA-30/50-TCCATTAT TCCTAGCTGCGGTATC-30). All qPCRwas run on ABIPRISM 7000 (Applied Biosystems).

Gene expression analysisGene expression analysis was performed using the nCounter

PanCancer Immune Profiling Panel (NanoString Technologies).ThenSolver AdvancedAnalysismodulewasused for data analysis,in accordance with NanoString guidelines. Background thresh-olding was performed, followed by normalization of the data viathe mean of the internal NanoString positive controls, and dif-ferential expression determined, with reference to uninfectedcells. A gene set's directed global significance score for a covariatemeasures the cumulative evidence for the up- or downregulationof genes in a pathway and is calculated as the square root of themean squared t-statistic of genes, with t-statistics generated fromthe linear regression algorithm within the nSolver AdvancedAnalysis module.

MicroscopyBrightfield and fluorescence images were obtained on a Zeiss

Axiovert 25microscope and capturedwith aNikonDS5Mcamera.For time-lapse sequences, images were obtained on a Nikon TE

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2000-E Eclipse inverted microscope and captured with a Hama-matsu Orca-ER C4742-95 using MetaMorph imaging software.Images were collected at 15-minute intervals with videos(12 frames/second) generated using ImageJ software (NIH,Bethesda, MD). Where appropriate, cells were stained with Cell-Tracker Orange CMTMR Dye (Thermo Fisher Scientific) andCellTrace Violet Cell Proliferation Kit (Thermo Fisher Scientific).Apoptosis was visualized using CellEvent Caspase 3/7 DetectionReagent (Thermo Fisher Scientific).

Statistical analysisWhere experiments produced two datasets, significance was

evaluated using a Student two-tailed t test. In all cases of morethan two experimental conditions, statistical analysis wasperformed using a one-way ANOVA test with Tukey post hocanalysis or two-way ANOVA test using Bonferroni post hocanalysis. All data are presented as mean � SD. Significancelevels used were P ¼ 0.01–0.05 (�), 0.001–0.01 (��), 0.0001–0.001 (��). Experiments were performed in biologicaltriplicate, unless stated otherwise.

ResultsFibroblast-targeted BiTE engineering

A FAP-targeted BiTE was engineered recognizing human FAPand CD3e. A BiTE specific for CD3e and an irrelevant antigen,filamentous hemagglutinin adhesin (FHA) of Bordetella pertus-sis, was used to control for unspecific binding. An N-terminalimmunoglobulin signal sequence and C-terminal decahisti-dine tag were added for mammalian secretion and detection.BiTE protein production was assessed by transfection ofHEK293A cells.

To confirm specificity of the FAP-BiTE for surface FAP, weestablished a human FAP-positive stable cell line using FAP-negative CHO cells. Peripheral blood mononuclear cell(PBMC)-derived T cells were activated 24 hours after cocultur-ing with CHO-FAP cells and FAP BiTE–containing supernatantsfrom transfected HEK293A (Fig. 1A) and mediated CHO-FAPcell lysis (Fig. 1B). Neither T-cell activation nor lysis wereobserved in cultures with parental CHO cells or control-BiTE,indicating that surface FAP expression is required for T-cellactivation, presumably via surface CD3 clustering and pseu-doimmunologic synapse formation.

FAP-BiTE–induced T-cell activationwas also evaluated in cocul-ture with NHDFs, which express surface FAP when cultured inhigh serum (10% FBS, Supplementary Fig. S1A). Incubation ofNHDF- and PBMC-derived T cells from six donors with FAP-BiTE-containing supernatants for 24 hours induced significant T-cellactivation and NHDF lysis, with the control-BiTE having no effect(Fig. 1C; EC50, 2.5 ng/mL). CFSE-labeled PBMC T cells coculturedwith NHDF and FAP-BiTE underwent multiple rounds of T-cellproliferation (Fig. 1D) and showed at least 10-fold increase inIFNg , IL2, TNFa, IL17F, IL22, and IL10 (Fig. 1E), with IFNgproduction 10-fold higher than that induced by physiologicanti-CD3/CD28 activation beads (Fig. 1F). Interestingly, FAP-BiTE induced activation and degranulation of CD4 and CD8 Tcells, directing both subsets to kill NHDF cells with similarpotency (Fig. 1G–I). Importantly, no induction of activationmarkers, proliferation and cytokines was observed with con-trol-BiTE or in the absence of NHDF target cells, confirming thatCD3 clustering is essential for T-cell activation.

Generation of BiTE-armed EnAdEnAd is a conditionally replicating chimeric group B adenovi-

rus generated by bioselection (Fig. 2A; ref. 32). The FAP-BiTE andcontrol-BiTE sequences were inserted downstream of the fibergene under transcriptional control of either an exogenous CMVpromoter or a splice acceptor (SA) site for the adenoviral majorlate promoter (MLP). The former drives immediate transgeneexpression upon successful cell infection, whereas MLP-drivenexpression occurs only in cells permissive to virus replication,such as human tumor cells. Viruses were rescued and purifiedfrom HEK293A cells (Supplementary Table S1).

Colorectal adenocarcinoma (DLD) cells were infected at100 vp/cell of the parental or recombinant viruses to assessreplication kinetics. Viral genome copies reached between 3–6� 1012 genomes/mL (Fig. 2B), indicating that BiTE expression didnot impair replication relative to the parental virus. Cytotoxicityof all recombinant viruses was also comparable with parentalEnAd (Fig. 2C). Therefore, modification of the viral genome toincorporate the BiTE transgene had little effect on viral replicationor oncolytic activity.

FAP-BiTE secretion by virus-infected HEK293A cells was dem-onstrated by immunoblotting of the supernatant (SupplementaryFig. S1B). Functionality of these secreted virus-encoded BiTEs wasassessed by adding supernatants to cocultures of PBMC-derivedCD3þ T cells and either CHOor CHO-FAP cells. T cells coculturedwith CHO-FAP cells showed strong CD25 induction and targetcell lysis when incubated with supernatants from EnAd-CMV-FAP-BiTE– or EnAd-SA-FAP-BiTE–infected cells (Fig. 2D and E).No activation or cytotoxicity was observed with supernatantsfrom cells infected with unmodified or control-BiTE–expressingEnAd, in the presence of parental CHOcells, or in the absence of T-cells. FAP-BiTE yield from DLD cells infected with modifiedviruses wasmeasured by comparing T-cell–mediated NHDF cyto-toxicity induced by 72-hour–infected DLD supernatants to astandard curve. Following a 24-hour cytotoxicity assay, we mea-sured FAP-BiTE at 9.8 and 49.2 mg/106 cells after 72 hours forEnAdCMV-FAP-BiTE and EnAdSA-FAP-BiTE, respectively. Thiswas consistent with previous reports, suggesting that while tran-scriptional initiation is delayed, there is superior total transgeneexpression when driven by the endogenous MLP compared withthe CMV promoter (30). The FAP-BiTE showed impressive poten-cy, with cytotoxicity detectable in supernatants diluted 10,000-fold (Supplementary Fig. S1C).

EnAd-FAP-BiTE–mediated oncolysis induces T-cell–mediatedfibroblasts killing

EnAd kills carcinoma cell lines by direct oncolysis (33), butdoes not effectively replicate in, or directly kill,fibroblasts or othernonepithelial stromal cells (23). However, FAP-targeted BiTEfrom infected tumor cells should allow T-cell activation andmediate targeted killing of FAP-expressing stromal fibroblasts.Cocultures of fibroblasts, moderately permissive SKOV3 ovariancarcinoma cells killed by EnAd 5–7 days postinfection (actingas BiTE producers), and PBMC-derived T cells were measured inreal-timeby cell index, a unitlessmeasure of cell viability (Fig. 2F).In the absence of T cells, tumor cells and fibroblasts cells persistedfor 100–120 hours, independent of virus infection. In thepresence of T cells, FAP-BiTE expression from infected SKOV3cells led to complete NHDF cytotoxicity, with lysis observedwithin 22 hours postinfection (hpi) by EnAd-CMV-FAP-BiTE and42 hpi for EnAd-SA-FAP-BiTE. Crucially, no cytotoxicity was






Figure 1.

FAP-BiTE–containing supernatants activate primary human T cells and target cytotoxicity to FAPþ cells. A, Activation (CD69þCD25þ) of primary humanCD3 cells cultured for 24 hourswith BiTE-containing supernatants andCHOorCHO-FAP cells.B, LDH release by target cells inA.C,CD25 expression on T cells (black)and NHDF lysis (LDH release, red) was assessed after 24 hours of coculture with BiTE- and PBMC-derived T cells from six healthy donors. D, Representativeproliferation of CFSE-stained T cells after 5-day coculture with BiTE and NHDFs. E, Cytokine production was evaluated by a multiple human Th cytokinepanel after coculturing T cells with BiTE and NHDFs for 48 hours. F, Secreted IFNg from cocultures of T cells with NHDF and BiTE-containing supernatants.Induction of CD25 (G) or degranulation (H) of CD4 and CD8 T cells following 24 hours coculture with BiTE and NHDFs. I, LDH release by NHDF cells after24 hours in cocultures with BiTE and either CD4- or CD8-purified T cells. Data show mean � SD of biological triplicates (A-C and E-I). Significance wasassessed using one-way ANOVA with Tukey post hoc analysis compared with "untreated" (A, C, E, and F) or "control-BiTE" (B) or using an unpaired two-tailedt test (G–I; � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ns, nonsignificant).

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Figure 2.

EnAd-expressing FAP-BiTE induces T-cell–dependent cytotoxicity of stromal fibroblasts. A, Genome of the BiTE-armed oncolytic adenovirus, EnAd. ITR,inverted terminal repeat; P, promoter; pA, polyadenylation site. B, Genome replication of parental EnAd- or BiTE-expressing viruses in DLD cells infected with100 vp/cell. C, Viability of DLD cells infected with EnAd or recombinant virus (MTS; 5 days postinfection). D and E, T-cell activation (D; CD25þ) and targetcell cytotoxicity (E; LDH release) in cocultures of T cells, target cells, and infected HEK293A supernatants 24 hpi. F,Viability of NHDF and SKOV3 (4:1) cells monitoredby xCELLigence in the absence or presence of CD3-purified PBMCs (5:1 effector:target). Mean (solid line) � SD (dotted line) of biological triplicates. G, T-cellactivation (CD25þ) in virus-infected cocultures of NHDF and SKOV3 cells. H, Representative images showing coculture of NHDF (red; stained with CellTrackerOrange CMTMR), DLD cells (unstained), and T cells (blue; stained with CellTrace Violet), followed by infection with EnAd or BiTE-armed virus. Apoptosis wasvisualized using CellEvent Caspase 3/7 Detection Reagent (green). White arrow, dead fibroblasts. Scale bar, 100 mm. Data show mean� SD of biological triplicates(D-G). Significance assessed using one-way ANOVAwith Tukey post hoc analysis compared with "EnAd" (B and C) or "uninfected" (D–G; � , P < 0.05; �� , P < 0.001).






observed in cultures infected with EnAd or EnAd expressing thecontrol-BiTE.

NHDF lysis was confirmed by lactate dehydrogenase (LDH)release in similar coculture experiments (Supplementary Fig.S1D). The kinetics of T-cell activation paralleled that of NHDFcytotoxicity (Fig. 2G; Supplementary Fig. S1E). Importantly,FAP-BiTE–encoded viruses failed to induce CD25 expressionin the absence of NHDF, further demonstrating the require-ment of FAPþ cells for BiTE-mediated T-cell activation (Sup-plementary Fig. S1F).

BiTE-induced cytotoxicity of stromal fibroblasts by T cells wasobserved by time-lapse microscopy using cocultures of T cells,fibroblasts, and DLD cells, which are more susceptible thanSKOV3 cells to EnAd-mediated lysis (Fig. 2H; SupplementaryMovie S1–S3). While infection with EnAd induced dramaticDLD killing within 48 hours, NHDFs remained viable through-out. In contrast, EnAd-CMV-FAP-BiTE infection induced bothdirect DLD killing and T-cell–mediated fibroblast cytotoxicity.Quantification of DLD and NHDF cells in parallel cultures

showed complete elimination of both cell types upon treatmentwith EnAd-CMV-FAP-BiTE or EnAd-SA-FAP-BiTE 72 hpi (Supple-mentary Fig. S1G).

EnAd-SA-FAP-BiTE–mediated T-cell activation and target celllysis is tumor selective

Conventional FAP-targeted therapeutics given intravenouslyare reported to induce FAPþ cell toxicity within the bonemarrow compartment (18). Coupling BiTE expression to virusreplication via the viral MLP restricts expression to the tumorcompartment, minimizing unwanted toxicity to FAPþ fibro-blasts in normal physiologic sites. To compare selectivity ofvirally encoded CMV- and MLP-driven BiTE expression, NHDFswere incubated with EnAd, EnAd-CMV-FAP-BiTE or EnAd-SA-FAP-BiTE in the presence of primary T cells only. At 72 hpi withEnAd-CMV-FAP-BiTE, we observed cytotoxicity in 80% ofNHDF cells (Fig. 3A). No lysis was observed in EnAd-SA-FAP-BiTE–infected cells, consistent with the inability of EnAdto complete its life cycle in nonepithelial tumor cells (23).

Figure 3.

EnAd-SA-FAP-BiTE does not induce T-cell activation or FAPþ cell lysis in the absence of tumor cells. A, Cytotoxicity in T-cell and NHDF cocultures was assessedby LDH release 72 hpi with BiTE-expressing viruses. B and C, T-cell activation in coculture with NHDF and NHBE or SKOV3 seeded at 5:1 and infected withBiTE-armed viruses. T cells were added 2 hpi. Activation (B, CD69þ; C, CD25þ) was assessed 72 hpi. D, LDH release in cocultures from B and C. T-cell activation(CD25þ; E) and LDH release (F) in cocultures of fresh bone marrow samples 5 days postinfection with FAP-BiTE–expressing EnAd. Prior to virus infection,bone marrow cells were plated with NHDF with or without SKOV3 (50:5:1). LDH release was calibrated against lysis of corresponding amounts of NHDF and SKOVcells without bone marrow cells, corrected for the amount released by healthy bone marrow cells. Data show mean � SD of biological triplicates. Significancebetween more than two conditions was assessed using one-way ANOVA with Tukey post hoc analysis compared with "uninfected" (A). Significance betweentwo conditions was assessed using an unpaired two-tailed t test (B–F; � , P < 0.05; �� , P < 0.001; ns, nonsignificant).

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To better simulate the multiple cell types present in a healthytissue, NHDFs were cultured with exogenous PBMC-derived Tcells and either NHBE cells or SKOV3 and subsequently infectedwith EnAd-SA-FAP-BiTE or EnAd-SA-control-BiTE. While EnAd-SA-FAP-BiTE infection of SKOV3 allowed T-cell activation andtarget cell lysis (Fig. 3B–D), NHBE cells did not.

Finally, for maximum clinical relevance, EnAd-SA-FAP-BiTEactivity was evaluated in three fresh human whole bone marrowsamples from healthy donors. Despite literature reports of bonemarrow toxicity of FAP-targeted antibodies, no FAPþ cells weredetected in any samples. Accordingly, FAPþ NHDF 'target' cellswere added prior to infection to determine whether our armedviruses triggered uncontrolled toxicity against FAPþ cells. NeitherEnAd-SA-FAP-BiTE nor EnAd-SA-control-BiTE induced endoge-nous T-cell activation (Fig. 3E) or targeted cytotoxicity (Fig. 3F) inthe absence of tumor cells. The addition of SKOV3 cells led to T-cell activation and cytotoxicity following EnAd-SA-FAP-BiTEinfection, with the latter thought to reflect predominantly BiTE-mediated T-cell lysis of FAPþ NHDFs. These data confirm thatMLP-driven FAP-BiTE production is restricted to tumor cells andsuggest there should be no systemic toxicity against FAPþ cellswithin normal bone marrow.

EnAd expressing FAP-BiTE activates tumor-associated T cells tokill endogenous fibroblasts within patient-derived malignantascites

Malignant peritoneal ascites are frequent in several advancedcarcinoma types, including ovarian, pancreatic, breast, and lungcancers (34), and often associated with a poor prognosis (34, 35).The fluid is routinely drained from some patients as a palliativetreatment, providing a convenient and informative liquid biopsy.There is mounting evidence that malignant ascites are sites ofsubstantial immunosuppression (36). We therefore assessed theeffects of cell-free ascites fluid on PBMC-derived T-cell activationusing anti-CD3/CD28 beads and FAP-BiTE. Bead-mediated T-cellactivation was significantly inhibited by 3 of 5 ascites samples(Fig. 4A). In contrast, FAP-BiTE-mediated T-cell activationwas notsuppressed by any ascites fluids compared with levels observed innormal serum (Fig. 4B).

Human ascites biopsy samples typically contain tumor cells,fibroblasts, lymphocytes, and macrophages, representing aunique tumor-like model system to assess endogenous tumor-associated T-cell activation. Figure 4C shows the cellular compo-sition of a representative sample containing CD3þ, EpCAMþ,CD11bþ, and FAPþ cells (see also Supplementary Fig. S2A andS2B; Supplementary Table S2). Ascites-associated CD3þ T cellswere, on average, 63% (up to 92.5%) positive for the exhaustionmarker PD1 compared with only 10%–20% of PBMC-derived Tcells (Fig. 4D). We assessed the ability of BiTE-encoding EnAd toinfect ascites cancer cells and secrete sufficient amounts of BiTE,leading to endogenous T-cell activation and killing of autologouscancer-associated fibroblasts within an ascites sample. Total asci-tes cells from four patient biopsies were incubated in 50% ascitesfluid with free FAP-BiTE or EnAd encoding FAP-BiTE. After fivedays, endogenous T cells were strongly activated in all ascitesbiopsy samples (30%–80% of total CD3þ cells; Fig. 4E), com-bined with CD3þ T-cell proliferation (Fig. 4F). Parental andcontrol-BiTE viruses did not induce T-cell activation orproliferation.

Ascites T-cell activation and cytotoxicity toward endogenousFAPþ fibroblasts was assessed by measuring the change in FAPþ

cell number during treatment (Fig. 4G). Free FAP-BiTE and EnAd-CMV-FAP-BiTE induced significant depletion of FAPþ fibroblastsin all samples, typically to levels below1%of those inuntreated orcontrol samples, consistentwithmarked falls in FAPmRNA,VEGFsecretion, and elimination of cells with a fibroblast-like morphol-ogy (Supplementary Fig. S3A–S3C). A similar trend was observedupon infection with EnAd-SA-FAP-BiTE, although one patient(patient biopsy 1) demonstrated neither T-cell activation norfibroblast depletion (Fig. 4E and G). Infection of this samplewith EnAd-SA-GFP also showed no GFP-positive cells (Supple-mentary Fig. S3D; Supplementary Table S2) and no EpCAMþ

tumor cells at the outset (Supplementary Fig. S3E). The samplelikely had insufficient tumor cells to support virus replication,demonstrating the strict necessity of tumor cells for virus repli-cation and MLP-drive BiTE expression, suggesting one predictorfor potency. A cytokine array of patient biopsy 1 demonstratedthat EnAd-CMV-FAP-BiTE infection induced at least 10-foldincreases in IL17A, IL17F, IL22, IFNg , and IL10 expression (Fig.4H). Parallel experiments using expanded mixed cultures ofascites-derived fibroblasts and tumor cells showed that fibroblastdepletion led to 50%–70% lower TGFb levels in supernatants(Fig. 4I), suggesting FAPþ cells to be a major source of immuno-suppressive TGFb within tumor ascites. Altogether, these datashow that treatment of malignant ascites with free or virallyencoded FAP-BiTE is able to polyclonally activate anergic T-cells,leading to targeted depletion of autologous tumor-associatedfibroblasts.

Global changes in immune pathway gene expression wereobserved in EnAd-SA-FAP-BiTE–treated samples

To assess the impact of CAF depletion, T-cell activation andvirolysis of cancer cells on the tumor microenvironment, we usedNanoString to determine changes in the expression of 730 cancerand immune pathway genes in three primary ascites samples,selected to represent the spectrum of clinical possibilities. Biopsy4 had a high ratio of FAPþ cells relative to EpCAMþ cells (likelyepithelial cancer cells; Supplementary Table S3), while biopsy 5had a similar proportion of EpCAMþ and FAPþ cells, and biopsy6, a relatively low ratio of FAPþ to EpCAMþ cells. All samples hadsimilar levels of CD3þ T cells and CD11bþ myeloid cells.

Significant T-cell activation was observed in all samples threedays postinfection with EnAd-SA-FAP-BiTE, but not EnAd-SA-control-BiTE (Supplementary Fig. S4A). Approximately 40% ofall genes showing significant changes in mRNA levels of at leasttwo-fold (Supplementary Fig. S4B); onlymRNAbasally expressedabove a minimum threshold level were included in the analysis.Considerably more genes showed changes following exposure toEnAd-SA-FAP-BiTE than EnAd-SA-control-BiTE, except biopsy 6,where the high number of EpCAMþ cancer cellsmay have resultedin extensive EnAd-specific gene changes due to BiTE-independentdirect virolysis. Changes were grouped by immune responsecategory as an average of three samples (Fig. 5A) or individualsamples (Supplementary Fig. S4C). Although individual biopsiesshowed some variation, all EnAd-SA-FAP-BiTE–infected samplesdemonstrated increased gene expression in numerous immunegroupings including cytotoxicity, pathogen defense, and T-cell, B-cell, and NK-cell function. While T-cell- and NK-cell–attractantchemokines (CXCL9, CXCL10, CXCL11) were also upregulated inall biopsies, strong decreases in fibroblast-associated CXCL6,CXCL12, and CXCL14 induced a downregulation in overall che-mokine expression for biopsy 4 (Supplementary Fig. S4D).






Figure 5B shows the dataset for biopsy 4, which had the highestnumber of FAPþ cells. The most highly upregulated genes (up to100-fold) following treatment with EnAd-SA-FAP-BiTE includedT-cell markers (GZMB, IFNG, IL2RA, PRF1, TNF). The greatest

decreases (up to 1,000-fold) were in fibroblast-associated genes,such as COL3A1, FN1, THY1, CXCL12, and IL13RA2. Similartrends were seen across all three samples, with the most modestchanges in fibroblastmarkers observed in biopsy 6, which had the

Figure 4.

EnAd-expressing FAP-BiTE activates endogenous T cells and induces FAPþ cell lysis in malignant exudate samples. A and B, Activation (CD69/CD25þ) ofPBMC-derived T cells cocultured with anti-CD3/CD28 Dynabeads (A) or NHDF and BiTEs (B) for 24 hours in normal serum or ascites fluid (50%). C, Representativeflow cytometry plot demonstrating the cellular composition for a typical ascites biopsy sample. D, PD-1 expression by T cells as a percentage of total CD3þ cells.E, CD25 expression on endogenous T cells five days posttreatment with free BiTEs or BiTE-expressing EnAd. Total unpurified cells were treated in 50%exudate fluid from the same biopsy sample. F andG,Relative quantity of CD3þ cells (F) and residual FAPþ cells (G). Effector:target ratioswere 79.4 (sample 1, black),2.27 (sample 2, blue), 31.6 (sample 3, red), and 2.44 (sample 4, gray). H, Cytokine production was evaluated by a multiple human Th cytokine panel. I, TGFb insupernatants harvested from ascites cells incubated with PBMC T cells and BiTE-expressing virus. Data show mean � SD of biological triplicates (A, B, and E-I).Significance was assessed using one-way ANOVA with Tukey post hoc analysis compared with "normal serum" (A and B) or "untreated" (E–I). D, Data showmean � SD. Significance assessed using an unpaired two-tailed t test (� , P < 0.05; �� , P < 0.01; �� , P < 0.001).

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Figure 5.

Global response of malignant ascites to infection with EnAd-SA-FAP-BiTE. A, Heatmap showing changes in mRNA counts (as global significance score, averageof three samples) within defined gene sets compared with untreated. Left, EnAd-SA-control-BiTE; right, EnAd-CMV-FAP-BiTE. B, Fold change plot showinggene-specific differences in mRNA counts following infection of biopsy 4 with EnAd-SA-FAP-BiTE. Five T-cell activation (blue) and fibroblast (gold) markersare shown. COL3A1, alpha1 (III) collagen; THY1, Thy-1; IL13RA2, IL13Ra2; FN1, fibronectin; GZMB, granzyme B; PRF1, perforin; IL2RA, CD25; IFNG, IFNg . C–E, Foldchange in mRNA counts (left, EnAd-SA-control-BiTE; right, EnAd-SA-FAP-BiTE; compared with untreated) in three ascites samples for fibroblast-specific genes (C)or genes involved in T-cell function (D) or antigen presentation and T-cell trafficking (E). Circle, biopsy 4; triangle, 5; square, 6. F and G, Expression levels ofCD163, CD206 (F), and CD64 and CD86 (G) on CD11bþCD64þ cells. gMFI, geometric mean fluorescence intensity. C–E, Data show mean of biological duplicates.Significant changeswere assessed using amultivariate linear regression algorithmwith three patient biopsies. Significance of changes in gene expression induced byeach virus versus uninfected is displayed adjacent the x-axis, and between EnAd-SA-control-BiTE or EnAd-SA-FAP-BiTE displayed above the plot. F and G,Data show mean � SD of biological triplicates. Significance was assessed using one-way ANOVA with Tukey post hoc analysis compared with "untreated"(� , P < 0.05; �� , P < 0.01; �� , P < 0.001).






lowest levels of FAPþ cells. Changes in expression of key genescomparedwithuntreated samples are shownby individual biopsyin Fig. 5C–F. For example, expression of the fibroblast markercollagen type III (COL3A1) is dramatically reduced upon infec-tion with EnAd-FAP-BiTE compared with EnAd-control-BiTE inall three samples, while THY1 and IL13RA2 (also used as fibro-blast markers; Fig. 5C) showed FAP-BiTE–dependent decreases intwo biopsies. Basal expression of these genes in biopsy 6 did notpass the minimum threshold for analysis. T-cell activation mar-kers (GZMB, PRF1, and IL2RA; Fig. 5D), checkpoint markers(PDCD1, CTLA4, and LAG3; Supplementary Fig. S4E), T-cell–recruiting chemokine CXCL9, and DC maturation/antigen pre-sentation markers LAMP3 and TAP1 (Fig. 5E) all increased in aFAP-BiTE–dependent manner. These latter findings are particu-larly encouraging, raising the possibility of immunosuppressionreversal in the tumormicroenvironment following EnAd-SA-FAP-BiTE infection.

Treatment of ascites samples with EnAd-SA-FAP-BiTE inducesrepolarization of resident tumor-associated macrophages

Macrophages are known to show plasticity between proinflam-matory "M1" and wound-healing "M2" phenotypes, with tumor-associated macrophages (TAM) usually skewed toward "M2." Toinvestigate the influence of FAP-BiTE on macrophage polariza-tion, patient-derived malignant ascites samples were treated withfree or EnAd-encoded FAP-BiTE to determine activation of endog-enous T cells and CD11bþCD64þ cells. Treatment with free FAP-BiTE or EnAd-SA-FAP-BiTE induced strong T-cell activation andIFNg secretion (Supplementary Fig. S4F). We observed simulta-neous induction of an activated M1-like macrophage phenotype,manifested by strong decreases in CD206 and CD163 (Fig. 5F)and increased CD64 expression (Fig. 5G). Infection with EnAd-SA-FAP-BiTE induced a large increase in CD86 expression, whilefree FAP-BiTE, EnAd-SA-control-BiTE, or IFNg alone had no effect(Fig. 5G).

EnAd-SA-FAP-BiTE activates TILs and induces BiTE-mediatedcytotoxicity in solid prostate tumor biopsies

We obtained seven fresh paired punch biopsies of malignantprostate tissue frompatients undergoing radical prostatectomy, cutinto thin sections for ex vivo cultures. Prostate tissue slices showed acharacteristically complex architecture,with glandular structuresof(malignant) EpCAMþ epithelial cells interspersed with largeregions of intervening stroma containing scattered CD8 T cells(Supplementary Fig. S5A). FAP expression was generally weak inbenign prostate tissue and high in malignant prostate tissue (Fig.6A). Fibroblasts showing the strongest FAP expression were oftenadjacent to malignant epithelial cells (Supplementary Fig. S5A).

To facilitate assessment of virus activity, we developed reporterviruses linking FAP-BiTE and RFP expression (Supplementary Fig.S5B). Following infection with EnAd-SA-FAP-BiTE-RFP, malig-nant tissue slices showed RFP expression, demonstrating success-ful viral infection, replication and BiTE expression (Supplemen-tary Fig. S5C). Positive staining for viral hexon confirmed EnAdreplication, apparently limited to malignant epithelial cells (Fig.6B). Malignant prostate tissue infected with EnAd-SA-FAP-BiTEshowed an increase in activated endogenous TILs seven dayspostinfection (Fig. 6C). Slices from all patients showed significantinduction of IFNg production, with IL2 levels also increasing infour samples (Fig. 6D and E); both cytokines are associated withactivated CD4 Th1 and CD8 cytotoxic T cells (37). Neither

untreated slices nor those infected with EnAd-SA-control-BiTE-RFP showed detectable T-cell activation, although some samplesdemonstrated modest increases in IFNg and IL2 following EnAd-SA-control-BiTE-RFP infection, likely a direct result of virolysis. Inbenign prostate tissue, there was little increase in IFNg or IL2 withany treatment (Supplementary Fig. S5D and S5E).

BiTE-mediated activation of T cells is expected to lead tofibroblast killing. Cells were visualized undergoing apoptosis inreal-time within ex vivo prostate tissues (Fig. 6F). EnAd-SA-FAP-BiTE-RFP–infected cells strongly associatedwith apoptotic nuclei,suggesting BiTE-mediated induction of proximal cytotoxicity ofsurrounding cells. FAP-BiTE–mediated cytotoxicity was observedin all patient biopsy samples, with intrinsic EnAd activity alsoinducing small increases, potentially due to a greater number ofcancer cells in some samples (Fig. 6G). Indeed, regions of highT-cell activation showed an absence or degradation of surround-ing tissue or stroma, with tissue architecture remaining intact inuninfected samples (Fig. 6H). Crucially, FAPlow benign prostatetissue showed negligible increases in cytotoxicity within theduration of the study (Supplementary Fig. S5F).

DiscussionHere, we have developed an armed oncolytic adenovirus com-

bining three distinct therapeutic strategies: direct virus-mediatedcytotoxicity toward cancer cells, creation of a proinflammatoryimmune environment, and removal of a key stromal cellmediatorof tumor immunosuppression. Encoding BiTEs within OVsexploits the strengths of both virotherapy and immunotherapywhile overcoming limitations of each agent alone. Whenexpressed locally, the short plasma half-lives of BiTEs will becomeadvantageous, minimizing systemic exposure and avoiding "on-target, off-tumor" toxicities (38).Conversely, arming anOVwith aBiTE provides an additional mechanism of cell killing and broad-ens the range of target cells to include OV-resistant stromal cells.

Combining OVs and BiTEs also has a potentially synergisticeffect on infiltrating T-cells. IncreasedCD8þ T-cell infiltration intothe tumor bed has been observed in several OV clinical trials,including studies using EnAdand FDA-approved Imlygic (24, 39),likely providing more effector cells for BiTE-mediated cytotoxic-ity. Simultaneously, BiTE-mediated redirection of TILs (poten-tially virus-specific) toward chosen targets may delay viral clear-ance and increase intratumoral spread.

For maximal translational relevance, our current and futurestudies will focus on primary human tumor biopsies maintainedex vivo, rather than compromise with imperfect murine modelsthat may not provide the desired tumor heterogeneity or realisticlevels of immune suppression (23, 40). Clinical samples retainthe heterogeneous and multifaceted cellular interactions ofadvanced human cancer, and, in the case of organotypic prostatetumor slices, the stromal architecture and extracellular matrix of asolid tumor. Treatment of both solid and liquid tumor biopsieswith EnAd-FAP-BiTE led to tumor-associated T-cell activation anddestruction of endogenous FAPþ fibroblasts, alongside secretionof large quantities of proinflammatory cytokines and chemo-kines, including IFNg , IL2, TNFa, IL17, and CXCL9. Crucially,this demonstrated that the patient's own tumor-associated T cellscan be used for therapeutic purposes in the realistic environmentof an advanced human tumor. It was particularly encouragingthat T cells within all tested patient biopsies, shown to be PD1þ

and likely anergic, were readily activated and rendered functional

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Figure 6.

EnAd-expressing FAP-BiTE activates tumor-infiltrating T cells and mediates cytotoxicity in malignant prostate slice cultures. A, FAP expression patterns inbenign and malignant prostate tissue slices. B, Expression of viral hexon protein in prostate tissue slice after EnAd infection. C, Representative image showingtumor-infiltrating T-cell activation (CD25þ) in prostate tissue slices 7 days postinfection. IFNg (D) and IL2 (E) levels in malignant prostate tissue slicesinfected with BiTE-expressing EnAd. F, Active caspase-3/7 (CellEvent Caspase 3/7 Green Detection Reagent, green) in malignant prostate tissue infected withEnAd-SA-FAP-BiTE-RFP (red). White arrows, dual-positive cells. Scale bar, 100 mm. G, LDH release of malignant prostate tissue slices from 5 patients infectedwith recombinant EnAd.H,Histologic architecture and T-cell activation (CD25þ) in EnAd-infected prostate slices. Data showmean� SD of technical triplicates (D, E,and G). For D and E, n ¼ 7; for G, n ¼ 5. Significance was assessed using one-way ANOVA with Tukey post hoc analysis compared with "uninfected"(� , P < 0.05; �� , P < 0.01; �� , P < 0.001).






by the BiTE to mediate cytotoxicity. This may reflect the highlevel of activating stimuli each T-cell can receive using a BiTE,where in principle every CD3 can be crosslinked to the targetantigen (>100,000 copies per cell) without being limited by therelatively small number of HLA-presented TCR-cognate peptidesavailable, likely to be less than 100 per cell. Indeed, the efficacy ofBiTE-mediated T-cell stimulation is augmented when targetinga high-density receptor like FAP, a result also seen with otherantigens (21, 22).

NanoString analysis confirmed the extensive depletion offibroblast-associated RNA in human malignant ascites samplestreated with EnAd-FAP-BiTE, together with strong induction ofgenes involved in T-cell function. Despite their varying cellularcompositions, similar immune-activating trends were seen in allsamples following EnAd-FAP-BiTE infection, with stimulation ofRNAs involved in leukocyte trafficking, dendritic cell maturation,and antigen presentation. Surface markers on ascites TAMsrevealed a clear shift from an M2-like phenotype to one that ismore proinflammatory. We expect that newly infiltrating mono-cytes, recruited byOV-mediated inductionofmonocyte-attractantchemokines, such as CCL2, CCL7, and RANTES, will acquire anM1 "activated" phenotype. Significantly, surface expression ofcostimulatory ligand CD86 on TAMs was only induced by thecombination of virus and FAP-BiTE (EnAd-FAP-BiTE). Wehypothesize that virus stimulation of pathogen-associatedmolec-ular patterns, IFN signaling, or STING and removal of CAF-mediated suppression are required for CD86 activation. Thesefindings indicate that coupling CAF depletion with potent acti-vatory stimuli (T-cell activation and viral-mediated immunogeniccell death) synergistically repolarize the tumormicroenvironmenttoward promotion of an effective anticancer immune response(41). Similarly, although we expect that suppressive markers willincrease in tandem with activation markers, this potential barrierto continued virus activity could be counteracted by combiningOVs with checkpoint inhibitors.

Using OVs for cancer-targeted transgene expression has nowbeen validated both preclinically and clinically (24, 42). Here, weregulated BiTE expression using the adenoviral MLP, limiting BiTEproduction to cells permissive to the virus life cycle. In the absenceof cancer cells, we observed no BiTE production or cytotoxicity(Fig. 3). This is particularly important in light of the "on-target off-tumor" toxicities observed with FAP-targeted antibodies or CAR-Tcells toward FAPþ bonemarrow cells. Infection of primary culturesof freshly isolated human bone marrow by EnAd-SA-FAP-BiTEshowed no T-cell activation or bone marrow cell toxicity in theabsence of tumor cells. Endogenous FAPþ cells also likely occur atfrequencies too low (�0.01%) to identify in themononuclear cellfraction.Hence, our elegant targeted expression strategy is expectedto avoid such toxicities while exploiting the potent effects of theFAP-BiTE within the microenvironment of each tumor deposit.

We therefore believe that armingOVs to express BiTEs targetingstromal elements, such as CAFs, can provide a powerful newmultimodal approach to cancer therapy. In this way, a single-

agent actively kills two different cell types using two distinct, yettargeted, cytotoxic mechanisms. EnAd provides a particularlypromising virus platform to achieve targeted BiTE expression indisseminated tumors, exploiting the blood stability and systemicbioavailability of the virus, which has been studied in several earlyphase clinical trials. This strategy to induce proinflammatory celldeath while reversing TME-mediated immunosuppression maybe what is ultimately required to turn intransigent, stromal-richcarcinomas into targets for a complete and durable immunother-apeutic response.

Disclosure of Potential Conflicts of InterestB.R. Champion is a chief scientific officer and has ownership interest

(including stock, patents, etc.) in PsiOxus Therapeutics. L.W. Seymour reportsreceiving a commercial research grant, has ownership interest (including stock,patents, etc.), and is a consultant/advisory board member for PsiOxus Thera-peutics. K.D. Fisher is a scientific advisor at PsiOxus Therapeutics and hasownership interest (including stock, patents, etc.) in PsiOxus Therapeutics.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: J. Freedman, M.R. Duffy, J. Hagel, B.R. Champion,L.W. Seymour, K.D. FisherDevelopment of methodology: J. Freedman, J. Hagel, R.J. BryantAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): J. Freedman, A. Muntzer, E.M. Scott, J. Hagel,L. Campo, R.J. Bryant, P. MillerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): J. Freedman, J. Lei-Rossmann, J. Hagel, L.W. SeymourWriting, review, and/or revision of the manuscript: J. Freedman,J. Lei-Rossmann, A. Muntzer, R.J. Bryant, C. Verrill, B.R. Champion,L.W. Seymour, K.D. FisherAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. LambertStudy supervision: J. Freedman, L.W. Seymour, K.D. FisherOther (sample processing): A. MuntzerOther (provided primary tumor material): C. Verrill

AcknowledgmentsThe authors gratefully acknowledge support from the Medical Research

Council (MRC-Oxford Doctoral Training Partnership, MR/K501256/1to J.D. Freedman) and Cancer Research UK (grant #C552/A17720 to J. Lei-Rossmann, K. Fisher, L. Seymour; studentship C5255/A20936 to E.M. Scott). M.R. Duffy is funded by the Kay Kendall Leukaemia Fund (grant KKL1050). J. Lei-Rossmann is supported by Linacre College, Oxford. pEnAd2.4 was kindlyprovided by PsiOxus Therapeutics. We are grateful to Egon Jacobus (UniversityofOxford,Oxford,UnitedKingdom) for the use of his primers. Special thanks toAlison Carr and her team for their helpful collection of ascites. C. Verrill'sresearch time is part-funded by the Oxford NIHR Biomedical Research Centre(Molecular Diagnostics Theme/Multimodal Pathology Subtheme). Weacknowledge the contribution to this study made by the Oxford Centre forHistopathology Research and the Oxford Radcliffe Biobank, which are sup-ported by the NIHR Oxford Biomedical Research Centre.

The costs of publication of this articlewere defrayed inpart 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 8, 2018; revised September 18, 2018; accepted October 16,2018; published first November 18, 2018.

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2018;78:6852-6865. Published OnlineFirst November 18, 2018.Cancer Res Joshua D. Freedman, Margaret R. Duffy, Janet Lei-Rossmann, et al. Targets Cancer and Immunosuppressive Stromal CellsAn Oncolytic Virus Expressing a T-cell Engager Simultaneously

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an oncolytic virus expressing a t-cell engager ...isolation kit (miltenyi biotec). for cd4þ and...

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