armed oncolytic adenovirus expressing pd-l1 mini-body ... · anti-human pd-l1 and isotype antibody...

Therapeutics, Targets, and Chemical Biology

Armed Oncolytic Adenovirus–Expressing PD-L1Mini-Body Enhances Antitumor Effects ofChimericAntigenReceptor TCells in Solid TumorsKiyonori Tanoue1,2, Amanda Rosewell Shaw1,2, Norihiro Watanabe1,2, Caroline Porter1,2,Bhakti Rana1,2, Stephen Gottschalk2,3, Malcolm Brenner1,2,3, and Masataka Suzuki1,2

Abstract

Chimeric antigen receptor–modified T cells (CAR T cells)produce proinflammatory cytokines that increase expression ofT-cell checkpoint signals such as PD-L1, which may inhibit theirfunctionality against solid tumors. In this study, we evaluated inhuman tumor xenograft models the proinflammatory proper-ties of an oncolytic adenovirus (Onc.Ad) with a helper-depen-dent Ad (HDAd) that expresses a PD-L1 blocking mini-antibody(mini-body; HDPDL1) as a strategy to enhance CAR T-cellkilling. Coadministration of these agents (CAd-VECPDL1)exhibited oncolytic effects with production of PD-L1 mini-bodylocally at the tumor site. On their own, HDPDL1 exhibited noantitumor effect and CAd-VECPDL1 alone reduced tumors only

to volumes comparable to Onc.Ad treatment. However, com-bining CAd-VECPDL1 with HER2.CAR T cells enhanced antitu-mor activity compared with treatment with either HER2.CAR Tcells alone or HER2.CAR T cells plus Onc.Ad. The benefits oflocally produced PD-L1 mini-body by CAd-VECPDL1 couldnot be replicated by infusion of anti-PD-L1 IgG plus HER2.CAR T cells and coadministration of Onc.Ad in an HER2þ

prostate cancer xenograft model. Overall, our data documentthe superiority of local production of PD-L1 mini-bodyby CAd-VECPDL1 combined with administration of tumor-directed CAR T cells to control the growth of solid tumors.Cancer Res; 77(8); 2040–51. �2017 AACR.

IntroductionIntratumoral treatment with oncolytic adenoviral vectors

expressing an immunomodulatory molecule (Armed Onc.Ads)is safe and has shown some clinical benefit in patients with solidtumors (1). However, local treatment with Armed Onc.Ad haslimited antitumor effect against metastasized tumors (1). Inaddition, Onc.Ads have low transgene capacity (2, 3), limitingthe potential to enhance antitumor immunity by addingmultiplegenetic modifications. We have shown that tumor cells co-infected with Onc.Ad and Helper-dependent Ads (HDAd), whichhave a cargo capacity of up to 34 kb, and therefore can expressmultiple immunomodulatory molecules in a single vector, rep-licate both Onc.Ad and HDAd. Infection with this dual Ad genetherapy (CAd-VEC) leads to multiple cycles of production andrelease of both the oncolytic and the immunogenic components(4). Although CAd-VEC significantly suppressed tumor growthcomparedwith treatmentwith eitherOnc.AdorHDAdalone in an

immunocompetent mouse model (4), it was insufficient to curebulky or metastasized tumors.

Chimeric antigen receptors (CAR) usually combine the extra-cellular antigen recognition domains of a monoclonal antibodyand a T-cell receptor signaling domain (CAR T cells; ref. 5). CAR Tcells can be systemically administered and home to both primaryand metastasized tumors (5), overcoming the limited systemicantitumor effects of locally administered Ad-based cancer immu-notherapies (1). Striking clinical successes against B-cell malig-nancies have been reported whenCAR T cells are directed to targetantigen CD19, which is highly expressed on both malignant andnormal B cells (6). Solid tumors have proven trickier, becausemany express a range of inhibitory cytokines (7) and immunecheckpoint ligands (8) that impair the recruitment and sustainedactivation of effector T cells. Thus, additional immunomodula-tion is likely required to increase CAR T-cell efficacy against solidtumors.

Recent clinical trials with immune-checkpoint inhibitors haveimproved tumor-specific T-cell responses (9). PD-L1 expressionon solid cancer cells is induced or increased in the presence of Th1cytokine IFNg (10), one of the cytokines expressed by activatedCAR T-cells (11). CAR-dependent activation of CAR T cells at thetumor site, therefore, may increase the expression of PD-L1 ontarget cancer cells, decreasing the antitumor effect of CAR T cellsthrough the PD-1:PD-L1 interaction (12).

As there are toxicities associated with systemic infusion of anti-PD-L1 antibody (13), we hypothesized that local secretion of ourfunctional checkpoint blockade through a single combinationagent, CAd-VEC, would be simpler, safer and perhaps moreefficacious than combining three separate treatment modali-ties—oncolytic viruses, checkpoint inhibitors and CAR T cells.

1Department of Medicine, Baylor College of Medicine, Houston, Texas. 2Centerfor Cell andGene Therapy, Baylor College ofMedicine, Texas Children's Hospital,Houston Methodist Hospital, Houston, Texas. 3Department of Pediatrics, BaylorCollege of Medicine, Houston, Texas.

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

CorrespondingAuthor:Masataka Suzuki, Baylor College ofMedicine, 1102 BatesAvenue, Suite 1770, Houston, TX 77030. Phone: 832-824-4807; Fax: 832-825-4732; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1577

�2017 American Association for Cancer Research.

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We hypothesized that a CAd-VEC–expressing anti-PD-L1 mini-antibody (PD-L1 mini-body) could block the PD-1:PD-L1interaction between CAR T cells and cancer cells locally whilelysing tumor cells, and that combining these treatment modal-ities would yield potent anti-tumor effects in solid tumors.Here, we demonstrate that CAd-VEC expressing a PD-L1 block-ing mini-antibody (CAd-VECPDL1) enhances the antitumoreffect of CAR T cells against human solid cancer cells in vitroand in vivo. Our "all-in-one" strategy proved more potent thanthe combination of anti-PD-L1 IgG and CAR T cells with orwithout additional Onc.Ad pretreatment in vivo.

Materials and MethodsAdenoviral vectors (HDAds and Onc.Ads)

The HDAd HDD28E4EGFP construct containing an EGFPtransgene driven by the CMV promoter (HDAdeGFP) was pro-duced as described elsewhere (4). HDAd without transgene(HDAd0) was produced as described elsewhere (14). To gener-ate the PD-L1 mini-body, the anti-human PD-L1 scFv encodinghuman IgG signal peptide and the single chain variable region ofthe YW243.55.S70 was fused with a hinge, CH2 and CH3regions of human IgG1 with a C-terminal HA tag. The PD-L1mini-body complementary DNA was inserted into the CMVpromoter with polyA signal sequences. After confirmation ofsequence and expression, this expression cassette was insertedinto the pHDD28E4 vector, and HDD28E4 PD-L1 mini-body(HDAdPD-L1) was rescued as described elsewhere (15). Onc.Ad5D24 was produced as described elsewhere (4, 16).

Cell linesHuman prostate cancer cell line PC-3, human non–small cell

lung carcinoma cell line A549, human hepatocellular carcinomacell line HepG2, and human squamous cell carcinoma line SiHawere obtained from the ATCC in 2014. Cell lines were authen-ticated by using short tandem repeat (STR) profiling by the ATCC.Cells were cultured under the recommended conditions.

Primary cellsHumanPBMCswere isolated using Ficoll-PaquePlus according

to the manufacturer's instructions (Axis-Shield). For preparationof mature dendritic cells (mDC), PBMCs were cultured in den-dritic cell medium (Cell Genix) for 2 hours at 37�C, and non-adherent cells were removed. The remaining monocytes werecultured in DC medium supplemented with 400 U/mL of IL4and 800 U/mL of GM-CSF. Fresh media with cytokines weresupplemented every 3 days. The mDCs were induced by additionof TNFa, PGE-1, IL1b and IL6 on day 6 and cultured for 48 hours(17). CD4þ T cells were isolated from PBMCs using MACScolumn according to the manufacturer's instructions (MiltenyiBiotec).

Mixed lymphocyte reactionCD4þ T cells were cultured in 96-well round bottom plates

together with allogeneic mDCs at a ratio of 10:1, using CTLmedium (18). Anti-human PD-L1 IgG, Isotype IgG (Biolegend),mediumofA549-infectedwithHDPD-L1orHDeGFPwere added,as described in Figure legends. Supernatants were collected at 5days after coculturing CD4þ T cells with allogeneic mDCs, andIFNg levels in media were measured using the BD cytokinemultiplex bead array system (BD Biosciences) according to the

manufacturer's instructions. Cells were labeled with 3H-thymi-dine for an additional 18 hours to measure T-cell proliferation.

Coculture experimentsHuman cancer cells genetically modified to express EGFP were

seeded in 12-well plates and infected with 1,000 viral particles(vp) per cell of HDAds or treated with 10 mg/mL of anti-humanPD-L1 IgG, Isotype IgG (Biolegend). HER2.CAR T-cells with aneffector to target ratio of 1:20 were added 48 hours after infectionand cultured for 5 additional days. Residual live EGFPþ cancercells and T cells were counted on the basis of EGFP and CD3expression with Counting Beads (Life Technologies). Cell num-bers were calculated per 5,000 microbeads.

PD-L1 ELISAAn Immulon 2 high binding 96-well plate (VWR) was coated

with 500ng/well of recombinant humanPD-L1 (BioVision). Afterblocking the plate with PBS-T containing 3% BSA, serially dilutedmedia of A549-infected with 1,000 vp/cell of HDegfp or HDPD-L1 mini were added and incubated at 4�C for 24 hours. Seriallydiluted anti-human PD-L1 antibody starting from 10 mg/well(BioLegend) was used as a positive control. After washing theplate with PBS-T, HRP-labeled anti-human IgG for PD-L1 mini-body detection or HRP-labeled anti-mouse IgG (Bio-Rad) foranti-human PD-L1 and isotype antibody detection were addedand incubated at room temperature for 1 hour. Then we devel-oped the washed plate. Absorbance was measured using Tecanreader (TECAN).

PD-L1 mini-body ELISAAmounts of PD-L1 mini-body in media of cancer cells

infected with 10 vp/cell of HDPD-L1 alone, Onc.Ad alone orwith CAd-VECPD-L1 (Onc.Ad:HDAd¼1:10) were quantifiedwith an ELISA-based assay. Media of cancer cells infected withAds were added to an Immulon 2 96-well plate (VWR). Recom-binant HA-tag fusion protein (Alpha Diagnostic Intl Inc.) wasused as a standard. After blocking with PBS-T containing 3%BSA, anti-HA monoclonal antibody (Clone 5B1D10; ThermoFisher Scientific) was added to the plate and incubated at 4�C for24 hours. After washing, HRP-labeled anti-mouse IgG (Bio-Rad)was added and incubated at room temperature for 1 hour, andthen we developed the washed plate.

Animal experimentsAfter counting, 1�106 harvested cancer cells (PC-3) were re-

suspended in 100mLof PBS and subcutaneously injected into 5-to6-week-old NU/J male mice. After the tumor size reached 100mm3, 1�108 of Onc.Ad, HDPDL1 or CAd-VECPD-L1 (Onc.Ad:HDAd;1:20) were intratumorally injected in a volume of 20 mL.Tumor size was followed and volumes were calculated using theformula:Width2 x Length x 0.5. For PD-L1mini-body detection intumor and serum samples, tumors and serumwere collected at thedates described in Results. Tumors were homogenized withmicropestles (VWR) at 4�C, and supernatants of tumor lysateswere isolated via centrifugation at 2,000 rpm for 5 minutes. Totalprotein concentration of serum was measured using the MicroBCA Protein Assay Kit (Thermo Scientific).

A total of 1�106 human cancer cells (PC-3 or SiHa) were re-suspended in a volume of 100 mL of PBS and subcutaneouslyinjected into 5-to 6-week-old NSG mice (PC-3: male mice, SiHa:female mice). After the tumor size reached 100 mm3, a total of

Local PD-L1 Blockade Enhances CAR T-cell Therapeutic Effect

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1�107 of Onc.Ad or CAd-VECPD-L1 (Onc.Ad:HDAd;1:20) wereintratumorally injected in a volume of 20 mL. Three days afterinjection of Ads, mice received 1�106 HER2.CAR T-cells intrave-nously. Tumor size was followed and volumes were calculatedusing the formula: Width2 x Length x 0.5. To track the migrationand survival of HER2.CAR T-cells in vivo, T cells were geneticallymodified to express eGFP.FFLuc (19). Biodistribution of HER2.CAR T-cells was assessed using the In Vivo Imaging System(Xenogen; ref. 19).

Isolation of tumor-infiltrating HER2.CAR T cells from tumorsAfter rinsing the collected tumors with PBS, tumors were

minced and incubated in RPMI media containing Collagenasetype IV (5mg/mL) and type I (1mg/mL; Thermo Fisher Scientific)at 37�C for 2 hours (20). Cells were passed through a 70-mm cellstrainer (BD Pharmingen) and stained with antibodies describedin Supplementary Material.

Quantification of vector genome DNA in Ad-infected cells andin Ad-injected tumors

Cells were infected with 10 Vp/cell of Onc.Ad, HDPD-L1 orCAd-VECPD-L1 (Onc.Ad:HDAd; 1:10) and harvested 48 hoursafter infection. Tumors were injected with a total of 1�108 Vp ofOnc.Ad, HDPD-L1 or CAd-VECPD-L1 (Onc.Ad:HDAd;1:20) andharvested at the indicated time points. Total DNA was extractedfrom infected cells or tumors, and vector copies were quantified asdescribed in Supplementary Material.

Statistical analysisData were analyzed by one-way ANOVA followed by Ranks

protected least significant difference test (SigmaPlot).

ResultsPD-L1 mini-body and anti-human PD-L1 IgG similarly blockthe PD-1:PD-L1 interaction

Cancer cells upregulate PD-L1 in the presence of IFNg (10),which is produced by activated T cells. We therefore coculturedT cells expressing second-generation HER2-specific CARs withCD28.z-signaling domains (HER2.CAR T cells), which wererecently reported to be safe in patients with sarcoma (11), withcancer cells. We evaluated the levels of PD-L1 on the HER2positive human prostate cancer cell line PC-3 and the squamouscell carcinoma cell line SiHa (Supplementary Fig. S1A), and theexpression of PD-1 on HER2.CAR T cells at different time points(Fig. 1A; Supplementary Fig. S1B). When cocultured with HER2.CAR T-cells, PC-3 (Pre: 60%, Post: 100%) and SiHa cells (Pre:20%, Post: 99%) upregulated PD-L1 expression to levels similarto those induced by recombinant IFNg treatment (SupplementaryFig. S1A). HER2.CAR T cells also expressed PD-1 within 24 hoursof coculture (Pre: 2%, Post: 30%), suggesting HER2.CAR T cellsexpress PD-1 upon activation, though PD-1 is one of multipleexhaustion markers expressed by T-cells (21).

To test whether blocking the PD-1:PD-L1 interaction betweencancer cells and HER2.CAR T cells increases cytotoxicity locally atthe tumor site, we constructed a HDAd encoding PD-L1 mini-body expression cassette (HDPDL1). We confirmed the dose-dependent expression of PD-L1 mini-body in media of non–small cell lung carcinoma A549 cells infected with HDPDL1 (Fig.1B). We next evaluated whether the PD-L1 mini-body secreted inmedia of A549 cells binds to recombinant human PD-L1 protein

(Fig. 1C). Although there was no binding of supernatant of A549cells infected with control HDAd (HDeGFP), the supernatant ofA549 cells infected with HDPDL1 had dose-dependent bindingto rPD-L1 similar to anti-human PD-L1 IgG. To evaluate whetherPD-L1 mini-body can promote T-cell responses by blocking thePD-1:PD-L1 interaction, supernatant from HDPDL1-infectedA549 cells was added to an allogeneic mixed lymphocyte reac-tion (MLR; Fig. 1D; refs. 18, 22). In the presence of PD-L1 mini-body or anti-human PD-L1 IgG, IFNg release increased 10-foldcompared with an MLR in the presence of isotype IgG orsupernatant of A549 cells infected with HDeGFP. The levels ofIFNg release were dependent on the dose of PD-L1 mini-body oranti-human PD-L1 IgG (Supplementary Fig. S2A). PD-L1 mini-body and anti-human PD-L1 IgG also enhanced T-cell prolifer-ation compared to controls (Supplementary Fig. S2B). Theseresults indicate that PD-L1 mini-body secreted from HDPDL1-infected A549 cells blocks the PD-1:PD-L1 interaction similarlyto anti-human PD-L1 IgG.

PD-1:PD-L1 blockade by PD-L1mini-body increases the killingeffect of HER2.CAR T cells in vitro

To evaluate whether PD-1:PD-L1 blockade by PD-L1 mini-body enhances HER2.CAR T-cell cancer cell killing, we coculturedHER2.CAR T cells with PC-3 and SiHa infected with HDPDL1 orcontrol HDAd. We also performed coculture experiments in thepresence of 10 mg/mL anti-human PD-L1 IgG or isotype IgG ascontrols (Fig. 2). Live cells were counted after 5 days coculture.Although there was no difference in live cancer cells betweenuntreated and PC-3 cells treated with control HDAd or isotypeIgG, PC-3 treated with anti-PD-L1 IgG or infected with HDPDL1enhanced cancer cell killing by HER2.CAR T cells 2- to 3-fold.PD-L1 blockade also increased HER2.CAR T-cell expansion 1.3- to2-fold (Fig. 2A) and proliferation compared with controls (Sup-plementary Fig. S3). During the same coculture experiment withSiHa cells, only those infected with HDPDL1 had 60% lower cellnumbers compared with control groups. HER2.CAR T-cells in thepresence of PD-L1 mini-body expanded 5-fold more than othergroups (Fig. 2B).

Importantly, we observed no toxicity due to HDAd infection,PD-L1 mini-body or IgG treatments compared with untreatedcells (Fig. 2). These results indicate that the cancer cell killing seenin our coculture experiments was dependent onHER2.CAR T cellsand that PD-1:PD-L1 blockade can enhance the killing effect ofthese cells despite increased expression of PD-L1 on cancer cells inthe presence of CAR T-cells (Fig. 1A).

CAd-VECPDL1 amplifies PD-L1 mini-body expression inhuman cancer cell lines in vitro and in vivo

To test whether coinfection of Onc.Ad with HDPD-L1 couldamplify PD-L1 mini-body in transduced human cancer cell lines,as previously shown with HDAd transgenes (4), we coinfectedHDPD-L1withOnc.Ad (CAd-VECPDL1) into human solid cancercell lines (non-small cell lung carcinoma A549, prostate cancerPC-3, squamous cell carcinoma SiHa, hepatocellular carcinomaHepG2), and evaluated the levels of PD-L1 mini-body in media48 hours after infection (Fig. 3A). A549, PC-3 and HepG2 cellsinfectedwith a CAd-VECPDL1had 4-fold (A549), 15-fold (PC-3),10-fold (HepG2) higher expression of PD-L1mini-body inmediacompared with cells infected with HDPDL1 alone. We confirmedthe amplified production of PD-L1 mini-body by Western blot-ting (Supplementary Fig. S4A). To test whether increased

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expression was dependent on the amplification of HDAd vectorDNA, we quantified both HDAd and Onc.Ad vector copies usingprimer sets for each backbone 48 hours after infection (Fig. 3B).Cells infected with the CAd-VECPDL1 had 1,000-fold moreHDAd vector copies than cells infected with HDAd alone. Toverify whether CAd-VECPDL1 induces both amplification ofHDAd and the lytic effects of Onc.Ad, cellular lysis of CAd-VECPDL1 was evaluated with MTS assay 96 hours after infection(Fig. 3C), and the LD50 of each treatment in each cell line wasdetermined (Supplementary Table S1). Although Onc.Ad alonewas more toxic than CAd-VECPDL1, CAd-VECPDL1 had dose-dependent lytic effects in infected cancer cell lines.

To examine the PD-L1 mini-body expression and anti-tumoreffect of CAd-VECPDL1 in vivo, nude mice were subcutaneously

transplanted with PC-3. After the tumor volume reached 100mm3, we injected 1�108 viral particles (Vp) of Onc.Ad, HDPDL1or CAd-VECPDL1 intra-tumorally. Tumor samples were collectedat different time points after injection, and the presence of PD-L1mini-body in tumors was evaluated byWestern blot analysis (Fig.3D). PD-L1 mini-body was detected in lysates from tumorsinjected with HDPDL1 alone or CAd-VECPDL1 over time. PD-L1mini-body was not detectable in the serum ofmice from eithergroup (Supplementary Fig. S4B). There was no difference intumor growth between untreated mice and those treated withHDPDL1 alone, indicating that PD-L1 mini-body expression atthe tumor site alone does not suppress tumor growth. Theseresults are consistent with those from our in vitro experiments(Fig. 2). Mice treated with CAd-VECPDL1 demonstrated 60%

Figure 1.

HDAd-derived PD-L1 mini-body functions similarly to commercial anti-PD-L1 IgG. A, PC-3 and SiHa were cocultured with HER2.CAR T cells generated from threedifferent donors (effector:target ratio of 1:20). Cells were harvested at 8, 24, 72, and 120 hours after coculture, and PD-1 on HER2.CAR T-cells and PD-L1 oncancer cells were analyzed by flow cytometry. Data are presented as means � SD (n ¼ 3). B, Schematic structure of HDAd encoding an anti-PD-L1 mini-bodyexpression cassette (HDPD-L1). A549 cells were infectedwith different dosages of HDPD-L1. Media and cells were collected at 48 hours after infection. Samples weresubjected to Western blotting, and PD-L1 mini-body in media was detected by anti-HA antibody. Human GAPDH in cells was detected by anti-human GAPDHantibody, HDeGFP encoding an EGFP expression cassette was used as a control. C, A549 media infected with 1,000 vp/cell of HDPD-L1 or HDeGFP werecollected at 48 hours after infection, and binding of PD-L1 mini-body to recombinant human PD-L1 was assessed by ELISA. Anti-PD-L1 IgG or isotype IgG werecontrols (10 mg/mL; highest concentration). Data are presented as means � SD (n ¼ 3). D, Purified CD4þ T cells were cocultured with irradiated allogeneicmature DCs in the presence of PD-L1 mini-body–containing medium, 10 mg/mL anti-PD-L1 IgG, or isotype IgG for 5 days (T-cell:DC ratio of 10:1). IFNg levels in themedium were measured by ELISA. Data are presented as means � SD (n ¼ 4); � , P ¼ 0.005. The experiment was triplicated with similar results.






lower tumor volume compared to control mice at 35 days afterinjection, as did those injected withOnc.Ad alone, indicating thatthe Onc.Ad-dependent lytic effects are maintained in vivo (Fig.3E). We also quantified Onc.Ad and HDPDL1 vector copies intumors at 35 days after injection (Fig. 3F) and found that tumorsinjected with CAd-VECPDL1 exhibited 100-fold more HDAdcopies than those injected with HDPDL1 alone.

Combinatorial treatment with CAd-VECPDL1 and CAR T cellsprolongs survival in vivo

To test whether pretreatment of CAd-VECPDL1 enhances theantitumor effects of adoptively transferred HER2.CAR T cells inxenograft models, NSG mice were subcutaneously transplantedwith PC-3, and 1�107 Vp of Onc.Ad or CAd-VECPDL1 wereintratumorally injected after the tumor reached100mm3.Controlmice were intratumorally injected with vehicle alone. A total of1�106 HER2.CAR T cells genetically modified to express fireflyluciferase (ffLuc) were systemically infused 3 days after Ad injec-tion (19). Although PD-L1mini-body increased HER2.CAR T-cellexpansion compared to HER2.CAR T-cells alone in vitro (Fig. 2A),there were no differences in ffLuc activity at tumor sites in animalstreated with HER2.CAR T cells alone or CAd-VECPDL1 withHER2.CAR T cells (Fig. 4A). In addition, we found that micetreated with Onc.Ad and HER2.CAR T cells initially had 50-90%less ffLuc activity post-HER2.CAR T-cell injection than those

treated with CAR T cells alone. The decreased activity reversed at14 days, such that mice from all three treatment groups hadsimilar ffLuc activity. We detected PD-L1 mini-body in tumorsamples 10 days after HER2.CAR T-cell injection (SupplementaryFig. S5A). Although we observed both adenovirus and T cells attumor sites 10 days after HER2.CAR T-cell injection, it was unclearwhether they colocalized there (Supplementary Fig. S5B). Theseresults suggest that adoptive HER2.CAR T-cell treatment has aminimal impact on Onc.Ad-dependent oncolysis.

Althoughwedid not observe increased T-cell expansion inmicepretreated with CAd-VECPDL1 compared with the other groups,mice pretreated with Ad gene therapy (either Onc.Ad or CAd-VECPDL1) had suppressed tumor growth compared to micetreated with HER2.CAR T cells alone (Fig. 4B). While mice treatedwith CAd-VECPDL1 had similar median survival to mice treatedwith HER2.CAR T-cells (60 days), mice treated with CAd-VECPDL1 and HER2.CAR T cells had 2-fold longer mediansurvival (110 days) than mice treated with a single agent (Fig.4C). These results indicate that PD-L1 mini-body-dependentHER2.CAR T-cell activation at the tumor site enhances the anti-tumor effect of HER2.CAR T cells.

To evaluate how Ad gene therapy phenotypically impactsHER2.CAR T cells and cancer cells, we collected tumors frommice 84 days after Ad injections.We phenotyped infiltrated T cellsand residual (recurrent) cancer cells (Fig. 4D and E) and found

Figure 2.

Blockade of PD-L1:PD-1 interaction enhances cancer cell killing by HER2.CAR T-cells. PC-3–expressing eGFP (A) and SiHa-expressing eGFP (B) were infectedwith 1,000 vp/cell of HDAd0 or HDPD-L1. HER2.CAR T cells were added at 24 hours after infection (effector:target ratio of 1:20). The same cocultureexperiments were performed in the presence of 10 mg/mL anti-PD-L1 IgG or isotype IgG. Cells were harvested 120 hours after coculture, and viable cells (7AAD�)per 5,000 counting beads were analyzed by flow cytometry. Data are presented as means � SD (n ¼ 4). � , P < 0.05; �� , P < 0.01; �� , P ¼ 0.006; �� , P ¼ 0.004;��, P < 0.001 for A. � , P < 0.03; �� , P < 0.01; �� , P < 0.005; �� , P < 0.0001 for B. The experiments were repeated with HER2.CAR T cells derivedfrom a different donor with similar results.

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approximately 60% of T cells isolated from tumor samples stillexpressedHER2.CAR (Fig. 4D).We also phenotypedHER2.CAR Tcells at different time points to confirm the T cells retained HER2.

CAR expression (Supplementary Fig. S5C). T cells isolated fromtumors treated with Ad gene therapy had more CD8þ T cells, butthere was no difference in memory phenotype (CCR7/CD45RO)

Figure 3.

Coinfection of Onc.Ad with HDPD-L1 (CAd-VECPD-L1) amplified PD-L1 mini-body while maintaining oncolysis in vitro and in vivo. A, A549, PC-3, SiHa, andHepG2 were infected with a total 10 Vp/cell of HDPD-L1, Onc.Ad or with an CAd-VECPD-L1 (Onc.Ad:HDAd; 1:10). Medium samples were collected at 48 hoursafter infection. Levels of PD-L1 mini-body in medium samples were quantified by ELISA-based assay for HA-tagged protein. Data are presented as means � SD(n ¼ 4); � , P ¼ 0.003; �� , P ¼ 0.002; �� , P < 0.001. B, DNA samples were extracted 48 hours after infection, and Onc.Ad and HDAd vector copy numberswere measured by quantitative PCR. Data were normalized with human genomic GAPDH. Data are presented as means � SD (n ¼ 4). � , P < 0.03; �� , P < 0.001.C, Cancer cell lines were infected with increasing doses of HDPD-L1, Onc.Ad, or with CAd-VECPD-L1 (Onc.Ad:HDAd¼ 1:10). Viable cells were analyzed at96 hours by MTS assay. Data are presented as means � SD (n ¼ 6). D, PC-3 cells were transplanted into the right flanks of nude mice. A total of 1�108 Vp ofOnc.Ad, HDAd, or CAd-VECPD-L1 (Onc.Ad:HDAd¼ 1:20) were injected intratumorally. Tumors were collected and harvested at 3, 7, and 21 days after injection.PD-L1 mini-body in tumor lysates was detected by Western blotting. E, Tumor volumes were measured at different time points. Data are presented asmeans � SD (n ¼ 4); � , P ¼ 0.006. F, Total DNA was extracted from tumors at 35 days after injection of Ads, and the copy number of each vector was determinedby quantitative PCR. Data were normalized with human genomic GAPDH. Data are presented as means � SD (n ¼ 4); � , P < 0.02; ��, P ¼ 0.008.






Figure 4.

CAd-VECPD-L1 enhanced the antitumor effects of HER2.CAR T-cells in vivo. A, PC-3 cells were transplanted into the right flanks of NSG mice. A total of 1�107 Vpof Onc.Ad or CAd-VECPD-L1 (Onc.Ad:HDAd¼ 1:20) was injected intratumorally. A total of 1�106 HER2.CAR T cells expressing firefly luciferase (ffLuc) was systemicallyadministered 3 days after injection of Ads. Bioluminescence of HER2.CAR T cells was monitored at different time points. Data are presented as means � SD(n¼ 8); � , P¼ 0.002; �� , P < 0.001.B, Tumor volumesweremeasured at different time points. Data are presented asmeans� SD (n¼ 8); � , P¼ 0.002.C,Kaplan–Meiersurvival curve after administration of Ad gene therapy. The end point was established as tumor volume of >1,500 mm3. Data are presented as means � SD(n ¼ 8); �P < 0.001. D, T cells at tumor site were isolated at 84 days after infusion, and phenotype was analyzed by flow cytometry. E, Tumor cells were isolated at84 days after infusion of HER2.CAR T cells and were cultured in vitro. Cells were harvested at 72 hours, and phenotype was analyzed by flow cytometry.

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of CD8þ or CD4þ T cells (Supplementary Fig. S5D). Althoughthere were similar expression levels of other exhaustion markers(Tim-3 and LAG-3; Supplementary Fig. S5D),HER2.CAR T cells inmice treated with CAd-VECPDL1 had 30% lower PD-1 expressioncompared to other groups. Since PD-1þ T-cell populations aremostly CD4þ (Supplementary Fig. S1C; ref. 23), the reducedCD4þ population in CAd-VECPDL1-treated mice may correlatewith fewer PD-1þ T cells. BecauseHER2.CAR T cells still expressedthe CAR, we analyzed HER2 expression on cancer cells fromtumors 84 days after Ad injection (Fig. 4E). We evaluated HER2expression on human CD47þ cells, which is highly expressed inhuman cancers, including prostate cancer, as tumor cells alsocontainedmouse stroma cells (Supplementary Fig. S5E; refs. 24,25). Human CD47þ cells had 80-90% lower HER2 expressioncompared with PC-3 cells cultured in vitro, indicating that PC-3cells downregulated HER2 expression. Interestingly, cancer cellsin a mouse pretreated with CAd-VECPDL1 showed 50% lessPD-L1 expression compared with other groups (Fig. 4E). Theseresults suggest that constitutive blockade of PD-L1 by PD-L1mini-body leads to downregulation of PD-L1 expression onremaining cancer cells.

We performed the same experiments with NSG mice subcuta-neously transplanted with SiHa cells (Fig. 5). Although 1�106

HER2.CAR T-cell alone minimally improved median survival (30days) compared to untreated mice (24 days), mice treated withCAd-VECPDL1 and HER2.CAR T cells had 2-fold longer mediansurvival (42 days) than untreated mice (Fig. 5C). We collectedtumors from mice treated with HER2.CAR T-cells with and with-out Ad treatments and phenotyped both the T cells and tumorcells (Fig. 5D and E). Tumor cells showed reduced HER2 expres-sion in the presence of HER2.CAR T cells, and only the tumorsample pretreated with CAd-VECPDL1 showed 50% less PD-L1expression compared with other groups, similar to what we see inthe PC-3 model.

Systemic treatment of PD-L1 IgG reduces the antitumor effectsof CAR T cells in vivo

As anti-PD-L1 IgG is FDA approved for use in humans (9), wecompared local blockade of the PD-1:PD-L1 interaction by PD-L1mini-body with systemic anti-PD-L1 IgG treatment in NSG micewith PC-3 tumors. We systemically injected anti-PD-L1 IgG intomice treated with Onc.Ad and HER2.CAR T cells andmice treatedwith HER2.CAR T cells alone (Fig. 6A). Because pretreatment ofCAd-VECPDL1 leads to PD-L1mini-body expression at the tumorsite before HER2.CAR T-cell infiltration, we infused anti-PD-L1IgGor isotype IgGbeforeHER2.CART-cell injection. Intratumoralinjection of CAd-VECPDL1 showed significantly better anti-tumor effects than systemic anti-PD-L1 IgG in mice treated withboth Onc.Ad and HER2.CAR T-cell and those treated with HER2.CAR T-cell alone (Fig. 6B). Overall, mice that received CAd-VECPDL1 had 2-fold longer median survival (110 days) com-pared with the IgG groups (59 days; Fig. 6C), indicating localexpression of PD-L1 mini-body is more effective than systemicanti-PD-L1 IgG treatment. Next, we examined HER2.CAR T-cellexpansion at tumor sites using ffLuc (Fig. 6D). Mice pretreatedwith anti-PD-L1 IgGhad30-90% lower ffLuc activity at tumor sitescompared to mice injected with CAd-VECPDL1 or mice injectedwith isotype IgG at 1, 3 and 7 days post-HER2.CAR T-cell injec-tion. Mice pretreated with anti-PD-L1 IgG also had less ffLucactivity at the ventral side 1 day post-HER2.CAR T-cell injection(Supplementary Fig. S6), suggesting that systemic administration

of anti-PD-L1 IgG before HER2.CAR T-cell infusion reduces thetotal number of circulating HER2.CAR T cells. Because intratu-moral CAd-VECPDL1 treatment has minimal distribution in theblood (Supplementary Fig. S4B), local PD-L1 mini-body expres-sion may not reduce the number of HER2.CAR T cells before theyreach the tumor site, as was seen inmice systemically treated withanti-PD-L1 IgG.

DiscussionHere,we demonstrate that PD-L1mini-body expressed byCAd-

VECPDL1 can block the PD-1:PD-L1 interaction between HER2.CAR T cells and cancer cells while maintaining cancer cell onco-lysis. The combinatorial effect of Onc.Ad-mediated oncolysis andPD-L1 mini-body–mediated blockade augments the antitumoreffect of adoptively transferred HER2.CAR T cells. Together, CAd-VECPDL1 andHER2.CART-cell treatment significantly prolongedanimal survival compared with treatment with HER2.CAR T cellsalone and toOnc.Ad with HER2.CAR T cells in a xenograft mousemodel of prostate cancer. Local blockade of the PD-1:PD-L1interaction by CAd-VECPDL1 also induced superior antitumoreffects compared to systemic administration of anti-PD-L1 IgG incombination with adoptive transfer of HER2.CAR T cells in ourxenograft mouse model.

Immune checkpoint inhibitors (e.g., PD-1, PD-L1 and CTLA-4)have been successful in treating multiple solid tumors, leading tomore robust and sustained T-cell responses (9); however, manypatients do not respond or subsequently relapse (26). To enhancehost immune responses to cancer cells, checkpoint inhibitors havebeen combined with other therapeutic agents including chemo-therapy, radiotherapy and oncolytic viral gene therapy (Onc.Vs)in preclinical models and clinical trials (26). Because Onc.Vsselectively lyse cancer cells and induce proinflammatoryresponses, combining immune checkpoint inhibitors with Onc.Vs seems to be a natural marriage (1). However, the therapeuticeffect of this combination relies on the patient's immuneresponse, andwhether this combination is safe remains unknown(1). To overcome these limitations, we aimed to concentratecheckpoint inhibition locally at the tumor site, thereby minimiz-ing off-target toxicities.We tested in twomurine xenograftmodelsthe combination of Onc.Ad-expressing an anti-PD-L1 checkpointinhibitor with adoptively transferred CAR-modified T cells, aslocal checkpoint blockade should increase their potency. Wehypothesized that this combination would create a proinflam-matory tumor microenvironment through oncolysis by Onc.Adand block the deleterious PD-1:PD-L1 interaction locally,enabling increased CAR T-cell activity.

We found that constitutive expression of PD-L1 mini-body atthe tumor site had superior antitumor effects than systemictreatment of anti-PD-L1 IgG in the presence of HER2.CAR T cells.Ten days after HER2.CAR T-cell injection, some mice that previ-ously received systemic infusion of anti-PD-L1 IgG, but not miceinfused with isotype IgG, had transient diarrhea. Similar sideeffects are seen in patients with renal cell carcinoma receivingatezolizumab (anti-PD-L1 antibody; ref. 13), suggesting thatsystemic treatment of anti-PD-L1 IgG with HER2.CAR T cellsleads to immune-related side effects in mice. In contrast, intra-tumoral administration of CAd-VECPDL1 followed by systemicHER2.CART-cell treatment causedno immune-related side effects(e.g., diarrhea, weight loss). Because PD-L1 mini-body expressedby CAd-VEC is localized at the tumor site with minimal






Figure 5.

CAd-VECPD-L1 enhanced the antitumor effects of HER2.CAR T cells in SiHa xenograft model. A, SiHa cells were transplanted into the right flanks of NSG mice.A total of 1 � 107 Vp of Onc.Ad or CAd-VECPD-L1 (Onc.Ad:HDAd¼ 1:20) was injected intratumorally. A total of 1 � 106 HER2.CAR T cells expressing firefly luciferase(ffLuc) was systemically administered 3 days after injection of Ads. Bioluminescence of HER2.CAR T cells was monitored at different time points. Data arepresented as means� SD (n¼ 5). B, Tumor volumeswere measured at different time points. Data are presented as means� SD (n¼ 5); � , P < 0.001. C, Kaplan–Meiersurvival curve after administration of Ad gene therapy. The end point was established as tumor volume of >1,500 mm3. Data are presented as means � SD (n ¼ 5);� , P ¼ 0.003. D, T cells at the tumor site were isolated at 32 or 42 days after infusion, and phenotype was analyzed by flow cytometry. E, Tumor cells were isolatedat 32 or 42 days post- HER2.CAR T-cell infusion and were cultured in vitro. Cells were harvested at 72 hours, and phenotype was analyzed by flow cytometry.

Tanoue et al.





circulation in the blood, our treatment may minimize toxicitiesrelated to systemic treatment of anti-PD-L1 IgG. Our results alsosuggest that systemic administration of anti-PD-L1 IgG beforeHER2.CAR T-cell infusion reduces the total number of circulatingHER2.CAR T cells. Stimulated na€�ve T cells express both PD-L1and PD-1, and blocking the PD-1:PD-L1 interaction leads toapoptosis through Fas:Fas ligand (FasL). HER2.CAR T cells inmicemay induce the expression of both PD-1 and PD-L1 throughtheir TCR and/or CAR, and therefore blockade of the PD-1:PD-L1interaction outside the tumor (e.g., in the lung) may induceunwanted Fas:FasL-dependent T-cell apoptosis (27).

Intratumoral administration ofOnc.Vs, includingOnc.Ads, haslimited distribution to metastasized tumors (28), and investiga-tors have been developing viral vectors to target both primary andmetastasized tumors systemically. However, patients whoreceived systemic Onc.Vs developed both Th1 and Th2 adaptiveimmune responses to the viruses after the first treatment, reducingthe antitumor efficacy of the second challenge due to rejection ofthe virus and infected cells (29). Because CAR T cells are generallygenerated from patients' own peripheral blood and are therefore

better tolerated thanOnc.Vs, CART cells can be infused repeatedlyto cancer patients (30). Our combination of intratumoral CAd-VEC with systemic CAR T-cell therapy could overcome the inher-ent limitation of Onc.Vs to create enhanced antitumor effects.

In this study, our "all-in-one" strategy attenuated the immu-nosuppressive effects of thePD-1:PD-L1 interactionon adoptivelytransferred CAR T cells at tumor sites, leading to superior antitu-mor effects and prolonged animal survival in a prostate cancerxenograft mouse model. This combinatorial treatment alsoshowed significant effects in mice with subcutaneous SiHatumors, although the potency was lower, suggesting that blockingthePD-1:PD-L1 interaction alonemaybe insufficient tomaximizethe antitumor effect of CAR T cells in particularly aggressive solidtumors. Because CAd-VEC is able to deliver multiple immuno-modulatory molecules (up to 34 kb) in a single HDAd vectorwith Onc.Ad-dependent lytic effect, in the future we can incor-porate additional molecules to maximize the anti-tumor effectof adoptively transferred CAR T-cells. A previous study showedthat Onc.Ad-expressing chemokine can enhance the infiltrationof adoptively transferred GD2.CAR T cells at the tumor site in

Figure 6.

CAd-VECPD-L1 had better antitumor effects than systemic PD-L1 IgG treatment. A, PC-3 cells were transplanted subcutaneously into the right flanks ofNSG mice. A total of 1 � 107 Vp of Onc.Ad or CAd-VEC (Onc.Ad:HDAd; 1:20) was injected intratumorally. A total of 1 � 106 HER2.CAR T cells expressing ffLucwas systemically administered at 3 days after injection of Ads. Mice treated with Onc.Ad with HER2.CAR T cells or HER2.CAR T cells alone received threeintraperitoneal injections of 100 mg anti-PD-L1 IgG at day 0, 3, and 6 after infusion of HER2.CAR T cells. B, Tumor volumes were measured at different timepoints. Data are presented as means � SD (n ¼ 8 or 7); � , P < 0.001. C, Kaplan–Meier survival curve after administration of Ad gene therapy. The end pointwas established as tumor volume of >1,500 mm3. Data are presented as means � SD (n ¼ 8 or 7); � , P < 0.001. D, Bioluminescence of HER2.CAR T cells wasmonitored at different time points. Data are presented as means � SD (n ¼ 7 or 8); � , P ¼ 0.001.






a neuroblastoma xenograft model, and Onc.Ad-expressing cyto-kine enhances proliferation of those CAR T cells, leading tosuperior antitumor effects than the combination of Onc.Ad(without transgene) with GD2.CAR T cells (19). We will inves-tigate whether combining PD-L1 mini-body with chemokinesand/or cytokines further enhances the antitumor effects of adop-tively transferred CAR T cells.

Overall, we demonstrate that combined CAd-VECPDL1 andCAR T-cell treatment has significant antitumor effects againstbulky solid tumors. Because intratumoral injection of Onc.Adshas been tested in patients with a range of solid tumors (31), ourconcept could readily be applied to other solid tumors and usedwith CARs targeting different surface molecules (e.g., GD2, PSCA;refs. 19, 32).

Disclosure of Potential Conflicts of InterestM.K. Brenner is an SAB in Tessa Therapeutics, Unum Therapeutics, Torque,

Nantkwest, Bluebird Bio., Turnstone, Cellmedica; has ownership interest(including patents) in and is a consultant/advisory board member for Patent.No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: A. Rosewell Shaw, M. Brenner, M. SuzukiDevelopment of methodology: K. Tanoue, A. Rosewell Shaw, C. Porter,M. SuzukiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Tanoue, A. Rosewell Shaw, N. Watanabe, C. Porter,B. Rana

Analysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):K. Tanoue, A. Rosewell Shaw,N.Watanabe, C. Porter,B. Rana, S. GottschalkWriting, review, and/or revision of the manuscript: K. Tanoue, A. RosewellShaw, S. Gottschalk, M. Brenner, M. SuzukiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Rosewell Shaw, S. GottschalkStudy supervision: M. Brenner, M. Suzuki

AcknowledgmentsThe authors would like to thank Dr. Brendan Lee in the Department of

Molecular andHumanGenetics at Baylor College ofMedicine for his support ofthe project and Catherine Gillespie in the Center for Cell and Gene Therapy atBaylor College of Medicine for her editing of the article.

Grant SupportThis work was supported by NIH (R00HL098692 to M. Suzuki),

T32HL092332 to A. Rosewell Shaw, BCM Head and Neck Seed Grant, andConcern Foundation to M. Suzuki. This work was also supported by NIH P01CA094237 to M.K. Brenner and S. Gottschalk, and we appreciate the use ofshared resources supportedbyNIHP30CA125123. K. Tanouewas supported byStrategic Young Researcher Overseas Visits Program for Accelerating BrainCirculation, Japan Society for the Promotion of Science.

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 11, 2016; revised January 12, 2017; accepted January 28, 2017;published OnlineFirst February 24, 2017.

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2017;77:2040-2051. Published OnlineFirst February 24, 2017.Cancer Res Kiyonori Tanoue, Amanda Rosewell Shaw, Norihiro Watanabe, et al. in Solid TumorsEnhances Antitumor Effects of Chimeric Antigen Receptor T Cells

Expressing PD-L1 Mini-Body−Armed Oncolytic Adenovirus

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