Systemic Immunotherapy of Non-Muscle Invasive Mouse ... · on the tumor cell surface (11). Enhanced...

Research Article

Systemic Immunotherapy of Non-Muscle InvasiveMouse Bladder Cancer with Avelumab, anAnti–PD-L1 Immune Checkpoint InhibitorAmanda J. Vandeveer1, Jonathan K. Fallon1, Robert Tighe2, Helen Sabzevari2,Jeffrey Schlom1, and John W. Greiner1

Abstract

Bacillus Calmette–Guerin (BCG) is the standard of carefor intravesical therapy for carcinoma in situ and non-muscleinvasive, nonmetastatic human urothelial carcinoma.Although the responsiveness to this immunotherapeutic isbelieved to be linked with (i) a high number of somaticmutations and (ii) a large number of tumor-infiltrating lym-phocytes, recent findings of the roles that inhibitory immunereceptors and their ligands play in tumor evasion may provideinsights into the limitations of the effectiveness of BCG andoffer new targets for immune-based therapy. In this study, anaggressive, bioluminescent orthotopic bladder cancer model,MB49 tumor cells transfected with luciferase (MB49luc),was used to study the antitumor effects of avelumab, anantibody to PD-L1. MB49luc murine tumor cells form multi-focal tumors on the mucosal wall of the bladder reminiscent

of non-muscle invasive, nonmetastatic urothelial carcinomas.MB49luc bladder tumors are highly positive for the expressionof PD-L1, and avelumab administration induced significant(P < 0.05) antitumor effects. These antitumor effects were moredependent on the presence of CD4 than CD8 T cells, asdetermined by in vivo immune cell depletions. The findingssuggest that in this bladder tumor model, interruption ofthe immune-suppressive PD-1/PD-L1 complex releases a localadaptive immune response that, in turn, reduces tumorgrowth. This bladder tumor model can be used to furtheridentify host antitumor immune mechanisms and evaluatecombinations of immune-based therapies for carcinoma in situand non-muscle invasive, nonmetastatic urothelial carcinoma,to provide the rationale for subsequent clinical studies.Cancer Immunol Res; 4(5); 452–62. �2016 AACR.

IntroductionEmerging experimental evidence and clinical evidence have

coalesced around the interrelationships between mutational het-erogeneity of different forms of cancer and the roles that immunecell checkpoint blockade might play in cancer immunotherapy(1, 2). Coincident with a better understanding of the roles ofinhibitory immune receptors and their ligands, collectivelytermed checkpoint inhibitors, has been an appreciation thatmanyhuman and mouse tumors are not blind to host immunity,but either express weak neo–tumor associated antigens or over-express self-antigens or fetal antigens that can be recognized byT cells (2–5). Although investigators realized that a large subset ofpatients had evidence of immune cell infiltrates within theirtumormicroenvironment, it was data highlighting CTLA-4 block-

ade with ipilimumab that provided the crucial connectionbetween an interruption of an immune cell checkpoint pathwayand significant antitumor responses (6, 7). Studies have alsofocused on other checkpoint inhibitors, particularly the pro-grammed death-1 (PD-1) receptor that interacts with distinctligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273;ref.8). PD-L1 is expressed on a wide variety of human and mousetumor cells and some immune cell populations, whereas PD-L2is mainly found on antigen-presenting cells (9, 10). PD-L1expression on tumors and tumor-infiltrating lymphocytes expres-sing IFNg colocalize in many tumors. With the accompanying T-cell–associated IFNg production, PD-L1 expression is increasedon the tumor cell surface (11). Enhanced PD-L1 expressionon tumor cells dampens tumor immunity by inducing antigen-specific T-cell apoptosis or anergy (12). Interruption, or blockade,of the PD-1/PD-L1 interaction by the administration of an anti-body to PD-1 or PD-L1 can overcome immune resistance inpreclinical models and has been correlated with positive clinicaloutcomes in patients diagnosed with metastatic bladder cancer(13–15). One such agent is avelumab, which is a fully humanIgG1 anti–PD-L1 antibody currently being studied clinically inmultiple tumor types.

For the last four decades, the standard of care for carcinomain situ and non-muscle invasive, nonmetastatic urothelial carci-noma has been immune-based: the intravesical instillation ofattenuated Bacillus Calmette–Guerin (BCG; refs.16, 17). Themech-anism of BCG action remains elusive, yet most investigatorsbelieve that the influx of immune cells is a crucial component(18). Approximately 30% to 45% of patients fail to respondinitially to BCG or relapse within 5 years of treatment (19). Thus,

1Laboratory of Tumor Immunology and Biology, Center for CancerResearch, National Cancer Institute, Bethesda, Maryland. 2EMD Ser-ono Research and Development Institute, Billerica, Massachusetts.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

A.J. Vandeveer and J.K. Fallon contributed equally to this article.

J. Schlom and J.W. Greiner contributed equally to this article.

Corresponding Author: Jeffrey Schlom, Laboratory of Tumor Immunology andBiology, National Cancer Institute, National Institutes of Health, 10 Center Drive,Room 8B09, MSC 1750, Bethesda, MD 20892. Phone: 301-496-4343; Fax: 301-496-2756; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-15-0176

�2016 American Association for Cancer Research.

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with the local production of IFNg by invading immune cells, thequestion arises as to whether the PD-1/PD-L1 axis might con-tribute to unresponsiveness or relapse following BCG therapy.Increasing PD-L1 expression predicts localized bladder cancerstage progression independent of tumor grade, and PD-L1 levelsare highest in carcinoma in situ andwithin granulomata of bladdertissues of patientswho failed BCG therapy (19–21). Therefore, thepresence of PD-L1 could conceivably play a role in abrogatinghost immune–related responses and result in bladder cancerprogression, which infers a biologic role for the PD-1/PD-L1interaction as a new immunotherapeutic target.

MB49 is a murine transitional cell bladder carcinoma line thatforms tumors when injected subcutaneously or orthotopicallyinto mouse bladders. The murine orthotopic bladder tumormodel provides an opportunity to study the immune-relatedevents involved in the use of immune cell checkpoint inhibitorsfor the treatment of carcinoma in situ and non-muscle invasive,nonmetastatic urothelial carcinoma and to establish scientificrationale for combining immune cell checkpoint inhibitors withother potential forms of therapy. Findings from the present studyclearly show that the successful targeting of PD-L1 on MB49bladder tumors with a PD-L1 antibody, avelumab, results insignificant antitumor effects that are associated with the expan-sion/generation of an adaptive immune response.

Materials and MethodsAnimals and cell lines

Female C57BL/6 mice were purchased from The JacksonLaboratory or Charles River Laboratories. F5 mice that aretransgenic (Tg) for nucleoprotein of influenza virus A/NT/60/68 (366ASNENMDAM374;NP68)-specific, H-2Db

–restricted T-cellreceptor were obtained fromTaconic Farms. Allmice were housedin microisolator cages in pathogen-free conditions. Mice used forthe in vivo antitumor studies were 16 to 18weeks old at the start ofstudy. Animal carewas in compliancewith TheGuide forCare andUse of Laboratory Animals (National Research Council).

The MB49 parental cell line (murine transitional cell carcino-ma) was kindly provided by Dr. Peter Pinto (Urologic OncologyBranch, CCR, NCI, NIH). Cells were grown, batch frozen, andused in the experiments described. The MB49 LucSHþ cells(MB49luc) were generated within our laboratory. All MB49 lineswere grown in DMEM with 10% heat-inactivated FBS supple-mented with 1 mmol/L nonessential amino acids, 1 mmol/Lsodium pyruvate, 2 mmol/L glutamine, and penicillin/strepto-mycin (100 U/mL). MB49luc growth medium also containedZeocin (200 mg/mL). MB49luc are parental MB49 cells transfectedwith a pSELECT-zeo-LucSh plasmid using Lipofectamine (Invivo-Gen) for luciferase expression detected by in vivo imaging.

In vitro F5 TCR.Tg T-cell activationBone marrow–derived dendritic cells (BMDC) were generated

from adult female C57BL/6 mice following in vitro growth for 6days in complete RPMI medium supplemented with 20 ng/mLmurine recombinantGM-CSF and10ng/mLmurine recombinantIL4.Medium and nonadherent cells were discarded on days 2 and4 and replaced with fresh medium containing GM-CSF/IL4. Onday 6, the nonadherent cells were collected, washed, and used forin vitro T-cell activation studies. PD-L1 expression on theseBMDCs was determined by cell surface staining with avelumab(data not shown).

BMDCs (50,000/well) were pulsed overnight with 10 to 1,000ng/mL NP68 peptide (ASNENMDAM, H-2Db) or control HYpeptide (WMHHNMDLI, H-2Db) in 24-well plates. After 24hours, splenic CD8aþ cells were purified from F5 TCR.Tg miceusing negative selectionmagnetic beads (Miltenyi Biotec) accord-ing to the manufacturer's instructions. Isolated F5 CD8aþ cellswere added to the 24-well plates at 10,000 cells/well alongwith 10mg/mLofHuIgG1or avelumab in1mL/well. After 5 days of in vitroT-cell activation, supernatantswere collected and stored at�20�C,and IFNg concentrations were later determined using a standardELISA kit (Thermo Fisher Scientific) according to the manufac-turer's instructions. Sample optical densities (ODs) at 450 nmwere measured using a Synergy HT plate reader (Bio-Tek).

Murine tumor modelsSubcutaneous tumor injections were carried out by inoculating

C57BL/6 mice with 1 � 105 MB49 parental cells on the rightshaved flank. Tumor growth was measured with calipers, and 8days after inoculation, mice were assigned to treatment groups.Treatments began on day 9, tumors were measured 2x/weekthroughout the study, and tumor volume was calculated as:Volume ¼ (width)2 � (length)/2. Mice were euthanized whenthe tumor size was greater than 1.5 cm3.

Intravesical instillation of orthotopic MB49luc bladder tumorswas carried out as previously described (22). Mice were anesthe-tized [ketamine (15 mg/kg) and xylazine (75 mg/kg)] and cathe-terized using Teflon catheters (SurFlash Polyurethane I.V. catheter24G x 3/4, SR �FF2419; Terumo Medical products). Orthotopicbladder tumors were established by instilling 7.5 � 104 MB49luc

cells for 45minutes intobladders thathadbeenpretreatedwith100mL of poly-L-lysine (0.1 mg/mL) solution [PLL, molecular weight(MW) 70,000 to 150,000; Sigma-Aldrich]. Tumor take was con-firmedby in vivo imaging 7 to10days later, atwhich timemicewereplaced into groups with equal tumor burden prior to treatment.

Immune cell subset depletionImmune cell subset depletions were carried out by administer-

ing anti-CD4 (100 mg GK1.5) or anti-CD8 (100 mg 2.43) T-cell–depletion antibodies eachday for 4 consecutive days and thereafterwith weekly injections. Natural killer (NK)-cell depletion wasachieved by weekly administration of 25 mL rabbit anti–asialo-GM1 (Cedarlane Laboratories). The administration of the deplet-ing antibodieswas begun1weekprior to the intravesical tumor cellinstillation. Depletions were confirmed with flow cytometric anal-yses for CD4 and CD8 cells and NK lysis of YAC-1 targets usingsplenocytes from representative mice (Supplementary Fig. S1).

TreatmentsAvelumab, a fully human anti–PD-L1 (IgG1), and an isotype-

control antibody,were kindlyprovidedbyEMDSerono.Avelumabbinds tobothhumanandmousePD-L1withhigh affinity,Kd¼ 0.3and 1.0 nmol/L, respectively. Both were diluted in Dulbecco'sphosphate buffered saline (DPBS) prior to injection. Tumor-bear-ingmicewere treatedwith 400mg per 100mL and injected i.p. threetimes, 3 days apart. Because avelumab is a human IgG1, threeinjections had to be compressed within a 7- to 9-day window (i.e.,days 9, 12, and 15 after tumor inoculation) to avoid the onset ofneutralizing mouse anti-human Ig (data not shown).

BCG (1.0 mg; TICE, Organon USA, Inc.) was administered in avolume of 100 mL instilled by catheterization for 1 hour, threetimes at weekly intervals starting on day 8 after tumor instillation.

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Bioluminescent imagingOrthotopic bladder tumor growthwas detected by IVIS Lumina

(Caliper Life Sciences) In-vivo Imaging. The abdominal regions ofthemicewere shaved, and themicewere injected i.p.with luciferinsalt (15 mg/kg), 10 minutes before imaging. Temporary anesthe-sia was administered by Forane (Isoflurane; USP; Baxter) inhala-tion at 2% for the duration of imaging. The acquired biolumi-nescence signals in a region of interest over the tumor site wereread out as total flux radiance [photons/sec/cm2/steradian (sr)]using the Living Image software, Version 4.1 (Caliper LifeSciences).

In vitro T-cell functional assaysSpleens were isolated fromMB49 subcutaneous tumor-bearing

mice that had been treated with avelumab. Splenocytes werewashed; red blood cells were lysed with ACK buffer and pulsedfor 6 to 8 days with p15E peptide (10 mg/mL) in 10 mL ofcomplete medium, consisting of RPMI 1640 supplemented with10% heat-inactivated FBS, 2 mmol/L L-glutamine, 50 mmol/Lb-mercaptoethanol, 0.1 mmol/L nonessential amino acids, 15mmol/L Hepes buffer (pH 7.4), 1 mmol/L sodium pyruvate, andpenicillin/streptomycin (100 U/mL). Splenocytes were harvestedand used in either cytotoxic T-cell lysis (CTL) and/or cytokinerelease assays.

For the cytokine release assay, splenocytes were plated in 24-well plates at 0.5�106 cells/well alongwith 5�106 irradiated (20Gy) peptide-pulsed splenocytes from na€�ve syngeneic mice. Pep-tide was added at 10, 1, or 0.1 mg/mL. Peptides included p15E(KSPWFTTL, H-2Kb) or control HY peptide (WMHHNMDLI, H-2Db). Total volume within the wells was 2mL. Supernatants werecollected at 48 hours, and IFNg concentration was measured byELISA as per themanufacturer's protocol (Pierce Protein ResearchProducts).

For the CTL assay, splenocytes were plated in triplicate wells ofround-bottom, 96-well plates along with EL-4 cells that had beenlabeledwith Indium-111andpulsedwithp15Eor control peptideVSV-NP (RGYVYQGL, H-2Kb). The plates were incubated over-night, and a gamma counter was used to measure Indium-111release. Resulting cytotoxicity was calculated as percent specificlysis ¼ [(experimental cpm � spontaneous cpm)/(total cpm �spontaneous cpm)] � 100. Triplicates are shown as mean � SE.

Histology and immunohistochemistryFor pathologic evaluation, tissues (i.e., bladder, lungs, liver,

kidneys, etc.) were removed for gross examination. Bladder tissuewas fixed in 10%neutral buffered formalin, embedded in paraffinblocks, and processed by routine histologic methods for hema-toxylin and eosin (H&E) staining. The remaining bladder tissuewas embedded in optimal cutting temperature (OCT) compoundfor staining ofCD3 (cloneOKT3; AbD-Serotec), CD4 (cloneRM4-5; BD PharMingen), CD8a (clone 53-6.7; BD PharMingen), andPD-L1 (clone AF1019; R&D Systems) as well as H&E. It has beenreported that instillation of a higher number of MB49luc tumorcells (i.e., 106) into mouse bladders previously traumatized withsilver nitrate resulted in lung metastases (23). Upon examinationof the H&E-stained slides, a board-certified pathologist found theurinary bladder pathology to be "carcinoma, transitional cell,malignant without metastasis" with no accompanying metastaticlesions in the lungs. Stained slides were scanned using the LeicaBiosystems anatomic pathology scanner. The Aperio Image Scopeanalyses were completed using positive pixel count (v8.1), which

determined the total number of positive pixels (Ntotal�Nnegative¼TNP) for each corresponding graph. TNP values were comparedon an individual treatment group basis and analyzed with respectto region (bladder/tumor interface, tumor) and size (cells/micronsquared; i.e., TNP cells/mm2). The bladder/tumor interface wasdefined as 50 mm in either direction from the bladder/tumorborder. Higher magnification images were taken on a LeicaDMI4000 microscope with LAS AF (Leica Microsystems Inc.).

Flow cytometry analysisMB49 cells were seeded in T75 flasks in complete DMEM-

supplementedmedium and treated with 10 ng/mL IFNg 24 hourslater. After 48 hours, cells were harvested and stained for theexpression of PD-L1 using avelumab or isotype-control antibo-dies (0.2 mg/106 cells) with a PE-conjugated, goat anti-human IgGsecondary antibody (0.5 mg/106 cells; Jackson Immunoresearch).Data were acquired with a BD FACS Calibur flow cytometerconnected to a Macintosh computer running BD Cellquest soft-ware. Data analysis was conducted using FlowJo software(FLOWJO, LLC).

Statistical analysisGraphPad Prism software (GraphPad Prism 6.01 forWindows,

GraphPad Software, Inc.) was used to perform all statisticalanalyses. Details of appropriate statistical analyses are foundwithin each figure legend. Differences were significant when theP value was less than 0.05.

ResultsInteraction between avelumab and mouse PD-L1

Initial studies determined the extent to which avelumab, ahuman IgG1, interacts with mouse PD-L1. Consistent with thebinding affinity studies (see Materials and Methods), avelumab,not the human isotype-control antibody, bound to PD-L1 on thesurface of the MB49 cells, a murine transitional cell carcinoma ofthe bladder. Constitutive PD-L1 expression on the surface of theMB49 tumor cells was high [approximately 96% positive, meanfluorescence intensity (MFI) ¼ 9.5] and was easily upregulated(approximately 99% positive, MFI ¼ 59.5) with the in vitroaddition of 10 units IFNg/mL (Supplementary Fig. S2A). PD-L1blockade can increase T-cell activation (21). Indeed, the additionof avelumab to an in vitro bioassay of BMDCs and peptide-pulsedT cells from F5 TCRTgmice increased IFNg production, indicatingthe ability of avelumab to not only recognize PD-L1, but also tointerrupt its signaling (Supplementary Fig. S2B).

Antitumor effects of avelumabSyngeneic C57BL/6 mice bearing subcutaneous MB49 tumors

(40–60 mm3) were treated with avelumab, isotype-control anti-body, or diluent on days 9, 12, and 15 after tumor inoculation(Fig. 1A–C). Measurement of individual tumors clearly showed aslowing of tumor growth in the avelumab-treated mice (Fig. 1C).By day 36 after tumor implantation, there was a significant (P <0.01) reduction in the average tumor volume of the avelumab-treated mice (Fig. 1D). Reduction in MB49 tumor growth inthe mice treated with avelumab was durable and led to a statis-tically significant (P < 0.05) improvement in percent survival (Fig.1E). Splenocytes isolated from the control and avelumab-treatedmice were analyzed for the presence of T cells recognizingtwo possible tumor-associated antigens—the transmembrane

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component of the retroviral envelope protein p15E and the Yantigens presented by the MB49 tumor cells implanted intofemale hosts. A low, but measurable p15E CTL (Fig. 1F) andIFNg (Fig. 1F, inset) release was found following the in vitropeptide stimulation of splenocytes from avelumab-treated mice.In agreement with previous findings (24), despite the expressionof male minor histocompatibility (HY) antigens by the MB49tumor cells, no HY-specific response was found in the tumor-bearing, control-treated female mice. Interestingly, control-trea-ted mice also had a low, but measurable p15E-specific CTL(Supplementary Fig. S2C).

Targeting PD-L1 in an orthotopic murine modelPD-L1 levels were measured using immunohistochemical

staining in bladders of mice following MB49luc instillation (Sup-plementary Fig. S3). As expected, the intravesical instillationof theMB49luc cells resulted innon-muscle invasive tumor formationonthe mucosal surface of the bladder (Supplementary Fig. S3A).Immunohistochemical staining revealed strong PD-L1 stainingon the tumor cell surface with accompanying staining in thetransitional epithelium, lamina propria, and submucosa, whichmay indicate a broad proinflammatory response to MB49luc

tumor cell instillation (Supplementary Fig. S3B). For comparison,some weak PD-L1 staining was found in the transitional epithe-lium of bladders from na€�ve mice (Supplementary Fig. S3C).

In an initial study, bladders of mice were instilled withMB49luc tumor cells and after 8 days were grouped according tothe amount of tumor-associated bioluminescence (Fig. 2A). Bio-luminescence determined by intravital imaging is helpful inassessing early tumor burden (i.e., tumor take), and the amountof radiance (photons) can be used to select mice for each treat-ment group based on equivalent tumor burden (22). Indeed, onday 8 following MB49luc instillation, tumor burden, based onimaging and radiance, was confirmed, andmicewere divided intotwo treatment groups: i.p. administration of avelumab or theisotype-control IgG (Fig. 2A). Based on ongoing clinical studiesusing 10 to 20mg/kg, 400mg (20mg/kg) avelumabor the isotype-control antibody was administered. Antibody treatments werecarried out on days 9, 12, and 15 after tumor instillation. Whenthe mice were imaged on day 21, a clear reduction in the biolu-minescence signal (i.e., radiance) was present in the bladders ofmice treated with avelumab (Fig. 2B). On day 29 after tumorinstillation, mice were euthanized and individual tumor burdenswere determined by bladder weights of the two treatment groupsand compared with that from na€�ve, non–tumor-bearing mice(average bladder weight: 22.0 � 1.7 mg; Fig. 2C). Mice treatedwith the isotype-control antibody had considerable tumor bur-den in that the average bladder weight was more than 7-fold thatof thena€�ve, non–tumor-bearingmice (161.3�56.1 vs. 22.0�1.7mg). Avelumab treatment significantly (P < 0.05) inhibited the

Figure 1.PD-L1 as a target for avelumab treatment in C57BL/6 mice bearing MB49 subcutaneous tumors. A–C, growth of individual s.c. MB49 tumors in PBS-treated (A),human Ig isotype-control (B), and avelumab-treated (C) C57BL/6 mice. Arrows indicate treatment days, and n indicates number of tumor-free mice.Data are representative of two independent experiments. D, average tumor volumes from C57BL/6 mice (n ¼ 10) bearing s.c. MB49 tumors shown in A–C. Micewere treated with PBS (solid circles), human Ig isotype control (open squares), or avelumab (3 � 400 mg; solid triangles) on days 9, 12, and 15 (arrows).�� , P < 0.01 (one-way ANOVA with Tukey test) versus untreated and human Ig control–treated groups. E, survival curves for the mice described in D. � , P < 0.05(log-rank test) avelumab-treated (solid triangles) versus isotype Ig control–treated group (open squares). F, splenocytes were isolated from 3 mice in whichavelumab treatment significantly reduced MB49 s.c. tumor growth in C. Splenocytes were stimulated in vitro in the presence of 10 mg/mL of either p15E(circles), HY (triangles), or VSV, a control peptide (squares). CTL killing was assayed as outlined inMaterials andMethods. Data are from a representative experimentthat was repeated with similar results. Inset, IFNg production by peptide pulsed splenocytes measured as described in Materials and Methods. Solid andopen bars represent the amount of IFNg generated by p15E and HY-stimulated splenocytes, respectively, over a 3-day incubation period. Error bars in D andF indicate SEM.






growth of MB49luc bladder tumors: the average bladder weight inthat treatment group, 34.1 � 4.5 mg, was not different than thebladder weights from na€�ve, untreated mice (Fig. 2C). In aseparate study, the reduction of MB49luc bladder tumor burdenwith avelumab administration also significantly (P < 0.01)improved percent survival when compared with isotype-con-trol–treated mice (Fig. 2D).

Because the current standard of care for carcinoma in situ andnon-muscle invasive nonmetastatic urothelial carcinoma is theintravesical instillation of BCG, it was important to comparesystemic avelumab versus BCG therapy in mice bearing orthoto-pic MB49luc bladder tumors. Once again, tumor burden wasassessed by changes in the amount of bioluminescence andoverall increase in individual bladder weights. By day 21 aftertumor instillation, there was a decrease in intravesical biolumi-nescence in the groups ofmice treated with BCG alone, avelumabalone, or avelumab combined with BCG (Fig. 3A). On day 29,

bladders taken from control-treated [i.e., isotype-control,i.p./saline intravesical (i.ves.)] mice instilled with the MB49luc

tumor cells had developed tumor burden in that their overallbladder weights were approximately 10-fold that of na€�ve,untreated B6 mice (225.6 � 79.1 vs. 22.0 � 1.7 mg; Fig. 3B).Bladder instillation of BCG (1.0 mg) on days 7, 14, and 22 aftertumor instillation reduced tumor growth (average bladderweight, 61.6� 13.6mg), whichwas somewhat surprising becausein previous studies, intravesical BCG instillation had little to noeffect on bladder tumor burden (22). Such variability could be aconsequence of leakage/voiding following intravesical BCG dos-ing and treatment duration as well as lot-to-lot differences in BCGbioactivity. In contrast, the antitumor effects of systemicallyadministered avelumabwere highly reproducible with an averagebladderweight just 25%higher than that ofna€�vemice (27.6�2.4vs. 22.0 � 1.7 mg, NS; Fig. 3B). The combination of systemicavelumab plus intravesical BCG instillation produced no

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additional reduction in tumor burden over that of avelumabalone (average bladder weight 30.6 � 1.8; Fig. 3B). Avelumabtherapywas also better tolerated as no overt toxicity was observed,whereas mice receiving BCG either alone or in combination oftenexhibited hunched habitus or ruffled fur, changes that could beregarded as transient grade 1/2 toxicities.

Immune components of avelumab therapyBladders frommiceof the four treatment groups shown inFig. 3

were removed, OCT-embedded, and analyzed for the presence ofCD4 and CD8 immune cell infiltrates at the bladder/tumorinterface and within the tumor mass based on immunohisto-chemical staining (Fig. 4; Supplementary Fig. S4 for higher mag-nification). For the control mice treated with the isotype-controlantibody (i.p.) and saline (i.ves.) 29 days after tumor installation,the normal bladder architecture was often severely disrupted,which made identification of the bladder/tumor interface diffi-cult. Nevertheless, in those evaluable isotype-control–treatedmice, there was a discernable gradient of CD4 T cells betweenthe bladder and tumor with appreciable accumulation at thebladder/tumor interface (Fig. 4A). No such gradient was foundfor the CD8 T cells, which were equally distributed betweenbladder/tumor interface and the tumor mass (Fig. 4B and C).Although intravesical BCG treatment did reduce tumor burden,all mice had enlarged bladders at the study endpoint, suggesting aweak ongoing antitumor response. Indeed, relatively low num-bers of CD4 and CD8 T cells were found either at the bladder/tumor interface or within the tumor mass (Fig. 4D–F). Avelumabtreatment of 10 mice with bladder tumors resulted in completetumor regression in 8 mice, confirmed by histopathology. In theremaining 2mice, small tumors remained, possibly indicating anactive antitumor response with increased numbers of CD4 andCD8 T-cell infiltrates located both at the bladder/tumor interface

and within the tumor mass (Fig. 4G–I). In mice treated with acombination of avelumab and intravesical BCG, increased num-bers of CD4 and CD8 T cells were found within the tumor mass(Fig. 4J–L). It should be noted that these analyses were done at thestudy end, and results were significantly affected by differenttumor burdens among the treatment groups. In future studies,the immune cell changes within the bladder, bladder/tumorinterface, and the tumor mass will be determined during a timeinterval encompassing an active, ongoing antitumor immuneresponse. Staining for PD-L1 expression revealed the presence ofhigh PD-L1 expression levels in the bladder tumor masses wheth-er or not they were being treated with avelumab (Fig. 5A–D).Interestingly, PD-L1 expression was lower in the bladders and atthe bladder/tumor interface in those mice in which avelumabtreatment successfully reduced tumor growth (Fig. 5C).

In a separate experiment, immune cell subsets were depletedusing specific antibodies for CD4, CD8, or NK cells prior toavelumab administration to mice bearing orthotopic bladdertumors. As shown in Fig. 6A, avelumab was highly effective inreducing the growth of bladder tumors in immune intact mice.However, in mice depleted of either CD4 or CD8 cells, avelumabtreatment was much less effective in controlling bladder tumorburden with tumor breakthrough occurring in a higher frequencyin mice depleted of CD4 T cells. One can surmise that CD4depletion would impede CD8 priming due to the lack ofT-helper (Th1) cytokines. But the fact that some antitumorefficacy of avelumab was maintained in those mice depleted ofCD8T cells suggests a role forCD4T cells in tumor growth control.These data were consistent with the immunohistochemical stain-ing showing intratumoral localization of CD4 and CD8 T cells inmice treated with avelumab (Fig. 4G and H). Avelumabmediatesantibody-dependent cell-mediated cytotoxicity in humans, butnot in mice (P. Kim, Laboratory of Tumor Immunology and

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Figure 3.Comparison of the antitumor effects of avelumab, BCG alone, or avelumab plus BCG. A, bioluminescence images and total flux radiance measurements of MB49luc

bladder tumors on day 21 after tumor instillation. C57BL/6 mice (n ¼ 10) bearing orthotopic MB49luc tumors were treated with DPBS/saline, DPBS/BCG (1.0 mg),avelumab (3 � 400 mg)/saline, or avelumab/BCG. BCG was administered intravesically on days 8, 15, and 22, whereas avelumab was injected i.p. ondays8, 11, and 14 after tumor instillation. B, allmicewere euthanized onday29, and individual bladderswereweighed to assess tumorburden. Solid black lines indicatethe mean bladder weights in each group. � , P < 0.05, avelumab/saline versus DPBS/BCG; �� , P < 0.01, DPBS/saline versus avelumab/BCG; �� , P < 0.0001,DPBS/saline versus avelumab/saline (Kruskal–Wallis test; Dunn test). Data are from a representative experiment that was done three times with similarresults. NS, not statistically significant.






Biology, Center for Cancer Research, NCI; personal communica-tion). Consistent with that findingwas that NK-cell depletion hadlittle or no adverse effects on the ability of avelumab to induce asignificant reduction in the growth of orthotopic bladder tumors.

It was of interest to determine whether successful treatment ofbladder tumors by avelumab also induced protective, long-termimmunememory. In a separate study, 17 of 38mice instilledwithMB49luc tumor cells and subsequently administered avelumab

had a complete resolution of their bladder tumors (based onimaging). Those 17mice remained tumor free for 36 days after theinitial tumor instillation, at which time 10 mice received anintravesical rechallenge with 7.5 � 104 MB49 tumor cells. At thesame time, a group of na€�ve mice received MB49 tumor cells viabladder instillation, and all developed large bladder tumors byday 29 (Fig. 6B). However, themice that were rendered tumor freeafter avelumab treatment were protected against the tumor

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Figure 4.Immunohistochemical detection of CD4 and CD8 immune cell subsets in the bladders of mice treated with avelumab, BCG, or avelumab plus BCG. C57BL/6 mice(n ¼ 10) bearing orthotopic MB49luc tumors were treated with DPBS/saline, DPBS/BCG (1.0 mg), avelumab (3 � 400 mg)/saline, or avelumab/BCG, asdescribed in Fig. 3. Mice were euthanized on day 29, and individual bladders from DPBS/saline (A, B), DPBS/BCG (D, E), avelumab/saline (G, H), and avelumab/BCG(J, K) were stained for CD4 (A, D, G, J) and CD8 (B, E, H, K) T cells as described in Materials and Methods. Bar ¼ 600 mm. Plots C, F, I, and L represent thenumber of CD4 and CD8 T cells enumerated at the bladder/tumor interface and within the tumor mass as described in Materials and Methods. Data are from arepresentative experiment that was repeated with similar results. T, tumor.

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rechallenge (Fig. 6B), suggesting that avelumab treatment gener-ated a strong protective immunologic memory response againstthe MB49 tumors.

DiscussionBladder cancer, the fifth most common cancer in the United

States, has a high prevalence, along with melanoma and lungcancer, for somaticmutations, presumably due toDNAdamage asa result of long-term exposure to extrinsic mutagens, such assmoking and carcinogenic chemicals (25, 26). Coincident withthose mutations, investigators have found a large number oftumor-infiltrating lymphocytes in bladder cancer (27–29).Indeed, immune-directed therapy utilizing intravesical BCGinstillation has been the standard of care for intravesical therapyfor carcinoma in situ and non-muscle invasive, nonmetastaticurothelial carcinoma for the past four decades (16–19). RepeatedBCG instillations have led to early accumulation of granulocytesfollowed bymacrophages and lymphocytes, predominantly CD4T cells, in the patients' bladders (30, 31). Investigators have shownthat BCG treatment induces a strong Th1 cytokine response, yetthe ability of BCG to mediate antitumor effects has been largelyattributed to NK-cell activation (32, 33). In the present experi-mental murine model, some of the characteristics found inpatients diagnosed with carcinoma in situ and non-muscle inva-sive, nonmetastatic urothelial carcinoma were recapitulated: (i)MB49luc tumor cell instillation resulted in a large number ofinfiltrating lymphocytes in the bladder, at the bladder/tumorinterface, and within the tumor mass. (ii) Mice treated with BCGhadmoreCD4 andCD8T cells at the bladder/tumor interface and

intratumorally, yet the potency of BCG to affect tumor reductionwas minimal. Those findings present multiple possibilities toexplain the limitations of BCG immunotherapy: (i) The immunecell infiltrate is broad-based, the tumor-associated antigen-spe-cific component is a minor component. (ii) There is an antigen-specific antitumor T-cell response, but it is dampened by PD-1/PD-L1 upregulation in response to release of high concentrationsof Th1 cytokines (i.e., IFNg) by NK cells in the tumor microen-vironment. The latter would explain the association between PD-1/PD-L1 overexpression with tumor grade and postoperativeprognosis in human bladder cancers (28). It would also explainwhy antibody blockade of the PD-1/PD-L1 immunosuppressivepathway would benefit patients diagnosed with metastatic blad-der cancer by releasing resident tumor-specific host immune cells,thus controlling tumor growth (34). Findings in the present studysupport the argument that this same approach, PD-1/PD-L1disengagement, might benefit patients diagnosed with carcinomain situ and non-muscle invasive, nonmetastatic urothelial carci-noma who either failed or relapsed following frontline BCGtherapy.

Murine MB49 bladder tumor cells are constitutively highlypositive for PD-L1, whichwas further upregulated following IFNgtreatment in vitro. High PD-L1 expression was maintained in vivofollowing the orthotopic instillation of MB49 cells that formedmultifocal non-muscle invasive tumors on the mucosal bladderepithelium. Systemic administration of avelumab, an antibody toPD-L1, significantly (P < 0.05) reduced tumor burden, resulting ina statistically significant (P < 0.01), durable improvement in long-term survival. Mechanistic studies clearly showed the need for anintact immune system, with depletion of either CD4 or CD8 T

T

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Figure 5.Immunohistochemical detection of PD-L1 in thebladders of mice bearing tumors and treated withPBS (A), BCG (B), avelumab (C), or avelumab plusBCG (D). Bar¼ 50mm,magnification,�100. T, tumor.






cells abrogating the antitumor effects of avelumab treatment.Depletion of NK cells had no adverse effect on the antitumorproperties of avelumab. Moreover, avelumab therapy alsoinduced strong T-cellmemory responses that protectedmice fromsubsequent tumor challenge.

A plausible working hypothesis is that intravesical or s.c.inoculation of MB49 tumor cells is accompanied by sufficienttumor cell disruption, resulting in the release of tumor-associatedantigens inducing a host immune response. Two possible tumor-associated antigens are the envelope protein p15E antigen asso-ciatedwith the active component of endogenous retroviruses and/or immune recognition of theHY antigens presented by theMB49tumor cells implanted into female hosts. Release of either/both ofthose tumor-associated antigens would provide appropriate sig-nals that support the influx of immune cells seen in the bladder, atthe bladder/tumor interface, and within the tumor mass ofuntreated mice. Although a low, but measurable splenic CTLactivity directed against the p15E epitope was found in untreatedmice, it had very little effect in impeding tumor growth. In eitherMB49 model, the presence of antitumor T cells in the tumormicroenvironment of untreated and isotype Ig control–treated

mice provided some short-term control on tumor growth. By day20 (s.c. model), immunosuppressive actions in the tumor micro-environment overrode any immune control, resulting in expo-nential tumor growth. The PD-1/PD-L1 axis seems to be a potentcomponent and contributor to local immunosuppression, asblockade of that axis by avelumab suppressed both subcutaneousand orthotopic MB49 tumor growth, leading to increased overallsurvival. These observations argue that the disruptionof the PD-1/PD-L1 immunosuppressive pathway is followed by expansion ofantigen-specific T cells and control over tumor growth. Supportfor that hypothesis comes from the increase in p15E-specificcytolysis in avelumab-treated mice, the loss of avelumab antitu-mor potency commensurate with either CD4 or CD8 T-celldepletions, and the generation of immunologic memory thatprotects tumor-free mice from subsequent tumor challenge.

The potent antitumor effects reported in the present studycoupled with those from early clinical trials in patients diagnosedwith metastatic bladder cancer have generated intense interest indeveloping a new paradigm in the immune-based treatment ofbladder cancer (13–15). Although the PD-1/PD-L1 axis seems tobe a crucial target for the treatment of metastatic bladder cancer,

Figure 6.Adaptive immunity mediates the antitumor effects of avelumab treatment of intravesical MB49luc bladder tumors. A, groups of C57BL/6 mice were treated ondays 1–4 and 6 with antibodies to deplete CD4 and CD8, or on days 1 and 6 to deplete NK cells. Weekly antibody injections maintained the depleted state foreach immune cell subset. Micewere instilledwith MB49luc tumor cells on day 4 and treated on days 8, 11, and 14with 400 mg of either the control human Ig (circles) oravelumab alone (squares). Avelumab was also administered to mice in which CD4 (triangles), CD8 (inverted triangles), or NK (diamonds) were depleted asdescribed above.Micewere euthanized on day29, and individual bladderweightsweremeasured for tumor burden. Solid lines indicate themean bladderweights foreach group. ��, P <0.001 (Kruskal–Wallis test, Dunn test); CD4-depleted groups versus intactmice. NS, not statistically significant. B, C57BL/6mice (n¼ 38) bearingorthotopic tumors were treated with avelumab, which resulted in complete tumor eradication in 17 mice (based on imaging) by day 32. On day 36, those "immune"mice that were cured with avelumab immunotherapy (based on bioluminescence) were rechallenged orthotopically with the original dose of MB49 cells(inverted triangles). Challenge with wild-type MB49 cells allowed the differentiation on whether any tumor breakout was from the primary MB49luc tumor cells(detected via imaging) or from the wild-type MB49 challenge. Age-matched na€�ve mice (triangles) were challenged using the same conditions. All mice wereeuthanized on day 29 after tumor instillation, and individual bladders were weighed to assess tumor burden. �� , P < 0.0001 (t test, Mann–Whitney test);na€�ve versus immune mice.

Vandeveer et al.





others have suggested that a focus on the immune-related eventsthat occur prior to the spontaneous initiation of a tumor-specificresponse would be worthwhile in understanding and designingnovel immune adjuvants to expand the immunotherapeuticapproach (35). There still remains an intriguing dichotomy withrespect to PD-L1 tumor expression. Although its expression isrelated to tumor escape via immune suppression, the targeting ofthose tumors with the highest expression of PD-L1 levels seems tocorrelate with better antitumor responses. Perhaps themost directexplanation is that higher PD-L1 expression is related to moreimmune cell accumulation in the tumor microenvironment.These phenomena might be tied to the innate immune systemand the events that trigger spontaneous generation of resident Tcells. Because the innate immune system is closely tied to proin-flammatory events, it would be interesting to examine the relativeamount of local tissue inflammatory responses when tumor cellsare instilled into the bladder. It is conceivable that a more robustinflammatory response generated at the mucosal surface of thebladder may facilitate T-cell priming, leading to a more potentantitumor response to subsequent anti–PD-L1 therapy. Indeed, asshown in Supplementary Fig. S3B, simple intravesical instillationof the MB49luccells seems to upregulate PD-L1 expression levelswithin the different mucosal and submucosal layers of thebladder.

The orthotopic intravesical murine bladder tumor model hasseveral characteristics, such as non-muscle invasive bladder tumorgrowth and responsiveness to anti–PD-L1 immune-based thera-pies, which argue for it to be suitable as an experimental model tostudy immune-related factors involved in non-muscle invasive,nonmetastatic bladder cancer. Use of the model might provideimportant insights into the cellular mechanisms involved inthe nonresponders to immune cell checkpoint immunotherapy,identification of effective immune-based combinations thatwould be expected to provide a more robust antitumor responsethan with immune cell checkpoint inhibitor monotherapy, andthe PD-L1 function within a tumor microenvironment devoid ofT cells. Such preclinical studies may provide the rationale for

localized bladder cancer clinical studies that combine immunecell checkpoint inhibitors with other forms of immunotherapy,chemotherapy, radiotherapy, and small-molecule inhibitors.

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

Authors' ContributionsConception and design: A.J. Vandeveer, J.K. Fallon, H. Sabzevari, J. Schlom,J.W. GreinerDevelopment of methodology: A.J. Vandeveer, J.K. Fallon, R. Tighe,J.W. GreinerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.J. Vandeveer, J.K. Fallon, J.W. GreinerAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A.J. Vandeveer, J.K. Fallon, J. Schlom, J.W. GreinerWriting, review, and/or revision of themanuscript: A.J. Vandeveer, J.K. Fallon,R. Tighe, H. Sabzevari, J. Schlom, J.W. GreinerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J.W. GreinerStudy supervision: J. Schlom, J.W. Greiner

AcknowledgmentsThe authors thank Garland Davis, Bertina Gibbs, LaJuan Chase, and Curtis

Randolph for their superior technical assistance, Dr. R. Ingrid Fernando forassistance with cell line transfections, Dr. Alfredo Molinolo for the use of slideimaging equipment, andDr.MiriamAnver for scoring and interpretation of IHCresults. They also thank Debra Weingarten for her expert editorial assistance.

Grant SupportThis research was supported by the Intramural Research Program of the

Center for Cancer Research, NCI, NIH (ZIA BC 010970), as well as through aCooperative Research and Development Agreement (CRADA) between EMDSerono and the NCI.

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

Received July 15, 2015; revised December 28, 2015; accepted January 19,2016; published OnlineFirst February 26, 2016.

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2016;4:452-462. Published OnlineFirst February 26, 2016.Cancer Immunol Res Amanda J. Vandeveer, Jonathan K. Fallon, Robert Tighe, et al. Inhibitor

PD-L1 Immune Checkpoint−Cancer with Avelumab, an Anti Systemic Immunotherapy of Non-Muscle Invasive Mouse Bladder

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