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Tumor Biology and Immunology

Targeting CCR8 Induces Protective AntitumorImmunity and Enhances Vaccine-InducedResponses in Colon CancerDaniel O. Villarreal, Andrew L'Huillier, Susan Armington, Cristina Mottershead,Elena V. Filippova, Brandon D. Coder, Robert G. Petit, and Michael F. Princiotta

Abstract

CCR8 is a chemokine receptor expressed principally onregulatory T cells (Treg) and is known to be critical for CCR8þ

Treg-mediated immunosuppression. Recent studies havedemonstrated that CCR8 is uniquely upregulated in humantumor-resident Tregs of patients with breast, colon, and lungcancer when compared with normal tissue-resident Tregs.Therefore, CCR8þ tumor-resident Tregs are rational targets forcancer immunotherapy. Here, we demonstrate that mAb ther-apy targeting CCR8 significantly suppresses tumor growth andimproves long-term survival in colorectal tumor mouse mod-els. This antitumor activity correlated with increased tumor-specific T cells, enhanced infiltration of CD4þ and CD8þ

T cells, and a significant decrease in the frequency of tumor-resident CD4þCCR8þ Tregs. Tumor-specific CD8þ T cellsdisplayed lower expression of exhaustion markers as well asincreased functionality upon restimulation. Treatment with

anti-CCR8 mAb prevented de novo induction and suppressivefunction of Tregs without affecting CD8þ T cells. Initialstudies explored a combinatorial regimen using anti-CCR8mAb therapy and a Listeria monocytogenes–based immunother-apy. Anti-CCR8mAb therapy synergizedwith L. monocytogenes–based immunotherapy to significantly delay growth of estab-lished tumors and to prolong survival. Collectively, thesefindings identify CCR8 as a promising new target for tumorimmunotherapy and provide a strong rationale for furtherdevelopment of this approach, either as a monotherapy or incombination with other immunotherapies.

Significance: Inhibition of CCR8 represents a promisingnew cancer immunotherapy strategy that modulates tumor-resident regulatory T cells to enhance antitumor immunity andprolong patient survival. Cancer Res; 78(18); 5340–8.�2018AACR.

IntroductionTumor-infiltrating CD4þFoxp3þ regulatory T cells (Treg) are a

major immune cell population that contribute to the establish-ment of an immunosuppressive tumor microenvironment (TME;refs. 1–4). Infiltration of large numbers of Tregs into the tumorshamper the development of effective antitumor immunity (1–4)and is often associated with poor prognosis (2–5). Treg modu-lation strategies have been shown to increase antitumor immu-nity and reduce tumor burden in both preclinical and clinicalsettings (4–7). However, although these strategies have demon-strated enhanced antitumor immune responses, there are draw-backs, such as autoimmunity and specificity of targeting (2–4,8–13). Because Tregs and activated effector lymphocytes bothexpress surface molecules that can be used as therapeutictargets (e.g., anti-CD25), there is the potential for ablation ofessential tumor-specific effector cells required to control tumorprogression in these types of antibody-mediated immunothera-pies (13, 14). Therefore, the development of a more effective

approach to specifically and selectively target tumor-infiltratingTregs is required.

In both humans and mice, the chemokine receptor CCR8 ispredominantly expressed on Tregs and on a small portionof Th2 cells, but not on Th1 cells (15, 16). This subset ofCD4þFoxp3þ Tregs expressing CCR8 (CCR8þ Tregs) has beendemonstrated to be a major driver of immunosuppression andis critical for Treg function and suppression (15–17). Morerecently, two independent studies characterizing the distinctmolecular signature of tumor-resident Tregs demonstratedthat CCR8 was a specific marker selectively upregulated bytumor-resident Tregs in several tumor types (18, 19). Inter-estingly, Plitas and colleagues highlighted that high expressionof CCR8þ Tregs was associated with poor prognosis in patientswith breast cancer (19). These studies suggest CCR8 may bean effective therapeutic target by which to selectively andspecifically modulate a subpopulation of tumor-resident Tregsin the TME to augment antitumor immunity. The extentof antitumor effects resultant from targeting CCR8 and itspotential as a promising cancer immunotherapy remains tobe determined.

Here, we demonstrate that an anti-CCR8 (aCCR8)–blockingmAb treatment impaired the suppressive character of the TME,markedly reducing tumor-resident CCR8þ Tregs. Ultimately,this contribution enhanced effector T-cell and antitumor immu-nity in the CT26 colorectal tumor model. Furthermore, ourstudy demonstrates that aCCR8 therapy synergizes with a Listeriamonocytogenes (Lm)–based immunotherapy to enhance optimalantitumor efficacy. Collectively, these novel findings highlight

Advaxis Immunotherapies, Princeton, New Jersey.

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

Corresponding Author: Daniel O. Villarreal, Advaxis, Princeton, NJ 08540.Phone: 609-423-2502; Fax: 215-540-4750; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-18-1119

�2018 American Association for Cancer Research.

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CCR8as apromisingnew target for cancer immunotherapy and itspotential for broad clinical application.

Materials and MethodsMice and tumor cell lines

Female, 6- to 8-week-old BALB/c and C57BL/6 (B6) mice werepurchased from The Jackson Laboratory. All mouse procedureswere performed in accordance with protocols approved byAdvaxis Immunotherapies IACUC. CT26.WT cells (CRL-2638)were purchased from the ATCC and authenticated by ATCC usingCOI analysis. The MC38 colon carcinoma cells were purchasedfrom Kerafast and were authenticated by simple sequence lengthpolymorphism. CT26 cells were maintained in RPMI mediumsupplemented with 10% FBS in a humidified atmosphere with5% CO2 at 37�C. The MC38 cell lines were maintained in DMEMmedium supplementedwith 10%FBS. All cell lineswere passagedtwice prior to storage and thawed and passaged twice prior toimplantation for all described tumor experiments. All cell lineswere determined to be free of Mycoplasma (Sigma-Aldrich).

Tumor models, tumor vaccine, and aCCR8 treatmentCT26 (3 � 105) and MC38 (3 � 105) cells were implanted

subcutaneously in the right flank of mice. Tumor vaccineconsisted of LmddA-274 (1 � 108) or AH1-21mer (1 � 108)diluted in PBS. Details of plasmid construction are described inthe Supplementary Materials and Methods. For therapeutictreatments, mice were treated with intraperitoneal injectionsof purified aCCR8 (4 mg; clone: SA214G2; BioLegend) andcontrol antibody (rat IgG2b,k, Clone RTK4530; BioLegend).Tumor growth was monitored using electronic calipers andcalculated according to the formula: V ¼ (length x width2)/2.Mice were immunized with 200 mL of vaccine intravenous onday þ12 and þ19 after tumor implantation. To analyze tumor-infiltrating T cells upon vaccine/anti-CCR8 combination treat-ment, tumors from all groups were harvested for analysis þ22days after tumor implantation. For survival experiments, micewere euthanized when tumor size reached 2,000 mm3 or whentumors become necrotic.

In vitro assaysTreg induction. Na€�ve 2 � 106 CD4þ T cells were isolated fromspleens of BALB/c mice through negative selection using theStemCell EasySepMouseNa€�ve CD4þ TCell Isolation Kit (catalogno. 19765) according to themanufacturer's instructions. The cellswere seeded on plates pretreated with 2 mg/mL aCD3. Cells wereincubated for 3 to 5 dayswithaCD28 (1mg/mL), IL2 (100U/mL),and TGFb1 at 5 ng/mL. The percent of converted Tregs wasevaluated by flow cytometry on the third day. For inhibition ofconversion, aCCR8 was added at 10 mg/mL.

Proliferation of CD8þ and CD4þ T cells from aCCR8-treated mice.A total of 2 � 106 CD8þ and CD4þ T cells were isolated fromspleens of BALB/c mice through negative selection using theStemCell EasySep Mouse CD8þ or Na€�ve CD4þ T Cell IsolationKits (catalog no. 19853; catalog no. 19765), respectively. Tomeasure proliferation, both CD8þ and CD4þ T cells were stainedwith carboxyfluorescein diacetate succinimidyl ester (CFSE) at aconcentration of 0.5 mmol/L for 10 minutes. Cells were thenquenched with RPMI medium supplemented with 10% FBS andwashed twice. The cells were seeded on pretreated aCD3 platesand incubated at 37�C for 3 days with aCD28 (1 mg/mL) and IL2

(100U/mL) and in the presence or absence ofaCCR8 (10 mg/mL)and TGFb (5 ng/mL).

Suppression assay. A total of 1 � 106 five-day culturally inducedTregs were incubated with freshly isolated CD8þ T cells (1� 106)in a 1:1 ratio on plates pretreated with 2 mg/mL of aCD3. Thefreshly isolated CD8þ T cells were stained with 1 mmol/L CFSEfor 10 minutes, quenched with RPMI medium supplementedwith 10% FBS, and washed prior to being plated. Coculturedcells were incubated for 3 days with aCD28 (1 mg/mL), IL2(100 U/mL), and indicated samples were treated with 10 mg ofaCCR8. Cells were then harvested and stained with viability dyefor 3 minutes, washed, and stained for CD3, CD4, and CD8 for30 minutes at 4�C. Cells were then washed and analyzed by flowcytometry. Proliferation was assessed on live CD3þCD8þCD4� Tcells only.

All in vitro assays are representative experiments of inde-pendent triplicate determinations.

Statistical analysisStatistical significance was determined either by unpaired

Student t test (two-tailed) or two-way ANOVA. For tumor survivalanalysis, Kaplan–Meier test was used. Error bars indicate SEM.All graphs and statistical analyses were generated using Prism 6software (GraphPad Software, Inc). All data are representative of2 to 3 independent experiments.

ResultsCCR8 mAb treatment impairs tumor growth in solid tumormodels

Considering that CCR8 is highly expressed on human colorec-tal tumor-resident Tregs (18, 19) and is principally expressed byTregs as part of their immunosuppressive arsenal (15–17), wefirstlooked at CCR8 expression on Tregs in CT26 tumor-bearingmice.We found that CCR8 was highly expressed mainly on tumor-infiltrating Tregs, with very little expression on splenic Tregs(Fig 1A), but is not expressed on CT26 tumor cells (data notshown). This suggests that CCR8þFoxp3þ Tregs within the TMEplay a role in suppressing tumor-specific immunity. Therefore,given that CCR8þ Tregs have been suggested to be potent driversof immunosuppression (17), we hypothesized that targetingCCR8þ Tregs with an aCCR8-blocking mAb would enhanceantitumor immunity. We examined the impact of CCR8 mAbtreatment in the CT26 colorectal tumor model. CT26 tumorcells were implanted into groups of na€�ve recipient BALB/c mice(n ¼ 10/group). Four days after tumor implantation, aCCR8 orIgG control therapy was initiated and given daily for a total of 8days as outlined in Fig. 1B. Interestingly, we found that treatmentwith aCCR8 generated significant antitumor activity andimproved long-term survival (P < 0.0001; Fig. 1B). Tumor growthcurves show that aCCR8 treatment markedly delayed tumorgrowth over control groups. To verify these results in a differenttumor model, we performed a similar experiment using theaCCR8 mAb therapy in the MC38 colon tumor model (Supple-mentary Fig. S1). We implanted C57BL/6mice withMC38 tumorcells, and aCCR8 or IgG therapy was administered from day 4 today 13 (8 days total) after tumor implantation (SupplementaryFig. S1A). Similarly, we found that treatmentwithaCCR8 showedsignificant MC38 tumor growth inhibition and prolonged sur-vival (Supplementary Fig. S1B). Collectively, these results show

CCR8 Is a Novel Target for Cancer Immunotherapy

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that blocking CCR8 can result in reduced tumor burden andimprove long-term survival in tumor-bearing animals.

CCR8 mAb therapy induces robust antigen-specifictumor-infiltrating CD8þ T cells

Our observation that aCCR8 treatment enhances antitumorimmunity prompted us to next examine the antigen (Ag)-specificCD8þ T responses systemically and in the tumors of aCCR8-treated tumor-bearing mice. For the immune analysis in tumor-bearing mice, total leukocytes were isolated from the spleens andtumors 17 days after tumor implantation. Splenocyte culturesfrom tumor-bearing mice treated with aCCR8 generated a robustsplenic Ag-specific IFNg ELISpot response when stimulated withthe classical MHC class I–restricted AH1 peptide used to monitorCD8þ T-cell responses (Fig. 2A). By direct staining with an AH1H2-Ld-restricted tetramer, aCCR8-treated mice significantlyamplified the frequency of AH1 tetramer-specific CD8þ T-cellresponses in the spleen (Fig. 2B). Similarly, aCCR8 therapyincreased the frequency of AH1 tetramer-specific CD8þ T cellsinfiltrating into the tumors, indicating trafficking of target effectorT cells to the site of malignancy and initiating effector function(Fig. 2C; ref. 20). CCR8 mAb therapy significantly increased thefrequency of IFNg-positive effector CD8þ T cells within the tumorcompared with the control group (Fig. 2D). Furthermore, asimilar trend was seen with the frequency of T cells secreting

IFNg when stimulated with PMA/ION, indicating that treatmentwith CCR8 mAb induces more functional CD4þ and CD8þ T-cellresponses overall (Fig. 2E). Finally, we observed that treatmentwithCCR8mAb significantly increased the level of polyfunctionaleffector CD8þ T cells coexpressing CD107a/IFNg within thetumor (Fig. 2E), indicating that these tumor-infiltrating T cellscomprised an effector CD8þ T-cell population with a cytolyticphenotype. These results demonstrate the ability of CCR8 mAbtherapy to alter the suppressive signature of the TME and promotetumor-specific effector T-cell function, which likely contributed tosignificantly delayed tumor growth.

CCR8 mAb therapy reprograms the tumor immune milieuTobetter define themechanismof action,wenext examined the

changes in the CT26 TME elicited by aCCR8 therapy. CCR8 mAbtreatment altered the tumor-infiltrating lymphocyte (TIL) com-position in the CT26 model. We observed that aCCR8 adminis-tration increased total CD45þ TILs compared with the controltreatment group (Fig. 3A). As expected, within the CD45þ TILpopulation, aCCR8 treatment profoundly increased the percent-age of tumor-infiltrating CD8þ and CD4þ T cells (Fig. 3A).Interestingly, both tumor-infiltrating CD8þ and CD4þ T cellsdisplayed significantly lower expression of PD-1 and CTLA-4exhaustion markers (Figs. 3B and C). This suggests that aCCR8therapy may help retain functional tumor-reactive effector T cells

Figure 1.

CCR8 mAb treatment mediates antitumor activity, suppressing CT26 tumor growth and improving long-term survival. A, Cells obtained at day 17postinjection from tumors and spleens. Representative density plots and graphs show frequency of CCR8þFoxp3þ cells gated on CD45þCD3þCD4þ Foxp3þ T cells.B, Data illustrate treatment regimen, group tumor measurements, and survival of CT26-implanted mice following treatment as indicated. �� , P < 0.0001.

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by limiting T-cell exhaustion. Furthermore, although not signif-icant, aCCR8mAb treatment slightly decreased the percentage ofG-MDSCs (CD11bþLyGC�Ly6Gþ) in the tumor (Fig. 3D).

Given that CCR8 is principally expressed on Tregs (17–19), wenext examinedwhetheraCCR8mAb treatment altered the tumor-resident Treg population and depleted the CCR8þ Treg targetpopulation. As expected, aCCR8 blockade could significantlyreduce the frequency of tumor-infiltrating CD4þ Tregs(Foxp3þCD4þ; Fig. 3E), and, more strikingly, deplete the fre-quency of infiltrating tumor-resident CCR8þFoxp3þCD4þ Tregs(Fig. 3F). Moreover, the CD8:Treg ratio within the tumorincreased significantly in aCCR8-treated tumors (Fig. 3E), furthersuggesting a tipping of immune balance in favor of antitumorimmunity (21). In addition, aCCR8 did not significantly reduceFoxp3þCD4þ, but did significantly reduce the CCR8þ Treg targetpopulation in the periphery, demonstrating the target-specificeffects of this agent (Supplementary Fig. S2A and S2B). Theseobservations indicate that aCCR8 can reprogram the suppressiveTME by specifically targeting and reducing suppressive tumor-

resident CCR8þCD4þ Tregs, thus inducing a more favorableantitumor TME with enhanced effector T-cell function.

CCR8 mAb reduces Treg induction and Treg functionStudies have demonstrated that the CCR8–CCL1 axis plays a

role in the process of Treg induction to a suppressive phenotypeas well as Treg survival (22, 23). We speculated that aCCR8-mediated blockadewas preventing de novo conversion of Tregs to asuppressive phenotype. To understand the mechanism of CCR8-mediated suppression, we first assessed whether aCCR8 played arole in Treg conversion. We demonstrated that aCCR8 mAbtherapy inhibited Treg induction, whereas the isotype Ab controlhad no effect (Fig. 4A). Nevertheless, we also observed that TILsharvested from CT26 tumors and cultured ex vivo in the presenceof aCCR8 demonstrated a substantial reduction of Tregs com-pared with control (45% vs. 10%, respectively) after 72 hours(Fig. 4B). We did observe an increase in the frequency of CD8þ

T cells within the ex vivo aCCR8-treated TILs (SupplementaryFig. 2C), likely due to the reduction of suppressive Tregs (Fig. 4B).

Figure 2.

CCR8 mAb treatment increases the frequency and function of specific inflammatory cytokine production of TILs and CT26 antigen-specific splenic andtumor-infiltrating CD8þ T cells. Spleens and TILs (n ¼ 5–8/group) from tumors of tumor-bearing CT26 mice were harvested 17 days after tumor implantation. A,The frequency of AH1-specific IFNg (spot-forming cells/106 splenocytes) responses induced was determined by IFNg ELISpot assay in response to thespecific CT26 AH1 epitope (SPSYVYHQF). B and C, Bar scatter plot graphs show the percentages of H2-Ld–restricted AH1-specific CD8þ T cells in the spleensof tumor-bearing mice (B) and the percentage of AH1 tetramer-specific CD8þ TILs of total CD45þ cells in the tumor (C). D, Column graphs show theintracellular cytokine staining for IFNg in CD8þ TILs following AH1 peptide stimulation. E, Shown are summary data of the intracellular cytokine staining forIFNg in CD4þ TILs and IFNg , and CD107a/IFNg in CD8þ TILS following PMA/ION stimulation 17 days after tumor implantation. � , P < 0.05; �� , P < 0.01.






We next evaluated whether aCCR8 treatment could inhibit thesuppressive function of already functionally suppressive Tregs.The presence of Tregs reduced CD8þ T-cell proliferation and theaddition of aCCR8 to Tregs/CD8þ T-cell cocultures significantlyattenuated the suppressive capacity of Tregs (Fig. 4C). To confirmthat targeting CCR8 did not directly affect T effector cells, we alsomeasuredCD8þ T-cell proliferation in the presence ofaCCR8 andfound no changes from basal proliferation rates (Fig. 4C), nor didthey affect CD4þ T-cell proliferation (Fig. 4D). Taken together,these data show that blocking CCR8 with an aCCR8 mAb resultsin both reduced Treg induction and diminished Treg-suppressivefunction, without affecting the proliferation of effector CD4and CD8 T cells.

CCR8mAb therapy with a Listeria-based tumor vaccine inducesantitumor immunity

We have demonstrated that Listeria-based vaccines can bepotent cancer immunotherapies by inhibiting suppressive cellfunction in the TME while simultaneously enhancing antitumoreffector immune cell activity (24–25). Given that aCCR8 therapyalone increases the frequencyofAH1-specific effectorCD8þT cellsin the TME (Fig. 2), we sought to increase the magnitude of thetumor-specific immune response by combining CCR8 mAb ther-apy with a Listeria-based cancer immunotherapy targeting theclassical AH1 tumor-associated antigen (Supplementary Fig. S3).Combination Lm-Ah1/aCCR8 therapy was administered in asequential dosing strategy: aCCR8 mAb treatment was firstadministered to modulate the tumor immune milieu (reduce

CCR8þ Tregs) as described in Fig. 1A with dosing started on day 4after tumor implantation followed by Lm-AH1 immunizationbeginning on day 11 after tumor implantation. Combinatorialtreatment showed significantly improved suppression of tumorgrowth and led to approximately 20% long-term survival com-pared with control group (Fig. 5A). Analysis of tumor-infiltratingleukocytes showed synergistically enhanced responses against theimmunizing AH1 antigenic target (Fig. 5B). Combinationtherapy significantly increased IFNg and TNFa production byeffector CD8þ and CD4þ TILs and reduced tumor-resident Tregs,indicating that the enhanced development of T cell–mediatedimmune responses might be critical for the efficacy of thecombination therapy (Fig. 5C and D). In addition, we observedthat the CD8:Treg ratio increased significantly within the tumor(Fig. 5C). This successful augmentation of effector CD4þ andCD8þ T cells in the TME indicates that the combination ofL. monocytogenes and aCCR8 could be an ideal strategy for immu-notherapy for a variety of cancers. These results set the stage foradditional studies designed to evaluate combination therapieswith aCCR8 mAb.

DiscussionAccumulating evidence indicates that effective cancer immu-

notherapy will need to control Treg infiltration into tumor tis-sues (2–7, 12–24). Tumor-infiltrating Tregs represent majorobstacles for driving effective tumor-specific immune responses.Several Treg depletion strategies, such as anti-CD25 and

Figure 3.

CCR8 mAb treatment alters the tumor immune microenvironment in the CT26 colorectal model. TILs (n ¼ 5–6/group) from tumors of CT26 mice wereharvested 17 days after tumor implantation. A, CD45þ leukocyte infiltrate and CD8þ and CD4þ TILs as percentage of total CD45þ cells are shown in treatedversus untreated group.B andC,Frequencyof CTLA-4 andPD-1 expressionon tumor-infiltrating CD8þT cells andCD4þT cells, respectively.D andE,TIL populations,including G-MDSCs (CD11bþLy6C�Ly6Gþ; D) and CD4þ Tregs (Foxp3þCD25þ), and the ratio of CD8/Tregs in the tumor (E) were identified by flow cytometry.F, CCR8 expression by Tregs (Foxp3þCD4þ) is shown by flow cytometry. � , P < 0.05; �� , P < 0.01; ns, not significant. Error bars, SEM of n ¼ 5–6/group.

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cyclophosphamide, have been explored to enhance immunother-apy (2–7, 12–14). However, there are serious drawbacks to thesetreatments such as (1) autoimmunity resulting from a reductionin Tregs systemically and/or (2) attenuation of antitumor immu-nity by depletion of effector T cells due to off-target effects.Therefore, effective new cancer immunotherapies are needed tospecifically target Tregs that are abundantly and specifically locat-ed in tumor tissues. Recent evidence indicates that CCR8þ Tregsare critical for Treg proliferation and suppression (17). Further-more, Plitas and colleagues and De Somine and colleaguesshowed that intratumoral Tregs of human patients with cancerpredominantly expressed CCR8, highlighting their therapeuticpotential as a target to manipulate tumor-resident Tregs (18, 19).In this study, we report for the first time that CCR8 can serve as anovel candidate target to inhibit tumor-resident Tregs, therebyenhancing the efficacy of immunotherapy.

In the current study, we demonstrated that CCR8 was predom-inantly upregulated in tumor-resident Tregs in comparison withperipheral Tregs (Fig. 1A). These data recapitulate those fromclinical studies, demonstrating in several different tumor types(colorectal, breast, lung, melanoma) that CCR8 is highlyexpressed predominantly on tumor-resident Tregs relative toperipheral tissue-resident Tregs (18, 19). As such,CCR8 representsa valuable target, as specifically limiting the CCR8 Treg popula-tion to the tumor in vivo can promote better-primed immuneresponses and effective antitumor immunity. The high expressionof CCR8 on Tregs within the TME suggests that tumor-residentCCR8þ Tregs suppressed tumor-infiltrating effector T cells. Thishypothesis is supported by the enhanced expansion, activation,and effector function of tumor-infiltrating T cells in aCCR8mAb-treatedmice (Fig. 2). The observation of higher tumor-infiltrating

CCR8þ Tregs in the tumors of nontreatedmice (Fig. 3F) correlatedwith poor tumor control. Plitas and colleagues support theseresults in a clinical setting, demonstrating that high CCR8 toFoxp3 mRNA ratios correlated with poor prognosis in breastcancer (19). We observed that CCR8 blockade altered the sup-pressive cellular signature of the TME by reducing tumor-infil-trating CCR8þFoxp3þ Tregs, while increasing the frequency ofinfiltrating tumor-specific effector T cells (Figs. 2 and 3). Recently,a study by Barsheshet and colleagues demonstrated that CCR8þ

Treg proliferation and suppression is dependent on interaction ofCCR8–CCL1 axis (17). Therefore, the number of CCR8þ Tregscould be reduced by targeting CCR8 with an aCCR8 antibody.Our results demonstrated that CCR8 blocking by aCCR8 mAbcould deplete the infiltratingCCR8þTregs and enhance antitumorimmunity. Thus, this mechanism of reducing suppressive CCR8þ

Tregs in the tumor may account for the robust increase in thefrequency of Ag-specific tumor-infiltrating effector CD8þ T-cellresponses with cytolytic potential. The increase of effector CD8þ Tcell-to-Treg ratios within the tumor is considered to be essentialfor controlling and eliminating established tumors (1–4, 21, 26).The fact that treatment with aCCR8 induced more functional Ag-specific CD8þ T cells (Fig. 2) suggests that aCCR8 treatmentreduced tumor-resident CCR8þ Tregs cells while sparing CD8þ

effector T cells. The selective effect on Tregs and not CD8þ T cellsmost likely results from the higher expression levels of CCR8 onTregs (17–19). Taken together, these results indicate that aCCR8therapy augments antitumor immunity by targeting CCR8þ Tregsin tumor tissues. Thus, these effects of aCCR8 treatment, alongwith the increase in the number of Th1 cytokine-producing T cells,facilitates the establishment of a proinflammatory TME, whichmay lead to improved antitumor immunity.

Figure 4.

Blocking CCR8 reduces Treg induction and Treg function. A, aCCR8 mAb reduces Treg induction, whereas the isotype control (IgG) does not. The 1 � 106 na€�veCD4þ T cells were cultured under specific conditions (including aCD28 and IL2) and Treg induction (frequency of Foxp3þCD4þ cells) was measured by flowcytometry. B, TILs were harvested from day 17 implanted CT26 nontreated tumors and cultured for 72 hours in the presence or absence of aCCR8 (10 mg)on plates coated with or without aCD3 in the presence of aCD28a and IL2. C, aCCR8 reduces the capacity of Tregs to suppress CD8 T-cell proliferation.Cultured induced Tregs were cocultured with purified CD8þ T cells (1:1 ratio) with or without aCCR8 (10 mg); control included CD8 T cells alone. The purifiedCD8þ T cells were cultured for 3 days on plates coated with aCD3 in the presence or absence of Tregs or aCCR8. Addition of aCCR8 significantly reversedthe suppressive function of Tregs. C and D, aCCR8 did not affect CD8þ T-cell proliferation (C), nor did it affect CD4þ T-cell proliferation (D). The effect of theaddition of aCCR8 antibody on proliferation was evaluated by measuring CFSE by flow cytometry. �� , P < 0.001; ��, P < 0.0001; ns, not significant.






Tregs have been shown to play a key role in controllingautoimmunity and in the regulation of pathologic and physio-logic immune responses (17, 23, 27). Therefore, the reduction of aparticular Treg cell population (in this case, CCR8þ Tregs) ratherthan the entirety of the Foxp3þ T-cell population can be exploitedto augment antitumor immunity without the risk of inducingautoimmunity. In this study, we showed that aCCR8 therapyselectively reduced intratumoral Tregs (Fig. 3E), but spared Tregsin the periphery, demonstrating the tumor-specific effects of theseagents. In our studies, no autoimmunity or toxicity were observedin the treatedmice. In fact, several studies that support this notionhave shown that CCR8-deficient mice do not exhibit any of thecharacteristics of severe autoimmune and lymphoproliferativedisorders resulting from Foxp3 deficiency (17, 23, 28–30). Inaddition, it is likely that, although aCCR8 therapy reducedCCR8þ Tregs during treatment, peripheral CCR8�Foxp3þ Tregscan be sufficient to prevent deleterious autoimmunity. CCR8blocking can therefore be a unique cancer immunotherapy aimedat depleting tumor-resident Tregs without the severe clinicaladverse events that would arise from systemic Treg depletion.Nevertheless, determining whether or not in vivo aCCR8 therapywill elicit autoimmunedisease in the clinicwarrants investigation.

We observed that blockade with CCR8 prevented the differen-tiation of naive CD4þ T cells into Tregs and inhibited theirsuppressive function (Fig. 4), which likely attenuated immuno-suppression, thereby enhancing efficacy of immunotherapy

(Fig 1). Furthermore, treatment with aCCR8 did not affect CD8þ

or CD4þ T-cell proliferation (Fig. 4C and D), nor did it affect theinduction of CD8þ effector T cells (Figs. 2 and 3). Taken together,these results indicate that treatment with aCCR8 exclusivelytargeted CCR8þ Tregs, disrupting the function of these cells,without affecting the function of effector CD8þ T cells. In addi-tion, how aCCR8 therapy may affect the immune function ofother cell populations that express CCR8 cannot be ruled out andrequires further study (17). Collectively, the results reveal thataccordant with the reduction of CCR8þ Tregs and enhancedeffector CTL responses, administration of aCCR8 led to stronginhibition of tumor growth and prolonged survival. Similarly,Hoelzinger and colleagues reports that neutralizing the CCR8ligand, CCL1, is also effective in blocking Treg conversion andinhibiting Treg-suppressive function (22). Overall, our resultsfurther add to the pool of information that the CCR8–CCL1 axisplays a role in Treg function and that targeting this axis canimprove antitumor immunity (17, 22).

CCR8 may have multiple effects on Treg biology (17, 23,28, 31, 32). Therefore, targeting CCR8 on Tregs may also havemultiple endpoints: (i) It has been reported that CCR8 has anti-apoptotic effects and is essential for Treg survival (23, 31). Thus,targeting CCR8 may increase Treg susceptibility to cell death viaapoptosis; (ii) given that CCL1 is unique in potentiating CCR8þ

Tregs (17–19, 22), targeting its receptor, CCR8, may block itsactivity for expansion and suppression; (iii) because CCL1 can

Figure 5.

CCR8mAb therapy with a Listeria-based tumor vaccine enhances antitumor immunity.A,Na€�vemice (n¼ 8/group) were inoculated with CT26 tumor cells (3� 105)on the right flank. Combination therapy was given in a sequential setting. On day þ4 postinoculation, mice were treated with aCCR8 (administered for8 consecutive days) and on day þ11 postinoculation mice were treated with Listeria-based vaccine or the combination as indicated. Mean tumor growth andsurvival are depicted.B, TILs (n¼4–5/group)were harvested at dayþ22, followed by analysis of frequency of CD45þ leukocyte infiltrate andCD8þ andCD4þ TILs aspercentage of total CD45þ cells. C, Summary data show the frequency of CD4þ Tregs (Foxp3þCD25þ) and the ratio of CD8 effector T cells to Tregs in the tumors.D, Summary data showing positive CD8 and CD4 T cells releasing IFNg , TNFa, and/or IL2 following AH1 peptide incubation with PMA/ION stimulation.Error bars, SEM of n ¼ 4–5/group. � , P < 0.05; ��, P < 0.01; ��, P < 0.001; �� , P < 0.0001.

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drive CCR8 Treg motility (32), it is plausible to speculate thatthe decrease in number of tumor-infiltrating regulatory cells bytargeting the CCR8–CCL1 axis could also be the result of analteration in their migration properties to the tumor (28, 32).Studies to elucidate the specific role of CCR8 on Treg biologywarrants further investigation and are currently under way inour laboratory.

Combining multiple immunotherapies for cancer will becritical for improving the therapeutic outcome for future clin-ical trials. In this study, our data show promising synergisticeffects of combining aCCR8 mAb with a Listeria-based immu-notherapy. We report that targeting CCR8 can increase theinfiltration of vaccine-induced effector T cells into tumors andincrease their functionality, thus leading to improved antitu-mor immunity, tumor regression, and prolonged survival inestablished tumor-bearing mice (Fig. 5). Our studies appear tobe supportive of previous work showing that eliciting highervaccine-induced effector T cells in the tumor correlated withbetter protective immunity for controlling tumor growth (33).In addition, the increased production of TH1 cytokine-produc-ing CD4þ and CD8þ tumor-infiltrating T cells likely helps shiftthe TME from a suppressive to a more inflammatory antitumorstate (34). In future studies, it will be interesting to test whethercombining aCCR8 with additional immunotherapies such ascheckpoint inhibitors or costimulatory molecules can achievebetter antitumor immunity. In addition, understanding howCCR8 functions, in what step of the cancer immunity cycle, willallow us to best design aCCR8 immunotherapies and targetthem to the patients who are most likely to respond. Overall,our findings provide initial in vivo evidence that a vaccine can becoupled with aCCR8 mAb and support the use of this combi-nation strategy for use in future clinical trials. We expect thatdata from this study can serve to leverage the design of clinicaltrials that would deliver aCCR8 as monotherapy or in combi-nation with other modalities such as vaccines or checkpointinhibitors for the treatment of cancer.

Our study provides the first compelling evidence for targetingCCR8 as a therapy to reduce tumor-resident Tregs to controlimmunity against cancer. The high specificity of CCR8 expressionon tumor-resident Tregs in various types of human cancers,such as colorectal, breast, lung, melanoma, and angiosarcoma

(18–19), highlights the broad clinical applicability for a CCR8blockade or depletion antibody in the treatment of a variety ofcancers. Overall, we present CCR8 as a promising new target forcancer immunotherapy, either as a single agent or in combinationwith other forms of cancer immunotherapies and, therefore, ithas direct relevance for the design of future therapeutic trialsin patients.

Disclosure of Potential Conflicts of InterestD.O. Villarreal has ownership interest (including stock, patents, etc.) in

Advaxis, Inc (Stock). C. Mottershead has ownership interest (including stock,patents, etc.) in Advaxis Stock. B.D. Coder has ownership interest (includingstock, patents, etc.) in Advaxis, Inc. R.G. Petit is the chief scientific officer,executive vice president at Advaxis Inc., and has ownership Interest (includingstock, patents, etc.) in Advaxis Inc. M.F. Princiotta has ownership Interest(including stock, patents, etc.) in Advaxis, Inc. No potential conflicts of interestwere disclosed by the other authors.

Authors' ContributionsConception and design: D.O. Villarreal, R.G. Petit, M.F. PrinciottaDevelopment of methodology: D.O. Villarreal, A. L'Huillier, S. ArmingtonAcquisition of data (provided animals, acquired and managed pati-ents, provided facilities, etc.): D.O. Villarreal, A. L'Huillier, S. Armington,C. Mottershead, E.V. Filippova, B.D. CoderAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D.O. Villarreal, A. L'Huillier, C. MottersheadWriting, review, and/or revision of the manuscript: D.O. Villarreal,A. L'Huillier, C. Mottershead, B.D. Coder, R.G. Petit, M.F. PrinciottaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): D.O. Villarreal, A. L'Huillier, S. Armington,B.D. CoderStudy supervision: D.O. Villarreal, R.G. Petit, M.F. Princiotta

AcknowledgmentsD.O. Villarreal would like to thank Rebekah Joy Siefert for her everlasting

support of my scientific innovation and creative progression. This work wassupported by Advaxis Immunotherapies, Inc.

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

Received April 20, 2018; revised June 4, 2018; accepted July 10, 2018;published first July 19, 2018.

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2018;78:5340-5348. Published OnlineFirst July 19, 2018.Cancer Res Daniel O. Villarreal, Andrew L'Huillier, Susan Armington, et al. Enhances Vaccine-Induced Responses in Colon CancerTargeting CCR8 Induces Protective Antitumor Immunity and

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