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Translational Cancer Mechanisms and Therapy

SMAC Mimetic Debio 1143 and Ablative RadiationTherapy Synergize to Enhance AntitumorImmunity against Lung CancerZhen Tao1, Neal S. McCall2, Norbert Wiedemann3, Gr�egoire Vuagniaux3,Zhiyong Yuan1, and Bo Lu2

Abstract

Purpose: Adaptive antitumor immunity following ablativeradiotherapy (ART) is attenuated by host myeloid-derivedsuppressor cell (MDSC), tumor-associated macrophage(TAM), and regulatory T-cell (Treg) infiltrates. We hypothe-sized treatment with ART and a secondary mitochondrial-derived activators of caspase (SMAC) mimetic could reversethe immunosuppressive lung cancer microenvironment tofavor adaptive immunity.

Experimental Design: To evaluate for synergy betweenART and the SMAC mimetic Debio 1143 and the depen-dence upon CD8þ T cells and TNFa, we used LLC-OVAsyngeneic mouse model of lung cancer and treated themwith Debio 1143 and/or ART (30 Gy) with or withoutanti-CD8, anti-TNFa, or anti-IFNg antibodies. Tumor-infiltrating OVA-specific CD8þ T cells, Tc1 effector cells,MDSCs, TAMs, and Tregs, were quantified by flow cyto-metry. Tc1-promoting cytokines TNFa, IFNg , and IL1b andthe immunosuppressive IL10 and Arg-1 within LLC-OVA

tumor tissue or mouse serum were measured by RT-PCRand ELISA.

Results: ART delayed tumor growth, and the addition ofDebio 1143 greatly enhanced its efficacy, which includedseveral complete responses. These complete respondersrejected an LLC-OVA tumor rechallenge. ART and Debio1143 synergistically induced a tumor-specific, Tc1 cellular andcytokine responsewhile eliminating immunosuppressive cellsand cytokines from the tumor microenvironment. Depletionof CD8þ cells, TNFa, and IFNg with blocking antibody abro-gated synergy between ART and Debio 1143 and partiallyrestored tumor-infiltrating MDSCs.

Conclusions: Debio 1143 augments the tumor-specificadaptive immunity induced by ART, while reversinghost immunosuppressive cell infiltrates in the tumor micro-environment in a TNFa, IFNg , and CD8þ T-cell–dependentmanner. This provides a novel strategy to enhance the immu-nogenicity of ART.

IntroductionIn patients with early-stage non–small cell lung cancer

(NSCLC), delivery of highly conformal, high-dose ablative radio-therapy (ART) has demonstrated local control and survival ratesthat rival those of surgery (1). Aside from establishing localcontrol of distant metastases, ART may, at times, contribute tosystemic disease control through generating antitumor immunityby stimulating tumor antigen and cytokine release (2–4). As such,ART is also being increasingly utilized in patients with oligometa-

static disease (5). However, 3 cell populations, myeloid-derivedsuppressor cells (MDSCs), tumor-associated macrophages(TAMs), and regulatory T cells (Tregs) can neutralize these favor-able host adaptive immune responses following radiation (6–9).Furthermore, the immune response following ART is not entirelyproinflammatory in nature, and, at times, ART may recruit andactivate the immunosuppressive cell compartment.

Secondary mitochondrial-derived activators of caspase(SMAC) mimetics belong to an investigational class of drugs thatantagonize inhibitor of apoptosis proteins (IAP), which inhibitcaspase function. By either direct inhibition or by triggeringautoubiquitination and IAP degradation, SMACmimetics restoreapoptotic function and sensitize cancer cells to chemotherapy andradiation (10–13). Furthermore, IAP depletion by SMACmimetics alters NFkB signaling such that autocrine or paracrineTNFa-mediated NFkB induction elicits cancer cell death ratherthan survival (14). IAP antagonism also modulates innate andadaptive immunity (15), increasing the activation of antigen-presenting cells (16), cell death in monocytes, maturation ofmonocyte-derived dendritic cells (17), and T-cell activation(18). One study has demonstrated that the combination ofoncolytic viruses and SMAC mimetic compounds robustly acti-vates the innate immune system against cancer cells (19). SMACmimetics also have proved to synergistically promote antitumorimmunity alongside programmed death-1 (PD-1) immunecheckpoint inhibitors in multiple mouse tumor models (20).These properties have led to early clinical trials investigating the

1Department of Radiation Oncology and Cyberknife Center, Tianjin MedicalUniversity Cancer Institute and Hospital, Tianjin, China. 2Department of Radi-ation Oncology, Thomas Jefferson University, Philadelphia, Pennsylvania.3Debiopharm International SA, Lausanne, Switzerland.

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

Z. Tao and N.S. McCall contributed equally to this article.

Corresponding Authors: Bo Lu, Thomas Jefferson University and Hospitals,G-301 BodineCancer Center, 111 South, 11th Street, Philadelphia, PA 19107. Phone:215-955-6705; Fax: 215-503-0013; E-mail: [email protected]; and ZhiyongYuan, Department of Radiation Oncology, Tianjin Medical University CancerInstitute and Hospital, Tianjin 300060, China. Phone/Fax: 8622-2334-1405;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-3852

�2018 American Association for Cancer Research.

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clinical activity of SMAC mimetics alongside chemoradiation inhead andneck cancer (21) and the anti-PD-L1 antibody avelumabin patients with advanced solid malignancies (22).

We hypothesized that the combination of ART and a SMACmimetic may synergize to promote an antitumor adaptiveimmune response. Here, we report a novel finding that treatmentof NSCLC with the combination of ART and the SMAC mimeticDebio 1143 displayed strong antitumor efficacy and generated aproinflammatory cascade, culminating in the CD8þ T-cell andTNFa-mediated elimination of immunosuppressive cells fromthe TME. Combination therapy with ART and Debio 1143 pro-moted a Tc1 effector, tumor-specific immune response, whichwassustained beyond the therapeutic time period.

Materials and MethodsCell lines, animals, and reagent

The murine Lewis lung carcinoma (LLC) cell line wasobtained from the ATCC. The LLC-OVA cell line expressing asingle polypeptide encoding H-2Kbb2-M, and the OVASIINFEKL peptide was kindly provided by Amer A. Beg (MoffittCancer Center, Tampa, FL) and was generated as describedpreviously (23). Cellswere cultured inDMEMsupplementedwith10% FBS, 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine,10 mg/mL penicillin–streptomycin, and 0.1 mmol/L nonessentialamino acids (Life Technologies). All mouse experimentswere approved by the Jefferson Institutional Animal Care & UseCommittee. Six- to 8-week-old female C57BL/6 mice were pur-chased from Jackson Laboratory. All mice were maintained underspecific pathogen-free conditions. SMAC mimetic Debio 1143was provided byDebiopharm International SA, andwas preparedin an aqueous solution and stored at 4�C within 1 week ofexperiments.

In vivo tumor growth experimentsCells were harvested after being cultured for less than 2 weeks

and counted. Six- to 8-week-old female C57BL/6 micewere injected subcutaneously at the right flank with 5 � 105

LLC-OVA cells in 200-mL serum-free DMEM. Tumors were

allowed to develop for 9 days, until tumor diameter wasapproximately 0.1–0.2 cm3, after which the mice were dividedinto 4 treatment groups (n ¼ 12 per group). On day 9, theC57BL/6 mice treated with either Debio 1143 (100 mg/kg forconsecutive 10 days) by oral gavage, ART of 30 Gy by using aPanTak 310keV X-ray machine at 0.25-mm Cu plus 1-mm Aladded filtration at 125 cGy/minute, or the combination. Tumordimensions were measured every 3 days, and volumes werecalculated using the formula: V ¼ L � W2 � 0.52, where L andW are the long and short diameters of the tumor, respectively.Tumor growth delay experiments were repeated 3 times toverify their accuracy. For the survival assay, mice were sacrificedwhen they showed signs of morbidity or when subcutaneoustumors reached 2,000 mm3 in size.

Flow cytometryTo obtain single-cell suspensions, 12 days after ART, tumor

tissues were minced into small fragments, and digested using1 mg/mL collagenase IV (Sigma-Aldrich) and 0.2 mg/mLDNaseI (Life Technologies) at 37�C for 45 minutes. Dissociatedcells were passed through a 70-mm cell strainer twice andwashed 3 times in DMEM. Spleens from tumor-bearing micewere homogenized and pass through a 70-mm cell strainer inice-cold PBS to achieve single-cell suspension. Red blood cellswere lysed using ACK Lysis Buffer (Life Technologies). Allsamples were pretreated with CD16/CD32 FcR blocker (BDBiosciences) before staining. Cells were stained with the fol-lowing antibodies obtained from eBioscience: CD11b-PE(clone M1/70, catalog No. 12-0112-83), Gr-1 APC (cloneRB6-8C5, catalog No. 17-5931), F4/80 (clone BM8, catalogNo. 17-4801), CD8a-PE (clone 53-6.7, catalog No. 12-0081),Mouse regulatory T-cell staining kit (clone FJK-16s, catalog No.88-8118), OVA peptide (SIINFEKL)-loaded H-2Kb tetramerlinked to APC (clone 25-D1.16, catalog No. 17-5743). Thefollowing antibodies were obtained from BioLegend: CD45-PerCP/Cy5.5 (clone 30-F11, catalog No. 103132), CD8a-FITC(clone 53-6.7, catalogNo. 100706), CD8a-Alexa Fluor 594 (clone53-6.7, catalogNo. 100758),CD4-FITC (cloneGK1.5, catalogNo.100406), CD11b-PE (clone M1/70, catalog No. 101208). Forintracellular cytokine staining, cells were stimulated with 50ng/mL PMA, 500 ng/mL ionomycin, and 10 mg/mL GolgiPlug(BD Biosciences) at 37�C for 4 hours. Cells were then harvested,fixed, and permeabilized using Cytofix/Cytoperm Fixation/Per-meabilization Kit (BD Biosciences) and stained with IFNg-FITC(eBioscience; clone XMG1.2, catalog No. 11-7311), and TNFa-Alexa Fluor 647 (BioLegend; clone MP6-XT22, catalog No.506314) to identify cytokine-secreting CD8þ T cells. Data werecollected on a BD LSRII Flow Cytometer, and analyzed usingFlowJo software (Tree Star Inc.).

Real-time PCRTotal RNA was isolated using TRIzol reagent (Life Technolo-

gies) according to the manufacturer's instructions. cDNAs weresynthesized with TaqMan Reverse Transcription Reagents(Applied Biosystems), following the manufacturer's instructions.The primer pairs used for iNOS, Arginase I, TNFa, IL10, andGAPDH are obtained from Taqman RT reagents (Applied Biosys-tems). The fold stimulation was determined using the compar-ative cycle threshold method (2�DDCt). All experiments wereperformed in triplicate.

Translational Relevance

Ablative radiotherapy (ART) can recruit and activate immu-nosuppressive host cells in the tumor microenvironment(TME), including myeloid-derived suppressor cells (MDSCs),tumor-associated macrophages (TAMs), and regulatory T cells(Tregs) that restrain antitumor immunity. Secondary mito-chondrial-derived activators of caspase (SMAC) mimetics,which are known to sensitize cancer cells to radiation-inducedcell death, can also promote antitumor immunity. The addi-tion of an investigational SMAC mimetic compound (Debio1143) to ART in a mouse model of lung cancer potentiatedantigen-presenting cell recruitment, T-cell priming, and anti-tumor immune responses. Furthermore, treatmentwithDebio1143 and ART significantly reduced the infiltration of immu-nosuppressive cell populations within the TME. These dataadvocate for early clinical trials to test the safety and efficacy ofthe combination Debio 1143 and ART in patients with lungcancer.

Tao et al.

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Cytokine ELISAQuantification of serum cytokines (TNFa, IFNg , IL10, IL1b)

was performed using R&DQuantikine ELISA Kit according to themanufacturer's instructions. The plates were read at 450 nmwithin 30 minutes. All standards, controls, and samples were runin triplicate.

In vivo antibody depletion experimentsC57BL/6 mice were inoculated subcutaneously with LLC-OVA

cells and treated with radiation and Debio 1143 according to theregimen described above. For CD8þ T-cell depletion experiments,200mgpermouse of anti-CD8 antibody (clone 2.43; Bio-XCell) orrat IgG2B isotype control were delivered via intraperitoneal injec-tion (i.p.) every 3 days beginning on day 9 for 4 times. For theTNFa blockade experiment, 500-mg anti-TNFa antibody (clone:XT3.11; Bio-XCell) or rat IgG1 isotype control was administeredintraperitoneally every 3 days beginning on day 9 for 4 times (4).For the IFNg-neutralizing experiment, 300-mg anti-IFNg antibody(clone: R4-6A2; Bio-XCell) or rat IgG1 isotype control was admin-istered intraperitoneally every 3 days beginning on day 9 for 4times (20). All antibody treatments were started from the day ofradiation or 1 day before radiation.

Statistical analysisSurvival analysis was done using the Kaplan–Meier method

with log-rank univariate analysis and Cox regression multivariateanalysis, with P < 0.05 considered statistically significant. Datawere analyzed using Prism 5.0 software (GraphPad Software).Data are represented as the mean � SD for all figure panels inwhich error bars are shown. The P values were assessed using2-tailed unpaired Student t tests. A P value of less than 0.05 wasconsidered statistically significant.

ResultsDebio 1143 and ART synergistically and durably inhibit lungcancer growth in vivo

We hypothesized that the SMAC mimetic compound Debio1143 could potentiate antitumor adaptive immune responses toradiation. LLC cells stably transfected with OVA-tetramer antigenin subcutaneous syngeneic tumor models were used to quantifytumor-specific immune responses (23). To assess for therapeuticsynergy between Debio 1143 and ART, LLC-OVA tumor-bearingC57BL/6 mice were treated with Debio 1143, ART, or both asshown in Fig. 1A. ART was delivered as a 30 Gy dose in a singlefraction, a regimen shown to maximize adaptive antitumorimmunity in multiple tumor models (24). Moderate decreasesin tumor size were observed following ART alone; however,treatment with the combination potently inhibited LLC-OVAtumor growth compared with Debio 1143 (P < 0.001) and ARTalone (P < 0.05), and 4 mice had a complete response to com-bination therapy (Fig. 1B). Furthermore, mouse survival wassignificantly increased in the ART (P < 0.01) and Debio 1143þ ART groups (P < 0.001) compared with controls (Fig. 1C).SMACmimetics, in the presence of type I IFN or oncolytic viruses,may stimulate long-term antitumor immunologic memory (20).To assess for immune memory after treatment with ART andDebio 1143, 4 complete responders in the ART þ Debio 1143group were rechallenged with 5 � 106 (i.e., 10 times the numberof cells of the initial challenge) LLC-OVA cells in the oppositeflank 37days after the initial treatmentwithARTwith 5 treatment-

na€�ve C57BL/6 mice serving as controls. While tumors grewrapidly in control mice after 12 days, none of the completeresponders established tumors 23 days after the rechallenge (Fig.1D), suggesting an induction of sustained, systemic antitumorimmunity with combination therapy.

The combination of ART and Debio 1143 produces anOVA-specific cytotoxic T-cell response in the tumormicroenvironment and reduces immunosuppressive cellpopulations

To assess for tumor antigen–specific immunity and the recruit-ment of adaptive immune cells after treatment with ART andDebio 1143, LLC-OVA tumors were probed for CD8þ T lympho-cytes expressing OVA 12 days after ART, as earlier time points didnot fully characterize the magnitude of changes in immune cellinfiltration (data not shown; ref. 24). Tumor-specific OVAþCD8þ

T cells were increased with ART alone (Fig. 2A), consistent withprevious reports (24). In the ART þ Debio 1143 group,OVAþCD8þ T cells were increased 2-fold compared with the ARTgroup (P < 0.05) and 7-fold over the Debio 1143 group (P <0.001), characterizing a robust tumor antigen–specific adaptiveimmune response (Fig. 2A). To assess whether combinationtherapy increased CD8þ T-cell activation and differentiation, weanalyzed tumors for OVAþCD8þ cells expressing Tc1 subtypemarkers IFNg and TNFa by flow cytometry. OVAþCD8þ T cellsfrom tumors treated with both Debio 1143 and ART expressedincreased levels of IFNg (P < 0.001 for Debio and ART vs. Debio,P < 0.01 for Debio and ART vs. ART; Fig. 2B) and TNFa (P < 0.001for Debio and ART vs. Debio, P < 0.05 for Debio and ART vs.ART; Fig. 2C). Given this induction in antigen-specific Tc1 effectorresponses in mice treated with ART and Debio 1143, we nextevaluatedwhether combination therapy affected antigen-present-ing cell infiltration by assessing tumors for dendritic cell markersCD45/CD11b/CD11c/CD86. Not surprisingly, we found signif-icantly more dendritic cells in mice treated with ART and Debio1143 compared with Debio 1143 (P < 0.001) and ART (P < 0.01)alone (Supplementary Fig. S1). Taken together, these data dem-onstrate that Debio 1143 potentiates the capability of ART toinduce tumor-specific Tc1 effector responses at the level of antigenpresentation and T-cell priming (25–28).

MDSCs, TAMs, and Tregs are 3 cell populations known tosuppress antitumor immunity or even promote tumor progres-sion (29–31). To determine the effects of ART andDebio 1143 onthe immunosuppressive cell compartment, LLC-OVA tumorswereharvested and assessed for cell-surfacemarkers of MDSC-like cells(CD45þCD11bþGr-1þ), TAMs (CD45þCD11bþF4/80þ), andTregs (CD4þCD25þFOXP3þ) by flow cytometry 12 days afterART. ART alone decreased Gr-1þCD11bþ cell infiltration, but theaddition of Debio 1143 dramatically decreased Gr-1þCD11bþ

populations among CD45þ cells in the TME (P < 0.05 for Debioand ART vs. ART, P < 0.001 for Debio and ART vs. Debio; Fig. 2D).Treatment with ART alone appeared to increase the TAM pheno-type among CD45þ cells, which was reversible with the additionof Debio 1143 (P < 0.05; Fig. 2E). As shown in Fig. 2F, thereductions in CD11bþGr-1þcells and TAMs with combinationtherapy coincided with a decline in Treg markers among CD4þ

cells (P < 0.001 for Debio and ART vs. Debio, P < 0.01 for Debioand ART vs. ART), consistent with the known role of MDSCs inTreg recruitment (32). Taken together, combination therapy withART and Debio 1143 facilitated the elimination of the immuno-suppressive cell compartment within the TME.

Debio 1143 and Ablative Radiation Enhance Antitumor Immunity

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Figure 1.

Debio 1143 andART synergistically anddurably inhibit lung cancer growth in vivo.A,C57BL/6micewere injectedwith 5� 105 LLC-OVAcells and tumorswere allowedto develop. On day 9, mice were divided into 4 groups (n ¼ 12 per group), and treated with Debio 1143, ART, or both, as shown. B, A tumor growth curve of eachtreatment group is shown (C) with corresponding survival data. D, Four mice that had a complete response to the combination of ART and Debio 1143 wererechallenged with 5 � 106 LLC-OVA cells (i.e., 10 times the number of cells of the initial challenge) injected into the opposite flank. Five treatment-na€�ve micewere used as the control. All data are expressed asmean� SD. Statistical differenceswere assessed using the unpaired Student t test (B) or log-rank test (C).P valuesare indicated as follows: � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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Figure 2.

The combination of ART and Debio 1143 reduces immunosuppressive cell populations and produces an OVA-specific cytotoxic T-cell response in the tumormicroenvironment.A,CD8þ T lymphocytes primedwith OVA-tetramer were quantified in LLC-OVA tumors using flow cytometry, and are expressed as a percentageof CD8þ cells. IFNg (B) and TNFa (C) expression among OVAþCD8þ lymphocytes was determined by flow cytometry. These cells are expressed as a percentage ofCD8þ cells. D, Tumors were analyzed for the MDSC markers CD45/Gr1/CD11b using flow cytometry. CD11bþGr-1þcells are expressed as a percentage ofCD45þ cells. E, LLC-OVA tumorswere assessed for the TAMmarkers CD45/F4/80/CD11b by flowcytometry. The percentage of TAMs is expressed as a percentage ofCD45þ cells. F, LLC-OVA tumors were assessed for the Treg markers CD4/FOXP3/CD25 by flow cytometry. Tregs are expressed as a percentage of CD4þ cells.All plots show a representative sample (left) and are expressed as a mean with 5 plotted replicates (right). Statistical differences were assessed using theunpaired Student t test. P values are indicated as follows: � , P < 0.05; �� , P < 0.01; �� , P < 0.001.






Combination treatment with ART and Debio 1143 generates aproinflammatory cytokine cascade

We next sought to correlate local cytokine expression withthese observations of Tc1 cell expansion and immunosuppres-sive cell compartment reductions after ART and Debio 1143.Using RT-PCR, we assessed LLC-OVA tumors for expression ofthe Tc1 cytokines TNFa and IFNg as well as the immunosup-pressive enzyme Arg-1 and cytokine IL10 (8, 33). As shownin Fig. 3A, we detected increases TNFa and IFNg mRNA tran-scripts after ART alone that were potentiated by the addition ofDebio 1143 (P < 0.01 for Debio and ART vs. Debio, P < 0.05 forDebio and ART vs. ART). ART alone increased IL10 and Arg-1mRNA, which was reversible with the combination of ART andDebio 1143 (Fig. 3A). To characterize systemic cytokineresponses, we measured serum levels of TNFa, IFNg , IL10, andIL1b, the latter of which is a cytokine crucial to the priming ofT cells with tumor antigen by ELISA (34). Again, serum TNFaand IFNg as well as IL1b were increased in the ART þ Debio1143 group compared with the Debio 1143 (P < 0.01) and ART(P < 0.05) groups (Fig. 3B), which were remarkably comparablein magnitude to the increases in cytokine transcripts. Similar toour observations of LLC-OVA tumors, ART alone increasedserum IL10 levels compared with vehicle control, but theaddition of Debio 1143 reversed this to levels below boththose of Debio 1143 (P < 0.05) and ART alone (P < 0.01).These data are consistent with previous studies describing themixed proinflammatory and anti-inflammatory cytokineresponse to radiation (7, 9, 33). The coadministration of Debio1143 with ART, in contrast, augmented proinflammatory cyto-kine production and curtailed anti-inflammatory cytokinesboth locally and systemically.

CD8þ T cells are required for the efficacy of Debio 1143/ARTcombination therapy

To evaluate the extent to which CD8þ T cells mediate ther-apeutic synergy between ART andDebio 1143, LLC-OVA tumorswere again grown in C57BL/6 mice and treated with ART, Debio1143, or both with or with anti-CD8 antibody as shownin Fig. 4A. Analysis of tumors treated with anti-CD8 antibodyconfirmed effective CD8 depletion (Supplementary Fig. S2).Four of 12 mice in the Debio 1143 group had a completeresponse; however, CD8þ T-cell depletion nullified synergybetween Deb 1143 and ART. The addition of anti-CD8 antibodyresulted in significantly larger tumors than those treated withcombination therapy (P < 0.05) and similar in size to thosetreated with ART alone (Fig. 4B). Deng and colleagues demon-strated that the clearance of CD11bþGr-1þ cells from the TMEwas dependent upon CD8þ cells and TNFa (4), and others haveshown that the depletion of CD11bþGr-1þ cells is adequateto reinstate T-cell immunity (6).To study the effects of CD8depletion on the immunosuppressive cell infiltrate, we againstudied the CD11bþGr-1þ cell population, which was the mostsensitive to combination therapy, using flow cytometry. Whilecombination therapy with ART and Debio 1143 reducedCD11bþGr-1þ cells compared with controls, CD8 depletionpartially restored this population (P < 0.05; Fig. 4C). TAM andTreg infiltration, however, was unaffected by CD8 depletion(data not shown). Collectively, these data suggest not only thatCD8þ T cells mediate the immunologic synergy between ARTand Debio 1143, but they also contribute to the elimination ofCD11bþGr-1þ cells from the TME.

TNF-a is essential for the efficacy of combination treatmentTo characterize the role of TNFa in combination therapy, we

performed neutralization experiments using anti-TNFa antibody

Figure 3.

Combination treatment affects cytokine levels in tumor tissues and in serum.A, Tumor tissues were assessed for TNFa, IFNg , IL10, and Arg-1 transcriptsby RT-PCR 12 days after treatment initiation. mRNA levels are expressed as aratio relative to the vehicle control. B, ELISA was used to measure TNFa, IFNg ,IL10, and IL1b in the serum of mice in various treatment groups. All data areexpressed asmean� SD of triplicatemeasurements. Statistical differenceswereassessed using the unpaired Student t test. P values are indicated as follows:� , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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(Fig. 5A). Again, 4 of 12 mice in the ART þ Debio 1143 grouphad a complete response. As expected, systemic blockade ofTNFa attenuated the therapeutic synergy between ART andDebio1143 (P < 0.01; Fig. 5B). TNFa has been shown to promoteCD11bþGr-1þ cell apoptosis in tumors treated with anti-PD-1antibodies and ART (4). We hypothesized the surge in expressionof TNFa following ART in the presence ofDebio 1143would havesimilar effects, which we evaluated by assessing tumors treated

with and without anti-TNFa antibody for CD11bþGr-1þ cells.While TAM and Treg infiltration were again unchanged in thepresence of anti-TNFa antibody (data not shown), TNFa deple-tion partially restored infiltration of tumors by CD11bþGr-1þ

cells treated with ART and Debio 1143 (P < 0.01; Fig. 5C). Thus,both the therapeutic synergy between ART and Debio 1143 aswell as the reductions in CD11bþGr-1þ cell infiltration weremediated by TNFa.

Figure 4.

CD8þ T cells are required for the efficacy of Debio 1143/ART combination therapy. A, LLC-OVA tumors were again allowed to grow in mice. On day 9, mice weretreated with anti-CD8 antibody, IgG (vehicle control), ART (30Gy), Debio 1143, or a combination of the 3 as shown (n ¼ 12 per group). B, A tumorgrowth curve of the various treatment groups is shown. C, Tumors from mice of the vehicle control, anti-CD8 antibody, ARTþ Debio 1143, and ARTþ Debio 1143þanti-CD8 antibody groups, were assessed for infiltration by CD11bþGr-1þ cells using flow cytometry. A representative sample (left) is shown, and data from5 replicates are expressed as mean � SD. Statistical differences were assessed using the unpaired Student t test. P values are indicated as follows: � , P < 0.05;�� , P < 0.01; �� , P < 0.001.






IFNg is indispensable in the synergy between ART andDebio 1143

Others have shown that IFNg plays a key role in priming anantitumor adaptive immune response after ART (24). To study therole of IFNg in the therapeutic synergy between ART and Debio1143, we depleted IFNg from treated or vehicle LLC-OVA tumor-bearingmice using blocking antibody as shown in Fig. 6A. Indeed,

tumors from mice treated with ART, Debio 1143, and anti-IFNgwere significantly larger (P < 0.05) than tumors frommice treatedwith just ART and Debio 1143 without anti-IFNg antibody(Fig. 6B). We next analyzed tumors for infiltration byCD11bþGr-1þcells to determine whether elimination of theimmunosuppressive cell compartment is dependent upon IFNg .Again, the addition of anti-IFNg to Debio 1143 and ART partially

Figure 5.

TNFa is essential for the efficacy of combination treatment. A, LLC-OVA tumors were again allowed to grow in mice. On day 9, mice were treated with anti-TNFaantibody, IgG (vehicle control), ART (30 Gy), Debio 1143, or a combination of the 3 as shown (n ¼ 12 per group). B, A tumor growth curve of the varioustreatment groups is shown. C, Tumors from mice of each group were assessed for infiltration by CD11bþGr-1þ cells using flow cytometry. A representativesample (left) is shown, and data from 5 replicates are expressed as mean. Statistical differences were assessed using the unpaired Student t test. P values areindicated as follows: � , P < 0.05; �� , P < 0.01; �� , P < 0.001.

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Figure 6.

IFNg is indispensable in the synergybetweenART andDebio 1143.A, LLC-OVA tumorswere again allowed to grow inmice. Onday9,micewere treatedwith anti-IFNgantibody, IgG (vehicle control), ART (30 Gy), Debio 1143, or a combination of the 3 as shown (n ¼ 12 per group). B, A tumor growth curve of the various treatmentgroups is shown. C, Tumors from mice of each group were assessed for infiltration by CD11bþGr-1þ cells using flow cytometry. A representative sample (left) isshown, and data from 5 replicates are expressed as mean. Statistical differences were assessed using the unpaired Student t test. P values are indicated asfollows: � , P < 0.05; �� , P < 0.01; �� , P < 0.001. D, The synergistic mechanism of action between ART and Debio 1143 is illustrated. Left, ART has mixed effects on theimmune system.While it increases CD8þ T effector cells and antigen-presenting cells and decreases MDSCs (CD11bþGr-1þ cells) and Tregs, TAM infiltration, and theproduction of Arginase-1 and IL10 are also increased. Right, the addition of Debio 1143, a SMAC mimetic, reversed the increases in TAMs, and further reducedinfiltration of MDSCs and Tregs within the TME. Furthermore, it reversed the increases in Arginase-1 and IL10 and potentiated the release of TNFa and IFNs aswell as the recruitment of antigen-presenting cells compared with ART alone. This coincided with an OVA-specific Tc1 effector antitumor immune response.






restored the CD11bþGr-1þ cells in the TME (P < 0.05; Fig. 6C).Collectively, these data indicate that both the therapeutic synergybetween ART and Debio 1143 and the associated clearance ofCD11bþGr-1þ cells from theTME is dependent uponCD8þT cellsas well as the 2major cytokinemodulators of adaptive immunity,TNFa and IFNg .

DiscussionThe immune system is now recognized as a keymediator of the

efficacy of ART (4). Aside from direct cancer kills, it also producesantitumor adaptive immunity; however, repopulation of the TMEby MDSCs, TAMs, and Tregs can stifle these responses (3, 35, 36).SMAC mimetic compounds, known to have radiosensitizingproperties (10–12), can also promote both innate and adaptiveimmune responses (19, 20). Using an LLC-OVA model, we havedemonstrated the SMAC mimetic Debio 1143 potentiates proin-flammatory cytokine release and the tumor-specific adaptiveimmune response to ART (Fig. 6), which included antitumorimmune memory in a subset of mice. The combination of Debio1143 and ART resulted in the elimination of the immunosup-pressive compartment from the TME in a manner dependentupon CD8þ T cells, TNFa, and IFNg .

Independent of immunity, combination treatment with SMACmimetics and radiation disinhibits apoptosis by degrading IAPsand promotes RIPK1/MLKL-mediated necroptosis via TNFa(10, 11). By increasing noncanonical NFkB signaling, SMACmimetics convert the TNFa produced by irradiation from aprosurvival to a cell death signal (14, 37, 38). Similarmechanismsmay partially account for therapeutic synergy between ART andDebio 1143 that we observed in vivo (Fig. 1B and C). Notably, inthe absence of host immunity, LLC-OVA cells were markedlyresistant to Debio 1143 alone in vitro (Supplementary Fig.S3A). Furthermore, Debio 1143 failed to sensitize LLC-OVA cellsto doses of ionizing radiation between 2 and 6 Gy (Supplemen-tary Fig. S3B). We performed a similar clonogenic assay usingablative doses of radiation. As shown in Supplementary Fig. S3C,the addition of Debio 1143 significantly reduced colony forma-tion at 30Gy, but not 10Gy or 20Gy (Supplementary Fig. S3C). Itis unclear why radiosensitization by Debio 1143 was onlyobserved at 30 Gy, although Debio 1143 only reduced colonysurvival by a modest 11.3%. Regardless, these data indicate thatdirect sensitization of LLC-OVA by Debio 1143 cell to ART-induced cell death could play aminor role in the synergy betweenART and Debio 1143.

Beyond direct cell death, the role of the adaptive immunesystem in mediating the synergy between ART and Debio 1143was clear. Combination therapy increased tumor infiltration bytumor antigen–specific, activated T cells (Fig. 2A–C), and com-plete responders to ART þ Debio 1143 group maintained thisadaptive immune response against LLC-OVA tumors upon rechal-lenge (Fig. 1D; refs. 16, 39). Furthermore, the therapeutic synergybetweenART andDebio 1143 on LLC-OVA tumorswas abrogatedby CD8 depletion (Fig. 4B). Notably, we found similar trends inan LLC tumor model without OVA expression, suggesting thatcombination therapy induces antitumor immunity even in theabsence of the artificial immunodominant OVA antigen (Sup-plementary Fig. S4). To our knowledge, this represents a first-timereport of immunogenic synergy betweenART and SMACmimicry.

Furthermore, our data suggest that antigen presentation andT-cell priming underlies the immunogenic synergy between ART

and Debio 1143. Indeed, SMAC mimetics and ART are known toactivate antigen presentation by nonredundant mechanisms.SMAC mimetic–induced NFkB activation in dendritic cells andmacrophages mimics CD40 engagement, driving tumor antigenpresentation (16, 18). Likewise, the roles of radiation in enhanc-ing tumor antigen release, in eliciting ATP and IFN release torecruit and activate antigen-presenting cells have been elaborated(24, 40–42). After irradiation, calreticulin, a phagocyte activator,undergoes cell membrane translocation in irradiated cancer cells,driving the production of IL1b from dendritic cells, which pro-motes T-cell priming and polarization at a transcriptional level(34). In keeping with the known potential of both SMACmimetics and ART to activate antigen-presenting cells, combina-tion therapy with ART and Debio 1143 augmented dendritic cellinfiltration (Supplementary Fig. S1) and IL1b production (Fig.3B) in the TME. This coincided with robust increases in tumorantigen–specific CD8þ T-cell responses (Fig. 2A) and Tc1 effectorcell infiltration (Fig. 2B and C) as well as the local and systemicproduction of Tc1-subtype cytokines (Fig. 3A and B), the end-products of antigen priming and presentation. This suggests thatthe immunogenic synergy between Debio 1143 and ART mayoccur at the level of antigen presentation and T-cell priming.

Negative regulators of antitumor immunity, MDSCs, TAMs,and Tregs within the TME restrict the immune response (6–9).Studies have reported conflicting effects of radiation on these cellpopulations with some reporting their mobilization to the TMEand others reporting their clearance from the TME in response toART alone (6, 9, 24). Similar to other studies of single fraction30 Gy ART, we found that ART alone decreased infiltration byTregs and CD11bþGr-1þ cells; however, contrary to that study,ART increased TAM infiltration (Fig. 2D–F; ref. 24). Despite this,ART monotherapy also increased IL10 and Arg-1 (Fig. 3A and B),yet it is unclear whether these data reflect increased TAM infiltra-tion or, alternatively, enhanced CD11bþGr-1þ cell immunosup-pression (43). These differences may partially be explained bydiffering radiation doses, tumor models, host immune back-grounds, timing of data collection, and immune cell markersanalyzed. For example, while the Cd11b and Gr-1 cell markersused in this study have been shown to identify cells with anMDSCphenotype in LLC syngeneic tumor models (44), they do notdistinguish between monocytic and polymorphonuclear distri-butions, which may have differing function (45). Regardless, ourdata are consistent with the overarching principle that ART, inaddition to promoting adaptive immunity, triggers compensatoryresponses among the immunosuppressive cell compartment thatrestrict the magnitude of the antitumor immune response. Mostnotably, our data indicate that the addition of Debio 1143 canreverse these immunosuppressive adaptations to ART, depletingthe TME of TAMs, Tregs, and CD11bþGr-1þ cells as well as Arg-1and IL10 (Fig. 6D).

We also found that the clearance of CD11bþGr-1þ cells fromthe TME was dependent upon these CD8þ T cells (Fig. 4C).Although the mechanisms by which MDSCs suppress T-cellimmunity are well described (8), the reciprocal mechanisms bywhichT cells eliminateMDSCs are poorly defined.Onepossibilityis that Fas-L interactions on T cells with the Fas death receptor onMDSCs promotes their demise. Studies demonstrate that MDSCsavidly express the Fas death receptor (46), and SMAC mimeticsupregulate Fas-L on T cells, shifting the T-cell killing mechanismfrom perforin/granzyme pathways toward Fas/FasL killing (39).However, therapeutic synergy as well as the clearance of


Tao et al.




CD11bþGr-1þ cells from the TMEwith combination therapywereboth dependent upon TNFa (Fig. 5B andC) and IFNg (Fig. 6B andC). Thus, it is possible that the role of T cells in clearingMDSCs inthe TME is limited to their production of these cytokines. Priorstudies reporting the effects of TNFa onMDSCs have beenmixed.Whereas some have suggested that TNFa blocks the differentia-tion and enhances the suppressive activity of MDSCs (47), ourresults corroborate those of Deng and colleagues, who reportedthat TNFa produced by CD8þ T cells induces apoptosis inMDSCsafter ART and PD-1 blockade (4). One possible explanation forthese different results is that the biologic consequences TNFasignaling and NFkB activation in of CD11bþGr-1þcells (i.e.,survival vs. cell death) are contextual and shifted in favor of celldeath in the presence of Debio 1143. Yet another possibility,TNFamay have differing effects on of CD11bþGr-1þ cells at highdoses generated by ART and PD-1 blockade or SMAC mimeticscompared with lower amounts produced in chronic inflamma-tory states (4).

Interestingly, the depletion of CD8 cells, TNFa, and IFNg failedto completely restore the CD11bþGr-1þ cell population in theTME to levels of vehicle controls. Although we observed onlyminor, statistically insignificant reductions in CD11bþGr-1þ cellsafterDebio 1143 alone (Fig. 2D), it is possible that the incompleterestoration of CD11bþGr-1þ cells in the 3 depletion experimentsis due to a direct cytotoxicity of SMACmimetics upon monocyticCD11bþGr-1þ cells (17). However, Deng and colleagues havealso reported that TNFa and, to a lesser extent, IFNg can mediateCD11bþGr-1þ cell apoptosis in a CD8-independent manner (4).This raises the possibility that any TNFa or IFNg-producing cell (i.e., CD4þ cells or even tumor cells) could play an indirect role theclearance of CD11bþGr-1þ cells from the TME after treatmentwith ART and Debio 1143. Nevertheless, while we studied theprimary modulators of CD8þ T-cell adaptive immunity, we can-not exclude the possibility of other cell types, such as natural killercells or neutrophils, participating in CD11bþGr-1þ cell clearance,as well.

MDSCs are well-known to upregulate programmed deathligand-1 (PD-L1) in response to radiation, which activates T-cellanergy and apoptosis upon ligation to its receptor, PD-1, onT cells(4, 48). PD-L1 expression on CD11bþ MDSCs also impedesthe early adaptive immune response to SMAC mimetic mono-therapy (16). Several groups have shown that the induction ofeffective adaptive antitumor immunity with either radiation or aSMAC mimetic compound requires blockade of the PD-1/PD-L1immune checkpoint (16, 20, 49), and Deng and colleagues

showed that this was dependent upon TNFa-mediated clearanceof CD11bþGr-1þcells (4). It is certainly plausible that additionaltherapeutic synergy could be achieved by triple therapy: ART,Debio 1143, and PD-1/PD-L1 inhibition. However, it is alsopossible that the TNFa-dependent clearance of MDSCs with thecombination of ART and Debio 1143 suffices to reduce PD-1/PD-L1 interactions in the TME, thereby disinhibiting adaptive immu-nity even without PD-1 blockade.

In summary, the combination of SMAC mimetic Debio 1143and radiotherapy resets the immunosuppressive nature of theTME in a TNFa, IFNg-, and CD8þ T-cell–dependent manner andproduces a systemic proinflammatory cytokine cascade. Thisrepresents a first-time report of remarkable therapeutic synergy,including durable systemic antitumor immunity, with the com-bination of ART and a SMAC mimetic. These data support thedesign of clinical trials evaluating the use of SMAC mimetics toharness the immunogenicity of ART.

Disclosure of Potential Conflicts of InterestB. Lu reports receiving commercial research grants from Debiopharm. No

potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: Z. Tao, N.S. McCall, N. Wiedemann, G. Vuagniaux,B. LuDevelopment of methodology: Z. Tao, N.S. McCallAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z. TaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): Z. Tao, N.S. McCall, B. LuWriting, review, and/or revision of the manuscript: Z. Tao, N.S. McCall,N. Wiedemann, G. Vuagniaux, B. LuAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): Z. Tao, B. LuStudy supervision: Z.-Y. Yuan, B. Lu

AcknowledgmentsThis work was supported by funding from Debio Pharma; National

Natural Sciences Foundation of China (grant nos. 81672524, 81602678,and 81502227); and Natural Science Foundation of Tianjin (grant No.17JCQNJC12300).

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 January 19, 2018; revised April 3, 2018; accepted October 18, 2018;published first October 23, 2018.

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2019;25:1113-1124. Published OnlineFirst October 23, 2018.Clin Cancer Res Zhen Tao, Neal S. McCall, Norbert Wiedemann, et al. Synergize to Enhance Antitumor Immunity against Lung CancerSMAC Mimetic Debio 1143 and Ablative Radiation Therapy

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