RG7212 Anti-TWEAK mAb Inhibits Tumor Growth through … · Cancer Therapy: Preclinical RG7212...

Cancer Therapy: Preclinical

RG7212 Anti-TWEAK mAb Inhibits Tumor Growth throughInhibition of Tumor Cell Proliferation and Survival Signalingand by Enhancing the Host Antitumor Immune Response

Xuefeng Yin1, Leopoldo Luistro1, Hua Zhong1, Melissa Smith1, Tom Nevins1, Kathleen Schostack1,Holly Hilton1, Tai-An Lin1, Theresa Truitt1, Denise Biondi1, XiaoqianWang1, Kathryn Packman1, Jim Rosinski1,Windy Berkofsky-Fessler1, Jian-Ping Tang1, Saumya Pant1, David Geho1, Suzana Vega-Harring2,Mark DeMario1, Hy Levitsky3, and Mary Simcox1

AbstractPurpose:Toexplore the role of TWEAK in tumor growthandantitumor immune response and the activity

and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models.

Experimental Design: TWEAK-induced signaling and gene expression were explored in tumor cell lines

and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor

xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features

correlated with antitumor activity were characterized.

Results: In tumor cell lines, TWEAK induces proliferation, survival, and NF-kB signaling and gene

expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible

CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and

macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor,

blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212

inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of

pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed

without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with

evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration

increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans

and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests

that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive

tumors. Clin Cancer Res; 19(20); 5686–98. �2013 AACR.

IntroductionTWEAK and the TWEAK receptor, Fn14, are upregulated

in cancer, and TWEAK-induced signaling promotes multi-ple processes known to contribute to tumor growth. ThisTNF superfamily ligand has been described as mediatingdiverse effects ranging from proliferation to apoptosis.Pathway upregulation in cancer suggests a tumor-promot-ing function and a rationale for inhibition as a therapeutic

strategy. Reports of TWEAK-induced apoptosis in tumor celllines argue against this.We therefore explored thebiology ofTWEAK by evaluating signaling, gene expression, and phe-notypic changes in TWEAK-stimulated cells and the effect ofa TWEAK blockade on tumor growth in mice. TWEAKmediates signaling through Fn14 to promote inflammation(1), cell proliferation (2–5), cell survival (6), and angio-genesis (7). TWEAKmRNA is detected in awide range of celltypes (8), and TWEAK protein has been shown to beexpressed by inflammatory and tumor cells (9–11). Fn14is expressed in multiple cell types, but expression levels arelow in normal cells and have been shown to increase withtissue injury, in inflammatory diseases, and in cancer (12).Pathway expression has been associatedwith poor outcomein patients with cancer. TWEAK and Fn14 expression arenegatively correlated with patient overall and disease-freesurvival in renal cell carcinoma (RCC; ref. 13), and Fn14 is anegative prognostic factor in breast cancer (14), gastriccancer (15), and glioma (16). TWEAK and Fn14 areexpressed in neuroblastoma cell lines and primary tumors,and increased levels of both are observed in high-stage

Authors' Affiliations: 1Roche Pharma Research and Early Development,Nutley, New Jersey; 2Roche Diagnostics GmbH, Pharma Research & EarlyDevelopment, Nonnenwald, Penzberg, Germany; and 3Roche PharmaResearch and Early Development, Roche Glycart AG, Schlieren,Switzerland

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

Corresponding Author: Mary Simcox, Forma Therapeutics, Inc. 500Arsenal Street, Watertown, MA 02472. Phone: 857-209-2853; Fax: 617-679-1977; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-13-0405

�2013 American Association for Cancer Research.

ClinicalCancer

Research

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tumors (17). In vitro, TWEAK stimulation of neuroblastomacell lines increased survival, partially via activation ofNF-kBsignaling, and induced the release of matrix metallopro-tease-9 (MMP-9), suggesting that TWEAKmay play a role ininvasiveness in neuroblastoma. Silencing of TWEAK andFn14 using siRNA significantly reduced survival. Fn14upregulation can occur as a consequence of overexpression(14) or activating mutations in oncogenic receptor tyrosinekinases (18).TWEAK induces proliferation and survival signaling in

tumor and endothelial cells via mitogen-activated kinasesand canonical and noncanonical NF-kB pathways associ-ated with tumor growth and survival. Here, we show theseeffects in tumor cell lines and that a TWEAK blockade withan anti-TWEAK mAb, RG7212, results in significant tumorgrowth inhibition (TGI) in multiple tumor models expres-sing Fn14. These in vitro and in vivo data are consistent withtumor-promoting effects shown with other TNF familyreceptors lacking death domains (19, 20) and with TWEAKdata in disease models in animals and pathologic condi-tions in humans (21, 22).In contrast to data showing TWEAK proinflammatory,

proangiogenic, and proliferation-inducing effects in diseasemodels in animals (5, 23–25) and association of pathwayactivation with human disease (21, 22, 26–28), somepublished in vitro studies have shown that TWEAK inducesapoptosis via upregulation of TNFa (29) or in combinationwith IFNg in a small number of tumor cell lines (8). In theabsence of IFNg , apoptosis induction was modest (30) orrequired relatively high TWEAKconcentrations (29). In vivo,surprisingly both RG7212 and agonistic anti-Fn14 antibo-dies result in tumor growth inhibition in xenograft tumormodels. RG7212, although sharing complex pathway biol-ogy with these agents, is herein characterized as a pathwayantagonist. RG7212 antitumor activity is accompanied bydownmodulation of TWEAK-induced proliferation andsurvival signaling, decreased transcription of proinflamma-tory, mitogenic, survival and immunomodulatory genesand by altered tumor immune cell composition. AlthoughTWEAK induced changes in multiple pathways, the dom-inant effect is on NF-kB signaling, inducing changes ingenes encoding secretedmitogens and immunomodulatorycytokines and receptors. These dominant effects point to a

mechanism of action of RG7212 that involves reconfigura-tion of the tumor microenvironment to promote tumorgrowth and escape from immune destruction. These phar-macodynamic effects in mice have also been observed inpatients in a phase I study.

Antitumor efficacy has been shown inmicewith agonisticanti-Fn14 antibodies, BIIB036 and PDL192 (enavatuzu-mab, ABT-361; refs. 12, 29, 30). These antibodies havedistinct properties, but each shows a complex mechanismof action including engagement of Fc receptors resulting inreceptor clustering effects and eliciting antibody-dependentcell-mediated cytotoxicity (ADCC) and activation of NF-kBsignaling (12, 16, 29, 31). In contrast, although pathwaybiology is complex due to TWEAK activation of multipledownstream signaling pathways and pathway expression inmultiple cell types, RG7212 acts by simply antagonizingTWEAK:Fn14 signaling. Here, we show that neither TWEAKnor RG7212 treatment impacted viability in 292 tumor celllines.

In malignancies, constitutive activation of NF-kB withinduction of survival and inflammation-promoting genes isa major event leading to the initiation and progression ofcancer (32).

Anti-Fn14 antibodies result in elevation of undesirableproinflammatory cytokines that are associated with humanpathology, including the development of pancreatitis (32–34). In the phase I study, pancreatitis was observed inpatients treated with PDL192 at a dose that achieved thedrug exposure target for antitumor efficacy in mice. Nocomplete or partial responses and 2 patients with stabledisease were reported.

We show that these cytokines are induced by TWEAK invitro and that RG7212, in contrast to PDL192, decreasesthese cytokines in the serum and tumors of treated mice.Importantly, RG7212-induced cytokine changes areobserved in tumormodels where complete TGI is observed.BIIB036 has also shown antitumor efficacy in mice that isdependent on full Fc effector function and is observedregardless of tumor cell line sensitivity to the antibody invitro (29) with minimal cytotoxicity only in combinationwith IFNg . In summary, both Fn14 antibodies have shownthat in vivo efficacy is at least partially mediated by ADCCand neither has shown significant cytotoxicity in vitro inmodels where antitumor activity was observed when grownas xenografts in mice, arguing direct cell killing is not thedominant mechanism of action. Expression of the pathwayin nontumor cells, including immune cells (9), and mod-ulation of immunomodulatory cytokines and chemokineswith tumor pathway modulation by either a TWEAK orFn14 antibody likely contribute to themechanismof actionof these agents.

TWEAK signaling promotes cell survival through induc-tion of AKT phosphorylation (31) and activation of NF-kBleading to upregulation of anti-apoptotic genes includ-ing BCL2L1(BCLXL) and BCL2L2(BCLW) (6). TWEAKinduces mitogen-activated protein kinase (MAPK) signal-ing in tumor and endothelial cells (32). TWEAK has beenshown to induce endothelial cell proliferation and tube

Translational RelevanceA blockade of TWEAK signaling with anti-TWEAK

mAb, RG7212, inhibits tumor growth in models withFn14 expression and pathway activation. RG7212decreased proliferation and serum immunomodulatorycytokines and chemokines, increased tumor apoptosis,and altered immune cell distribution in mice. Similarpharmacodynamic changes, including decreases intumor pERK and altered tumor immune cell content,were observed in patients with evidence of tumor shrink-age in phase I clinical testing.

TWEAK Blockade with Anti-TWEAK mAb Inhibits Tumor Growth

www.aacrjournals.org Clin Cancer Res; 19(20) October 15, 2013 5687




formation in vitro (2) and to promote angiogenesis (33)and tumor growth (34) in vivo. TWEAK plays a role ininnate and adaptive immune antitumor response. Itsabsence in TWEAK-knockout mice was shown to enhanceboth innate and adaptive immune responses to tumorchallenge (35).

As the biology of TWEAK in tumor cells is controversialand has not been broadly profiled, we examined signalingand gene expression changes in a panel of tumor celllines, investigating the impact of TWEAK on viability invitro. The reversal of these TWEAK-induced effects andtumor growth inhibition with an antibody blockade wasthen explored in vivo in xenograft models in immunode-ficient and in transplanted tumors in immunocompetentmice. Because of the potential complexity of host andtumor cell interactions in mouse tumor models, weimmunized hamsters to generate a fully cross-reactiveantibody directed against soluble TWEAK that washumanized to produce RG7212. The impact of immunefunction on antitumor efficacy was explored by using arange of mouse genetic backgrounds and by testing theeffects of specific immune cell depletion on antibodyactivity. RG7212 acts as a TWEAK antagonist, blockingTWEAK-induced signaling and cytokine production, pro-moting apoptosis, altering immune cell distribution, andinhibiting tumor growth without toxicity in mice. Inphase I testing, evidence of tumor regression, with phar-macodynamic changes consistent with those observed inmice and without toxicity, was observed in patients (36).

Materials and MethodsDetailed experimental procedures are available in Sup-

plementary Experimental Procedures.

Cell linesHuman U2OS, HCT116, ACHN, AsPC1,Calu-3, Caki-1,

AsPC-1, Panc-1, SJSA, SK-MES-1, MDA-MB-231, KPL4,U118MG,MDA-MB-436, HEC1A, 22Rv1 andmurine LewisLung carcinoma (LLC1) were purchased from the AmericanType Culture Collection and identities were confirmed viasequencing. Human umbilical vein endothelial cells(HUVEC) were purchased from Clonetics. Human NCI-H332M and murine tumor cell lines, B16BL6, B16F10,Pan02, and RENCA were obtained from the NCI CellRepository (Frederick, MD).

MiceAthymic nu/nu (nude)micewere purchased fromCharles

River Laboratories, and SCID-beigemice were from TaconicFarms. C57BL6/J and Balb/c mice were obtained fromJackson Laboratory.Mice used in studies were approximate-ly 6 to 8 weeks old. All procedures conducted according toprotocols approved by the Roche Animal Care and UseCommittee.

Generation of anti-TWEAK mAbs and ELISAsRG7212 was generated using conventional immuniza-

tion and hybridoma screening techniques. ELISA details

(TWEAK binding, TWEAK:Fn14 blocking, TWEAK quanti-tation, cytokine, and cleaved cytokeratin) are described inSupplementary Information.

Gene expression profilingAffymetrix gene array and PCR array studies described in

Supplementary Experimental Procedures.

Profiling of immune cells in mouse spleen, wholeblood, and Pan02 tumors

Single-cell suspensions were prepared from spleen andmouse tumors using gentleMACS dissociator and corre-sponding kits following manufacturer’s recommendations(Miltenyi Biotec). Dissociated cells and leukocytes wereincubated with fluorescence-labeled antibodies followedby washes using PBS containing 0.5% bovine serum albu-min (BSA). Labeled cells were fixed in 1% formaldehydesolution, and the differential cell populations were ana-lyzed using the imaging flow cytometer ImageStreamX(Amnis Corporation). Antibodies are listed in Supplemen-tary Information.

Tumor and tumor cell lysatesProtein lysate preparation for immunoblotting andELISA

and antibodies used are in Supplementary Information.

Statistical analysisIn all studies, values are expressed as mean � SEM or

box plot summaries, as indicated. Statistical analyses wereconducted by unpaired Student t test, or Mann–WhitneyU test, or linear regressions, as indicated. Differences wereconsidered statistically significant at P < 0.05. Additionaldetails can be found in the Supplementary ExperimentalProcedures.

ResultsAnti-TWEAK mAb properties

RG7212 was generated using conventional immuniza-tion and hybridoma screening techniques (SupplementaryMethods). As cross-reactive antibodies were of interest formodeling host and tumor-derived TWEAK effects in mice,Armenian hamsters were immunized with human solubleTWEAK, followedby aboostwith solublemurine TWEAK. Ahamster antibody with high affinity and strong neutralizingactivity was selected for generation of a chimeric antibody.RG7212, a humanized IgG1kmAb, was generated from thechimeric antibody using standard humanization method-ologies. mRG7212 is a murine IgG2a chimeric version ofRG7212. Four IgG1 monoclonal anti-TWEAK mAbs wereused in these studies. Identical binding and neutralizationproperties are shown in Supplementary Table S1. For sim-plicity, mAbs in this report are designated RG7212 andmRG7212.

RG7212 is a fully cross-reactive, binding human, rat, andmurine TWEAK with high affinity (Fig. 1A and B) andblocking TWEAK binding to Fn14 and TWEAK-inducedproliferation and cytokine secretion (Fig. 1B). The aminoacid sequence of Cynomolgus monkey TWEAK is identical

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Clin Cancer Res; 19(20) October 15, 2013 Clinical Cancer Research5688




to human TWEAK. Human TWEAK binds mouse Fn14 (34)and mouse TWEAK binds to human Fn14 (Fig. 1C). Cross-functionality and the cross-reactivity of RG7212 supportmodeling of the host- and tumor-derived TWEAKbiology inanimal studies. A RG7212 pharmacokinetic study inACHN-tumor bearing mice was conducted for use in com-bination with efficacy data to estimate the target drugexposure resulting in an antitumor effect (Fig. 1D). In safetystudies, animals were dosed to achieve exposures exceedingthe drug levels required for antitumor efficacy, based onsingle and multiple dose-level pharmacokinetic (PK) stud-ies, monkeys and rats were selected for toxicology studiesbased on RG7212 cross-reactivity and health authorityguidelines for rodent and non-rodent species selection.In repeat dose good laboratory practice (GLP) studies,intravenous administration of RG7212 at doses up to100 mg/kg/wk [rats; area under curve (AUC) 204 mg�h/mLCmax 2,670 mg/mL] and 50 mg/kg/wk (monkeys; AUC 211mg�h/mL, Cmax 2,110 mg/mL) for 13 weeks was well-toler-ated, with no test article-related findings. These resultssuggested a favorable safety profile for pursuing RG7212in clinical studies.

RG7212 inhibits growth in human tumor xenograft,transplanted syngeneic mouse tumor, and patient-derived tumor graft models

RG7212 antitumor efficacy was shown inmultiple tumormodels, including cell line- and patient-derived humanxenografts and transplantedmouse tumor xenograftmodels(Supplementary Table S2). Complete tumor growth inhi-bition to regression was observed in ACHN (RCC), MDA-MB-231(breast), Caki-1 (RCC), andCalu-3 (non–small celllung cancer, NSCLC) models with the highest level of Fn14expression and detectable TWEAK expression (Fig. 2A–Dand Supplementary Table S2). The antitumor effect ofRG7212 is dose-dependent after twice weekly administra-tion. RG7212 treatment resulted in statistically significantTGI with biweekly dosing �3.75 mg/kg (Fig. 2A). Togetherwith the pharmacokinetic data from the same study(Fig. 1D), the estimated average steady state concentration(Css avg) and estimated trough concentration to achieve a90% antitumor activity are 261 � 88.2 mg/mL and 167 �56.8 mg/mL, respectively.

No antitumor activity was observed with RG7212 treat-ment in either 22Rv1 prostate or KPL4 breast cancermodels

Figure 1. RG7212 properties. A, high-affinity RG7212 binding to recombinant mouse and human TWEAK (ELISA). B, RG7212 binding and neutralizingproperties. IC50 values in mouse, rat, and human TWEAK-binding ELISA, murine and human TWEAK:Fn14 blocking ELISA, TWEAK-stimulated IL8secretion (ELISA) and U2OS and HUVEC proliferation (MTT). C, murine and human TWEAK have similar activity in human Fn14-expressing U2OS cells(5-day MTT assay). D, RG7212 pharmacokinetics in ACHN tumor–bearing mice, where dose-dependent antitumor efficacy was observed. See Fig. 2A. SeeSupplementary Methods for antibody generation and characterization (Supplementary Table S1) and PK sampling protocol for modeling and efficacysummary (Supplementary Table S2).






lacking Fn14 expression (Fig. 2D and Supplementary TableS2), nor was RG7212 active in HEC1A human endometrialcarcinoma xenografts (Supplementary Fig. S1A), a cell linereported to undergo TWEAK-induced apoptosis in vitro(29). RG7212 did not inhibit and, more importantly, didnot accelerate tumor growth as might be expected with aTWEAK blockade if TWEAK signaling was a negative regu-lator of tumor growth.

RG7212 antitumor efficacy was assessed in additionalxenograft models (Supplementary Table S2) with a range ofFn14 expression (Supplementary Fig. S1B), including eval-uation of the activity of murine RG7212 in 5 transplanted,syngeneic tumor models (Supplementary Fig. S1C). Signif-icant antitumor activity was observed in syngeneic Pan02and RENCA, models with the highest level of Fn14 expres-sion (Supplementary Fig. S1D). Activity in the aggressivePan02 model was modest but comparable to the bestresponse observed with standard-of-care cytotoxic agentgemcitabine (37) or immunomodulating agents (38).Tumor growth and tumor-induced immunosuppression ismultifactorial and single-agent therapies are unlikely to becurative, but RG7212 monotherapy activity and lack of

toxicity suggest that combination therapies should beexplored.

Combination studies were conducted in Fn14-expressingsyngeneic, cell line- and patient- derived tumor models(Supplementary Table S2). In Pan02 tumors, mRG7212increased the level of antitumor efficacy of gemcitabinefrom 51% to 67% TGI (P < 0.05; Supplementary Fig.S1E). RG7212 increased the activity of cisplatin from33% TGI (P < 0.05) to 61% (P < 0.05) in patient-derivedxenograft (PDX; Champions Oncology, Inc.) PULM009(Supplementary Fig. S1F) and increased the activity ofcisplatin from 34% to 50% TGI (P > 0.05) in the paclitax-el-resistant PDX ST013 ovarian model.

In cell line–derived xenograft models, addition ofRG7212 did not antagonize standard of care agentsor targeted therapies (Supplementary Table S2). InCalu-3, combination of RG7212 with cisplatin, B20 4.1(cross-reactive anti-VEGF mAb), or both increased anti-tumor efficacy compared to the monotherapy treatments.Docetaxel efficacy was increased in both the MDA-MB-436 breast and Calu-3 models when combined withRG7212.

Figure 2. RG7212 inhibits tumor growth in tumors with high-level Fn14 expression. A, RG7212 antitumor efficacy in ACHN xenografts. TGI is dose-related andstatistically significant (P� 0.004) with�3.75mg/kg biweekly dosing. B, immunoblot and IHC show high levels of Fn14 protein in tumor cell lines that respondtoRG7212,whengrownasxenografts inmice.C, TWEAKconcentrations in conditionedmedia from tumor cell linesmeasuredbyELISA.D,RG7212 is inactivein Fn14-nonexpressing 22Rv1 prostate cancer model. Statistical analysis of xenograft data was determined by the rank-sum test and one-way ANOVA and apost hoc Bonferroni t test (10 mice per group). P values are comparisons of RG7212 to vehicle-treated mice. See also Supplementary Fig. S1 andSupplementary Table S2.

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Monotherapy and combination therapies, where combi-nation agents were dosed at the respective maximum tol-erated dose, were well tolerated with no significant bodyweight loss (�20%, P < 0.05) or evidence of gross toxicity.Exome sequencing data showed that RG7212 was active

in models from cells expressing either mutant or wild-typeBRAF, CDKN2A, KRAS, TP53, and TRAF2 (SupplementaryFig. S1G). Mutations were not identified in TWEAK or Fn14genes in responders or nonresponders. All responsive mod-els express wild-type MAP3K14(NIK), and a mutation(T763A) was found in nonresponder AsPC1. All modelsassessed in vivo expressed wild-type PIK3CA. Lack of activityin models was not correlated with mutations in CD40,LTBR, TRAF3, or TAC1, associated with constitutively acti-vated NF-kB (39). A TRAF2 mutation of uncertain func-tional significance was identified in responder Caki-1.Expression analysis suggests that downstream pathwayexpression andactivationmarkersmaybeuseful in additionto Fn14 in predicting antitumor efficacy.Quantitative PCR was used to characterize cell lines that

respond, or are refractory, to RG7212 treatment whengrown as xenografts in mice. mRNA expression with andwithout TWEAK stimulation was compared using a customPCR array (Supplementary Fig. S2A) that included NF-kBgenes and TWEAK- modulated genes. Additional candidateresponse prediction markers significantly elevated at base-line in responders were: IKBKG(NEMO), MAP3K14(NIK),STAT3, STAT5B, and TRAF1 (Supplementary Fig. S2B).NIKand TRAF1 are TWEAK-inducible, suggesting that the path-way is activated at baseline in responders possibly reflectingpathway dependence. NEMO is required for canonical andNIK for noncanonical NF-kB activation (40), suggestingthat both pathways are critical and activated in responsive,TWEAK-dependent tumors.

TWEAK induces proliferation and survival signaling intumor cellsAs there are conflicting reports in the literature regarding

the functional consequences of TWEAK stimulation in cells,proliferation, survival, and gene expression profiles wereassessed in multiple Fn14-expressing cells. These studieswere also useful for generating pharmacodynamic biomark-er candidates for evaluation in efficacy studies and clinicaltesting. TWEAK stimulation induces proliferation of U2OSosteosarcoma cells and HUVECs up to 2-fold but in tumorcell lines that are most responsive to RG7212, when grownat tumor xenografts in mice and in a large panel of tumorcell lines, no significant increase or decrease in cell numberoccurred with ligand treatment (Supplementary Fig. S3A–S3D). Neither TWEAK nor RG7212 treatment affected theviability of 299 tumor cell lines assessed in CellTiterGlo(Supplementary Fig. S3D) or in efficacy lines in MTT assays(Supplementary Fig. S3C). TWEAK did not induce apopto-sis in tumor cells, as measured by caspase-3 activation,cytokeratin-18 or caspase-3 cleavage. or detection of cyto-plasmic nucleosomes (Supplementary Fig. S3E–S3H).Signaling changes in TWEAK-stimulated tumor cells are

consistent with the magnitude of these observed effects on

proliferation and survival. TWEAK stimulation leads tomodest increases in ERK and AKT phosphorylation butdramatic increases in NF-kB signaling with induction ofIKBa and NF-kB p65 phosphorylation (Fig. 3A and Sup-plementary Fig. S3I and S3J). TWEAKsignaling is blockedbyRG7212 in tumor cells that respond to the antibody whengrown as xenografts in vivo (Fig. 3A) and is not induced intumor cells lacking Fn14 expression (Supplementary Fig.S3K). In contrast, activation of NF-kB and significant, largefold-changes in TWEAK-induced gene expression wereobserved (Fig. 3B and C, Supplementary Table S3), suggest-ing that these are likely to be important drivers of tumorgrowth and maintenance in Fn14-expressing tumors.TRAF1- and IAP-encoding genes are TWEAK inducible (Fig.3B and Supplementary Table S3 and Supplementary Fig.S3L and S3M), and levels of these proteinswere decreased intumors from RG7212-treated mice. TRAF and IAP com-plexes modulate signaling via TNF receptor superfamilymembers, including Fn14, by activating NF-kB signalingvia ubiquitin ligase activity (19, 41). TRAF-1, -2, -3, and -5have been shown to bind Fn14 (11). Ligand bindinginduces ubiquitin-mediated degradation of these proteinsand activation of NIK. TWEAK stimulation of cells leads torapid degradation of TRAF1 and c-IAP-2 proteins, transcrip-tion of both genes, and then increases in both TRAF1 and c-IAP-2protein levels (Fig. 3D). TWEAKstimulation increasedlevels of NIK and pIKBa, enhanced by addition of theproteasome inhibitor MG132. TRAF1 and IAP are amongseveral TWEAK induced proteins that are downmodulatedin tumors inhibited by RG7212 treatment in mice.

TWEAK-induced expression changes were assessed inAffymetrix gene arrays in time course experiments in 4tumor cell lines (Supplementary Table S3) and by quanti-tative PCR (qPCR) in 14 tumor cell lines. Cell lines wereselected using a Roche internal gene expression database toidentify high and non-Fn14–expressing tumor lines,derived from histologically distinct tumor types. Datadescribe the biology of TWEAK and were used to monitorpathway inhibition in mice treated with RG7212. Withrespect to understanding the role of TWEAK in drivingtumor growth, it is notable that the most strongly, consis-tently induced gene changes across a range of tumor celllines regulate cellular proliferation, survival, NF-kB path-way activity, immune cell recruitment, and function andhere we show that a number of these, at the mRNA and/orprotein level, are decreased in mice treated with RG7212.

TWEAK induces multiple mitogens (Fig. 3B and C andSupplementary Table S3). TWEAK induces genes encodingcritical protein regulators of NF-kB signaling: TRAF1,BIRC3, NFKB2, and NFKBIE with TRAF1 expression maxi-mally induced up to 10-fold (Fig. 3B and SupplementaryTable S3) andmore than 2-fold in themajority of tumor celllines tested (Supplementary Fig. S3L).

Cellular inhibitors of apoptosis, c-IAP-1 and -2 proteins(encoded by BIRC2 and BIRC3 genes), are required forTWEAK-induced activation of NF-kB and MAPK signaling(19). BIRC3 was increased up to 97-fold in gene arraystudies (Fig. 3B) and more than 2.5-fold in 13 of 14






Fn14-expressing human tumor cell lines by qPCR (Supple-mentary Fig. S3M) NF-kB inhibitor epsilon (NFKBIE),encoding an NF-kB inhibitory protein regulator of canon-ical signaling (40),was induced inbyTWEAK inmost tumorcell lines (Fig. 3B and Supplementary Fig. S3N).

TWEAK induced gene expression shapes the tumormicroenvironment and may promote escape fromantitumor immune mechanisms

TWEAK-induced gene changes include increases inexpression of critical regulators of tumor immune cell com-position and function (Fig. 3B and C and SupplementaryTable S3). In vitro, TWEAK was shown in Affymetrix arrays(Supplementary Table S3) andbyqPCR(Fig. 3C) to increaseexpression of cytokine and chemokine genes in tumor celllines. Among these were CCL2, CXCL3 (mouse orthologCXCL2), CCL5, CCL7, CSF1, CSF2, CCL20, IL8, and IL6.

In addition to these factors that modulate immune cellrecruitment, TWEAK was shown to induce CD274 (PDL1,B7-H1), encoding the ligand for the inhibitory T-cell recep-tor, CD279 (programmed death-1, PD-1, PDCD1; Fig. 3C).

TNFa, a suggested factor in TWEAK-induced apoptosis, inthese expression studies across multiple cell lines was onlyminimally modulated at the first time point in U2OS, cellsthat do not die but proliferate in response to TWEAK. Acrossmultiple cell lines in Affymetrix arrays, TWEAK decreasedexpression of only 2 genes, SORBS2 and PRICKLE, inmore than 1 cell line andmore than 2-fold (SupplementaryTable S3).

TWEAK-induced effects are reversed in RG7212-treatedmice

Expression of a number of TWEAK-induced genes wassignificantly downregulated in tumors fromRG7212-treated

Figure 3. TWEAK induces proliferation, survival, and immune signaling and gene expression. A, RG7212 blocks TWEAK-induced signaling. B, most highlyTWEAK-induced genes across 4 Affymetrix gene arrays promote survival, TNFAIP3, VCAM1, BIRC3, TNFRSF9; regulate NF-kB activation: NFKB2, TRAF1,TNFAIP3, BIRC3, NFKBIE; leukocyte recruitment: CSF2, IL8, CCL2; proliferation: HBEGF. Data shown for models with in vivo efficacy data, ACHN,MDA-MB-436, and Pan02 and for U2OS that proliferates in response to TWEAK in vitro. Expression changeswith 8-hour TWEAK stimulation shown; changesat 1, 4, 8, and 24 hours in Supplementary Table S1. �, BIRC3 mRNA 97-fold increase with TWEAK stimulation in U2OS cells was not plotted to maintainscale. C, heatmap representation of TWEAK-induced cytokines by qPCR in Fn14-expressing cells. Values shown represent highest fold changewith 4-, 8-, or24-hour TWEAK stimulation. All changes are significant by P < 0.05. Uncolored squares represent no statistical increase. No changes in any genes innonexpressing Fn14 line 22Rv1 by qPCR or in B16BL6 by Affymetrix gene array with TWEAK stimulation (data not shown). Five-fold increase in CXCL3 inACHN not quite significant P¼ 0.06. Not included: Panc-1 only increases: <1.5-fold in CSF1 and IL8 and H322M only increase: 2.5-fold in CCL5 (all changesP < 0.05). D, TRAF1 and c-IAP-2 proteins initially degraded then increased in TWEAK-stimulated cells. Immunoblots: Caki-1 and ACHN cell lysates fromTWEAK (100ng/mL)-stimulated cells. TWEAK increasesNIKandpIKBa; pretreatmentwithMG132 (25mmol/L) for 1hour at 37�C leads to accumulation of bothbut does not lead to increases in TRAF1 or c-IAP-2. See also Supplementary Fig. S3C, S3J, S3B, and Table S3.

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mice (Fig. 4A and Supplementary Table S3). Tumor mRNAlevels of TWEAK-inducible genes: CCL2, CXCL3, CXCL11,CXCL10, PD-L1, IL8, TNFAIP3, TNFRSF9, andNFKBIE andmRNAs encoding genes not induced by TWEAK: CCR5,CXCL9, and PD1 were decreased 24 hours following thefirst dose of RG7212 (Fig. 4A). These genes encoding cyto-kines, chemokines, and receptors regulate immune cellnumbers, distribution, and function. The roles of theseregulators of immune cell distribution and function inmediating the antitumor activity of RG7212 are an activearea of current investigation.RG7212 modulation of several additional pharmacody-

namic serum and tumormarkers was shown inmice. Dose-related decreases in serum and tumor levels of TWEAK (Fig.4B, Supplemental Figure S4A) and levels of human CCL2,IL8, and IL6 and murine EGF, bFGF, and MMP9 proteins

were significantly decreased in the serum from RG7212-treated tumor-bearing mice (Fig. 4C and D and Supple-mentary Fig. S4B–S4E). Of the murine proteins decreased,only MMP9 expression is TWEAK inducible. Host tissueexpression changes are intriguing as EGF and bFGF areknown to be key tumor-promoting mitogens secreted bytumor-associated macrophages (42) and TWEAK induces,and RG7212 treatment reverses, expression of several mac-rophage recruiting chemokines.

Downregulation of TWEAK-induced signaling wasshown in tumors from RG7212 treated mice includingdecreases in pERK, pAKT, c-IAP-1, and c-IAP-2 (Fig. 5A).Tumor shrinkage and decreases in pERK (Fig. 5B) and Ki67were also observed in a patient with melanoma afterRG7212 treatment (36), supporting that proliferation isdecreased in tumors with RG7212 treatment. Levels of

Figure 4. RG7212-induced pharmacodynamic changes in tumor-bearing mice. A, RG7212 treatment decreases expression of TWEAK induced genes. Foldchanges in ACHN tumor cell mRNA with TWEAK stimulation (blue). Fold changes in mRNA with RG7212 treatment 24 hours after first dose (red). �, all mRNAchanges in ACHN cells or xenografts except PDL1 induced in MDA-MB-436 in vitro with TWEAK stimulation and CXCL3 mRNA decreased in Caki-1xenografts after RG7212 treatment. AllP and fold change values are in Supplementary Table S3. B, levels of humanTWEAKare decreased inCaki-1 xenografttumors (per 100mgprotein) and serum fromRG7212-treatedmice. Decreaseobservedwithin 4hours in tumors and ismaintainedwith repeat dosing in serum.Free TWEAK was quantitated by ELISA. Murine TWEAK was below the limit of detection in serum and decreased in tumors with RG7212 treatment (data notshown). C, RG7212 treatment decreases serum levels of human CCL2 protein� 3.5-fold 24 hours (P ¼ 0.002) and 18-fold 72 hours (P ¼ 0.0002) followingthe first RG7212 dose in ACHN tumor–bearing mice.�� D, MMP9 decreased in serum from ACHN, Caki-1, or Calu-3 tumor–bearing mice 24 hoursafter first dose (P<0.05)��. �MyriadRBMassays. Serawere collected 4, 8, 24, and72hours following the first dose and6hours following thedosesday 7, 14, or21.n¼5per timepoint.MultiplexedELISA (MyriadRBM,Human inflammationMAP, andRodentMAPversion 2.0). ��, differencebetween serum fromRG7212and vehicle-treated mice. Data are mean � SEM (n ¼ 5 mice/serum samples per time point). Statistical analysis by Mann–Whitney U test. See alsoSupplemental Fig. S4A–S4F for other serum and tumor PD effects.






TWEAK-inducible TRAF1 mRNA and protein (Fig. 5C andSupplemental Fig. S4F) and IAP proteins (Fig. 5A) weredecreased in tumors from RG7212-treated mice. By quan-titative immunohistochemistry (IHC), TRAF1 protein wasdecreased more than 50% in tumors in mice and in patienttumor biopsies in the phase I study following RG7212dosing (36).

Both murine NFKBIE mRNA (Fig. 4A) and levels of acti-vated NFKB2 (p52) protein (Fig. 5D) were decreased relativeto control tumors showing RG7212 modulation of bothcanonical and noncanonical NF-kB signaling, respectively.

Contrary to reports of TWEAK-inducing apoptosis in vitro,we observed evidence of apoptosis inductionwith a TWEAKblockade by RG7212 in our tumor models. Apoptoticsignaling, assessed using a cleaved caspase-3 (CC3)-specificIHC antibody and image quantitation and PARP cleavagewith immunoblotting, was increased in tumors fromRG7212-treated mice (Fig. 6A). Increased ACHN tumorCC3 staining was detected 8 and 24 hours following thefirst dose. No changes in CC3 or in PARP cleavage were

detected in tumors from B20.4.1 or erlotinib-treated mice(data not shown). PARP cleavage was more dramatic inCaki-1 tumor lysates in a study where tumor regression wasobserved compared to that observed in the ACHN model,where treatment resulted in tumor stasis. These and genearray data are consistent with a prosurvival, tumor-promot-ing function for TWEAK. TWEAKwas shown to dramaticallyincrease the expression of inhibitors of apoptosis (Fig. 3B)including BIRC3 (19) and TNFAIP3 (43), encoding c-IAP-2and A20 proteins, respectively.

RG7212 efficacy is dependent on immune cell functionThe results of these investigations aswell as the published

data in TWEAK knockout mice suggest a role of TWEAKin modulating antitumor immune response. Preliminary,intriguing results showing that higher level immune func-tion in tumor-bearing mice increases the level of antitumoractivity of RG7212 (Supplementary Table S2), that treat-ment with RG7212 in immunocompetent mice altersimmune cell distribution (Fig. 6B), and that specific

Figure 5. RG7212 blocks TWEAK-induced signaling in tumor-bearing mice and phase I patient. A, RG7212 decreases pERK, pAKT, c-IAP-1, andc-IAP-2 proteins in tumor lysates by immunoblotting. B, pERK IHC from melanoma patient biopsy collected 7 days following first dose. C, immunoblotsshowing TRAF1 protein is decreased in Caki-1 tumors from RG7212-treated mice throughout the course of treatment. IHC showing TRAF1 decreasefollowing second RG7212 dose. See also BIRC3 and TRAF1 mRNA induced before protein increases with TWEAK stimulation (Supplementary Table S3,Fig. S3L and S3M). D, RG7212 inhibits noncanonical NF-kB signaling in ACHN tumors. Levels of activated NFKB2 (tumor p52 are decreased 24 and72 hours following the first dose of RG7212.

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immune cell depletion dramatically alters the level of anti-tumor efficacy of RG7212 (Fig. 6C).TWEAK knockout mice showed enhanced antitumor

immune effects. In the MDA-MB-231 model, TGI was91% in nude and 48% in SCID beige mice. This loss ofantitumor activity was observed in 2 additional models inSCID beige mice. Nude mice have functional natural killer(NK) cells, lack thymic function, and have decreased num-bers of T cells. Mice with both SCID and beige mutationslack T, B, and NK cells. Both strains have functionalmacrophages.In C57BL6 immunocompetent mice with Pan02 tumors,

at the end of RG7212 treatment, CD3þ T cells weresignificantly increased in blood and spleen and slightly,but not significantly increased in tumors (Fig. 6B). Mono-

cytes/macrophages (CD11bþ/F4/80þ) were significantlydecreased in blood and significantly increased in tumorsfrom mRG7212-treated mice. A significant increase in totalleukocytes (CD45þ) andno significant changes inB cells,NKcells, or monocytes (CD11bþ/F4/80�) with RG7212 treat-ment were detected. Increases in tumor and spleen T-cellnumbers were reported for TWEAK-knockout mouse studies(35). These data show the impact of RG7212 treatment onimmune cell distribution after chronic treatment but maynot reflect earlier changes coincident with dramatic cytokineand chemokine changes observed early in efficacy studies.

To examine the role of specific immune cells in antitumorefficacy of mRG7212, immune cell depletion experimentswere carried out with in C57BL6 mice with Pan02 tumors.The efficacy ofmRG7212was evaluated in 3 groups ofmice:

Figure 6. Apoptosis induction and immunemodulation are components of themechanism of action of RG7212. A, apoptosis induced in tumors fromRG7212-treated mice: PARP cleavage by immunoblotting with total anti-PARP antibody (ACHN tumor lysates, day 21 of study). PARP cleavage detectedthroughout treatment course (RG7212 20 mg/kg twice weekly) in Caki-1 lysates prepared from tumors collected from treated mice. CC3 quantitated inspecimens by IHC with imaging (n ¼ 5). B, RG7212 treatment alters immune cell distribution. Immune cell populations in blood, spleen, and Pan02tumors quantitated using ImageStream Flow cytometry. mRG7212 treatment increases CD3þ T cells in blood and spleen, decreases blood, and increasestumor macrophages (CD11bþ/F4/80þ) and increases total tumor leukocytes (CD45þ). Each dot is sample from one mouse. C, antitumor efficacy ofmRG7212 is dependent on CD8þ T and NK cells and is increased with depletion of CD4þ T cells. mRG7212 efficacy with immune cell depletion study design.All antibodies dosed at 20 mg/kg. Tumor growth curves with RG7212 treatment with specific immune cell depletion. D, RG7212 increases tumor T-cellinfiltration. Representative CD3 IHC from phase I patient biopsies.






depleted ofCD4þ, CD8þ, orNK cells before RG7212dosingand throughout the study (Fig. 6C). Depletion of NK cellsand CD8þ T cells led to a complete loss of mRG7212antitumor efficacy, suggesting a role for TWEAK in suppres-sing the antitumor immune response through suppressionof these immune cell populations. Conversely, depletion ofCD4þ T cells appears to improve the antitumor efficacy ofmRG7212. Clearly, antitumor activity with RG7212 isimpacted by immune system components and multipleimmunomodulatory factors are modulated with antibodytreatment.

As in the mouse tumors, increased tumor T-cell infiltra-tion (Fig. 6D) was observed after treatment in the patientwith melanoma where other pharmacodynamics changespresented here were observed (Fig. 5B). Consistent with theexpected changes in immune cell recruitment from thecytokine changes inmice, a decrease in tumormacrophageswas observed the melanoma tumor (36).

DiscussionRG7212has shown antitumor efficacy inmultiple diverse

tumor models in mice with efficacy associated with tumorFn14 expression and pathway activation. Expression ofFn14 has been shown to be upregulated in many humancancers, andRG7212 efficacy is independent of TP53, BRAF,and KRAS mutational status, suggesting that RG7212 mayprovide broad clinical benefit in patients with TWEAK-driven tumors.

Our data show that TWEAK induces signaling and geneexpression to promote tumor cell proliferation, survival,andNF-kB signaling and these effects are reversed in tumorsfromRG7212-treatedmice. TWEAK also induces expressionof a number of mitogens and cytokines and chemokinesthat interact with host cells within and beyond the tumormicroenvironment.

TWEAK-induced factors modulate T-cell and monocyterecruitment, T-cell activation, and macrophage differentia-tion and these were downregulated in RG7212-treatedmice. PDL1, CCL2, CXCL-9, -10, and -11 may providemechanisms of escape from an antitumor immuneresponse, suggesting that RG7212 may represent a novelapproach to overcoming these ligands. Antagonistic PD1andPDL1 antibodies show evidence of antitumor activity inpatients with melanoma, kidney, and NSCLCs (44). T-cellregulating CXCL-9, -10, and -11, CXCR3 ligands may playimmunosuppressive roles in cancer (42). This has beenshown for CXCL11, a chemoattractant for T cells. In RCCspecimens, its expression was correlated with increasedtumor Foxp3þ T cells with expression characteristics offunctional Tregs (45). TWEAK strongly induces CCL2 andCSF1 that recruit and induce differentiation of tumor-pro-motingmacrophages, respectively. TAMS also impact T andNK cell tumor infiltration and activation to suppress adap-tive immune responses (42).

Blocking TWEAK signaling inmice and humans alters thetumor immune cell composition including tumor macro-phages that express both TWEAK and Fn14 (9). RG7212-induced changes in the microenvironment may influence

both TWEAK signaling in macrophages, as well as theirphenotype (cytotoxic M1 vs. tumor-promotingM2). Futurestudies will look at the evolution of the immune cellchanges during treatment to define initiating events and tounderstand how a TWEAK blockade remodels the immunesystem over time in treated mice.

Consistent with decreased tumor growth and enhancedantitumor immune response in TWEAK knockout mice,RG7212 activity is dependent on immune cells. Immunecell redistribution with RG7212 treatment and depletionstudies show that TWEAK plays a role in immune cellrecruitment, defining the tumor immune cell content, andRG7212 antitumor efficacy is dependent on NK and CD8þ

T-cell function and is diminished by CD4þ T cells.Further studies are needed to assess the effects of RG7212

monotherapy and combination therapies on T-cell func-tion, macrophage phenotype, and NK activation to specif-ically ask whether treatment enhances tumor cell killing byCD8þ cells and to characterize CD4þ cells and tumormacrophage populations.

Together our results suggest that TWEAK signaling pro-motes tumor growth and survival, establishes a tumormicroenvironment that supports tumor growth, and inter-acts with the host immune system to suppress tumordestruction. RG7212 treatment reverses these effects andinhibits tumor growth in mice.

Preliminary results from the phase I study of RG7212show decreased tumor cell proliferation and pathway activ-ity plus immune cell changes in patients that are consistentwith the mechanism of action for RG7212 emerging fromthese mice studies.

Disclosure of Potential Conflicts of InterestT.A. Lin is employed as Research Leader/Principal Scientist in Hoffmann-

La Roche, Inc. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: X. Yin, L. Luistro, K. Packman, J. Rosinski, M.E.SimcoxDevelopment ofmethodology:X. Yin, L. Luistro, D. Biondi, J. Rosinski, W.Berkofsky-Fessler, S. PantAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): X. Yin, L. Luistro, M. Smith, T. Nevins, H. Hilton,T.-A. Lin, T. Truitt, D. Biondi, X. Wang, S. Pant, S. Vega-HarringAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):X. Yin, L. Luistro,H. Zhong,H.Hilton, T.-A. Lin, T. Truitt, D. Biondi, X. Wang, K. Packman, J. Rosinski, W. Berkofsky-Fessler, J.-P. Tang, S. Pant,D.Geho, S. Vega-Harring,H. Levitsky,M.E. SimcoxWriting, review, and/or revision of themanuscript: L. Luistro, H. Zhong,K. Schostack,H.Hilton, X.Wang, J. Rosinski,W. Berkofsky-Fessler, J.-P. Tang,S. Pant, D. Geho, M. DeMario, H. Levitsky, M.E. SimcoxAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): L. LuistroStudy supervision: L. Luistro, K. Packman

AcknowledgmentsThe authors thank Susanne Arbogast, Antje-Christine Walz, Lisa Culton,

MartinWeisser, Tobias Bergauer, Alvin Stern, Julie Hang, andUeli Gubler forhelp with experiments and Wei Xu for his careful review of the article.

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 February 12, 2013; revised July 24, 2013; accepted August 7,2013; published OnlineFirst August 23, 2013.

Yin et al.





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2013;19:5686-5698. Published OnlineFirst August 23, 2013.Clin Cancer Res Xuefeng Yin, Leopoldo Luistro, Hua Zhong, et al. Enhancing the Host Antitumor Immune ResponseInhibition of Tumor Cell Proliferation and Survival Signaling and by RG7212 Anti-TWEAK mAb Inhibits Tumor Growth through

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