combined immunostimulatory monoclonal antibodies extend...

Cancer Therapy: Preclinical

Combined Immunostimulatory Monoclonal AntibodiesExtend Survival in an Aggressive Transgenic HepatocellularCarcinoma Mouse Model

Aizea Morales-Kastresana1, Miguel F. Sanmamed1,2, Inmaculada Rodriguez2, Asis Palazon1, Ivan Martinez-Forero1, Sara Labiano1, Sandra Hervas-Stubbs1, Bruno Sangro3, Carmen Ochoa1, Ana Rouzaut1,Arantza Azpilikueta1, Elixabet Bola~nos1, Maria Jure-Kunkel4, Ines G€utgemann5, and Ignacio Melero1,2

AbstractPurpose: Immunostimulatory monoclonal antibodies (ISmAb) that unleash antitumor immune

responses are showing efficacy in cancer clinical trials. Anti-B7-H1 (PD-L1) monoclonal antibodies (mAb)

block a critical inhibitory pathway in T cells, whereas anti-CD137 and OX40 mAbs provide T-cell

costimulation. A combination of these ISmAbs (anti-CD137 þ anti-OX40 þ anti-B7-H1) was tested using

a transgenic mouse model of multifocal and rapidly progressing hepatocellular carcinoma, in which c-myc

drives transformation and cytosolic ovalbumin (OVA) is expressed in tumor cells as a model antigen.

Experimental Design: Flow-cytometry and immunohistochemistry were used to quantify tumor-

infiltrating lymphocytes (TIL) elicited by treatment and assess their activation status and cytolytic potential.

Tolerance induction and its prevention/reversal by treatmentwith the combinationof ISmAbswere revealed

by in vivo killing assays.

Results: The triple combination of ISmAbs extended survival of mice bearing hepatocellular carcinomas

in a CD8-dependent fashion and synergized with adoptive T-cell therapy using activated OVA-specific TCR-

transgenic OT-1 and OT-2 lymphocytes. Mice undergoing therapy showed clear increases in tumor

infiltration by activated and blastic CD8þ and CD4þ T lymphocytes containing perforin/granzyme B and

expressing the ISmAb-targeted receptors on their surface. The triple combination of ISmAbs did not result in

enhanced OVA-specific cytotoxic T lymphocyte (CTL) activity but other antigens expressed by cell lines

derived from such hepatocellular carcinomas were recognized by endogenous TILs. Adoptively transferred

OVA-specific OT-1 lymphocytes into tumor-bearing mice were rendered tolerant, unless given the triple

mAb therapy.

Conclusion: Extension of survival and dense T-cell infiltrates emphasize the translational potential of

combinational immunotherapy strategies for hepatocellular carcinoma. Clin Cancer Res; 19(22); 6151–62.

�2013 AACR.

IntroductionImmunotherapies with cytokines, vaccines, and T-cell

adoptive therapy have been tested in early clinical trials

for hepatocellular carcinoma with minor impact on thecourse of the disease (1–4), with the exception of a pilotstudy using adoptive T-cell therapy (5). Immunostimu-latory monoclonal antibodies (ISmAb) have recentlyemerged as a new therapeutic tool in oncology (6, 7).Their overall mode of action is the enhancement of theweak ongoing immune responses present in patients withcancer. Two types of ISmAbs can be categorized depend-ing on the receptors bound on immune system cells:agonist monoclonal antibodies (mAb) for activatingreceptors (7) and antagonist mAbs for coinhibitory recep-tors (8).

Antagonist ISmAbs directed to coinhibitory receptors areattracting much attention following approval of an anti-CTLA-4 mAb (9) to treat metastatic melanoma and theunprecedented clinical responses elicited by antibodiesdirected to PD-1 (CD279; ref. 10) and B7-H1 (PD-L1 orCD274) in melanoma, renal cell carcinoma, and lungcancer (11).

Authors' Affiliations: 1Centro de Investigaci�on M�edica Aplicada (CIMA),Universidad de Navarra; 2Department of Oncology, Clinica Universidad deNavarra; 3Liver Unit, Clínica Universidad de Navarra and Centro deInvestigaci�on Biom�edica en Red de Enfermedades Hep�aticas y Digestivas,Pamplona, Spain; 4Oncology Drug Discovery division, Bristol-MyersSquibb, Lawrenceville, New Jersey; and 5Department of Pathology, Uni-versity of Bonn, Bonn, Germany

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

I. Gutgemann and I. Melero share credit for senior authorship.

Corresponding Author: Ignacio Melero, CIMA Avenida PIO XII, 55 31008Pamplona. Spain. Phone: 34-948 194700; Fax: 34-948-194717; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-13-1189

�2013 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 6151

on December 28, 2019. © 2013 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst September 12, 2013; DOI: 10.1158/1078-0432.CCR-13-1189

http://clincancerres.aacrjournals.org/

Treatment of transplantable mouse models with agonistantibodies as monotherapies has shown clear signs ofefficacy in the case of anti-CD137 (12), anti-OX40 (13),anti-CD40 (14), and anti-GITR (15) mAbs. Beyond mono-therapies, these agents can be used in combinatorialapproaches, in which synergy is often observed againsttransplantable tumors (7, 16). Moreover, synergy has alsobeen observed on carcinogen-induced sarcomas using acombination that included anti-CD40 and anti-CD137mAbs (17).

Spontaneous carcinomas which arise in oncogene-trans-genic mice are highly resistant to immunotherapyapproaches (18, 19) and are likely to represent a morepredictivemodel for translational research. To date no signsof efficacy have been reported beyond early precancer stagesof development (20). A model of multifocal hepatocellularcarcinoma has been generated in which transgenic humanc-myc and cytosolic chicken ovalbumin (OVA) are underthe transcriptional control of a tet-off system (21). Ifdeprived of doxycycline around birth, such mice developlethal multifocal hepatocellular carcinoma clearly seenupon microscopic examination at 3 weeks of age. Almost100% of suchmice die of massive tumor burden within thefirst 10 weeks of life. Importantly, deprivation of doxycy-cline later in life does not result in such rapid and aggressivemalignancies and tumors occur with lower penetrance andhigher latency. It has been demonstrated in thismodel, thatrestingOVA specificCD8þOT-1T cells are rendered tolerantupon adoptive transfer into hepatocellular carcinoma-bear-ing mice (21). Strains of c-myc transgenic mice that do notcoexpress OVA developed tumors with similar penetranceand age onset (21). As various treatment attempts usingimmunization strategies have failed in these mice (ref. 21and data not shown), we tested a combination of ISmAbstargeting CD137, OX40, and B7-H1 (PD-L1) administeredat 3 weeks of age. Surprisingly, the survival of suchmice wasclearly extended and the presence of an abundant andactivated T-cell infiltrate in tumor nodules was observed.

Materials and MethodsMice

For tumor induction experiments, c-myc OVA mice werecrossed with tTALAP transgenic mice to generate c-myc-OVA-tTALAP double transgenic mice (c-myc OVA tgþ)maintaining the presence of doxycycline in drinking waterduring pregnancy as described (21). Doxycycline wasremoved at birth, to induce the expression of the c-myconcogene and ovalbumin in the liver. Rag1�/� transgenicmice and T-cell receptor transgenic mice specific for H-2Kb

OVA257-264 (OT-1) and H-2IAb OVA323-339 (OT-2) werepurchased from Jackson Laboratories. C57Bl/6 mice werepurchased fromHarlan Laboratories. All animal procedureswere conducted under institutional guidelines that complywith national laws and policies (study number 054/10).

Reagents and cell linesEL-4 thymoma cell line was obtained from the American

TypeCultureCollection.OVA-transfected EL-4 cell line, EG-7, was a kind gift fromDr. Claude Leclerc (Institut Pasteur).MC38 was provided by Dr. Karl E. Hellstr€om (University ofWashington, Seattle). These cell lines were authenticated byIdexx Radil (Case 6592-2012). Hepa 1.6 hepatoma cell linewas a kind gift of Dr. Rub�en Hern�andez (CIMA, Pamplona,Spain) and MC38OVA was generated by lentiviral tranfec-tion (SanchoD. and colleagues;manuscript in preparation)and provided by Dr. Sancho (Centro Nacional de Investi-gaciones Cardiovasculares, Madrid, Spain). Hybridomas,antibody production and generation of JMJ cells aredescribed in supplementary methods.

Adoptive T-cell therapy and antibody treatmentSpleens from OT-1 and OT-2 transgenic mice were

mechanically disrupted and cell suspensions were culturedwith 5mg/ml of the cognate OVA peptides (described insupplementary methods) for 48 hours (OVA257-264 andOVA323-339, respectively). A total of 2 � 106 OT-1 cells and2 � 106 OT-2 cells were intravenously transferred to c-mycOVA tgþmice on day 21 or 28 after birth, depending on theexperiment. For survival experiments, a dose of 2 � 104 IUof i.p. IL-2 was administered to mice transferred withactivated OT-1 and OT-2 on day 21 or 28. For treatmentwith mAbs, Combo3 (100 mg of each anti-CD137, anti-OX40 and anti-B7-H1) or control rat IgG was administeredintraperitoneally on the indicated days. For depletionexperiments, 3 doses of 200 mg of anti-CD4 or anti-CD8bmAbs were injected intraperitoneally starting one daybefore Combo3 treatment onset and every 3 days.

Phenotypic analyses of tumor-infiltrating lymphocytesFor analysis of tumor-infiltrating lymphocytes,micewere

treated with two doses of control rat IgG, the single immu-nostimulatory mAbs, pairs of immunostimulatory mAbs orthe Combo3 (anti-CD137, anti-OX40 and anti-B7-H1)antibody combination and sacrificed on the indicated days.Livers were excised, weighted, and enzymatically disruptedwith DNase I and collagenase D (both from Roche) for

Translational RelevanceAdvanced hepatocellular carcinoma remains an

unmet need in cancer therapy. Immunostimulatorymonoclonal antibodies anti-PD-1, B7-H1 (PD-L1),CD137, and OX40 are under intensive developmentin clinical trials in which these novel agents are testedas monotherapies for patients with cancer with veryencouraging results. This study provides evidence forsynergistic therapeutic effects of a combination ofthese antibodies against autochthonous liver cancerarising in c-myc transgenic mice. Evidence for a moreintense and active immune infiltrates in the sponta-neous tumors and interference with immune toleranceestablishment provide a strong rationale for testingthese feasible combination strategies in patients withhepatocellular carcinoma.

Morales-Kastresana et al.

Clin Cancer Res; 19(22) November 15, 2013 Clinical Cancer Research6152




15 minutes at 37�C. To obtain unicellular cell suspensions,livers were mechanically disrupted and passed through a70-mm cell strainer (BD Falcon, BD Bioscience) pressingwith a plunger. To remove non-mononuclear cells, unicel-lular cell suspensions were pelleted, resuspended in a 35%Percoll gradient and centrifuged for 10 minutes, 500 � gat room temperature. Erythrocytes were lysed with ACKbuffer (Gibco). Single-cell suspensions were treated withFcR-Block in a PBS-based buffer containing 10% of fetalcalf serum to avoid unspecific staining. Flow cytometryand immunohistochemistry analysis were performed asdescribed in Supplementary Methods.

Statistical analysisPrism software (GraphPad Software) was used to analyze

tumor infiltrating lymphocytes, specific lysis, and IFN-gproduction between groups by applying unpaired Studentt tests or U-Mann–Whitney tests. Survival curves wereanalyzed by Kaplan–Meier plots and log rank tests. P values< 0.05 were considered significant.

ResultsExpression of target molecules for anti-CD137, OX40,and B7-H1 (PD-L1) mAbs in the tumormicroenvironment of a transgenic c-myc–drivenhepatocellular carcinoma modelImmunotherapy with ISmAbs needs the target molecules

to be expressed at the cell surface as a requirement forbiologic effects. CD137 and PD-1 are not constitutivesurface proteins on T cells but become expressed followingantigen-elicited activation (22, 23). OX40 is also upregu-lated fromdim levels of expression tohigh levels uponT-cellactivation (24). Indeed, we confirmed with OT-1 (CD8þ)and OT-2 (CD4þ) TCR transgenic T cells that, if theselymphocytes are cultured in the presence of their cognateovalbumin peptides, they acquire bright expression of PD-1, B7-H1 (PD-L1), CD137, and OX40 (Supplementary Fig.S1A).To explore the potency of the therapeutic effects of

ISmAbs directed to such targets (CD137, OX40 and B7-H1) in vivo, we selected an aggressivemodel of spontaneoushepatocellular carcinoma: c-myc OVA tgþ transgenic mice(21). In these mice, concurrent c-myc and ovalbuminexpression can be induced specifically in the liver in atime-dependent manner. In the C57Bl/6 background, ifdoxycycline is removed from the drinking water at birth,mice develop multifocal tumors resembling human hepa-tocellular carcinomas within 2 to 3 weeks (SupplementaryFig. S2A). OVA-encoding mRNA is detectable at day 5following doxycycline deprivation from themother’s drink-ing water (Supplementary Fig. S2B). Carcinomas progressrapidly with tumor cells that characteristically express theOVA protein in the cytoplasm, while the surroundinghealthy liver cells do not express OVA protein by immu-nohistochemistry (21).Cell suspensions from the livers of suchmice at 3weeks of

age show that FACS-gated CD8þ and CD4þ lymphocytes

express PD-1 and B7-H1 at bright levels. We also documen-ted that CD4þ T cells expressedCD137 andOX40,while theexpression on CD8þ T cells was barely detectable or absent(Supplementary Fig. S1B).

Moreover, immunohistochemical data indicated B7-H1expression on stromal and endothelial cells in situ (I.G€utgemann and colleagues; Manuscript in preparation).Hence, concomitant expression of the target molecules forthree ISmAbs at tumor microenvironment led us to testthese ISmAbs in combination as an immunotherapy strat-egy for autochthonous liver tumors.

A combination of anti-CD137, anti-OX40, and anti-B7-H1 (PD-L1) mAbs extends survival and synergizes withadoptive T-cell therapy

To assess the therapeutic effects of these combinations, c-myc OVA tgþmice were deprived of doxycycline at birth toinduce hepatocellular carcinomas and treated on day 21and 25 of extrauterine life with two doses of 100 mg ofanti-CD137 (1D8), anti-OX40 (OX86), and anti-B7-H1(10B5) mAbs (Combo3). Of note, mice at this age havehistologic evidence of multiple tumor nodules (Supple-mentary Fig. S2A). As can be seen in Fig. 1A, miceundergoing Combo3 treatment had a significantlyextended survival if compared with those treated withcontrol antibody, and 2 of 10 mice were still alive 15months later.

In the same experiment, we also treated a group ofmice on day 21 with 2 � 106 activated OT-1 cells plus2 � 106 activated OT-2 cells i.v. followed by 2 � 104 IUof IL-2 i.p. This group of mice did not experience anyimprovement in terms of overall survival (Fig. 1A).However, if mice received both such adoptive T-celltransfer and the Combo3 regimen, survival was dramat-ically extended in comparison with the Combo3 group.In this group, 4 of 11 mice were alive 15 months aftertreatment.

Figure 1B shows representative data of ultrasound exam-inations performed on mice from each experimental treat-ment group at week 6. Photographs of the abdomen showliver enlargement and abdominal distension with lessabdominal distension in treated animals.

Next, we set up an independent experiment to analyse thecontribution of each antibody given separately versus theCombo3 regimen. As can be seen in Fig. 2A, 10 of 37 (27%)mice survived in the Combo3 group. In contrast, none ofthe groups treated with single antibodies showed any sta-tistically significant improved survival. Nonetheless, anti-B7-H1 mAb monotherapy showed a marginal survivalbenefit with 2 of 17 (11.8%) mice alive at the end of thisexperiment.

We have recently carried out a clinical trial with theanti-CTLA-4 mAb tremelilumab in advanced hepatocel-lular carcinoma patients with signs of clinical activity (3partial responses in 17 patients evaluated; ref. 25). As aconsequence, we tested two doses of 100 mg of anti-CTLA-4 mAb in the transgenic mice with no evidence forany contribution to extended survival either as a single

Combined Immunostimulatory mAbs for Spontaneous Hepatocellular Carcinoma

www.aacrjournals.org Clin Cancer Res; 19(22) November 15, 2013 6153




agent or when given concomitantly on top of the Com-bo3 regimen (Combo4; Supplementary Fig. S3).

To explore the immune cell requirements for the thera-peutic activity observed upon Combo3 treatment we selec-

tively depleted from the animals CD8bþ T lymphocytes orCD4þ T cells at the time of therapy instigation (Fig. 2B).Only CD8þ T cells were absolutely required to extendsurvival in this setting.

Figure 2. The triple combinationof ISmAbs is efficacious anddependent on CD8þ lymphocytes,whereas each mAb givenseparately does not extendsurvival. A, experiments performedas in Fig. 1A, but in this case 100 mgof individual antibodieswere dosedto the indicated groups. B, micetreated as in Fig. 1A were depletedwith anti-CD8b or anti-CD4 mAbsgiven on days 20, 23, and 26.Fractions of surviving mice at day250 (A) and 150 (B) in the indicatedgroups are provided in the legend.P values were analyzed by log ranktest. ns, non significant; � P < 0.05;��, P < 0.001.

Figure 1. Survival benefit of c-mycOVA tgþ mice developinghepatocellular carcinomas uponcombined treatment with anti-CD137, OX40, and B7-H1 ISmAbs,and its enhancement by adoptiveT-cell therapy. A, Kaplan–Meiersurvival follow-up of c-myc OVAtgþ mice whose cages weredeprived of doxycycline in drinkingwater at birth and were treated onday 21 and 25 of life either with acombination of anti-CD137, OX40,andB7-H1 (Combo3), or control ratIgG. The indicated mice receivedadoptive T-cell therapy (ACT) withboth 2 � 106 activated OT-1 and2 � 106 activated OT-2 cellscomplemented with 2 � 104 IUof IL-2 i.p. B, representativeultrasound images at 6 weeks ofage from mice in each treatmentgroup with the hepatocellularcarcinoma contour marked by ablack line and providing theestimated area. Photographs of theabdominal regions of these miceare provided. Fraction of survivingmice per group is indicated in thelegend. P values refer to rat IgG-treated control group analyzed bylog rank test. ��, P < 0.01;��, P < 0.001.






Infiltration of hepatocellular carcinomas by activatedCD4þ and CD8þ T lymphocytes upon combinationaltreatment with ISmAbsIf the mechanism of action is cellular immunity, stronger

T-cell infiltrates would be expected in treated c-myc OVA

tgþmice. For this reason, livers frommice under treatmentwere thoroughly examined by histology and immunohis-tochemistry. Indeed, Combo3 treatment resulted in moreintense tumor infiltration byCD3þT lymphocytes (Fig. 3A).Immunohistochemical staining results showing livers from

Figure 3. Immunohistochemistrystudies of the livers of miceundergoing treatment with thetriple combination or singleISmAbs. A,Histologic images (H&Eand the indicated monostaining) ofthe livers from c-myc OVA tgþmice analyzed at week 5 afterreceiving on day 28 treatment withone dose of indicated mAb or itscombination. Immunostainingswere performed for CD3 andTUNELþ apoptotic nuclei. The barsrepresent 50 mm, and these images(� 400 magnification) arerepresentative of multiplemicroscopic fields from five or sixlivers analyzed each. B,quantitative analysis of the numberof CD3þ T lymphocytes in thetumor versus non-tumoral liverfrom the indicated treatmentgroups automatically counted fromwhole liver tissue sections. Datashow the mean � SD of five or sixmice per group. #, absolutenumber of cells. ��, P < 0.01.






mice which had only received one dose on day 28 andwhich were sacrificed on day 34 are shown in Fig. 3A.Interestingly, the tumor nodules show intense lymphocyticinfiltrates, whereas normal surrounding non-neoplastictissue shows less increase in T cells (Fig. 3B). Slightlyincreased apoptotic tumor cells were observed in miceundergoing immunotherapy, indicating cytolytic attack bythe lymphocytic infiltrate. Ki-67 staining demonstratedproliferating CD3þ lymphocytes in addition to tumor cells(data not shown).

In parallel, cell suspensions from these livers were ana-lyzed by immunofluorescence and flow cytometry. As canbe observed in Fig. 4A, Combo3 led to a higher content ofCD8þ and CD4þ T lymphocytes.

A separate set of experiments, comparing Combo3 andcontrol antibody given on days 21 and 25 (administered asin Fig. 1), confirmed the higher lymphocyte content withcomparable organ weight (Fig. 4B). More detailed FACSanalyses shown in Fig. 4C andD, clearly indicate that CD8þ

and CD4þ T cells readily infiltrated these tumors. Thesewere both increased in number and also showed anenlarged (blastic) phenotype (Supplementary Fig. S4Aand Fig. 4C and D). Accordingly, CD8þ and CD4þ Tlymphocytes exhibited a higher intracellular content ofgranzyme B (Fig. 4C and D). It is of interest, that infiltratingT lymphocytes instigated by Combo3 showed brightersurface expression of CD137, OX40, and PD-1. This is veryimportant because surface expression permits the continu-ous effect of the ISmAb combination, in contrast with thecontrol treated mice, in which expression of CD137 wasalmost absent on the infiltrating CD8þ T lymphocytes.OX40was also expressed atmuch lower levels in the controlgroup than in the Combo3 group.

Given the fact that depletion of CD8þ T lymphocytesspoiled the therapeutic activity of Combo3, we studied theexpression of cytolytic effectormechanisms in this subset ofTILs. Expression was analysed in Combo3 treated mice incomparison to the corresponding doublets and controlantibody. As can be seen in Supplementary Fig. S5, perforinand granzyme B were stimulated by all the doublets with aclear further enhancement in the triple combination. Sur-face FasL is also slightly upregulated while TRAIL is not. As apotential drawback, this regimen also elicited infiltrates ofFoxp3þ regulatory CD4þ T cells (Tregs) that were alsomoreabundant than in the control group (Supplementary Fig.S4B), albeit CD8þ/Treg ratios were still clearly increasedupon Combo3 treatment (Supplementary Fig. S4B).

Hepatocellular carcinoma-derived cell lines expressOVA and are recognized by OVA-specific CD8þ Tlymphocytes

Explanting tumors allowed for the establishment of con-tinuously growing cell lines that died off if exposed todoxycycline in culture as expected for transformed cells inthis transgenic model (data not shown). After repeatedattempts, several cell lines named JMJ (JMJ7 and JMJ9) wereestablished from a mouse suffering bulky liver tumors withslightly different morphologies by phase-contrast micros-

copy (Supplementary Fig. S6A). These cells expressedEpCAM and B7-H1, albeit with dim surface levels of MHCclass I molecules (Supplementary Fig. S6B). However, bothB7-H1 and the MHC class I and II antigen presentingmolecules were upregulated when these cell lines wereexposed in culture to IFN-g for 48 hours (SupplementaryFig. S6B).

Retrotranscription-PCR (RT-PCR) profiling of these cellsand lysates from liver explants showedmRNA expression ofalbumin andHNF4 (indicating hepatocyte lineage; Supple-mentary Fig. S6C). Ovalbumin and human c-myc geneswere also expressed. More importantly, as shown in Sup-plementary Fig. S6D, OT-1 T-cell blasts readily killed JMJcells in chromium release assays, supporting adequate pre-sentation of the immunodominant OVA epitope by tumorcells, even without IFN-g preincubation.

Hepatocellular carcinoma-bearing mice tolerizeactivated OT-1 T-cell blasts unless the triplecombination of ISmAbs is given

None of the JMJ cell lines grafted as tumors either inimmunocompetent or immunodefficient Rag1�/� mice(Fig. 5A). However, small �5 � 5 mm pieces of tumor ifsurgically implanted under a skin flap of Rag1�/� immu-nodeficient mice rendered progressive lethal tumors thatwere larger than 15 mm of diameter by day 30 (Fig. 5B).However, in immunocompetent syngeneic mice, tumorswere rejected in every experiment, indicating that an anti-tumor T-cell response was responsible for this observation.

In fact, if WT-na€�ve mice were implanted with suchhepatocellular carcinoma explants these mice developedOVA (SIINFEKL)-specific in vivo killing activity when chal-lenged 7 days later. Interestingly, if the same experiment isperformed in c-myc OVA tgþmice, the in vivo killing abilityis not observed, indicating induction of T-cell tolerance(Fig. 5C).

To explore whether the Combo3 treatment can interferewith T-cell tolerance, we performed the experiments shownin Fig. 5D. Activated OT-1 lymphocytes were adoptivelytransferred into c-myc-OVA tgþ tumor bearingmice on day21 followed by Combo3 or control antibody treatments ondays 21 and 25. No in vivo killing activity by these trans-ferred OT-1 cells was observed unless Combo3 had beenadministered.

Tumor infiltrating T lymphocytes elicited by the ISmAbcombination treatment recognize JMJ cell lines

Specific T-cell responses require antigen. If tolerizedagainst OVA, we theorized that other antigens were rec-ognized by endogenous tumor-infiltrating CTLs. Indeed,isolated tumor infiltrating lymphocytes released IFN-g ,when cocultured with irradiated JMJ7 cells, while they didnot when cocultured with control cell lines (Fig. 6A).Moreover, gated CD8þ TILs from Combo3 treated micedegranulated and expressed surface CD107a upon expo-sure to JMJ7 cells (Fig. 6B). An additional series of similarexperiments with immunomagnetically isolated CD8þ

TILs from Combo3-treated mice indicated that these






Figure 4. The ISmAb triple combinationattainsmorenumerous and robust T lymphocyte infiltrates in hepatocellular carcinomas.A,AbsolutenumbersofCD3þ,CD8þ, andCD4þ lymphocytes fromcell suspensions retrieved from individual livers ofmice treated atweek 4 (day 28) as in Fig. 3with the indicated antibodies.B, liver weights and absolute numbers of CD45þ leukocytes per gram of tissue in the liver of c-myc OVA tgþmice treated with control antibody or the triplecombination (Combo3) as in Fig. 1 on days 21 and 25 of life. C and D, flow cytometry results of samples as in B, analyzed for CD8þ (C) and CD4þ (D)percentages, frequency of lymphoblastic cells according to FSC/SSC, and the percentage of positive cells and MFI for CD137, OX40, PD-1 and granzyme Bimmunostainings, as indicated in the corresponding graphs. Apool of three independent experiments (with 2–6mouse per group in each experiment) is shownin (B), (C), and (D). #, absolute numbers. �, P < 0.05; ��, P < 0.01; ��, P < 0.001; ns, non-significant.






lymphocytes recognized JMJ cells while the control cellswere not recognized including MC38 cells expressingOVA (Supplementary Fig. S7A). Similar observationswere made with CD8 splenocytes from the same mice(Supplementary Fig. S7B).

Our next goal was to find out if such lymphocyteswould recognize H-2Kb and H-2Db fitting epitopesderived from OVA, human c-myc, or the transcriptionalregulator of bacterial origin (tTA). Our hypothesis wasthat these transgenes could be immunogenic and acti-vate TILs from Combo3-treated mice. However, none ofthe corresponding synthetic peptides elicited IFN-g pro-duction, including subdominant epitopes of OVA,sequences of human c-myc dissimilar to mouse c-mycand the transcriptional regulator of bacterial origin(tTA; Fig. 6C). At this point of time the identity of therecognized peptides is unclear, although c-myc tran-scriptional targets are likely candidates (26).

In summary, we have identified a potent triple combi-nation of ISmAbs that can surpass established tumorimmune tolerance mechanisms in such a way that survivalof otherwise rapidly progressing multifocal hepatocellularcarcinoma tumor bearingmice is extended.Upon treatmentwe found an efficient endogenous T-cell response againsttumor cells.

DiscussionOur study shows that a combination of ISmAbs achieves

partial efficacy in an aggressive and T-cell–tolerizing mousemodel of hepatocellular carcinoma (21). The rationale wasthat blockade of the B7-H1-PD-1 interaction would tran-siently release a key T-cell repressor system (27), whereasagonist anti-CD137 and OX40 mAbs would enhance theantitumor T-cell response (13, 22). The improved survivalwith only two doses of the mAbs given on days 21 and 25 isquite remarkable. Repeated treatments for longer periods oftime are problematic, as rat and hamster immunoglobulinsare immunogenic inmice and elicit neutralizing antibodies.In the past, treatment of animals injected subcutaneouslywith tumor cell lines has been successful, although thisseems not to reflect the situation in patients. Possiblereasons include the organ- and tumor-specific microenvi-ronment that develops when a solid tumor grows over aperiod of time in a given organ (28). To better predictclinical potential, preclinical evidence of immunotherapyefficacy on spontaneous transgenic tumors is to be consid-ered in conjunction with that on orthotopically implantedtumors (29).

The rapid progression and technical demands of thistumor model prevent testing of sequential or differentdosage treatments using ISmAbs, although this question

Figure 5. The ISmAb triple combination therapy acting on CTLs determines the immunonogenic versus tolerogenic behavior of hepatocellularcarcinoma arising in c-myc OVA tgþ mice. A, failure to graft of JMJ cells in C57Bl/6 Rag1�/� and WT mice. Numbers of grafting attempts with 106 cellsper mouse are plotted. Both JMJ7 and 9 subcultures were tested with identical negative grafting results. B, individual follow-up of the size ofhepatocellular carcinoma explants (�5 � 5 mm) onto C57Bl/6 Rag1�/� and WT mice. C, conventional in vivo killing assay against SIINFEKL-loadedsplenocytes in syngeneic mice implanted subcutaneously with tumor cubes following the time course indicated in the time flow scheme. D, doxycyclinedeprived c-mycOVA tgþmice were treated on day 21 with 2� 106 activated OT-1 T cells and with control rat antibody or Combo3 on days 21 and 25. On day29, as indicated in the time flow scheme, an in vivo killing assay measuring CTL activity against SIINFEKL was performed. Data were summarized from threeindependent experiments (with 2–3 mice per group in each experiment) and displayed as a dot graph. ��, P < 0.01.






remains relevant regarding clinical trial designs (30). To ourknowledge, this is the first report of combined ISmAbefficacy in a spontaneous autochthonous tumor modelarising in oncogene-transgenic mice. A related workincludes a combination of anti-DR5, anti-CD137 andanti-CD40 mAbs that has shown partial efficacy in carcin-ogen-induced sarcomas (17). In addition, an anti-CD40mAbhas shown activity against transgenic pancreatic ductaladenocarcinomas in combination with gemcitabinethrough an effect mediated by macrophages without Tlymphocyte involvement (31).Oncogene transgenic mice are thought to pose much

more serious difficulties to immunotherapy than trans-planted models, as they tend to be less immunogenic(32) andmore tolerogenic (33). In the model studied here,multiple independent carcinoma foci develop at the sametime (21) and demonstrate the hepatocellular carcinomatypical dense neovascular microenvironment. In thesetumors antigen-specific CD8þ immunity is downmodu-lated and tumors progress at a fast pace with T-cell tolerancerepresenting the major hurdle (21). Triple combination ofISmAbs makes sense since synergistic effects of the com-bined treatment consisting of anti-CD137 plus anti-B7-H1(34, 35) mAbs or anti-CD137 plus anti-OX40 mAbs (36)have been reported in mouse models of transplantabletumors. As expected from the mechanism of action of thesethree antibodies, we observe an increase in T-cell infiltratesin tumor tissue composed of both activated CD4þ andCD8þ T lymphoblasts. Indeed, CD8þ T lymphocytes arean absolute requirement to achieve an extended overall

survival. Furthermore, CD8þ TILs express more intenselythe perforin-granzyme B cytolytic machinery upon Com-bo3. The effector mechanisms of tumor cell destruction arelikely to involve both cytolytic granules and FasL. Of note,JMJ cells express functional Fas and IFNgR and thereforethese molecules could play a synergistic role in destroyingthe tumor as previously reported for immunotherapy withanti-CD137 mAb (37).

Our goal was to boost CTLs with anti-CD137 and bothCD8þ and CD4þ T lymphocytes with anti-OX40, while atthe same time removing PD-1 negative influences onlymphocyte activation. The reported functional effects ofanti-OX40 mAb repressing regulatory T cells would alsobe an advantage (38). Individual contributions of eachmAb to the overall therapeutic effects are difficult to teaseout, as each antibody may modify the biologic activityand receptor expression of the other partner antibodies inthe combination.

The important role of CD4þ T cells in antitumor immu-nity is a matter of active research (39, 40). In our model,depletionofCD4-expressing T lymphocytes didnot hamperthe observed treatment benefit. However, we show thatCD4þ cells are infiltrating the tumors and bear the receptorsto be stimulated by the combination of ISmAbs. It will beimportant to identify in future experiments the exact role ofCD4þ T cells and the cell presenting antigen to suchCD4þ Tcells, as tumor cells poorly express MHC class II moleculesunless exposed to IFNg . CD4þ T lymphocytes can cooperatewith the elicitation ofmore powerful CTLs and alsomediatedirect antitumor effects (41). It should be noted that in our

Figure 6. TILs in hepatocellular carcinoma tumors recognize specific tumor antigens in JMJ cells. A, TILs isolated from tumor-bearingmice 4weeks of age thathad been treated with Combo3 on days 21 and 25 were cultured with irradiated JMJ7 and MC38 cells that had been IFN-g pretreated for 48 hours toupregulate MHC-I and II expression. Concentration of IFN-g in the supernatants was measured 72 hours later. n ¼ 5 mice, repeated twice. B, TILs as in (A),restimulated for 5 hours to assess CD107a surface expression by flow cytometry on gated CD8þ T lymphocytes. The graph shows a pool of twoexperiments (with 3–5miceper group in each).C, TILs isolated as inA in a separate experimentwerepooledand restimulated in vitrowith JMJ7or the indicatedsoluble 8-9 mer synthetic peptides predicted to fit H-2Kb and H-2Db antigen presenting molecules in the sequences of chicken OVA, human-c-myc, and tTAproteins. ��, P < 0.01; ��, P < 0.001.






depletion experiments we also eliminate protumoral Tregsand that might be a confounding factor to interpret theresults upon depletion of CD4þ lymphocytes.

Selective expression of OVA in the tumor tissue allowedtwo types of experiments: (i) combinations with antigen-specific adoptive T-cell therapy and (ii) studies on the T-celltolerance-inducing microenvironment in tumor bearingtransgenic mice.

A clear synergy was observed between the triple com-bination of ISmAbs and adoptive transfer of activatedCD8þ and CD4þ T lymphocytes recognizing OVA. Pre-vious studies have demonstrated that infiltration byOVA-specific T cells in the c-myc OVA tgþ model is notsufficient for tumor regression, as these T cells arerendered tolerant within the tumor microenvironment(21). Here, we clearly demonstrate that combinedISmAb therapy prevents tolerization of adoptively trans-ferred OVA-specific T cells and is able to improve sur-vival of the mice. In spite of their tolerizing ability,tumors were immunogenic, as tumor explants wererejected in immunocompetent syngeneic mice, whereastumors did engraft in Rag1�/� immunodeficient mice.

Cultured tumor infiltrating lymphocytes retrieved fol-lowing treatment degranulated and released IFN-g in thepresence of cell lines derived from c-myc OVA tgþ tumorsbut not in the presence of control tumor cell lines. As wecould not identify antigenic determinants from the threetransgenes (OVA, the tTA transcriptional regulator orhuman c-myc), the identity of the antigens recognized byendogenous TILs still remains to be elucidated.

A more detailed picture of the function of the infiltratinglymphocytes would demand in vivo microscopy or in vivoimaging (42, 43). It is clear that treatment increases thenumber of activated effector TILs but we know little abouttheir performance in vivo. Future research is aiming atvisualizing and comparing the liver tumor microenviron-ment upon therapy using fluorescent lymphocytes.

To date, ISmAbs as those in Combo3 have not beenstudied in patients with hepatocellular carcinoma.We haveperformed a clinical trial with the anti-CTLA-4 mAb treme-limumab in advanced hepatocellular carcinoma with signsof clinical activity and evidence for an enhancement ofimmune responses anti-hepatitis C virus (25). In our mod-el, we did not observe efficacywith two 100 mg doses of anti-CTLA-4 mAb as monotherapy and addition of anti-CTLA-4mAb to the triple combination.

At present, a phase I clinical trial with anti-PD-1 mAb forhepatocellular carcinoma is ongoing (NCT01658878),whose results are eagerly awaited. Combination with adop-tive T-cell therapy is more difficult to be translated topatients since no suitable TCRs or chimeric antigen recep-tors (CARs) are yet available for hepatocellular carcinomaantigens; and TILs are extremely difficult to culture fromhepatocellular carcinoma tissue (Alfaro C and colleagues;Unpublished data). Therefore, it is extremely encouragingthat endogenous CD8 and CD4 T-cell responses areobserved in this model system, where hepatic tumors prog-ress rapidly and render T cells tolerant.

Anti-CD137 mAb triggers liver inflammation in healthymice and human patients (44, 45). This could both beadvantageous and deleterious, because patients with hepa-tocellular carcinoma could be prone to suffer from hepaticside effects, while they also may benefit from the inflam-matory response in this organ (44, 46). Here, we found noevidence of adverse autoimmune hepatitis in the surround-ing liver tissue, based on immunohistochemical findings atthe doses tested. In fact, T-cell infiltrates were much moreintense in the malignant tissue than in the surroundingnormal liver tissue.

Overall, our experiments provide promising results forfurther preclinical and clinical trials using combinations ofmore than two ISmAbs in human primary and metastaticliver cancer, showing evidence for synergistic efficaciousimmunotherapy in mice bearing aggressive hepatocellularcarcinomas.

Disclosure of Potential Conflicts of InterestB. Sangro is a consultant/advisory board member of Medimmune. I.

Melero has a commercial research grant andhonoraria from speakers’ bureaufrom Bristol Myers Squibb and is a consultant/advisory board member ofMerck, Bristol Myers Squibb, and Medimmune. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: A. Morales-Kastresana, M.F. de Sanmamed,I. MeleroDevelopmentofmethodology:A.Morales-Kastresana,M.F. de Sanmamed,I. Rodriguez, A. Palazon, S. Labiano, S. Hervas-Stubbs, M.C. Ochoa,A. Roauzaut, A. Azpilikueta, E. Bola~nosAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): A. Morales-Kastresana, M.F. de Sanmamed,M.C. Ochoa, A. Roauzaut, M. Jure-Kunkel, I. G€utgemannAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): A. Morales-Kastresana, M.F. de San-mamed, I. Martinez-Forero, B. Sangro, A. Roauzaut, M. Jure-Kunkel,I. G€utgemann, I. MeleroWriting, review, and/or revision of the manuscript: A. Morales-Kastre-sana, M.F. de Sanmamed, I. Martinez-Forero, B. Sangro, M. Jure-Kunkel,I. G€utgemann, I. MeleroAdministrative, technical, or material support (i.e., reporting ororganizing data, constructing databases): A. Morales-Kastresana,I. G€utgemannStudy supervision: I. Melero

AcknowledgmentsThe authors thank H. Kanishka for assistance using the 3D Histech midi

analysis system, V. Belsue and P. Sarobe for peptide synthesis and D.Corbacho for ultrasound analyses assistance. The authors also thank LiepingChen and Mario Colombo for their generous gift of mAb-producing hybri-domas and David Sancho for MC38-OVA transfectants.

Grant SupportThis work was financially supported by grants from MICINN (SAF2008-

03294, SAF2011-22831; to I. Melero). I. Melero was also funded by theDepartamento de Educaci�on del Gobierno deNavarra andDepartamento deSaluddelGobierno deNavarra, Redes tem�aticas de investigaci�on cooperativaRETIC (RD06/0020/0065), European commission VII framework program(ENCITE and IACT), SUDOE-IMMUNONET, and "UTE for project FIMA". A.Morales-Kastresana and S. Labiano are recipients of predoctoral scholarshipsfrom Ministerio de Economia. M.F. de Sanmamed receives a Rio Hortegacontract from ISCIII. A. Palazon is a recipient of an FIS predoctoral schol-arship from ISCIII. I. Gutgemann was funded by Deutsche Krebshilfe.

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 30, 2013; revised August 9, 2013; accepted August 27, 2013;published OnlineFirst September 12, 2013.






References1. Korangy F, Hochst B, Manns MP, Greten TF. Immunotherapy of

hepatocellular carcinoma. Expert Rev Gastroenterol Hepatol 2010;4:345–53.

2. Palmer DH, Midgley RS, Mirza N, Torr EE, Ahmed F, Steele JC, et al. Aphase II study of adoptive immunotherapy using dendritic cells pulsedwith tumor lysate in patients with hepatocellular carcinoma. Hepatol-ogy 2009;49:124–32.

3. Pardee AD, Butterfield LH. Immunotherapy of hepatocellular carcino-ma: unique challenges and clinical opportunities. Oncoimmunology2012;1:48–55.

4. Sangro B, Mazzolini G, Ruiz J, Herraiz M, Quiroga J, Herrero I, et al.Phase I trial of intratumoral injection of an adenovirus encodinginterleukin-12 for advanced digestive tumors. J Clin Oncol 2004;22:1389–97.

5. Takayama T, Sekine T, Makuuchi M, Yamasaki S, Kosuge T, Yama-moto J, et al. Adoptive immunotherapy to lower postsurgical recur-rence rates of hepatocellular carcinoma: a randomised trial. Lancet2000;356:802–7.

6. Melero I, Grimaldi AM, Perez-Gracia JL, Ascierto PA. Clinical devel-opment of immunostimulatory monoclonal antibodies and opportu-nities for combination. Clin Cancer Res 2013;19:997–1008.

7. Melero I, Hervas-Stubbs S, Glennie M, Pardoll DM, Chen L. Immu-nostimulatory monoclonal antibodies for cancer therapy. Nat RevCancer 2007;7:95–106.

8. Pardoll DM. The blockade of immune checkpoints in cancer immu-notherapy. Nat Rev Cancer 2012;12:252–64.

9. Robert C, Thomas L, Bondarenko I, O'Day S, M DJ, Garbe C, et al.Ipilimumab plus dacarbazine for previously untreated metastatic mel-anoma. N Engl J Med 2011;364:2517–26.

10. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDer-mott DF, et al. Safety, activity, and immune correlates of anti-PD-1antibody in cancer. N Engl J Med 2012;366:2443–54.

11. Brahmer JR, Tykodi SS, Chow LQ, HwuWJ, Topalian SL, Hwu P, et al.Safety and activity of anti-PD-L1 antibody in patients with advancedcancer. N Engl J Med 2012;366:2455–65.

12. Melero I, Shuford WW, Newby SA, Aruffo A, Ledbetter JA, Hell-strom KE, et al. Monoclonal antibodies against the 4-1BB T-cellactivation molecule eradicate established tumors. Nat Med 1997;3:682–5.

13. Weinberg AD, Rivera MM, Prell R, Morris A, Ramstad T, Vetto JT, et al.Engagement of the OX-40 receptor in vivo enhances antitumor immu-nity. J Immunol 2000;164:2160–9.

14. French RR, Chan HT, Tutt AL, Glennie MJ. CD40 antibody evokes acytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help. Nat Med 1999;5:548–53.

15. Cohen AD, Diab A, Perales MA, Wolchok JD, Rizzuto G, MerghoubT, et al. Agonist anti-GITR antibody enhances vaccine-induced CD8(þ) T-cell responses and tumor immunity. Cancer Res 2006;66:4904–12.

16. TakedaK,KojimaY,UnoT,HayakawaY, TengMW,YoshizawaH, et al.Combination therapy of established tumors by antibodies targetingimmune activating and suppressing molecules. J Immunol 2010;184:5493–501.

17. Uno T, Takeda K, Kojima Y, Yoshizawa H, Akiba H, Mittler RS, et al.Eradication of established tumors in mice by a combination antibody-based therapy. Nat Med 2006;12:693–8.

18. Boggio K, Di Carlo E, Rovero S, Cavallo F, Quaglino E, Lollini PL, et al.Ability of systemic interleukin-12 to hamper progressive stages ofmammary carcinogenesis in HER2/neu transgenic mice. Cancer Res2000;60:359–64.

19. Zabala M, Lasarte JJ, Perret C, Sola J, Berraondo P, Alfaro M, et al.Induction of immunosuppressive molecules and regulatory T cellscounteracts the antitumor effect of interleukin-12-based gene therapyin a transgenic mouse model of liver cancer. J Hepatol 2007;47:807–15.

20. Lollini PL, Cavallo F, Nanni P, Forni G. Vaccines for tumour prevention.Nat Rev Cancer 2006;6:204–16.

21. Ney JT, Schmidt T, Kurts C, Zhou Q, Eckert D, Felsher DW, et al.Autochthonous liver tumors induce systemic T cell tolerance associ-

ated with T cell receptor down-modulation. Hepatology 2009;49:471–81.

22. Melero I,MurilloO, Dubrot J, Hervas-Stubbs S, Perez-Gracia JL.Multi-layered action mechanisms of CD137 (4-1BB)-targeted immunothera-pies. Trends Pharmacol Sci 2008;29:383–90.

23. Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands intolerance and immunity. Annu Rev Immunol 2008;26:677–704.

24. Croft M. Control of immunity by the TNFR-related molecule OX40(CD134). Annu Rev Immunol 2010;28:57–78.

25. Sangro B, Gomez-Martin C, de la Mata M, Inarrairaegui M, Garralda E,Barrera P, et al. A clinical trial of CTLA-4 blockade with tremelimumabin patients with hepatocellular carcinoma and chronic hepatitis C.J Hepatol 2013;59:81–88.

26. Dang CV, O'Donnell KA, Zeller KI, Nguyen T, Osthus RC, Li F. The c-Myc target gene network. Semin Cancer Biol 2006;16:253–64.

27. Topalian SL, DrakeCG, Pardoll DM. Targeting the PD-1/B7-H1(PD-L1)pathway to activate anti-tumor immunity. Curr Opin Immunol 2012;24:207–12.

28. Willimsky G, Blankenstein T. The adaptive immune response to spo-radic cancer. Immunol Rev 2007;220:102–12.

29. HoffmanRM.Orthotopicmetastaticmousemodels for anticancer drugdiscovery and evaluation: a bridge to the clinic. Invest New Drugs1999;17:343–59.

30. Martinez Forero I, Okada H, Topalian SL, Gajewski TF, Korman AJ,Melero I. Workshop on immunotherapy combinations. Society forImmunotherapy of Cancer annual meeting Bethesda, November 3,2011. J Transl Med 2012;10:108.

31. Beatty GL, Chiorean EG, Fishman MP, Saboury B, Teitelbaum UR,Sun W, et al. CD40 agonists alter tumor stroma and show efficacyagainst pancreatic carcinoma in mice and humans. Science 2011;331:1612–6.

32. DuPage M, Mazumdar C, Schmidt LM, Cheung AF, Jacks T. Expres-sion of tumour-specific antigens underlies cancer immunoediting.Nature 2012;482:405–9.

33. Willimsky G, Blankenstein T. Sporadic immunogenic tumoursavoid destruction by inducing T-cell tolerance. Nature 2005;437:141–6.

34. Hirano F, Kaneko K, Tamura H, Dong H, Wang S, Ichikawa M, et al.Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiatescancer therapeutic immunity. Cancer Res 2005;65:1089–96.

35. Palazon A, Martinez-Forero I, Teijeira A, Morales-Kastresana A, AlfaroC, Sanmamed MF, et al. The HIF-1alpha hypoxia response in tumor-infiltrating T lymphocytes induces functional CD137 (4-1BB) for immu-notherapy. Cancer Discov 2012;2:608–23.

36. Gray JC, French RR, James S, Al-Shamkhani A, Johnson PW,Glennie MJ. Optimising anti-tumour CD8 T-cell responses usingcombinations of immunomodulatory antibodies. Eur J Immunol2008;38:2499–511.

37. Morales-Kastresana A, Catalan E, Hervas-Stubbs S, Palazon A,Azpilikueta A, Bola~nos E, et al. Essential complicity of perforin-granzyme and Fas-L mechanisms to achieve tumor rejection fol-lowing treatment with anti-CD137 mAb. J Immunother Cancer2013;1:3–6.

38. Piconese S, Valzasina B, Colombo MP. OX40 triggering blocks sup-pression by regulatory T cells and facilitates tumor rejection. JExpMed2008;205:825–39.

39. Hunder NN, Wallen H, Cao J, Hendricks DW, Reilly JZ, Rodmyre R,et al. Treatment ofmetastaticmelanomawith autologousCD4þ T cellsagainst NY-ESO-1. N Engl J Med 2008;358:2698–703.

40. Perez-Diez A, Joncker NT, Choi K, Chan WF, Anderson CC, Lantz O,et al. CD4 cells can bemore efficient at tumor rejection than CD8 cells.Blood 2007;109:5346–54.

41. Braumuller H, Wieder T, Brenner E, Assmann S, Hahn M, Alkhaled M,et al. T-helper-1-cell cytokines drive cancer into senescence. Nature2013;494:361–5.

42. Hoffman RM. The multiple uses of fluorescent proteins to visualizecancer in vivo. Nat Rev Cancer 2005;5:796–806.

43. Suetsugu A, Katz M, Fleming J, Truty M, Thomas R, Saji S, et al. Non-invasive fluorescent-protein imaging of orthotopic pancreatic-cancer-






patient tumorgraft progression in nude mice. Anticancer Res 2012;32:3063–7.

44. Dubrot J, Milheiro F, Alfaro C, Palazon A, Martinez-Forero I, Perez-Gracia JL, et al. Treatment with anti-CD137 mAbs causes intenseaccumulations of liver T cells without selective antitumor immunother-apeutic effects in this organ. Cancer Immunol Immunother 2010;59:1223–33.

45. Ascierto PA, Simeone E, SznolM, Fu YX,Melero I. Clinical experienceswith anti-CD137 and anti-PD1 therapeutic antibodies. Semin Oncol2010;37:508–16.

46. Niu L, Strahotin S, Hewes B, Zhang B, Zhang Y, Archer D, et al.Cytokine-mediated disruption of lymphocyte trafficking, hemopoiesis,and induction of lymphopenia, anemia, and thrombocytopenia in anti-CD137-treated mice. J Immunol 2007;178:4194–213.






2013;19:6151-6162. Published OnlineFirst September 12, 2013.Clin Cancer Res Aizea Morales-Kastresana, Miguel F. Sanmamed, Inmaculada Rodriguez, et al. Mouse ModelSurvival in an Aggressive Transgenic Hepatocellular Carcinoma Combined Immunostimulatory Monoclonal Antibodies Extend

Updated version

10.1158/1078-0432.CCR-13-1189doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2013/09/13/1078-0432.CCR-13-1189.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/19/22/6151.full#ref-list-1

This article cites 46 articles, 13 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/19/22/6151.full#related-urls

This article has been cited by 12 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/19/22/6151To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-13-1189

http://clincancerres.aacrjournals.org/content/suppl/2013/09/13/1078-0432.CCR-13-1189.DC1

http://clincancerres.aacrjournals.org/content/19/22/6151.full#ref-list-1

http://clincancerres.aacrjournals.org/content/19/22/6151.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/19/22/6151


combined immunostimulatory monoclonal antibodies extend...

Documents