BRAF Inhibitor Vemurafenib Improves the Antitumor Activity of Adoptive Cell Immunotherapy · BRAF Inhibitor Vemurafenib Improves the Antitumor Activity of Adoptive Cell Immunotherapy

Microenvironment and Immunology

BRAF Inhibitor Vemurafenib Improves the Antitumor Activityof Adoptive Cell Immunotherapy

Richard C. Koya1,6, Stephen Mok1,2,3, Nicholas Otte2, Kevin J. Blacketor2, Begonya Comin-Anduix1,6,Paul C. Tumeh4,6, Aspram Minasyan5, Nicholas A. Graham3, Thomas G. Graeber3,6, Thinle Chodon2, andAntoni Ribas1,2,3,6

AbstractCombining immunotherapy with targeted therapy blocking oncogenic BRAFV600 may result in improved

treatments for advanced melanoma. In this study, we developed a BRAFV600E-driven murine model of melanoma,SM1, which is syngeneic to fully immunocompetent mice. SM1 cells exposed to the BRAF inhibitor vemurafenib(PLX4032) showed partial in vitro and in vivo sensitivity resulting from the inhibition of MAPK pathway signaling.Combined treatment of vemurafenib plus adoptive cell transfer therapywith lymphocytes geneticallymodifiedwitha T-cell receptor (TCR) recognizing chicken ovalbumin (OVA) expressed by SM1-OVA tumors or pmel-1 TCRtransgenic lymphocytes recognizing gp100 endogenously expressed by SM1 resulted in superior antitumorresponses compared with either therapy alone. T-cell analysis showed that vemurafenib did not significantly alterthe expansion, distribution, or tumor accumulation of the adoptively transferred cells. However, vemurafenibparadoxically increased mitogen-activated protein kinase (MAPK) signaling, in vivo cytotoxic activity, andintratumoral cytokine secretion by adoptively transferred cells. Taken together, our findings, derived from 2 inde-pendent models combining BRAF-targeted therapy with immunotherapy, support the testing of this therapeuticcombination in patients with BRAFV600 mutant metastatic melanoma. Cancer Res; 72(16); 3928–37. �2012 AACR.

IntroductionTargeted therapies that block driver oncogenicmutations in

BRAFV600 result in unprecedentedly high response rates andimproved overall survival in patients with advancedmelanoma(1–4). However, these responses are usually of limited dura-bility, which is a common feature of most oncogene-targetedtherapies for cancer. Conversely, many tumor immunotherapystrategies induce low-frequency, but extremely durable, tumorresponses, frequently lasting years (5–7). The ability to com-bine both treatment approaches could merge the benefits ofhigh response rates with targeted therapies and durableresponse rates with immunotherapies.

Combining immunotherapy with BRAF inhibitors, such asvemurafenib (formerly PLX4032 or RG7204) or dabrafenib(formerly GSK2118436), 2 highly active agents for the treat-ment of BRAFV600 mutant melanoma, is supported by concep-tual advantages and emerging experiences (8–10) that warrantthe testing of such combinations in animal models. It has beenreported that BRAF inhibitors may synergize with tumorimmunotherapy by the increased expression of melanosomaltumor-associated antigens following inhibition of themitogen-activated protein kinase (MAPK) pathway (8). In addition,there are potential theoretical limitations to such a combina-tion, because blocking signaling through the MAPK pathwaymay alter lymphocyte activation or effector functions. How-ever, when tested at a wide range of concentrations in vitro andin vivo, BRAF inhibitors do not have significant adverse effectson human T lymphocyte functions (9, 11), and patients treatedwith BRAF inhibitors have increased intratumoral infiltrates byCD8þ T cells soon after therapy (10). Further, RAF inhibitorscan have a paradoxical effect of activating the MAPK pathwaythrough the transactivation of CRAF by a partially blockedwild-type (WT) CRAF–BRAF dimmer (12–14). This phenom-enon of paradoxicalMAPK activation is themolecular basis forthe development of cutaneous squamous cell carcinomas inpatients treated with BRAF inhibitors (15), and it could beevident in activated T cells as upstream activation of TCRs hasa potent effect of activating RAS-GTP leading to enhancedCRAF–BRAF dimerization.

Previously, no implantable syngeneic BRAFV600E-drivenmurine melanoma model able to grow progressively in a fullyimmunocompetent and widely used mouse strain had been

Authors' Affiliations: 1Department of Surgery, Division of Surgical Oncol-ogy; 2Department ofMedicine, Division ofHematology/Oncology; 3Depart-ment of Molecular and Medical Pharmacology, Crump Institute for Molec-ular Imaging; 4Department of Medicine, Division of Dermatology; 5UCLABiomedical Physics Interdepartmental Graduate Program, Los Angeles;and 6the Jonsson Comprehensive Cancer Center, University of CaliforniaLos Angeles, Los Angeles, California

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

R.C. Koya and S. Mok contributed equally as first authors.

Corresponding Authors: Richard C. Koya, Division of Surgical Oncology,54-140 CHS, Los Angeles, CA 90095-1782. Phone: 310-825-2676; Fax:310-825-2493; E-mail: [email protected]; andAntoni Ribas, Divisionof Hematology-Oncology, 11-934 Factor Building. UCLA Medical Center,10833 Le Conte Avenue, Los Angeles, CA 90095-1782. Phone: 310-206-3928; Fax: 310-825-2493; E-mail: [email protected].

doi: 10.1158/0008-5472.CAN-11-2837

�2012 American Association for Cancer Research.

CancerResearch

Cancer Res; 72(16) August 15, 20123928

on June 30, 2020. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 12, 2012; DOI: 10.1158/0008-5472.CAN-11-2837

http://cancerres.aacrjournals.org/

described. We derived such a cell line frommice transgenic forthe BRAFV600E mutation with restricted expression in melano-cytes, resulting in a murine melanoma model syngeneic toC57BL/6 mice. This model allowed us to test the concept ofimmunosensitization (16) by combining the vemurafenib-induced inhibition of driver oncogenic BRAFV600E signalingwith adoptive cell transfer (ACT) immunotherapy. Vemurafe-nib meets most of the criteria as an immune-sensitizing agent(16). In humans, it selectively inhibits a driver oncogene incancer cells (17), which is neither present nor required for thefunction of lymphocytes (9). It results in rapid melanoma celldeath in humans as evidenced by a high frequency of earlytumor responses in patients (1, 18). The antitumor activitymayincrease the expression of tumor antigens directly by tumorcells (8), or enhance the cross-presentation of tumor antigensfrom dying cells to antigen-presenting cells. In addition, theprofound and selective antitumor effects of vemurafenibagainst BRAFV600 mutant melanoma cells may result in a morepermissive tumormicroenvironment allowing for an improvedeffector function of CTLs, which may be further enhanced by adirect effect of paradoxical MAPK activation. Using 2 differentTCR transgenic cell ACT models, we tested the concept ofimmunosensitization with vemurafenib, showing an improve-ment of the antitumor effects using the combination overeither single-agent therapy alone.

Materials and MethodsMice, cell lines, and reagentsBreeding pairs of C57BL/6 (Thy1.2, Jackson Laboratories,

Bar Harbor, ME), pmel-1 (Thy1.1) transgenic mice (kind giftfrom Dr. Nicholas Restifo, Surgery Branch, National CancerInstitute, Bethesda, MD), NOD/SCID/g chainnull (NSG) mice(NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ, Jackson Laboratory, BarHar-bor, ME), and mice transgenic for BRAFV600E mutation expres-sion inmelanocytes (kind gift fromDrs. Philip Hinds and FrankHaluska, Tufts Medical Center, Boston, MA) were bred andkept under defined-flora pathogen-free conditions at the Asso-ciation for the Assessment & Accreditation of LaboratoryAnimal Care-approved animal facility of the Division of Exper-imental Radiation Oncology, UCLA, and used under the UCLAAnimal Research Committee protocol #2004-159. The SM1murinemelanomawas generated froma spontaneously arisingtumor in BRAFV600E mutant transgenic mice. The tumor wasminced and first implanted into NSG mice, and then seriallyimplanted into C57BL/6 mice for in vivo experiments. Part ofthe minced tumor was plated under tissue culture conditionsfor deriving the SM1 cell line. When used in vitro, SM1 wasmaintained in RPMI (Mediatech) with 10% fetal calf serum(Omega Scientific), 2mmol/L L-glutamine (Invitrogen), and 1%(v/v) penicillin, streptomycin, and amphotericin (Omega Sci-entific). Sequencing for BRAFV600Emutation was carried out aspreviously described (19). SM1-OVA was generated by stableexpression of OVA through lentiviral transduction as previ-ously described (20). A plasmid expressing the 2 chains of anMHC–I-restricted TCR specific for OVA (OT-1) was a kind giftof David Baltimore (California Institute of Technology, Pasa-dena, CA; ref. 21). The plasmid was recloned to express a F2A

picornavirus sequence between the TCR chains for highexpression upon transduction of murine splenocytes using amurine stem cell virus retroviral vector as previously described(22, 23).M202,M229, andM233arepreviously describedhumanmelanomacell lines (19). Vemurafenib (alsoknownasPLX4032,RG7204, or RO5185426) was obtained under a materials trans-fer agreement with Plexxikon Inc. (Berkeley, CA). Vemurafenibwas dissolved in dimethylsulfoxide (DMSO; Fisher Scientific)and used for in vitro studies as previously described (19). Forin vivo studies, vemurafenib was dissolved in DMSO, followedby PBS (100mL),whichwas then injecteddaily intraperitoneally(i.p) into mice at a dose of 10 mg/kg. Because the originalformulation of vemurafenib is poorly bioavailable (1, 15), weused a dosing regimen i.p. that has been shown to haveadequate pharmacokinetic parameters in blood (24).

SM1 oncogenic analysisBRAFV600E sequencing was carried out as previously

described (19). Copy number analysis was conducted usinga mouse high-density genotyping array (The Jackson Labora-tory, Bar Harbor, Maine) and data was genotyped with the RMouseDivGeno (v1.03) software package (25). To detectregions of copy number alteration, we selected the subset of"chippable" invariant genomic probes (i.e., exon 1 probes;ref. 25). Normalized and log2 transformed data was segmentedusing circular binary segmentation algorithm in the R DNA-copy package (26). The minimum number of markers for achanged segmentwas set at 5, with a 0.0005 significance level toaccept a change-point. The segmented data was visualized inthe Integrative Genomics Viewer (27). For comparison tohuman melanoma, we compared this data to copy numberalterations observed in 108 human melanoma short-termcultures and cell lines (28).

Cell viability assaysMurine and human melanoma cells, na€�ve C57BL/6 spleno-

cytes, or activated pmel-1 splenocytes were seeded in 96-wellflat-bottom plates (5� 103 cells/well) with 100 mL of 10% fetalcalf serummedia and incubated for 24 hours. Graded dilutionsof vemurafenib or DMSO vehicle control, in culture medium,were added to eachwell in triplicate and analyzed following theMTS assay (Promega).

ACT therapy in vivo modelsFor the OVAmodel, SM1-OVA tumors were implanted s.c. in

C57BL/6 mice. When tumors reached 5 to 8 mm in diameter,mice were conditioned for ACT with a lymphodepleting reg-imen of 500 cGy of total body irradiation and then received 1�106 C57BL/6-derived splenocytes i.v. that had been geneticallymodified to express the OT-1 TCR by retroviral transduction aspreviously described (22, 23). For the pmel-1 model, C57BL/6mice with previously implanted SM1 tumors were treated withlymphodepleting total body irradiation, i.v. injection of 1� 106

gp10025-33 peptide-activated pmel-1 splenocytes and subcuta-neous vaccination with gp10025-33 peptide-pulsed dendriticcells when tumors reached 5 to 8mm in diameter as previouslydescribed (29, 30). In both cases, the ACT was followed by3 days of daily i.p. administration of 50,000 IU of interleukin

Immunosensitization with BRAF Inhibitors

www.aacrjournals.org Cancer Res; 72(16) August 15, 2012 3929




2 (IL-2). Tumors were followed by caliper measurements 3times per week.

Flow cytometric analysisSM1 tumors harvested from mice were digested with col-

lagenase and DNase (Sigma-Aldrich). Splenocytes and tumor-infiltrating lymphocytes (TIL), obtained from digested SM1tumors, were stained with antibodies to CD8a (CalTag), CD3,CD4, Thy1.1 (Becton Dickinson Biosciences), OVA/H-2Kb tet-ramer, or gp10025-33/H-2D

b tetramer (Beckman Coulter), andanalyzed with LSR-II or FACSCalibur flow cytometers (BectonDickinson Biosciences), followed by analysis using Flow-Josoftware (Tree-Star) as previously described (30). Intracellularinterferon gamma (IFN-g) staining was done as previouslydescribed (30).

Immunofluorescence imagingStaining was carried out as previously described (23). Briefly,

sections of OCT (Sakura Finetek) cryopreserved tissues wereblocked in donkey serum/PBS and incubated with primaryantibodies to CD8 or Thy1.1 (Becton Dickinson Biosciences),followedby secondary donkey anti-rat antibodies conjugated toDyLight 488 (Jackson Immunoresearch Laboratories) or strep-tavidin-conjugated FITC (Invitrogen). Negative controls con-sisted of isotype matched rabbit or rat immunoglobulin G inlieu of the primary antibodies listed above. Then, 4,6-diami-dino-2-phenylindole (DAPI) was used for the visualization ofnuclei. Immunofluorescence images were taken in a fluores-cence microscope (Axioplan-2; Carl Zeiss Microimaging).

In vivo cytotoxicity assayThe assay was conducted as previously described (30). In

brief, splenocytes from na€�ve WT C57BL/6 mice were pulsedwith 50 mg/mL of gp10025-33 peptide or the same amountof control OVA257-264 peptide. After 1 hour of incubation,gp10025-33-pulsed WT splenocytes were labeled with6 nmol/L CFSE for 10 minutes at 37�C, whereas controlOVA257-264-pulsed splenocytes were differentially labeledwith a 10-fold dilution of CFSE (0.6 nmol/L). Cells wereinjected i.v. into experimental mice at 16 days after pmel-1adoptive cell transfer. After 10 hours, 3 mice per group weresacrificed and their spleens examined for the presence ofCFSE-labeled cells. Percent cytotoxic activity was calculatedas number of live gp10025-33 pulsed splenocytes divided bythe number of live OVA257-264 pulsed splenocytes, whichwere distinguished on the basis of the 10-fold difference inCSFE fluorescence by flow cytometry.

Bioluminescence imagingPmel-1 splenocytes were retrovirally transduced to express

firefly luciferase as previously described (29), and used for ACT.Bioluminescence imaging was carried out with a Xenogen IVIS200 Imaging System (Xenogen/Caliper Life Sciences) as pre-viously described (22, 23).

Micro-PET/computed tomography imagingMice were anesthetized with 2% isoflurane. PET was con-

ducted 1 hour after intravenous administration of 200 mCi of

[18F]FDG, [18F]D-FAC, or [18F]FHBG and mice were scannedusing a FOCUS 220 micro-PET scanner (Siemens; energywindow of 350 to 750 keV and timing window of 6 ns) aspreviously described (23).

Statistical analysisData were analyzed with GraphPad Prism (version 5) soft-

ware (GraphPad Software). AMann–WhitneyU test or ANOVAwith Bonferroni post-test was used to analyze experimentaldata. Survival curves were generated by actuarial Kaplan–Meier method and analyzed with the Jump-In software (SAS)with log-rank test for comparisons from the time of tumorchallenge towhenmice were sacrificed due to tumors reaching14 mm in maximum diameter, or the end of the study periodhad been reached.

ResultsDerivation of a BRAFV600E mutated murine melanomasyngeneic to C57BL/6 mice

The cell line SM1 was derived from a spontaneously arisingmelanoma from a mouse with the BRAFV600E oncogene spe-cifically expressed by melanocytes. These mice had beengenerated by germline insertion of the BRAFV600E gene down-stream of the murine tyrosinase locus control region (promot-er enhancer) as described previously (31). These melanocytes-specific BRAFV600E transgenic mice had been backcrossed formore than 20 generations with C57BL/6 mice. It has beenpreviously described that mice carrying transgenic BRAFV600E

develop melanocytic hyperplasia histologically reminiscent ofhuman nevi, and develop spontaneous melanomas with lowpenetrance due to the dominant oncogenic senescence effectof BRAFV600E (31). Cross-breeding them with CDKN2A or p53-deficient mice increases the frequency of melanoma develop-ment (31), but we found that the resulting tumors could not begrown in C57BL/6 mice (data not shown) possibly due toinnate responses against mixed-background minor antigensfrom the 2 transgenic strains. To optimize the chances ofestablishing a progressively growing tumor, we first passagedthe original SM1 cells in deeply immune-deficient NSG mice,and from there we were able to implant and develop progres-sively growing tumors in fully immunocompetent C57BL/6mice.

SM1 is a vemurafenib-moderately sensitive BRAFV600E

mutant melanomaSequencing of the hotspotT1799Amutation inBRAF showed

the presence of the BRAFV600E transversion in SM1 cells (Fig.1a). Whole-genome copy number analysis showed multiplegenomic aberrations in SM1, with frequent deletions andamplifications (Supplementary Fig. S1A), which is a commonfinding in human melanomas. Among target genes of interest,SM1 has deletion of CDKN2A and amplification of BRAF andMITF genes (Supplementary Fig. S1B), events that are alsofrequently observed in human melanoma (Supplementary Fig.S1C). We tested the antitumor effects of single-agent vemur-afenib against SM1 by in vitroMTS cell proliferation assay after72 hours of treatment. The 50% inhibition concentration (IC50)

Koya et al.

Cancer Res; 72(16) August 15, 2012 Cancer Research3930




of vemurafenib was 14 mmol/L, which is approximately one loghigher than the sensitivity of M229 (IC50 of 0.5 mmol/L), aBRAFV600E mutant human melanoma cell line highly sensitiveto vemurafenib, and at a similar range as the relatively resistantBRAFV600Emutant human melanoma cell line M233 (IC50 of 15mmol/L). SM1 was more sensitive to vemurafenib than theNRASQ61L mutant (and BRAF WT) M202 and M207 cell lines(IC50 >200 mmol/L; Fig. 1B). Despite its relative resistance inMTS assays, SM1 responded to vemurafenib in vitro as showedby a profound G1 arrest effect (Fig. 1C), and evidence ofapoptotic cell death with increasing concentrations (Fig.1D). Further, the exposure of SM1 to vemurafenib resulted inthe expected effects of inhibiting downstream MAPK pathwaysignaling with additional inhibition of the PI3K/AKT signaling(Fig. 1E), similar to that previously described in BRAFV600E

mutant human melanoma cell lines (19, 32). SM1 tumors

established subcutaneously in C57BL/6 mice responded tosingle-agent vemurafenib with a growth delay compared withthe progressive tumor growth in mice treated with vehiclecontrol (Fig. 1F). As with our prior results in tests conductedon human lymphocytes (9), increasing concentrations ofvemurafenib did not negatively alter the viability of murinelymphocytes (Fig. 2A). Further, analysis for pERK showedparadoxical MAPK activation, shown by increase in pERK,most notably at 1 and 5 mmol/L, when murine splenocyteswere exposed to vemurafenib and assayed 24 hours later(Fig. 2b). Because the response to single-agent vemurafenibwas not complete and this BRAF inhibitor did not negativelyaffect murine splenocytes, we hypothesized that SM1 wouldbe a useful model to test the potential beneficial effects ofadding an immunotherapy strategy to the treatment withvemurafenib.

Figure 1. Effects of vemurafenib inthe murine SM1 BRAFV600E mutantmelanoma cell line. A, detection ofBRAFV600E mutation (T1799A) inSM1 by DNA sequencing. B, humanmelanoma cell-lines andmurine SM1melanoma cells were exposed toincreasing concentrations ofvemurafenib for IC50 determinationusing an MTS assay. C, cell-cyclearrest in SM1 cells induced byvemurafenib (15 mmol/L) after a72-hour exposure analyzed by flowcytometry. D, apoptosis markeranalysis of SM1 cells exposed tovemurafenib for 72 hours at15 mmol/L analyzed by flowcytometry. E, immunoblotting foranalysis of signaling molecules aftervemurafenib exposure of SM1 cellsat increasing concentrations for 1 or24 hours. F, SM1 tumor implantedmice were injected i.p. daily with10 mg/kg of vemurafenib or vehiclecontrol and followed for tumor-sizechanges over time.

A

C

D

p-Akt(Ser)

Akt

p-Erk

Erk

p-Mek

Mek

p-S6K(Thr/Ser)

p-S6K(Thr)

S6K

PTEN

Tubulin

24 h1 h

PLX (µmol/L) VCVC

1 155

PLX (µmol/L)

1 155

E

F

BA

0

SM-1

Vehicle

SM-1

PLX4032

M202

Vehicle

Pro

pid

ium

iodid

e

M202

PLX4032

Vehicle PLX4032

Vehicle

PLX4032

0

1,500

1,000

500

04 8

Day

Ave

rag

e tu

mo

rvo

lum

e (m

m3 )

12 16

5

G0/G

1 = 65.95%500

400

300

200

100

0

600

400

200

0

G2/M = 24.5%

S = 9.22%

G0/G

1 = 79.99%

G2/M = 7.51%

S = 4.55%

G0/G

1 = 82.31%

G2/M = 9.47%

S = 7.47%

G0/G

1 = 78.41%

G2/M = 9.43%

S = 7.25%

10

SM-1M233M249M202M207

15 20

150

100

50

0

C A G

PLX4032 concentration (μmol/L)

% o

f vi

abili

ty

A A A AG

Annexin V

Co

urt

200

150

100

50

0

Co

urt

500

400

300

200

100

0

Co

urt

Co

urt

0 200 400 600 800 1K

FL2-A

0 200 400 600 800 1K

FL2-A

0 200 400 600 800 1K

FL2-A

0 200 400 600 800 1K

FL2-A






Combined therapy with vemurafenib and ACTimmunotherapy improves antitumor responses againstSM1 tumors

We generated a mouse model targeting the model tumorantigenOVA.We stably expressedOVA in SM1cells to generateSM1-OVA for studies of ACT with splenocytes expressing aTCR specific for OVA (Fig. 3A and 3B). LymphodepletedC57BL/6 mice with established subcutaneous SM1-OVAtumors received ACT of splenocytes obtained from C57BL/6mice genetically modified with a retroviral vector expressingthe 2 chains of the OVA-specific TCR (Fig. 3C and 3D). Wetitrated the conditions of this immunotherapy to provide asuboptimal antitumor activity (similar in range to single-agentvemurafenib) to allow the testing of the benefits of the com-bination. In 2 replicate experiments, the combined therapy ofvemurafenib and OT-1 TCR-engineered splenocyte ACT wasconsistently superior to either therapy alone (Fig. 3E), and itimproved survival (Fig. 3F; P ¼ 0.0004 by log rank test).

As the OVA model is based on the recognition of a foreignantigen, we decided to confirm the results in the pmel-1 ACTmodel (Fig. 4A). The pmel-1 model is based on T cells trans-genic for a TCR recognizing the murine melanosomal antigengp100 (33), which is endogenously expressed by SM1 (Fig. 4B).In 3 replicate experiments, the combined therapy with pmel-1ACT and vemurafenib provided superior antitumor activitycomparedwith either therapy alone,which had been titrated toprovide a suboptimal response against established SM1

tumors (Fig. 4C). As with the OVA model, Kaplan–Meieranalysis of actuarial survival curves generated with the com-bined data from 3 replicate experiments showed that thecombined therapy improved survival compared with singletherapies (Fig. 4D; P < 0.0001 by log rank test).

Vemurafenib does not alter the tumor antigen or MHCexpression by SM1 cells

A hypothesized mechanism of improved antitumor activityof combining BRAF targeted therapy with immunotherapy isan increase in tumor antigen or MHC expression by cancercells (8). Therefore, we tested whether exposure to vemurafe-nib increased the expression of the gp100 melanoma tumorantigen or the expression of surface MHCmolecules, as well asthe recognition by TCR transgenic cells specific for gp100.However, vemurafenib did not significantly alter gp100 tumorantigen expression by SM1 cells (Fig. 4B). The baseline expres-sion of the MHCmolecule H2-Db was very low in cultured SM1cells, and it did not significantly change on exposure tovemurafenib (Supplementary Fig. S2).

No difference in T-cell number, distribution, and tumortargeting of ACT therapy when combined withvemurafenib

It has been reported that biopsies of some patients treatedwith BRAF inhibitors have increased CD8 infiltrates (10). Toanalyze whether vemurafenib expanded or changed the dis-tribution of adoptively transferred cells in vivo with higheraccumulation in tumors, we analyzed their presence in spleens,tumor-draining lymph nodes, and tumors. However, in ourmodel there was no evidence of either a systemic (spleen orlymph nodes) or local (tumor) increase in the quantity ofadoptively transferred antitumor T cells with treatment withvemurafenib (Fig. 5A and 5B; Supplementary Fig. S3A and 3B).To rule out the possibility that weweremissing an effect by notanalyzing the whole animal, we genetically labeled the adop-tively transferred cells with the firefly luciferase transgene toallow their in vivo tracking using bioluminescence imaging.Again, there was no evidence of a differential expansion or invivo distribution and tumor targeting by the adoptively trans-ferred pmel-1 cells when mice were treated with vemurafenib(Fig. 5C). The quantitative analysis of luciferase activity overtime in mice treated with pmel-1 ACT alone or pmel-1 ACTcombined with vemurafenib showed similar in vivo distribu-tion to lymphoid organs and to the antigen-matched tumors(Fig. 5D). Further, we employed a higher resolution method tovisualize a differential systemic immune response using thePET probe [18F]FAC, which has preferential uptake by acti-vated murine lymphocytes (34). Again, there was no differencein the PET scan images with or without systemic treatmentwith vemurafenib (Supplementary Fig. S4).

Increased functional activation of intratumorallymphocytes with exposure to vemurafenib

The in vivo cytotoxicity assay allowed testing of whethervemurafenib had a direct effect of enhancing lymphocytecytotoxicity in vivo, independent of its effects on SM1 tumorcells, as the targets are syngeneic splenocytes devoid of the

pERK

ERK

VC 1 155

Tubulin

PLX4032 (µmol/L)

PLX4032 (µmol/L)

% o

f via

bilit

yA

B

Figure 2. A, effects of vemurafenib on murine splenocyte viability. Cellviability assay (MTS) at different time points and doses of vemurafenib inex vivo activated pmel-1 splenocytes. B, immunoblotting for analysis ofphosphorylated ERK (pERK) and total ERK after vemurafenib exposure ofex vivo gp-100 peptide activated pmel-1 splenocytes at increasingconcentrations for 24 hours.

Koya et al.





BRAFV600E mutation. In 3 replicate experiments the ACT ofpmel-1 cells induced potent cytotoxic effects against spleno-cytes pulsedwith the gp100 peptide, but not against the controlOVA peptide. The cytotoxicity increased with systemic treat-ment with vemurafenib when analyzed at limiting numbers ofadoptively transferred pmel-1 cells (Fig. 6A), but not when thenumber of pmel-1 cells adoptively transferred was 1 log higherand the pmel-1 cells already had a very high lytic activityagainst gp100 peptide pulsed splenocytes (Supplementary Fig.S5). We then analyzed the activation state of TILs by detectingcytokine production. In 2 replicate experiments, TIL collectedfrom mice treated with the combination showed a higherability to respond to short-term ex vivo restimulation with thegp100 antigen, as assessed by IFN-g secretion (Fig. 6B; Sup-

plementary Fig. S6). Therefore, the addition of vemurafenibincreased the functionality of adoptively transferred pmel-1cells in terms of their ability to release an immune stimulatingcytokine and intrinsic antigen-specific lytic activity.

DiscussionThe two approaches with high response rates for the treat-

ment of patients with metastatic melanoma are BRAF inhibi-tors and lymphocyte ACT therapies with ex vivo expandedmelanoma-specific T cells (1, 18, 35). However, in both cases,tumors frequently relapse after an initial response (18, 36).Our data supporting the combination of ACT with vemurafe-nib provides a strong rationale to translate combined

Figure 3. Combined antitumoractivity of TCR engineered adoptivecell adoptive cell transfer (ACT)immunotherapy and vemurafenib inthe ovalbumin (OVA) model. A,schematic of the OT-1 TCRengineered ACT model based onadoptively transferring C57BL/6splenocytes, stably transduced toexpress a TCRspecific forOVAusingretroviral transduction, intolymphodepleted mice withpreviously established flank SM1cells stably expressing OVA (SM1-OVA). Vemurafenib or DMSO vehiclecontrol was started on day þ2 fromthe tumor, and on day þ7, micereceived the ACT of OVA-specificTCR-transduced splenocytes withlymphodepleting radiation therapythe day before, followed by 3 days ofsystemic IL-2 therapy. B, Westernblot analysis for OVA expression inparental SM1 cells (line 1), SM1-OVAcells (line 2), and SM1-OVA tumorgraft (line 3). C, schematic of themurine stem cell virus-basedretroviral vector co-expressing thealpha and beta chains of the OT-1TCR linked by a F2A picornavirussequence. D, tetramer analysis forOVA-specific surface TCRexpression on splenocytes fromC57BL/6 untransduced (left panel) ortransduced with the OT-1 TCR-expressing retrovirus. E, tumorgrowth curves in C57BL/6 mice withestablished SM1-OVA tumors. F,Kaplan–Meier actuarial plot of time tomouse sacrifice due to large tumorburden or to study termination whentumor size was less than 14 mm inmaximum diameter, combiningresults from 2 replicate experimentsin the OVA TCR engineered ACTmodel.






immunotherapy and targeted therapy for patients withBRAFV600 mutant metastatic melanoma. The scientific ratio-nale for combinations of targeted therapies and immunother-apy is based on the notion that pharmacologic interventionswith specific inhibitors of oncogenic events in cancer cellscould sensitize cancer cells to immune attack, which has beentermed immunosensitization (16). An immune-sensitizingagent should ideally block key oncogenic events in cancercells, resulting in an increase in cell-surface ligands for immuneeffector cells, and induce an intracellular pro-apoptotic cancercellmilieu, whichwould enhance the ability of immune effectorcells such as CTLs and natural killer cells to recognize and killcancer cells. At the same time, immune-sensitizing agentsshould not impair the viability or function of immune effectorcells (16). Most of these desired features could be fulfilled byspecific BRAF inhibitors currently used in patients withBRAFV600 mutant metastatic melanoma (8–10).

We explored the potential mechanisms by which vemura-fenib could improve the antitumor activity of adoptivelytransferred T cells in 2 animal models. Our studies show thatthis BRAF inhibitor does not change the cell expansion ordistribution of adoptively transferred cells bymorphologic andmolecular imaging studies. However, lymphocytes exposed tovemurafenib have higher pERK, which is a key feature of anactivated MAPK signaling pathway. Further, we noted animmune cell-intrinsic ability to increase the cytotoxic functionof antigen-specific T cells, and TILs from vemurafenib-treatedmice had higher functional activation with increased ability torelease the immune-stimulating cytokine IFN-g following anti-gen re-exposure. These immune-activating effects of vemur-afenib can be explained by the ability of RAF inhibitors toparadoxically activate the MAPK pathway in cells that are WTfor BRAF but have strong upstream signaling (12–15). There-fore, it is possible that, in this model with a moderately

Figure 4. Combined antitumoractivity of TCR engineeredadoptive cell adoptive cell transfer(ACT) immunotherapy andvemurafenib in the pmel-1 model.A, schematic of the pmel-1 model,where C57BL/6 mice withestablished SM1 tumors receivedvemurafenib or DMSO vehiclecontrol from day þ2 after tumorimplantation, lymphodepletingradiation therapy on day þ7, andthe adoptive transfer of pmel-1splenocytes activated in vitro withgp100 peptide on dayþ7. This wasfollowedwith 3daysof systemic IL-2 therapy and gp100 peptidepulsed dendritic cell (DC) vaccinesondayþ7. B,Western blot analysisfor gp100 expression in parentalSM1 cells exposed to DMSOvehicle control or vemurafenib(PLX4032) at 3 differentconcentrations for 1 or 24 hours.Protein loading was normalized totubulin. C, tumor growth curves inC57BL/6 mice with establishedSM1 tumors. D, Kaplan–Meieractuarial plot of time to mousesacrifice due to large tumor burdenor to study termination when tumorsize was less than 14 mm inmaximum diameter, combiningresults from 2 replicateexperiments in the pmel-1 ACTmodel.

Koya et al.





sensitive tumor target, the main beneficial effects of vemur-afenib are derived from the ability of this agent to directlyimprove immune effector functions independent of the effectsagainst the BRAFV600E mutant tumor.One of the potentialmechanisms of combinatorial activity of

tumor-damaging agents and immunotherapy, leading toincreased TIL activation, is an increased antigen presentationby the tumor cells themselves (8). However, in our studies wecould not readily detect an increase in tumor antigen or MHCmolecule expression by SM1 cells exposed to vemurafenib. Analternative approach leading to increased antigen presentationwould be an increased tumor antigen cross-presentation byhost antigen-presenting cells picking up antigen released bydying cancer cells. However, it is hard to develop directevidence of tumor antigen cross-presentation in these animalmodels, which may be further explored. In addition, it ispossible that vemurafenib could alter the tumor microenviro-ment inhibiting the production of immune suppressive factorsby the melanoma cells, leading to increased adoptively trans-ferred lymphocyte activationwithout increasing antigen cross-presentation. A slower tumor growth and blocking the onco-genic MAPK pathway signaling would favorably modulate the

tumor microenviroment allowing antitumor lymphocytes tobe better activated and produce IFN-g as we have detected.

It is possible that themechanism of improved combinatorialeffects may be different in a BRAFV600 mutant tumor withhigher sensitivity to vemurafenib. In our models based on theSM1 cell line, single-agent vemurafenib had a mainly anti-proliferative effect in vivo, as opposed to the induction ofrapid tumor regression. SM1 is relatively resistant to single-agent vemurafenib in vitro and in vivo, possibly because ofthe multiple genomic alterations in this cell line, includingdeletion of CDKN2A and amplification of BRAF and MITF. Infact, amplification of BRAFV600E is a bona fide mechanism ofresistance to BRAF inhibitors in the clinical setting (37), andpossibly the main reason why SM1-established tumors inmice do not regress with the treatment with vemurafenib. Ifnew murine melanoma cell lines driven by BRAFV600E aredeveloped in the future with higher in vitro and in vivosensitivity to BRAF inhibitors, it is possible that even moresynergistic effects of BRAF inhibitors with immunotherapymay be detected. A rapid tumor response may be more likelyto induce tumor antigen-specific T-cell activation by antigencross-presentation, or inhibition of the immunosuppressive

Figure 5. Effects of vemurafenib onthe number or distribution ofadoptively transferred lymphocytes.A, pmel-1 transgenic T cells wereused for ACT in the pmel-1 combinedtherapy model. Tumors wereharvested on day þ5 after ACT andrepresentative H&E (left) andimmunofluorescence for pmel-1cells stained with anti-Thy1.1-FITC(green, right), and nuclei stained withDAPI (blue, right). B, splenocytes andTILs harvested at day5were countedand analyzed by flow cytometry forgp100 tetramer/Thy1.1/CD3/CD8staining. C, in vivo biolumine-scenceimaging of TCR transgenic T-celldistribution. Pmel-1 transgenic Tcells were transduced with aretrovirus-firefly luciferase and usedfor ACT. Representative figure at day5 depicting 3 replicate mice pergroup. D, quantitation ofbioluminescence imaging of serialimages obtained through day 14post-ACT of pmel-1 transgenic cellsexpressing firefly luciferase with 3mice per group.






tumor microenvironment, and the responding tumor mayenlist inflammatory cells producing chemokine attractantsfor lymphocytes, resulting in increased intratumoralinfiltration.

In conclusion, combined therapy with the BRAFV600-specificinhibitor vemurafenib and TCR-engineered ACT resulted insuperior antitumor effects against a fully syngeineic BRAFV600E

mutant melanoma. Although the absolute number of T cellsinfiltrating the tumor was not increased by vemurafenib, thecombination increased the functionality of antigen-specific Tlymphocytes. Therefore, our studies support the clinical testingof combinations of BRAF targeted therapy and immunother-apy for patients with advanced melanoma.

Disclosure of Potential Conflicts of InterestA. Ribas has received honoraria from consulting with Roche-Genentech,

which is the maker of vemurafenib. No potential conflicts of interest have beendisclosed by the other authors.

AcknowledgmentsThe authors thank Ashley Cass for assistance with bioinformatic analyses.

Vemurafenib (PLX4032) was generously provided by Dr. Gideon Bollag fromPlexxikon Inc.

Grant SupportThis work was funded by the NIH grants P50 CA086306 and P01 CA132681,

The Seaver Institute, the Louise Belley and Richard Schnarr Fund, the WesleyCoyleMemorial Fund, theGarcia-Corsini Family Fund, the Fred L.Hartley FamilyFoundation, the Ruby Family Foundation, the Jonsson Cancer Center Founda-tion, and the Caltech-UCLA Joint Center for Translational Medicine (all to A.Ribas). N.A. Graham was supported by the UCLA Tumor Biology Program, USDepartment of Health andHuman Services, and Ruth L. Kirschstein InstitutionalNational Research Service Award T32 CA009056. A. Minasyan was supported bythe Eugene V. Cota-Robles Fellowship and the Competitive Edge Fellowshipfunded by NSF AGEP. P.C. Tumeh was supported by K08 AI091663.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 21, 2011; revised April 28, 2012; accepted May 19, 2012;published OnlineFirst June 12, 2012.

BA

pmel-1 ACT: TILs

VemurafenibVehicle

IFN

γ

#V2

#V3

#P2

#P3

#V1 #P1

IFN

γ

Vehicle/Mock ACT

OVA peptide

pulsed

gp100 peptide

pulsed

Ve

hic

le +

pm

el-1

AC

Tve

mu

rafe

nib

+ p

me

l-1

AC

T

Mouse

Ve3

Mouse

Ve4

Mouse

PLX3

Mouse

PLX4

Figure 6. Effects of vemurafenib on the cytotoxic and cytokine-producing functions of adoptively transferred lymphocytes. A, effects on cytotoxicity with thein vivo cytotoxic T cell assay. C57BL/6 mice received ACT of 5 � 104 pmel-1 splenocytes and daily vemurafenib or vehicle administered intraperitoneally.On day 16, mice received an intravenous challenge with CFSE-labeled target cells (splenocytes pulsed with gp100 peptide or control OVA peptide).Ten hours later, splenocytes were harvested and analyzed by flow cytometry. B, effects on cytokine production upon antigen restimulation. SM1tumor-bearing C57BL/6 mice received pmel-1 ACTwith or without vemurafenib. At day 5 post-ACT, tumors were harvested and TILs isolated for intracellularIFN-g staining analyzed by intracellular staining by flow cytometry on 5 hour ex vivo exposure to the gp10025-33 peptide.

Koya et al.





References1. Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA,

et al. Inhibition of mutated, activated BRAF inmetastatic melanoma. NEngl J Med 2010;363:809–19.

2. Kefford R, Arkenau H, Brown MP, Millward M, Infante JR, Long GV,et al. Phase I/II study of GSK2118436, a selective inhibitor of onco-genic mutant BRAF kinase, in patients with metastatic melanoma andother solid tumors. J Clin Oncol 2010;28:611s.

3. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J,et al. Improved survival with vemurafenib in melanoma with BRAFV600E mutation. N Engl J Med 2011;364:2507–16.

4. Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS,et al. Survival in BRAF V600-mutant advanced melanoma treated withvemurafenib. N Engl J Med 2012;366:707–14.

5. AtkinsMB, LotzeMT, Dutcher JP, Fisher RI, Weiss G,Margolin K, et al.High-dose recombinant interleukin 2 therapy for patients with meta-static melanoma: analysis of 270 patients treated between 1985 and1993. J Clin Oncol 1999;17:2105–16.

6. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, HaanenJB, et al. Improved survival with ipilimumab in patients with metastaticmelanoma. N Engl J Med 2010;363:711–23.

7. 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.

8. Boni A, Cogdill AP, DangP, UdayakumarD,NjauwCN, SlossCM, et al.Selective BRAFV600E inhibition enhances T-cell recognition of mel-anoma without affecting lymphocyte function. Cancer Res 2010;70:5213–9.

9. Comin-Anduix B, Chodon T, Sazegar H, Matsunaga D, Mock S, Jalil J,et al. The oncogenic BRAF kinase inhibitor PLX4032/RG7204 does notaffect the viability or function of human lymphocytes across a widerange of concentrations. Clin Cancer Res 2010;16:6040–8.

10. Wilmott JS, LongGV,Howle JR,Haydu LE, SharmaRN, Thompson JF,et al. Selective BRAF inhibitors induce marked T-cell infiltration intohuman metastatic melanoma. Clin Cancer Res 2012;18:1386–94.

11. Hong DS, Vence L, FalchookG, Radvanyi LG, Liu C, Goodman V, et al.BRAF(V600) inhibitor GSK2118436 targeted inhibition ofmutant BRAFin cancer patients does not impair overall immune competency. ClinCancer Res 2012;18:2326–35.

12. Heidorn SJ, Milagre C, Whittaker S, Nourry A, Niculescu-Duvas I,Dhomen N, et al. Kinase-dead BRAF and oncogenic RAS cooperateto drive tumor progression through CRAF. Cell 2010;140:209–21.

13. Poulikakos PI, ZhangC, Bollag G, Shokat KM, RosenN. RAF inhibitorstransactivate RAF dimers and ERK signalling in cells with wild-typeBRAF. Nature 2010;464:427–30.

14. Halaban R, ZhangW, Bacchiocchi A, Cheng E, Parisi F, Ariyan S, et al.PLX4032, a selective BRAF(V600E) kinase inhibitor, activates the ERKpathway and enhances cell migration and proliferation of BRAF mel-anoma cells. Pigment Cell Melanoma Res 2010;23:190–200.

15. Su F, Viros A, Milagre C, Trunzer K, Bollag G, Spleiss O, et al. RASmutations in cutaneous squamous-cell carcinomas in patients treatedwith BRAF inhibitors. N Engl J Med 2012;366:207–15.

16. Begley J, Ribas A. Targeted therapies to improve tumor immunother-apy. Clin Cancer Res 2008;14:4385–91.

17. Bollag G, Hirth P, Tsai J, Zhang J, Ibrahim PN, Cho H, et al. Clinicalefficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 2010;467:596–9.

18. Ribas A, Kim K, Schuchter L, Gonzalez R, Pavlick AC, Weber J, et al.BRIM-2: an open-label, multicenter phase II study of RG7204(PLX4032) in previously treated patients with BRAF V600E muta-tion-positive metastatic melanoma. J Clin Oncol 2011;29:Abstr 8509.

19. Sondergaard JN, Nazarian R, Wang Q, Guo D, Hsueh T, Mok S, et al.Differential sensitivity of melanoma cell lines with BRAFV600Emutation to the specific RAF inhibitor PLX4032. J Transl Med2010;8:39.

20. Koya RC, Kasahara N, Pullarkat V, Levine AM, Stripecke R. Transduc-tion of acute myeloid leukemia cells with third generation self-inacti-vating lentiviral vectors expressing CD80 and GM-CSF: effects onproliferation, differentiation, and stimulation of allogeneic and autolo-gous anti-leukemia immune responses. Leukemia 2002;16:1645–54.

21. Yang L, Baltimore D. Long-term in vivo provision of antigen-specific Tcell immunity by programming hematopoietic stem cells. Proc NatlAcad Sci U S A 2005;102:4518–23.

22. Brusko TM,KoyaRC,ZhuS, LeeMR,PutnamAL,McClymont SA, et al.Human antigen-specific regulatory T cells generated by T cell receptorgene transfer. PLoS One 2010;5:e11726.

23. KoyaRC,MokS, Comin-Anduix B, Chodon T, RaduCG, NishimuraMI,et al. Kinetic phases of distribution and tumor targeting by T cellreceptor engineered lymphocytes inducing robust antitumorresponses. Proc Natl Acad Sci U S A 2010;107:14286–91.

24. Lee JT, Li L, BraffordPA, vandenEijndenM,HalloranMB,Sproesser K,et al. PLX4032, a potent inhibitor of the B-RAF V600E oncogene,selectively inhibits V600E-positive melanomas. Pigment Cell Melano-ma Res 2010;23:820–7.

25. YangH, Ding Y, Hutchins LN, Szatkiewicz J, Bell TA, Paigen BJ, et al. Acustomized and versatile high-density genotyping array for themouse.Nat Methods 2009;6:663–6.

26. Olshen AB, Venkatraman ES, Lucito R, Wigler M. Circular binarysegmentation for the analysis of array-based DNA copy number data.Biostatistics 2004;5:557–72.

27. Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES,Getz G, et al. Integrative genomics viewer. Nat Biotechnol 2011;29:24–6.

28. Beroukhim R, Mermel CH, Porter D, Wei G, Raychaudhuri S, DonovanJ, et al. The landscape of somatic copy-number alteration acrosshuman cancers. Nature 2010;463:899–905.

29. Prins RM, Shu CJ, Radu CG, Vo DD, Khan-Farooqi H, Soto H, et al.Anti-tumor activity and trafficking of self, tumor-specific T cells againsttumors located in the brain. Cancer Immunol Immunother 2008;57:1279–89.

30. Vo DD, Prins RM, Begley JL, Donahue TR, Morris LF, Bruhn KW, et al.Enhanced antitumor activity induced by adoptive T-cell transfer andadjunctive use of the histone deacetylase inhibitor LAQ824. CancerRes 2009;69:8693–9.

31. Goel VK, Ibrahim N, Jiang G, Singhal M, Fee S, Flotte T, et al.Melanocytic nevus-like hyperplasia and melanoma in transgenicBRAFV600E mice. Oncogene 2009;28:2289–98.

32. Nazarian R, Shi H,WangQ, Kong X, Koya RC, Lee H, et al. Melanomasacquire resistance to B-RAF(V600E) inhibition by RTK or N-RASupregulation. Nature 2010;468:973–7.

33. Overwijk WW, Theoret MR, Finkelstein SE, Surman DR, de Jong LA,Vyth-Dreese FA, et al. Tumor regression and autoimmunity afterreversal of a functionally tolerant state of self-reactive CD8þ T cells.J Exp Med 2003;198:569–80.

34. Radu CG, Shu CJ, Nair-Gill E, Shelly SM, Barrio JR, Satyamurthy N,et al. Molecular imaging of lymphoid organs and immune activation bypositron emission tomography with a new [18F]-labeled 20-deoxycy-tidine analog. Nat Med 2008;14:783–8.

35. RosenbergSA,RestifoNP,Yang JC,MorganRA,DudleyME.Adoptivecell transfer: a clinical path to effective cancer immunotherapy.NatRevCancer 2008;8:299–308.

36. JohnsonLA,MorganRA,DudleyME,CassardL, YangJC,HughesMS,et al. Gene therapy with human and mouse T-cell receptors mediatescancer regression and targets normal tissues expressing cognateantigen. Blood 2009;114:535–46.

37. Shi H, Moriceau G, Kong X, Lee MK, Lee H, Koya RC, et al. Melanomawhole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance. Nat Commun2012;3:724.






2012;72:3928-3937. Published OnlineFirst June 12, 2012.Cancer Res Richard C. Koya, Stephen Mok, Nicholas Otte, et al. Adoptive Cell ImmunotherapyBRAF Inhibitor Vemurafenib Improves the Antitumor Activity of

Updated version

10.1158/0008-5472.CAN-11-2837doi:

Access the most recent version of this article at:

Material

Supplementary

http://cancerres.aacrjournals.org/content/suppl/2012/06/12/0008-5472.CAN-11-2837.DC1

Access the most recent supplemental material at:

Cited articles

http://cancerres.aacrjournals.org/content/72/16/3928.full#ref-list-1

This article cites 36 articles, 11 of which you can access for free at:

Citing articles

http://cancerres.aacrjournals.org/content/72/16/3928.full#related-urls

This article has been cited by 10 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://cancerres.aacrjournals.org/content/72/16/3928To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/lookup/doi/10.1158/0008-5472.CAN-11-2837

http://cancerres.aacrjournals.org/content/suppl/2012/06/12/0008-5472.CAN-11-2837.DC1

http://cancerres.aacrjournals.org/content/72/16/3928.full#ref-list-1

http://cancerres.aacrjournals.org/content/72/16/3928.full#related-urls

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

mailto:[email protected]

http://cancerres.aacrjournals.org/content/72/16/3928


Documents

BRAF Inhibitor Vemurafenib Improves the Antitumor Activity of Adoptive Cell Immunotherapy · BRAF Inhibitor Vemurafenib Improves the Antitumor Activity of Adoptive Cell Immunotherapy