combined alloreactive ctl cellular therapy with prodrug ... · ated prodrug activator gene therapy...

Cancer Therapy: Preclinical

Combined Alloreactive CTL Cellular Therapy with ProdrugActivator Gene Therapy in a Model of Breast CancerMetastatic to the Brain

Michelle J. Hickey1, Colin C. Malone1, Kate L. Erickson1, Amy Lin1, Horacio Soto1, Edward T. Ha1,Shuichi Kamijima2, Akihito Inagaki2, Masamichi Takahashi2, Yuki Kato3, Noriyuki Kasahara2,3,Barbara M. Mueller4, and Carol A. Kruse1

AbstractPurpose: Individual or combined strategies of cellular therapywith alloreactive CTLs (alloCTL) and gene

therapy using retroviral replicating vectors (RRV) encoding a suicide prodrug activating gene were explored

for the treatment of breast tumors metastatic to the brain.

Experimental Design: AlloCTL, sensitized to the HLA of MDA-MB-231 breast cancer cells, were

examined in vitro for antitumor functionality toward breast cancer targets. RRV encoding the yeast cytosine

deaminase (CD) gene was tested in vivo for virus spread, ability to infect, and kill breast cancer targets when

exposed to 5-fluorocytosine (5-FC). Individual and combination treatments were tested in subcutaneous

and intracranial xenograft models with 231BR, a brain tropic variant.

Results: AlloCTL preparations were cytotoxic, proliferated, and produced IFN-g when coincubated with

target cells displaying relevant HLA. In vivo, intratumorally placed alloCTL trafficked through one estab-

lished intracranial 231BR focus to another in contralateral brain and induced tumor cell apoptosis. RRV-CD

efficiently spread in vivo, infected 231BR and induced their apoptosis upon 5-FC exposure. Subcutaneous

tumor volumes were significantly reduced in alloCTL and/or gene therapy–treated groups compared to

control groups. Mice with established intracranial 231BR tumors treated with combined alloCTL and RRV-

CD had a median survival of 97.5 days compared with single modalities (50–83 days); all experimental

treatment groups survived significantly longer than sham-treated groups (median survivals 31.5 or 40 days)

and exhibited good safety/toxicity profiles.

Conclusion: The results indicate combining cellular and suicide gene therapies is a viable strategy for the

treatment of established breast tumors in the brain. Clin Cancer Res; 19(15); 4137–48. �2013 AACR.

IntroductionBreast cancer is themost commonmalignancy in women

in the United States and metastasis is a major cause ofmorbidity and mortality in these patients. With improve-

ments in the control of visceral and bone metastasis, theincidence of brainmetastasis is rising (1–3). The progressiveneurological disabilities associated with brain metastasisnot only impair the quality of life but also decrease thesurvival. The biology of the brain and blood–brain barrier,and the fact that brain metastases can present as multiplelesions often characterized by nests of infiltrating tumorcells surrounding the larger brainmetastases (4, 5)make themanagement of brain metastases very challenging. Currenttherapeutic approaches include whole brain radiation ther-apy and stereotactic radiosurgery usually administered aspalliative care (6–8).

Newer approaches include the use of tumor-targeted CTLfor adoptive immunotherapy. A unique form of adoptive T-cell therapy for brain tumors involves the use of alloreactiveCTL (alloCTL), potent cytotoxic T-cell effectors that aretrained to recognize non- or aberrant-self class I and II HLA.They are generated by sensitization of peripheral bloodmononuclear cells (PBMC), isolated from a healthy donor,to the HLA of the tumor-bearing host (9–11). The HLAprimarily acts as tumor-directed antigen in the brain,because its expression, especially class I, is absent onnormal

Authors' Affiliations: Departments of 1Neurosurgery, 2Medicine, and3Molecular & Medical Pharmacology, University of California Los Angeles,Los Angeles; and 4Torrey Pines Institute for Molecular Studies, San Diego,California

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

Current address for A. Lin: Tocagen, Inc., 3030Bunker Hill Street, Ste #230,San Diego, CA 92109.

N. Kasahara, B.M. Mueller, and C.A. Kruse jointly participated in oversightof this research.

Corresponding Author: Carol A. Kruse, Department of Neurosurgery,University of California Los Angeles, 695 Charles E. Young Drive, South,Gonda 1554B, Mail Code 176122, Los Angeles, CA 90095. Phone: 310-267-2535; Fax: 310-206-2093; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-12-3735

�2013 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 4137

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Published OnlineFirst June 18, 2013; DOI: 10.1158/1078-0432.CCR-12-3735

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neuroglia, that is, neurons, oligodendrocytes, and astro-cytes (12–14), but is highly expressed on brain tumor cells(13, 15–18).

Another new therapeutic approach is the use of replicat-ing virus vectors for gene therapy or oncolytic virotherapy.Recently, it has been demonstrated that retroviral replicat-ing vectors (RRV), unlike their replication-defective coun-terparts, exhibit sustained transduction in dividing cancercells and can efficiently transfer transgenes throughout solidtumors (19–24). Glioma-bearing rats show significantlyprolonged survival when given RRV coding for the prodrugactivator gene, yeast cytosine deaminase (CD), followed bymultiple administrations of 5-fluorocytosine (5-FC) pro-drug (20–22). The lack of detectable spread of the RRV tonormal tissues additionally provides an element of safety inthis approach (21, 23, 25).

Both alloCTL adoptive immunotherapy and RRV-medi-ated prodrug activator gene therapy have been studiedindividually in patients with primary brain tumors. Nota-bly, the clinical feasibility and safety of intratumoralalloCTL treatment was initially tested in a small pilot study(9, 11), in which 3 recurrent grade 3 glioma patientsreceiving this treatment exhibited survival longer thanexpected. These data led to the initiation of a phase Idose-escalation study at University of California LosAngeles (UCLA), now accruing patients (www.clinical-trials.gov, NCT 01144247). As well, RRV encoding cytosinedeaminase for prodrug activator gene therapy has advancedto the clinic for recurrent high-grade gliomas (www.clin-icaltrials.gov, NCT 01156584 and NCT 01470794).

Clinically, brain metastases are more common thanprimary brain tumors (26), and represent a dire clinicalsituation in need of effective therapies. Here, we evaluatedboth of these cellular and gene therapy approaches, indi-vidually and in combination, for the treatment of intracra-

nial breast tumors. Our results show that alloCTL generatedto the breast tumor cell line MDA-MB-231 (231) showedcytotoxicity, proliferation, and IFN-g production inresponse to coincubation with the parental target cell line,as well as with 2 metastatic sublines (27, 28). Intracraniallyplaced alloCTL were capable of migrating from one estab-lished intracranial tumor focus to another in contralateralbrain over a relatively short time span (6 hours) in immune-incompetentmice, and apoptotic tumor cells were observedin proximity to these CTL. Using subcutaneous and intra-cranial human breast tumor xenograft models, we foundthat combining prodrug-activating gene therapy withalloCTL adoptive transfer resulted in improved outcomecompared to the benefit seen with each therapy individu-ally. Our findings thus support clinical translation of thismultimodal approach.

Materials and MethodsCell lines and HLA expression

The human breast tumor cell line MDA-MB-231 [231;American Type Culture Collection (ATCC), Manassas, VA]was grown in L15 medium supplemented with 10% FBS(Aleken). The brain-seeking 231 subline, 231BR (27), thebone-seeking 231 subline, 231-1833 (28), and 293Thuman embryonic kidney cells (ATCC) were maintainedin Dulbecco’s Modified Eagles Medium supplemented with10% FBS, sodium pyruvate, and penicillin/streptomycin(Sigma). Class I HLA display was determined on breastcancer cell lines by flow cytometric analysis using RPE-conjugated mouse anti-human HLA-ABC antibody and amouse RPE-conjugated immunoglobulin G served as iso-type control (eBiosciences). The percentage of positive cellsand relative antigen density indicated bymean fluorescenceintensity (MFI) were determined (13). Specific HLA-ABCtypes were determined by low-resolution molecular HLAtyping (University of California, San Diego, Torrey PinesClinical Laboratory, San Diego, CA).

Generation of alloCTL-enriched cultures by one-waymixed lymphocyte tumor reaction

PBMC were isolated from leukopaks of healthy donorsundergoing leukapheresis (UCLA Institutional ReviewBoard approved protocol) using density gradient centrifu-gation as previously described (10). Before one-way mixedlymphocyte tumor reaction (MLTR), 231 cells underwent48-hour incubation in culture medium with 500 IU/mLrecombinant human IFN-g (RD Systems Inc.) to upregulatetheir HLA expression (13). The stimulator (S) 231-cellmonolayers were then washed with PBS and detached with2 mmol/L EDTA containing 1% bovine serum albumin,washed and inactivated with 7000 rad (X-ray Irradiator,Gulmay Medical, Inc.). Responder (R) PBMC were mixedwith inactivated S cells at a R:S ratio of 10:1. The cellmixtures were placed into Aim-V medium (Life Technolo-gies, Inc.) containing 5% heat-inactivated autologous plas-ma and 60 International Units (IU) of recombinant humaninterleukin-2 (IL-2)/mL (Proleukin; Novartis). Growth wasmonitored daily and cultures were fed and maintained as

Translational RelevanceMetastasis to the central nervous system is common in

advanced breast cancer. With limited treatment optionsavailable to patients with metastatic foci, we exploredcellular and gene therapy approaches as potential treat-ments for breast cancer metastatic to brain. This reportdescribes work to indicate the individual, and especiallythe combined immunogene treatment modalities areeffective in vitro and in in vivo xenograft models. Thesenovel therapies are well tolerated, brain sparing, andprovide multiple mechanisms of tumor cell–targetedcytotoxicity, including CTL effector-mediated and che-motherapeutic-mediated cytolysis with suicide vector/prodrug that may be further promoted with bystandereffects. Preclinical studies of the individual treatmentmodalities have warranted their advance to the clinic fortreatment of gliomas. Those data and that within thisstudy support accelerated translation of the therapies fortreatment of brain metastases.

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Clin Cancer Res; 19(15) August 1, 2013 Clinical Cancer Research4138




previously described (10). AlloCTL preparations were usedin assays between 12 and 14 days post-MLTR.

Plasmid constructs and viral vector productionThe pAC3-yCD2 and pAC3-GFP plasmid constructs,

encoding amphotropic Moloney murine leukemia viruscarrying expression cassettes consisting of an internal ribo-some entry site followed by either cytosine deaminase orGFP transgenes, respectively, have been described previous-ly (23, 24).Viral vector stocks were generated by transfection of 293T

cells using the FuGENE6 transfection agent (Roche Molec-ular Biochemicals) per manufacturer instructions (22).Conditioned medium (viral supernatant) was collected48 hours after transfection with either the pAC3-GFP orpAC3-yCD2 plasmids (23, 24).Concentrated viral vector supernates were prepared from

293T stable vector producing cells. Conditioned mediumwas harvested after 72-hour incubation, and clarified bycentrifugation before 100-fold volume concentration usingaVivaspin-20 (Sartorius Stedim)permanufacturer protocol.Control supernatant from nontransduced, nonvector pro-ducing 293T cells was harvested and concentrated in paral-lel. Concentrated viral titers were approximately 106 trans-ducing units/mL when tested using either 293T or 231BRcells. The titer was determined 48 hours after adding 50mmol/L of 30-azido-deoxythymidine (Sigma Aldrich) to thecultured cells toprevent further virus replicationasdescribed(20, 29). For subcutaneous tumors, 10 mL of concentratedvector producing or nonvector producing supernatantswereadmixed with tumor cells immediately before injection.

Antitumor functionality assessments of alloCTLCytotoxicity assay. Lytic activity of alloCTL was assessed

at various effector-to-target ratios (E:T) by 51Chromiumrelease cytotoxicity assays at 12 to 14 days post-MLTR(9). Target cells were labeled with 100 mCi Na2

51CrO4

(Amersham) and the assay was conducted as previouslydescribed (30). Maximal release was obtained from targetsincubated with 0.1 M HCl, and spontaneous release wasthe radioactivity released from targets in assay mediumalone. The percentage specific release was calculated by theformula: [(51Crexperimental � 51Crspontaneous)/(

51Crmaximal �51Crspontaneous)] � 100%. Mean specific release � SEM wasdetermined from triplicate wells.Cytokine production. Human IFN-g concentrations

were measured in clarified supernates collected 48 hoursafter coincubation of day 12 alloCTL with relevant tumortarget cells. Aliquots of the alloCTL from the same prepa-ration either were or were not restimulated at day 12 post-MLTR with 231 or sublines, 231-1833 or 231BR, at a R:S of10:1. Manufacturer instructions were followed for a Quan-tikine ELISA kit specific for human IFN-g (R&D Systems)with a sensitivity of 2 pg/mL (30).Immunophenotype and proliferation of restimulated

alloCTL. At 12 days post-MLTR, alloCTL were restimu-lated with 231 cells or sublines for 72 hours at a R:S ratio of10:1 in culture medium containing bromodeoxyuridine

(BrdU; BD Biosciences). GolgiStop (BD Biosciences) wasadded to block protein transport during the last 5 hours ofculture. Cells were washed and surface stained with APC-conjugated anti-CD4, PeCy5-conjugated anti-CD8 for 15minutes at 4�C, washed again, fixed, and permeabilizedaccording to instructions for the BD BrdU Flow Kit (BDBiosciences). Cells were then intracellularly stained withphycoerythrin-conjugated anti-IFN-g and fluorescein iso-thiocyanate-conjugated anti-BrdU for 30 minutes at 4�C,washed, resuspended, and immunophenotyped using a BDLSRII flow cytometer running BD-DIVA acquisition soft-ware. Dot blots were analyzed using FlowJo flow cytometricanalytic software.

In vivo studies with human 231BR xenograftsRag2�/�gc�/� mice were purchased from Taconic Farms

(Hudson; ref. 31). All animal experiments were conductedaccording to institutional guidelines under approvedprotocols.

Subcutaneous tumor model. 231BR cells (2 � 106) werecoinjected with concentrated RRV-GFP, RRV-CD, or con-centrated nonviral control supernate subcutaneously in theright flank of 6-week-old mice. After allowing tumors toestablish for 15 days, mice were randomized to treatmentgroups (n ¼ 4–6). Certain groups were treated with intra-tumoral alloCTL (1.2 � 107). All mice were treated with 5-FC (500mg/kg) intraperitoneally (i.p.) every day from days21 to 27. Caliper measurements of tumor width and lengthwere conducted every 3 to 4 days. Tumor volumes werecalculated for individual animals using the formula: volume¼ (4/3)p(width/2)2(length/2) (32). Mean tumor volumesfor each treatment group � SEM were plotted over time.

Intracranial vector spread. 100% RRV-GFP transduced231BR (231BR-GFP) cells were admixed with nontrans-duced 231BR at 2% or 8% of the total tumor cell inoculum(2� 105/3 mL). Cells were then placed into the right side ofthe brain through a burr hole, 2 mm lateral and 1 mmanterior to the bregma, at a depth of 3 mm. Groups of mice(n ¼ 3) were sacrificed on days 7, 14, and 20 post-tumorinstillation, and brains were harvested. Tumor tissue at theinjection site was excised (approximately 5 � 5 � 5 mm3),minced, and digested in collagenase/dispase (1 mg/mL;Roche) as described (22). Single cell suspensions werestained with fluorescent APC anti-HLA-ABC (eBiosciences)and flow cytometrically analyzed.

Intracranial alloCTL motility studies. 231BR cells (2 �105 each) were stereotactically injected into the abovecoordinates and its enantiomeric position, such that rightand left hemispheric tumor foci could develop. Tumorswere allowed to establish 18 or 21 days before alloCTL (2�106) were injected into the left tumor foci. Mice weresacrificed 6 hours later, and brains were harvested, placedinto 10%buffered formalin, paraffin-embedded, sectioned,and diaminobenzidine immunostained using rabbit anti-human CD3 (Clone SP7, Genway Biotech Inc.) with hema-toxylin counterstain and evaluated by light microscopy.

Intracranial efficacy studies. Mice underwent surgery forplacement of intracranial cannulas (Plastics One) that were

Immuno-Gene Therapy of Breast Metastases to Brain

www.aacrjournals.org Clin Cancer Res; 19(15) August 1, 2013 4139




placed through a burr hole in the right side of the skull atthe above coordinates. The cannulas extended 3 mm intothe brain and affixed to the skull with resin. Six days later,tumor cells (2� 105 total cells in 3 mL), consisting of either100% 231BR, or 98% 231BR cellsþ 2% 231BR-GFP or 2%231BR-CD cells, were infused through the cannulas and theanimalswere placed into various treatment groups. AlloCTLor unstimulated PBMC (2� 106/3 mL) or PBS were infusedthrough the cannulas into the tumor on days 9 and 16posttumor instillation. All groups of mice (n ¼ 9–10) weretreated with 3 cycles of 5 daily i.p. 5-FC (500 mg/kg)injections (per cycle) beginning on days 12, 26, and 47posttumor instillation. Mice were monitored for signs ofmorbidity and weighed every 3 to 4 days for the duration ofthe experiment.

Analysis of RRV biodistributionTo harvest brains, coronal cuts were made at the site of

cannula implantation and 4 mm posterior to that cut. Theanterior sectionswere snap-frozen for quantitative real-timePCR (qRT-PCR) to determine RRV biodistribution; theposterior 4 mm sections were fixed in formalin, paraffin-embedded, and the blocks were sectioned (5 mm) beforestaining with hematoxylin and eosin (H&E).

Genomic DNA was extracted using the DNeasy tissue kit(Qiagen) from tissues (liver, lung, spleen, kidney, bonemarrow, and brain) of all long-term survivors and repre-sentative animals derived from control and experimentalgroups that succumbed to tumor after intracranial treat-ment with RRV and/or alloCTL. To detect integrated RRVsequences, qRT-PCR amplification of genomic DNA wascarried out in duplicate with TaqManUniversal PCRMasterMix (PE Applied Biosystems) using a My-iQ2 Biorad Ther-mal Cycler. The primers and probe were designed to targetthe 4070A amphotropic env gene (4070A-F, 50-GCGGAC-CCGGACTTTTGA-30; 4070A-R, 50-ACCCCGACTTTACG-GTATGC-30; probe, FAM-CAGGGCACACGTAAAA-NFQ).Human RNase P (hRNase P) andmouse b-actin (mb-actin)were also quantified as reference genes using TaqManRNasePDetectionReagents andCustomTaqManGene ExpressionAssays (Applied Biosystems), which is designed to targetmouse b-actin gene (b-actin-forward, 50-GGTCGTACCA-CAGGCATTGT-30; b-actin-reverse, 50-CTCGTAGATGGGC-ACAGTGT-30; probe, FAM-CCCGTCTCCGGAGTCC-NFQ).A standard curve for copy number was prepared by ampli-fying serial dilutions of plasmid pAC3-yCD2 in a back-ground of genomic DNA from nontransduced cells.

Statistical analysesGraphPad Prism software (version 4; GraphPad Soft-

ware) was used to analyze the in vitro and in vivo data. TheStudent t test determined significant differences betweenthe percentage lysis of target tumor cells with and withoutthe addition of anti-HLA; P-values �0.05 were significant.Experimental and control groups for the in vitro restimula-tion experiments measuring proliferation, immunopheno-type, and cytokine production, and the in vivo subcutaneoustumor volume comparisons at given times were compared

using a two-way ANOVA with Bonferroni correction andP-values �0.05 divided by the number of comparisons (3)in experiments described in Figs. 2 and 4 were consideredsignificant. Median survival times (MST) from the Kaplan–Meier curves were analyzed by nonparametric log-ranktests.

Results and DiscussionHLA class I onbreast tumor cells andupregulationwithIFN-g

Flow cytometric analysis showed that nearly all (94–99%) of the cells in the parental 231 line, as well as 231-1833 and 231BR sublines, expressed HLA class I and theirincubation with IFN-g resulted in 1.4- to 1.5-fold increasesin MFI (Supplementary Table S1). HLA-ABC expressionafter IFN-g induction was highest on the parental 231 cellline (MFI 1402 � 49.7) compared with slightly lowerexpression by each of the sublines, thusmaking the parental231 cells most desirable as stimulator cells in a one-wayMLTR for alloCTL generation. The low-resolution molecu-lar type of HLA-ABC loci in 231 cells was HLA-A�02,B�41,�40, and C�17,�02 (Supplementary Table S2).

AlloCTL exhibit antitumor function in vitroThe standard method for generating alloCTL is by one-

way mixed lymphocyte reaction where inactivated stim-ulator PBMC from the cancer patient are combined withresponder PBMC from healthy donors genetically distinctfrom the patient (10). However, because PBMC are notavailable from the patient from whom 231 breast tumorcells were derived, to generate alloCTL we used a one-wayMLTR where 231 tumor cells were incubated with IFN-gto upregulate HLA class I expression as stated earlier,then inactivated and used as stimulator cells with re-sponder PBMC isolated from HLA-mismatched allogeneicdonors (33).

Four different donors provided PBMC that served assources of precursor alloCTL for these experiments. Alldonors weremismatched at 4 to 5 class I HLA-ABC antigenscompared to that displayed by the 231 stimulator cells(Supplementary Table S2). The HLA types of alloCTL usedfor in vitro and in vivo experiments are provided and asso-ciated with data in particular figures. Note these prepara-tions will contain CTL directed to minor tumor-associatedantigens as well as HLA, albeit the precursor frequency forCTL within PBMC would be higher to major than to minorantigens (34–36).

To determine if the alloCTL made by MLTR had antitu-mor function in vitro, we conducted cytotoxicity assays onday 14 following one-wayMLTR. AlloCTL effector cells weremixed with 51Cr-loaded 231, 231-1833, or 231BR targetcells at a range of E:T ratios for 4 hours. As expected, and in adose-dependent manner, the ability of alloCTL to kill eachof the 3 target cell lineswas similar; therewere no significantdifferences in the percentages of lysis obtained at each E:Tratio (Fig. 1). Furthermore, the cytolysis induced by alloCTLto 231 and its sublines is in part HLA-restricted, as indicat-ed by significant inhibition of cytotoxicity (33.3–41.3%

Hickey et al.





reduction in percentage lysis of 231 and variants, P < 0.006)upon antibody-mediated blockade of HLA. We replicatedthe cytotoxicity findings with alloCTL (with and withoutblocking antibody to HLA) using 2 other breast cancer celllines, BMC Brain2 and MDA-MB-361 (unpublished data).

AlloCTL subset proliferation and IFN-g productionfollowing restimulationThe ability of the CD4þ and CD8þ T-cell subsets within

the alloCTL preparations to proliferate and produce IFN-gfollowing restimulation with 231, 231-1833, or 231BRtarget cells was evaluated. AlloCTL that had been restimu-lated onday 12 after one-wayMLTR for 72 hours at a 10:1 R:S ratio with one of the 3 relevant cell lines, or left unsti-mulated, were surface stained for CD4 and CD8, and thenfixed, permeabilized, and stained for proliferation marker(BrdU) and proinflammatory IFN-g production. Figure 2shows data obtained from a representative alloCTL prepa-ration. The percentages of CD8þ T cells that incorporatedBrdU upon restimulation and thus proliferated upon stim-ulationwith relevant target cells were significantly higher (P< 0.017) compared to those that were unstimulated (Fig.2A). Lymphocyte proliferation occurred equally well withparental 231 cells and the metastatic sublines, 231-1833 or231BR. In contrast, there were no significant differences inBrdU incorporation between CD4þ T cells that were resti-mulated versus those that were unstimulated (Fig. 2A),indicating lack of CD4þ subset proliferation, regardless of

whether restimulationwas conductedwith the parental 231cells or the 231-1833 or 231BR subline counterparts.

The total percentages of CD8þ T cells were significantlyhigher (P < 0.017) within alloCTL populations that wererestimulated with relevant target cells (51.8–56%) com-pared to unstimulated alloCTL (30.9%; Fig. 2B). Becausethe CD8þ cells proliferated when restimulated with 231,231-1833, and 231BR cells and the CD4þ cells did not,the total percentages of CD4þ T cells were significantlyreduced (P � 0.017) compared to the unstimulatedcounterpart (Fig. 2C). The data show that restimulationof alloCTL with 231, 231-1833, or 231BR breast cancercells results in a significant shift in T-cell subsets thatmake up the alloCTL culture.

Expression of IFN-g following 72-hour restimulationwith 231, 231-1833, and231BR, compared to unstimulatedcells, was also evaluated. Protein transport was blockedduring the last 5 hours of culture to allow intracellularaccumulation of IFN-g . Expression of IFN-g was significant-ly higher (P � 0.017) within CD8þ T cells that wererestimulated with 231, 231-1833, or 231BR, compared tocells that were unstimulated (64.6, 45.1, 43.8 vs. 27.5,respectively; Fig. 2D). Production was more robust whenrestimulation was conducted with the parental cells versusthe sublines, correlating with the relative antigen densities(MFIs) of cell-surface class IHLA (Supplementary Table S1),suggesting that this parameter may have some influenceover response. In contrast, production of IFN-g in CD4þ Tcells was not affected following restimulation with any ofthe cell lines (data not shown).

To confirm the earlier observations, IFN-g secretion byalloCTL at day 12 post-MLTRwas analyzed from supernatescollected 48 hours following restimulation with relevanttarget cells, and compared with IFN-g secreted by unstimu-lated alloCTL. The average secretion of IFN-g by restimu-lated alloCTL coincubated with 231, 231-1833, and 231BR(17.2 � 1.2, 16.9 � 0.50, and 13.2 � 0.97 ng/mL, respec-tively) was significantly elevated (P < 0.017) compared tothat of unstimulated alloCTL (7.4 � 0.21 ng/mL).

AlloCTL migrate through and to distant tumor fociin vivo and induce apoptosis

We examined the migratory capacity of alloCTL in thecontext of multifocal intracranial tumors. As shown in Fig.3A, bilateral 231BR tumors were established intracraniallyin the right and left hemispheres of Rag2�/�gc�/�mice, andallowed to grow for 21 days. AlloCTL specific for 231 HLAwere then stereotactically injected into the tumor bedon theright side (Fig. 3A), and coronal sections of brain wereprepared 6 hours later and immunostained with a humanCD3-specific antibody (Fig. 3B–G). Figure 3B shows arepresentative low power photomicrograph of a coronalbrain section with bilateral tumor foci. Intermediate powermagnification of the tumor (Fig. 3C) in the right side of thebrain shows numerous DAB-stained (rust-color) humanCD3þ T lymphocytes (black arrows) penetrating the tumormass at the instillation site. A higher power magnificationphotomicrograph (Fig. 3D) from the same area shows

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Figure 1. In vitro cytotoxicity of alloCTL to target cells displaying relevantHLA antigen with alloreactivity demonstrated by inhibition of lysis withanti-HLA-ABC. Four-hour 51Cr-release assays show that alloCTLgenerated following one-way MLTR with irradiated stimulator 231 tumorcells are cytotoxic toward the parental 231 cells (black bars) or tometastatic sublines, 231-1833 (gray bars) and 231BR (white bars), atvarious E:T ratios. HLA restriction of the cytotoxicity to the 3 target celltypes (231, black angled stripes; 231-1833, gray angled stripes; 231BR,horizontal stripes) was shown (15:1 E:T) by adding antibody toHLA-ABC.The data are representative of 3 separate experiments; they are averagesof the percentage lysis from triplicate wells � SEM (�, P � 0.006).






CD3þ cells (black arrows) in proximity to a necrotic tumorcell with a fragmented nucleus indicating cell death (whitearrows). In addition, CD3þ alloCTL have trafficked andlocalized to established tumor in contralateral brain (Fig.3E–G). Rust-colored CD3þ cells are shown having perme-ated the tumor mass (t) on the left side and also appear inperivascular spaces (asterisks), which is a path for tumorinvasion (Fig. 3E), whereas no CD3þ cells are visible innormal brain. Intermediate and higher power magnifica-tion photomicrographs (Fig. 3F and G) similarly showlarger, activated CD3þ T-cell phenotypes (black arrows) inclose juxtaposition to apoptotic tumor cells (white arrows),indicating cytotoxic functionality may be retained by thetrafficking alloCTL.

Cellular andgene therapies show therapeutic benefit insubcutaneous 231BR xenograft models

To determine if cellular and gene therapy approachesindividually or in combination would be efficacious in vivo,Rag2�/�gc�/� mice were injected subcutaneously with231BR (initial inoculum 2 � 106 cells) admixed withRRV-GFP, RRV-CD, or control nonviral supernatant.Tumors were allowed to establish for 15 days before someexperimental groups were treated with intratumoralalloCTL (1.2 � 107 cells). All groups (n ¼ 4–6) receivedthe suicide gene therapy prodrug, 5-FC (500 mg/kg i.p.) ondays 21 to 27. Tumor volumesmonitored over time showedsignificant differences between the control and experimen-tal groups starting from day 25 (P < 0.017; Fig. 4). The

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Figure 2. CD4/CD8 T-cell subsetproliferation (BrdU incorporation)and IFN-g production followingalloCTL restimulation. A, BrdUincorporation by CD8þ and CD4þ

T-cell subsets within alloCTL aftertheir restimulation. Unstimulated(angled stripe), 231 (black bar),231-1833 (gray bar), 231BR (whitebar). The percentages of CD8þ Tcells that incorporated BrdU weresignificantly higher (�, P � 0.017)in the CD8þ T cells that wererestimulated compared tothose that were unstimulated.Representative data from 1 of 3separate experiments; data are theaverage percentage of live cellsfrom triplicate wells � SEM. Flowcytometric plots of alloCTLsensitized to 231 and stained for(B) CD8 or (C) CD4 72 hours afterrestimulation with 231, or 231-1833 or 231BR sublines, withunstimulated cells as a control.Data are representative from 1 of 3separate experiments. D,percentage of CD8þ/IFN-gþ cellsalloCTL after restimulation.Representative data are shownfrom triplicate wells of 1 of 3separate experiments. Expressionof IFN-g was significantly higher(�, P � 0.017) in CD8þ T cells thatwere restimulated with 231, 231-1833, and 231BR compared tocells that were unstimulated.

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2 control groups that received 231BR with nontherapeuticRRV-GFP or control nonviral naive 293 cell supernatant atthe start of the experiment both exhibited progressive tumorgrowth in vivo, with average group tumor volumes at day 28of 759 � 243 mm3 and 659 � 114 mm3, respectively.Therapeutic benefit was noted for all experimental treat-ment groups (groups that received RRV-CD, alloCTL, or acombination of the 2) compared to mice that receivedtumor with concentrated nonviral supernatant. Tumorstreated with RRV-CD alone, without alloCTL injection,showed significant (P � 0.017) tumor growth inhibitionupon application of 5-FC prodrug treatment between days25 and 27 compared to control groups. Tumors treatedwithnonviral supernatant but receiving intratumoral alloCTLalso showed significant (P � 0.017) tumor growth inhibi-tion compared to control groups, following cellular thera-py, but as expected, did not show response to prodrugadministration. Notably, the greatest reduction in meantumor volumes was obtained when intratumoral cellulartherapy with alloCTL was combined with prodrug activatorgene therapy with RRV-CD/5-FC (P� 0.017). We obtainedsimilarly efficaciousfindings in a second tumormodel usingsubcutaneous U-87MG glioma xenografts (K. Haga et al.,in preparation). In those experiments, a more clinicallyrelevant scenario was tested where tumors were allowedto establish for 1-week before infusing with RRV-CD super-natant and/or alloCTL. This experimental paradigmrequired an in situ transduction of preestablished tumor to

obtain a beneficial effect fromadministered prodrug. Again,efficacy was observed for the individual as well as thecombination therapies.

RRV effectively spreads through intracranial breasttumor xenografts

RRV can achieve significantly enhanced transductioncompared to their replication defective counterparts (19).RRV exhibit an amplification process that is inherent to thewild-type virus life cycle, so although viral replicationkinetics and lag times for exponential growth phase repli-cation can vary, even a slower replication will allow pro-gressive vector spreadwithin a tumor until prodrug is given.We have previously demonstrated RRV replication andgene expression in subcutaneous mammary tumors (29).To evaluate RRV spread in intracranial breast tumors,Rag2�/�gc�/� mice were intracranially injected with 2 �105 naive 231BR cells mixed supplemented with either 2%or 8% RRV-GFP transduced 231BR cells (231BR-GFP).Transduction of the tumor by RRV was evaluated by flowcytometry on days 7, 14, and 20 post-tumor instillation(Fig. 5A and B). To distinguish the human 231BR tumorcells from mouse brain cells, enzyme-digested tumortissues were stained with fluorescently labeled human-specific anti-HLA-ABC and the percentages of GFPþ cellswithin this population were quantified. When the initialtumor inoculum contained either 2% or 8% 231BR-GFPcells, by day 7 post-tumor instillation, approximately

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Figure 3. AlloCTL trafficking in vivo through and to tumor foci. Brains were collected 6 hours after alloCTL injection into a 3-week established 231BR tumorfocus as seen by human anti-CD3 positivity in ipsilateral and contralateral tumor foci within immune incompetent mouse brain. A, coronal brain sectionschematic of tumor placement and alloCTL at ipsilateral injection site. B, gross coronal brain section that was DAB immunostained with anti-CD3 thatshows 2 tumor foci (ipsilateral-red and contralateral-blue boxes, respectively). C, low power photomicrograph of rust-colored CD3þ cells (black arrows) intumor (t) focus #1 and alloCTL instillation site. D, higher power view showing 3 CD3þ cells in juxtaposition to an apoptotic tumor cell (white arrow).E–G, tumor focus #2 in brain contralateral to alloCTL injection. E, low power photomicrograph shows abundant CD3þ cells (black arrows) primarily within thetumor mass (t) but not in normal brain (n) located in the hemisphere opposite to alloCTL instillation. Yellow asterisks show pockets of tumor cellswithin Virchow–Robin perivascular spaces. (F) Intermediate and (G) high-powermagnification photomicrographs at that site again show apoptotic tumor cells(white arrows) in proximity to CD3þ cells (black arrows). The brain sections are counterstained with hematoxylin. Representative photomicrographs areshown from 1 of 10 mice; 2 experiments were analyzed after H&E staining and 2 experiments after DAB immunostaining. Bars ¼ 0.8 mm in B, 30 mm in C,8 mm in D, 35 mm in E, 15 mm in F, and 10 mm in G.






57% of the human class I HLA-ABCþ tumor cells hadbeen transduced with RRV-GFP. Subsequently, tumortransduction levels rose to �75% to 83% at 20 daysfollowing tumor instillation. Figure 5B shows represen-tative flow cytometric dot-plots from day 14, indicating71.2 � 3.7% and 78.9 � 1.1% GFP transduction of HLA-ABCþ human 231BR cells after initial tumor inoculationwith 2% and 8% 231BR-GFP cells, respectively.

Individual and combined cellular (alloCTL) and gene(CD) therapies are safe and exhibit therapeutic benefitin intracranial 231BR xenograft models

The efficacy of combined cellular alloCTL immunother-apy with RRV prodrug activator gene therapy was tested inRag2�/�gc�/� mice bearing established intracranial 231BRtumors. Indwelling intracranial cannulas were used toestablish intracerebral tumors in 7 groups (n ¼ 9–10) ofmice, consisting of either 100% 231BR or 98% 231BRmixed with either 2% 231BR-GFP or 2% 231BR-CD cells(Fig. 6). Control groups of mice were injected with non-therapeutic 231BR-GFP tumor cells instead of 231BR-CD,or were infused with PBS or unstimulated PBMC in place ofalloCTL. On days 9 and 16 post-tumor instillation, effectoralloCTL, or control PBMC or PBS was infused into theestablished tumor bed through the cannula. All mice weretreated with 5-day cycles of 5-FC (500 mg/kg, i.p.); up to 3cycleswere possible, spaced 2or 3weeks apart beginning onday 12 post-tumor instillation (Fig. 6A). Kaplan–Meier

survival plots and MST for the control and experimentalgroups are shown in Fig. 6B. The MST of the untreatedcontrol group (no RRV, no alloCTL effector cells, group 1)was 31.5 days, whereas theMST of the sham-treated controlgroup given nontherapeutic 231BR-GFP vector producingcells with the tumor inoculum andunstimulated PBMCwas40 days (group 3). The MSTs for mice treated with individ-ual experimental modalities ranged from 50 to 83 days. TheMSTs of the 2 groups receiving alloCTL therapy alone (withRRV-GFP infection or no RRV infection) were 65 and 83days respectively (groups 4 and 2), whereas the MSTs of the2 groups receiving prodrug activator gene therapy alone(with PBMCorPBS, insteadof alloCTL) andup to 3prodrugcycles were 50 and 57 days, respectively (groups 6 and 5).Statistical significance was reached (P < 0.05) for all of theindividual immune or gene therapy–treated groups com-pared to eachof the control groups (Fig. 6C). TheMSTof thegroup receiving the combined intratumoral effector alloCTLand RRV prodrug activator gene therapies (group 7) was97.5 days, thus exhibiting a highly statistically significantsurvival benefit compared to eachof the control groups (Fig.6C). Furthermore, the group receiving combined immuneand gene therapies showed a statistically significant addi-tional survival advantage compared to those groups receiv-ing gene therapy alone (P < 0.0001 and 0.0123, groups 7 vs.6 and 5, respectively). The combination treatment alsoshowed a trend toward statistical significance compared tothe groups receiving alloCTL cellular therapy alone (P <0.0511 and 0.6380, groups 7 vs. 4 and 2, respectively).

The individual animal weights of Fig. 6 animals weremonitored every 3 to 4days andplotted over the duration ofthe experiment as one indication of treatment toxicity and/or morbidity due to tumor progression. The plots for thecontrol groups, and the single or combined modalitygroups are shown starting on day 12 to 106 with prodrugcycle administration indicated on the abscissa (Supplemen-tary Fig. S1). The plots not only show the extension ofsurvival of animals in the experimental groups, but as well,indicate the health of the 11 long-term survivors present ingroups 2, 4, 5, and 7. Except in sham-treated controlanimals (groups 1 and 3), generally little to no weight lossoccurred during or shortly after the first prodrug cycle andsecond alloCTL infusion. Progressive weight loss at latertime points was likely associatedwith higher tumor burden,and notably, stabilization or reversal of weight loss wasobserved following prodrug administration cycles in indi-vidual animals in groups 2, 5, 6, and 7; this was mostapparent after the last prodrug cycle in groups 2 and 7, andmost of those animals went on to thrive as indicated bystable or increasing weight gains. Thus, treatment withintracranial infusions of alloCTL as well as prodrug admin-istration in RRV-CD–treated animals both contributed pos-itively to overall clinical response to therapy asmeasured byaverage body weight, and mice treated with combinedimmunogene therapy had the highest overall averageweight throughout the course of treatment.

Histopathologic findings of the brains from the long-term survivors (n¼11, fromFig. 6 experimental groups 2, 4,

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Figure 4. Subcutaneous tumor volumes to evaluate individual orcombined cellular and gene therapy treatment strategies. Rag2�/�gc�/�

mice were subcutaneously injected with 2 � 106 231BR cells withconcentrated RRV-GFP (white triangles), RRV-CD, or concentratednonviral supernate. Tumor growth was allowed to establish for 15 daysbefore some groups received intratumoral injections of 1.2� 107 alloCTL(downward arrow; white circles and white diamonds). All mice weretreated with 500 mg/kg 5-FC i.p. from days 21 to 27 post subcutaneoustumor injection (upward arrows). Data are represented as the averagetumor volume � SEM. Asteriks indicate P � 0.017 by two-way ANOVAwith Bonferroni posttests between mice who received MDA-231BR þconcentrated supernate (black diamonds) and the following experimentalgroups: �MDA-231BR þ concentrated supernate þ day 15 alloCTL(white diamonds); ��MDA-231BR þ RRV-CD þ day 15 alloCTL (whitecircles); ��MDA-231BR þ RRV-CD (black circles). Similar findings wereobtained from 2 tumor volumetric experiments; one experiment was aWinn-type assay (n ¼ 4 mice/group) and the second experiment shownhere was an established tumor assay (n ¼ 6 mice/group).

Hickey et al.





5, and 7) showed no evidence of tumor in H&E-stainedsections, whereas there was consistent presence of tumor inanimals that succumbed before the end of the survivalexperiment (Supplementary Fig. S2).Tomonitor the spread of RRV-GFP or RRV-CDwithin the

brain and to extratumoral sites, genomicDNAwas extractedfrom brain/brain tumor, lung, liver, spleen, kidney, andbone marrow tissues, then analyzed by qRT-PCR usingprimers and probe sequences specific for the 4070A ampho-tropic envelope. Data are shown as RRV copy number/5 �104 cells and calculated as detailed in Supplementary TableS3. In the long-term survivors from the experimental treat-ment groups (Fig. 6), there were low or no detectable levelsof RRV signals, which correlated with the apparent absenceof tumor by histopathology. In tumor-bearing animals,there were also higher levels of virus; representative dataare shown for one animal in group 7 that succumbed totumor on day 90. As expected in immunodeficient animalmodels, RRV signals indicating extratumoral vector spreadwere largely restricted to hematopoietic tissues, and in othertissues, the lymphoid cell content within them (37).Overall, our results show proof-of-concept that a unique

combination regimen, consisting of cellular therapy withalloCTL and gene therapy using RRV encoding a prodrugactivator gene, represents a promising strategy to treat breasttumors metastatic to the brain. We postulate that themechanisms at work benefiting the combined treatment

might involve alloCTL favorably improving the dissemina-tion of RRV to metastatic foci. This could either be by (i)adsorption of virus particles to the surface of trafficking Tcells that then transduce tumor cells, that is, a "hitch-hiking" mechanism without involving actual infection ofT cells (38), or (ii) a small percentage of alloCTL thatbecome transduced and then act asmotile vector producingcells to carry the vector to other tumor foci. Suicide genetherapymay also augment the immediate effects of adoptivecellular therapy, because tumor cell injury (combined apo-ptosis þ lysis) induced by effector T cells or suicide genetherapy could improve endogenous immune function bypresentation of fragmented tumor cells and antigens to Tcells. It is possible that further therapeutic gains can berealized, particularly in immunodeficient xenograftmodels,by administering additional cycles of prodrug, as previouslyreported (20), and by adjusting the timing and sequencingof alloCTL infusion versus prodrug administration.Although the xenograft model used in these studies did notallow a determination of the degree of inflammationinduced by the viral vector interacting with endogenousimmune cells in the brain, prior studies in a syngeneicanimal model indicate that this is not a problem (21). Infact, recent studies in syngeneic intracranial glioma modelsshow that an intact endogenous immune system cancontribute to complete tumor eradication after RRV-medi-ated prodrug activator gene therapy, as well as to restriction

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Figure 5. In vivo RRV-GFP spread through intracranial 231BR tumor. Rag2�/�gc�/� mice were intracranially injected with a total inoculum of 2 � 105 231BRtumor cells containing either 2%or 8%231BR-GFP vector producing cells. A, vector spread in nontransduced tumor cells was evaluated at 7, 14, and 20 daysposttumor instillation using enzymatically digested tumor cells that stained positive for GFP and were also positive for anti-human HLA-ABC. Data areaverages of GFPþ/HLA-ABCþ cells from triplicate wells � SEM. B, representative flow cytometric data from day 14 post-tumor instillation showing 71.2 �3.7%GFPþ cells from the 2% (left), and 78.9� 1.1% from the 8% 231BR-GFP (middle) supplemented populations, respectively. Tumor cells isolated from amouse intracranially injected with 100% 231BR are shown as a negative control (right). One experiment; n ¼ 3 mice/group/timepoint.






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Figure 6. Immunogene treatment schema, control, and experimental treatment groups, Kaplan–Meier survival plots with median survival times. A, thetreatment schema along with the 7 control or experimental groups are detailed. Mice were first surgically implanted with cannulas. Six days later they wereintracranially infused with either nontransduced 231BR cells or 2%/98% mixtures of RRV-transduced/nontransduced 231BR cells. The transduced 231BRcells had RRV coding for either nontherapeutic GFP marker gene (control) or therapeutic CD gene. AlloCTL (therapeutic cells) or PBMC (unstimulatedcontrol cells) were infused into the tumor site on days 9 and 16 post-tumor infusion. All animals received the nontoxic prodrug, 5-FC, which wasintraperitoneally injected daily for 5 days (one cycle; up to 3 cycles were possible spaced 2–3 weeks apart). Groups 1 and 3were controls for the suicide genetherapy and cellular therapy treated groups. The experimental groups testing cellular therapy with alloCTL were groups 2 and 4. The experimentalgroups testing suicide gene therapy were groups 5 and 6. The combination immunogene therapy was group 7. B, the Kaplan–Meier survival plots along withthe MST are shown for all 7 control or experimental groups (n ¼ 9–10/group) as observed to day 112 posttumor injection. Long-term survivors wereobtained for all individual or combined cellular and gene therapeutic groups. The P values of significance (P < 0.05) calculated by nonparametric log-rank(Mantel–Cox) tests were as follows from this singular experiment:

Groups Treatments P value

Groups 1,2 PBS vs. alloCTL 0.0011Groups 1,4 PBS vs. GFPþalloCTL 0.0365Groups 3,2 GFPþPBMC vs. alloCTL 0.0038Groups 3,4 GFPþPBMC vs. CDþalloCTL 0.0101Groups 1,5 PBS vs. CD 0.0078Groups 1,6 PBS vs. PBMCþCD 0.0051Groups 3,5 GFPþPBMC vs. CD 0.0093Groups 3,6 GFPþPBMC vs. CDþPBMC 0.0004Groups 1,7 PBS vs. alloCTLþCD 0.0001Groups 3,7 GFPþPBMC vs. CDþalloCTL 0.0001Groups 5,7 CD vs. CDþalloCTL 0.0123Groups 6,7 CDþPBMC vs. CD+alloCTL 0.0001

Three separate survival experiments were conducted; 2 pilot studies with n ¼ 4 to 6/group gave similar findings.

Hickey et al.





of systemic viral spread to normal tissues (23). Activationof endogenous antitumoral immune responses due todestruction of the immunosuppressive tumor environmentand release of immunostimulatory cytokines from adop-tively transferred alloCTL may also contribute to furthertherapeutic benefit. As alloCTL and RRV therapies have nowindividually reached the clinical testing stage, we can envi-sion the clinical design for combination immunogenetherapy of breast cancer metastatic to brain to be feasible,as shown in Supplementary Fig. S3. In summary, continuedpreclinical and clinical investigation of combined, localcellular, and gene therapy regimens are warranted for brainmetastases.

Disclosure of Potential Conflicts of InterestN. Kasahara has ownership interest (including patents) and is a consul-

tant/advisory board member of Tocagen Inc. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design: M.J. Hickey, H. Soto, N. Kasahara, B.M. Mueller,C.A. KruseDevelopment of methodology: M.J. Hickey, C.C. Malone, A. Lin, A.Inagaki, N. Kasahara, C.A. KruseAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): M.J. Hickey, K.L. Erickson, H. Soto, E.T. Ha, S.Kamijima, M. Takahashi, Y. Kato, B.M. MuellerAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis):M.J. Hickey, C.C. Malone, K.L. Erickson,

H. Soto, S. Kamijima, A. Inagaki, Y. Kato, N. Kasahara, B.M. Mueller, C.A.KruseWriting, review, and/or revision of the manuscript: M.J. Hickey, K.L.Erickson, S. Kamijima, A. Inagaki, Y. Kato, N. Kasahara, B.M. Mueller, C.A.KruseAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): M.J. Hickey, C.C. Malone, K.L.Erickson, H. Soto, S. Kamijima, A. Inagaki, Y. KatoStudy supervision: M.J. Hickey, N. Kasahara, B.M. Mueller, C.A. Kruse,H. Soto

AcknowledgmentsThe MDA-MB-231BR and MDA-MB-231-1833 human breast cancer cell

lines were kindly provided by Drs. T. Yoneda (University of Texas HealthScience Center, San Antonio, TX) and J.Massague (Memorial Sloan KetteringCancer Center, NY), respectively.

Grant SupportFinancial support for the study was supplied in part by USAMRMC

W81XWH-08-1-0734 (C.A. Kruse), CBCRP14IB-0114A (B.M.Mueller), NIHRO1 CA121258 (N. Kasahara), NIH/NCATS UCLA CTSI Grant NumberUL1TR000124 (C.A. Kruse/N. Kasahara), the Joan S. Holmes MemorialResearch Fund (C.A. Kruse), the Joan S. Holmes Memorial PostdoctoralFellowship (M.J. Hickey), and an unrestricted gift donation from Tocagen,Inc., to the Regents of theUniversity of California. Y. Kato is the recipient of apredoctoral fellowship from the Japan Student Services Organization. A.Inagaki is the recipient of a postdoctoral fellowship from the SusanG.KomenBreast Cancer Foundation.

The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked advertise-ment in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 4, 2012; revised April 22, 2013; accepted May 23,2013; published OnlineFirst June 18, 2013.

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2013;19:4137-4148. Published OnlineFirst June 18, 2013.Clin Cancer Res Michelle J. Hickey, Colin C. Malone, Kate L. Erickson, et al. Gene Therapy in a Model of Breast Cancer Metastatic to the BrainCombined Alloreactive CTL Cellular Therapy with Prodrug Activator

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