her2-specific t cells target primary glioblastoma stem ... · n. ahmed and v.s. salsman contributed...

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Published OnlineFirst January 12, 2010; DOI: 10.1158/1078-0432.CCR-09-1322

Cancer Therapy: Preclinical Clinical
Cancer
Research
HER2-Specific T Cells Target Primary Glioblastoma StemCells and Induce Regression of AutologousExperimental Tumors
Nabil Ahmed1,2,3, Vita S. Salsman1,2,3, Yvonne Kew6, Donald Shaffer1,2, Suzanne Powell6,7,Yi J. Zhang8, Robert G. Grossman8, Helen E. Heslop1,2,3,4, and Stephen Gottschalk1,2,3,5

Abstract

Authors' AChildren'sand 5Imm6PathologyHouston, T

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N. Ahmed

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Purpose: Glioblastoma multiforme (GBM) is the most aggressive human primary brain tumor and iscurrently incurable. Immunotherapies have the potential to target GBM stem cells, which are resistant toconventional therapies. Human epidermal growth factor receptor 2 (HER2) is a validated immunother-apy target, and we determined if HER2-specific T cells can be generated from GBM patients that will targetautologous HER2-positive GBMs and their CD133-positive stem cell compartment.Experimental Design: HER2-specific T cells from 10 consecutive GBM patients were generated by

transduction with a retroviral vector encoding a HER2-specific chimeric antigen receptor. The effectorfunction of HER2-specific T cells against autologous GBM cells, including CD133-positive stem cells,was evaluated in vitro and in an orthotopic murine xenograft model.Results: Stimulation of HER2-specific T cells with HER2-positive autologous GBM cells resulted in

T-cell proliferation and secretion of IFN-γ and interleukin-2 in a HER2-dependent manner. Patients'HER2-specific T cells killed CD133-positive and CD133-negative cells derived from primary HER2-positive GBMs, whereas HER2-negative tumor cells were not killed. Injection of HER2-specific T cellsinduced sustained regression of autologous GBM xenografts established in the brain of severe combinedimmunodeficient mice.Conclusions: Gene transfer allows the reliable generation of HER2-specific T cells from GBM patients,

which have potent antitumor activity against autologous HER2-positive tumors including their putativestem cells. Hence, the adoptive transfer of HER2-redirected T cells may be a promising immunotherapeu-tic approach for GBM. Clin Cancer Res; 16(2); 474–85. ©2010 AACR.

Glioblastoma multiforme (GBM) is the most aggressiveprimary brain tumor in adults (1). Currently, the best ther-apy consists of gross total surgical resection, which is fre-quently not possible due to location in eloquent areas ofthe brain, followed by radiotherapy and chemotherapy.Long-term survival is occasionally seen in rare cases thatoccur in young adults, generally under age 30. However,in the majority of cases, which occur in middle and oldage, radiotherapy and chemotherapy only slow but donot stop tumor growth, resulting in 5-year survival rates

ffiliations: 1Center for Cell and Gene Therapy, 2TexasCancer Center, and Departments of 3Pediatrics, 4Medicine,unology, Baylor College of Medicine; Departments of, 7Medicine, and 8Neurosurgery, The Methodist Hospital,exas

lementary data for this article are available at Clinical Cancernline (http://clincancerres.aacrjournals.org/).

and V.S. Salsman contributed equally to this work.

ding Author: Nabil Ahmed or Stephen Gottschalk, Center forne Therapy, Baylor College of Medicine, 6621 Fannin Street,, Houston, TX 77030. Phone: 832-824-4723; Fax: 832-825-il: [email protected] or [email protected].

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of <4% (2, 3). The recent identification of chemotherapy-and radiotherapy-resistant CD133-positive tumor stemcells in GBMs (4, 5) may help explain why conventionaltherapies are ineffective. Although the exact mechanism oftumor stem cell resistance to conventional therapies re-mains elusive, their quiescent state and increased capacityto eliminate cytotoxic drugs and repair damaged DNA arethought to be key contributing factors (4, 5). Immu-notherapy may be able to benefit GBM patients becauseimmune-mediated killing relies neither on tumor cell pro-liferation nor on the aforementioned cytotoxic pathways.Results from completed phase I/II immunotherapy clin-

ical trials with tumor cell or dendritic cell vaccines wereencouraging, showing disease stabilization and suggestingprolonged patient survival (6–9). However, these trialshave also highlighted some of the limitations of dendriticcell vaccines (10), particularly their failure to reproduciblyand effectively expand tumor antigen–specific T cells,which may be present at low frequency or are anergized.One means of overcoming this limitation is adoptive T-cell transfer, in which tumor-specific T cells are preparedex vivo and then transferred to affected individuals. Geneticmodification of T cells with chimeric antigen receptors(CAR) can reliably generate tumor-specific T cells ex vivo

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Translational Relevance

The outcome for patients with glioblastoma multi-forme (GBM) remains unchanged over the last 30years. Thus, there is a need for new targeted therapies,and T-cell immunotherapy clearly has the potential tofulfill this need. We show here that T cells from GBMpatients can readily be modified with human epider-mal growth factor receptor 2 (HER2)–specific chimericantigen receptors to produce effector cells, which re-lease immunostimulatory cytokines in response to,and kill, autologous primary HER2-positive GBM tu-mor cells, including CD133-positive GBM stem cells.These HER2-specific T cells also had a potent antitu-mor activity against autologous tumors in an orthoto-pic xenogeneic severe combined immunodeficientmouse model. Hence, the adoptive transfer of HER2-redirected T cells may be an attractive immunothera-peutic approach for GBM.

Targeting Stem Cells in Glioblastoma


for clinical use (11, 12). CARs are synthetic molecules thatconsist of an extracellular antigen binding domain thatusually contains the heavy and light chain variable regionsof a monoclonal antibody joined to transmembrane andcytoplasmic signaling domains derived from the CD3-ζchain and from costimulatory molecules such as CD28.CARs recognize antigens expressed on the surface of tumorcells, and in this study, we targeted the human epidermalgrowth factor receptor 2 (HER2), a tumor-associated anti-gen that is expressed by up to 80% of GBMs but not bynormal postnatal neurons or glia (13–16).We now show that T cells from GBM patients can read-

ily be modified with HER2-specific CARs to produce effec-tor cells, which release immunostimulatory cytokines inresponse to, and readily kill, autologous primary HER2-positive GBM tumor cells, including CD133-positiveGBM stem cells. These HER2-specific T cells had a potentantitumor activity against autologous tumors in an ortho-topic xenogeneic severe combined immunodeficient(SCID) mouse model.

Materials and Methods

Blood donors, primary tumor cells, and cell lines. Bloodsamples and primary tumor cells were obtained from sub-jects with GBM on a protocol approved by the Institution-al Review Board of Baylor College of Medicine at BaylorCollege of Medicine and The Methodist Hospital. TheGBM and medulloblastoma cell lines (U373 and Daoy)and the breast cancer cell line (MDA-MB-468) were pur-chased from the American Type Culture Collection. All celllines were grown in DMEM (Invitrogen) with 10% FCS(HyClone) supplemented with 2 mmol/L GlutaMAX-I,1.5 g/L sodium bicarbonate, 0.1 mmol/L nonessential

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amino acids, and 1.0 mmol/L sodium pyruvate (all medi-um supplements from Invitrogen). T cells were maintainedin RPMI 1640 with 10% FCS containing 2 mmol/LGlutaMAX-I.Tumor tissues from patients with GBM undergoing sur-

gical resection were processed aseptically, and primary cellcultures were initiated using DMEM high-glucose medium(Invitrogen) supplemented with 15% heat-inactivatedFCS, 2 mmol/L GlutaMAX-I, 1% insulin-transferrin-seleni-um-X supplement, and 1% penicillin-streptomycin mix-ture (all medium supplements from Invitrogen). Cellswere used within 7 d of plating or established as primarycell lines.Immunohistochemistry.Mice were euthanized by CO2 in-

halation and fixed with intracardiac perfusion of 4% para-formaldehyde. The brain tissue was postfixed overnightand embedded in paraffin, and histology was done on10-μm serial horizontal sections. Tissue sections obtainedfrom mouse xenografts and from paraffin-embedded sur-gical excision samples were stained by a standard H&Etechnique. HER2 expression in GBM xenografts was de-tected by phospho-HER2 immunohistochemistry as previ-ously described (17).Generation of retroviral constructs. The HER2-specific

CAR with a CD28.ζ signaling domain was constructedby subcloning the HER2-specific single-chain variable frag-ment FRP5 into a SFG.CD28.ζ retroviral vector as previ-ously described (18). A retroviral vector encoding thefusion protein eGFP-firefly luciferase (eGFP.FFLuc) wasused to generate firefly luciferase–expressing GBM cellsfor the in vivo studies (18).Retrovirus production and transduction of T cells. To pro-

duce retroviral supernatant, 293T cells were cotransfectedwith an FRP5.CD28.ζ retroviral vector containing plasmid,Peg-Pam-e plasmid encoding the sequence for MoMLVgag-pol, and plasmid pMEVSVg containing the sequencefor VSV-G using GeneJuice transfection reagent (EMDBiosciences; ref. 19). Supernatants containing the retrovi-rus were collected 48 and 72 h later. VSV-G pseudotypedviral particles were used to transduce the PG-13 producercell line for the production of viral particles.OKT3/CD28 activated T cells were transduced with ret-

roviral vectors as described (19). Briefly, peripheral bloodmononuclear cells were isolated by Lymphoprep (GreinerBio-One) gradient centrifugation. Peripheral blood mono-nuclear cells (5 × 105 per well) in a 24-well plate were ac-tivated with OKT3 (OrthoBiotech) and CD28 monoclonalantibodies (BD Biosciences) at a final concentration of 1μg/mL. On day 2, recombinant human interleukin (IL)-2(Chiron) was added at a final concentration of 100 units/mL, and on day 3, cells were harvested for retroviral trans-duction. For transduction, we precoated a nontissueculture–treated 24-well plate with a recombinant fibronec-tin fragment (FN CH-296; RetroNectin; Takara Bio USA).Wells were washed with PBS (Sigma) and incubated twicefor 30 min with retrovirus. Subsequently, 3 × 105 T cellsper well were transduced with retrovirus in the presence of100 units/mL IL-2. After 48 to 72 h, cells were removed

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and expanded in the presence of 50 to 100 units/mL ofIL-2 for 10 to 15 d before use.Flow cytometry. We used a FACSCalibur instrument

(Becton Dickinson) and CellQuest software (BectonDickinson) for all flow cytometric analyses, analyzing>10,000 events; in all cases, negative controls includedisotype antibodies. Cells were washed once with PBScontaining 2% fetal bovine serum and 0.1% sodiumazide [fluorescence-activated cell sorting (FACS) buffer;Sigma] before addition of antibodies. After 15 to 30 minof incubation at 4°C in the dark, the cells were washedonce and fixed in 0.5% paraformaldehyde/FACS bufferbefore analysis.T cells were analyzed with anti-CD8 FITC, anti-CD4

phycoerythrin, and anti-CD3 peridinin chlorophyll pro-tein, and tumor cell lines with anti-HER2 phycoerythrin.All monoclonal antibodies (except CD133; Miltenyi Bio-tec) were obtained from BD Biosciences. To determine cellsurface expression of the HER2 CAR transgene, a recombi-nant HER2-Fc fusion protein (R&D Systems) was used.Bound HER2-Fc was detected with a goat anti-Fc FITC sec-ondary antibody (Chemicon; ref. 19).Cytotoxicity assays. Cytotoxicity assays were done as pre-

viously described (20). Briefly, 1 × 106 target cells were la-beled with 0.1 mCi (3.7 MBq) 51Cr and mixed withdecreasing numbers of effector cells to give E:T ratios of40:1, 20:1, 10:1, and 5:1. Target cells incubated in com-plete medium alone or in 1% Triton X-100 were used todetermine spontaneous and maximum 51Cr release, re-spectively. After 4 h, we collected supernatants and mea-sured radioactivity in a gamma counter (CobraQuantum; Perkin-Elmer). The mean percentage of specificlysis of triplicate wells was calculated according to the fol-lowing formula: (test release − spontaneous release)/(max-imal release − spontaneous release) × 100.Analysis of cytokine production and T-cell expansion. Ef-

fector T cells (FRP5.CD28.ζ.CAR-expressing T cells or non-transduced T cells) from GBM patients or healthyvolunteers were cocultured with primary autologousGBM cells in short-term culture (<14 d). HER2-positiveand HER2-negative cell lines at a 1:1 E:T ratio were platedin a 24-well plate. After 24 to 48 h of incubation, culturesupernatants were harvested and the presence of IFN-γand IL-2 was determined by ELISA as per the manufac-turer's instructions (R&D Systems). T-cell expansion wasdetermined by counting viable cells (trypan blue exclu-sion) 7 d after stimulation.Whole-genome gene expression profiling. The genome-

wide expression analysis was done by the Dan L. DuncanCenter Genomics and Proteomics Core Laboratory atTexas Children's Hospital using the HumanWG-6 v3BeadChips (Illumina, Inc.). This array contains >48Ktranscript probes (representing 42,620 genes). Briefly,RNA was isolated from primary tumors and the cor-responding primary cell lines were established in vitro.Total RNA concentrations were measured using theNanoDrop 1000 spectrophotometer (NanoDrop Scientific).RNA integrity was checked in electrophoresis using 2%

Clin Cancer Res; 16(2) January 15, 2010


agarose gel with 6% formaldehyde. Total RNA (0.5 μg)was used to synthesize biotinylated cRNA using TotalPrepRNA Amplification kit (Ambion, Inc.), and 1.5 μg of bio-tinylated cRNA were applied to the HumanWG-6 v3 Bead-Chips and processed according to the vendor's instruction.The BeadChips were scanned using Beadstation 500 GXscanner. The image files were imported into the Bead Stu-dio software version 3.2.7 (Illumina), and the data wereprocessed using the quantile normalization algorithm.This method assumes that the distribution of the expres-sion values does not change dramatically between arrays.All arrays were adjusted so that they had an almost iden-tical overall intensity distribution. All 42,620 genes wereused to calculate the correlation coefficient between sam-ples in the BeadStudio software. All samples that con-tained genes with detection P value of <0.001 weresubjected to a differential analysis using Illumina's owntest with false discovery correction.Orthotopic xenogeneic SCID mouse model of GBM. All an-

imal experiments were conducted on a protocol approvedby the Baylor College of Medicine Institutional AnimalCare and Use Committee. Recipient nonobese diabetic–SCID mice were purchased from Taconic (C.B-Igh-1b/Icr-Tac-Prkdcscid; FOX CHASE CB-17 SCID ICR). Nine- to12-wk-old male mice were anesthetized with rapidsequence inhalation isoflurane (Abbot Laboratories) fol-lowed by an i.p. injection of 225 to 240 mg/kg of Avertinsolution and then maintained on isoflurane by inhalationthroughout the procedure. The head was shaved, and thenthe mice were immobilized in a Cunningham Mouse/Neo-natal Rat Adaptor (Stoelting) stereotactic apparatus fittedinto an E15600 Lab Standard Stereotaxic Instrument(Stoelting) and then scrubbed with 1% povidone-iodine.A 10-mm skin incision was made along the midline. Thetip of a 31-gauge, 0.5-inch needle mounted on a Hamiltonsyringe served as the reference point. A 1-mm burr holewas drilled into the skull, 1 mm anterior to and 2 mmto the right of the bregma.Firefly luciferase–expressing primary GBM cells (5 × 104

in 2.5 μL) from patients 2, 3, or 5 were injected 3 mm deepto the bregma, corresponding to the center of the right cau-date nucleus over 5 min. The needle was left in place for 3min, to avoid tumor cell extrusion, and then withdrawnover 5 min. All GBMs engrafted and started to grow asjudged by exponential increments in their bioluminescencesignals. Six days after tumor cell infection, mice containingmalignant cells derived from each individual patient wererandomly assigned to receive no therapy, HER2-specific Tcells, or nontransduced T cells. T-cell therapy consisted of2 × 106 autologous HER2-specific or nontransduced T cellsinjected locally in 5 μL to the same tumor coordinates. Theincision was closed with two to three interrupted 7.0 Ethi-con sutures (Ethicon, Inc.). A s.c. injection of 0.03 to 0.1mg/kg of buprenorphine (Buprenex RBH) was given forpain control. For the experiment with CD133-positiveGBM stem cells, cells were isolated by high-speed cell sort-ing and injected intomice as described above (1 × 104 in 2.5μL per mouse). On day 8, mice were treated with 2 × 106

Clinical Cancer Research





autologous HER2-specific or nontransduced T cells in 5 μLto the same tumor coordinates.Bioluminescence imaging. Isoflurane-anesthetized ani-

mals were imaged using the IVIS system (Xenogen Corp.)10 min after 150 mg/kg D-luciferin (Xenogen) was injectedi.p. The photons emitted from luciferase-expressing cellswithin the animal body and transmitted through the tissuewere quantified using “Living Image,” a software programprovided by the same manufacturer. A pseudocolor imagerepresenting light intensity (blue, least intense; red, mostintense) was generated and superimposed over the gray-scale reference image. Animals were imaged every otherday for 1 wk after injections, then twice weekly for 2 wk,and then weekly thereafter. They were regularly examinedfor any neurologic deficits, weight loss, or signs of stressand euthanized according to preset criteria in accordancewith the Baylor College of Medicine Center for Compara-tive Medicine guidelines.



Statistical analysis. For the bioluminescence experi-ments, intensity signals were log transformed and summa-rized using mean ± SD at baseline and multiplesubsequent time points for each group of mice. Changesin intensity of signal from baseline at each time point werecalculated and compared using paired t tests or Wilcoxonsigned-ranks test.

Results

Generation of functional HER2-specific T cells from sub-jects with GBM. To redirect patients' lymphocytes toHER2, we used a second-generation HER2-specific CARconsisting of an extracellular domain derived from thehigh-affinity HER2 monoclonal antibody, FRP5 (21),and signaling domains from the costimulatory moleculeCD28 and the CD3 ζ-chain (FRP5.CD28.ζ CAR; Supple-mentary Fig. S1A). CD3/CD28 activated T cells from 10

Fig. 1. Functionality ofHER2-specific T cells fromsubjects with GBM. HER2-specificand nontransduced (NT) T cells,generated from newly diagnosedGBM patients, were coculturedwith HER2-positive (U373 orDaoy) or HER2-negative(MDA-MB-468) cells. A, onlyHER2-specific T cells killedHER2-positive targets in a4-h 51Cr release cytotoxicity assay;nontransduced T cells did not.The HER2-negative cell lineMDA-MB-468 was not killed byHER2-specific or nontransducedT cells. Points, mean of all patients;bars, SD. Results fromexperiments done for nine GBMpatients in triplicates are shown.B, in coculture assays doneat a 1:1 T-cell to tumor cellratio, the IFN-γ and IL-2concentration was determined inthe coculture supernatant 24 to48 h after stimulation. OnlyHER2-specific T cells producedIFN-γ and IL-2 after exposure toHER2-positive cells in comparisonwith nontransduced T cells.Results from experiments done induplicates are shown.




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consecutive newly diagnosed GBM patients were trans-duced with RD114-pseudotyped retroviral vectors encod-ing FRP5.CD28.ζ CAR, and 4 to 7 days after transduction,we determined the expression of HER2-specific CARs byFACS analysis. On average, 79% (SD, ±15%) of GBM pa-tients' T cells expressed HER2-specific CARs, and bothCD4- and CD8-positive T cells were transduced (Supple-mentary Fig. S1B and C).To evaluate the in vitro function of patients' HER2-

specific T cells, we used standard cytotoxicity assays



and coculture experiments with both HER2-positive tar-gets (GBM cell line U373 and medulloblastoma cellline Daoy) and HER2-negative controls (MDA-MB-468). All patients' HER2-specific T cells killed HER2-positive target cells and secreted immunostimulatoryTh1 cytokines (IFN-γ and IL-2), whereas nontransducedT cells from the same patients did not (Fig. 1A and B).In addition, HER2-negative target cells were not killedand did not induce cytokine production. Hence, geneticmodification of T cells from GBM patients allows rapid

Fig. 2. HER2 protein expression on primary GBM. A, FACS analysis. Primary GBM cells from freshly excised tumors in short-term culture were stained forHER2 expression. Open curves, isotype control; solid curves, HER2. Nine of 10 tumor cell lines expressed HER2 on the cell surface. B, usingthe HER2-specific mouse monoclonal antibody NCL-L-CB11 (Novocastra), HER2 expression was confirmed on the corresponding paraffin-embeddedsections. One tumor had no detectable HER2 protein expression using both methods (patient 8). Magnification, ×200.

Fig. 3. HER2-specific T cells kill autologous HER2-positive GBM and are activated in coculture. A, the cytolytic activity of T cells expressing HER2 CAR wasdetermined in a standard 4-h chromium release assay. There was always an increase of cytolytic activity of HER2-specific T cells above background(nontransduced T cells) against autologous HER2-positive GBMs. As controls, the HER2-positive GBM cell line U373 and the HER2-negative MDA-MB-468were used. HER2-specific T cells from all patients killed U373 cells, whereas MDA-MB-468 was not killed (shown for patient 4). B, HER2-specificT cells (black columns) or nontransduced T cells (white columns) from GBM patients were cocultured with autologous tumor cells or HER2-negative controlcells (MDA-MB-468; hatched columns) in a 1:1 ratio. Twenty-four to 48 h after stimulation, the cytokine concentration in the medium was determinedby ELISA. HER2-specific T cells produced IFN-γ and IL-2 after stimulation with eight of nine HER2-positive tumor samples. No cytokine release wasseen with nontransduced T cells. Median cytokine levels for all patients are shown for U373 (HER2-positive control) and MDA-MB-468 (HER2-negativecontrol). Results of experiments done in duplicates are shown.






and reliable generation of functional HER2-specific Tcells.HER2-specific T cells recognize and kill autologous GBM

cells. To investigate if patients' HER2-specific T cells rec-



ognize and kill autologous HER2-positive GBM cells, weused short-term cultured autologous GBM cells as tar-gets. Nine of 10 tumor cell lines expressed HER2 onthe cell surface as judged by FACS analysis (Fig. 2A).




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HER2 expression of the corresponding primary tumor wasconfirmed by immunohistochemistry (Fig. 2B). Althoughwe observed moderate heterogeneity of HER2 expressionwithin individual GBMs, there was concordance betweenFACS analysis and immunohistochemistry, with one tu-mor having no detectable HER2 protein expression usingboth methods (patient 8).In standard 4-hour 51Cr release assays, there was always

an increase of cytolytic activity of HER2-specific T cells incomparison with nontransduced T cells against autolo-gous HER2-positive GBMs, confirming HER2 specificity(Fig. 3A). Besides tumor cell killing, cytokine productionof T cells is critical for their activation and sustained anti-tumor activity. In coculture experiments, HER2-specific Tcells from eight of nine patients with HER2-positive GBMssecreted substantial amounts of Th1 cytokines (IFN-γ andIL-2) denoting T-cell activation (Fig. 3B). No IFN-γ or IL-2production was detected when HER2-specific T cells werecultured with HER2-negative targets (MDA-MB-468) orfrom cocultures with autologous nontransduced T cells.These results indicate that HER2-specific T cells recognizeand kill autologous HER2-positive GBM cells in a HER2-specific manner.HER2-specific T cells kill autologous CD133-positive GBM

stem cells. We next evaluated if HER2-specific T cells alsokill CD133-positive GBM stem cells, which are resistant tocurrent standard therapies, including radiotherapy andchemotherapy. We separated the CD133-positive andCD133-negative cell populations from primary GBM celllines of three patients by high-speed cell sorting. Three per-cent to 5% of the total cell population was CD133 positive(Fig. 4A). This GBM stem cell population expressed 2.8- to4-fold higher levels of HER2 in comparison with theirCD133-negative counterparts (Fig. 4B). In a 4-hour 51Crrelease assay, HER2-specific T cells of all three patientskilled autologous CD133-positive as well as CD133-nega-tive cells (Fig. 4C). Autologous nontransduced T cells in-duced no significant killing of either cell populations.These results show that HER2-specific T cells are able totarget the treatment-resistant CD133-positive compart-ment, which may contribute to tumor recurrence in GBM.Regression of primary GBM xenografts after administra-

tion of autologous HER2-specific T cells. To determine thein vivo antitumor activity of HER2-specific T cells, weused an orthotopic autologous GBM xenograft model. Pri-mary GBM cell lines from GBM patients were establishedin vitro, and their identity with the primary tumor wasconfirmed using a 48,000-probe gene chip array [Pearsoncorrelation coefficient (r2) = 0.77-0.83; SupplementaryFig. S2]. To allow serial bioluminescence imaging in vivo,we transduced primary GBM cell lines from three patientswith a retroviral vector encoding an eGFP-firefly luciferasefusion gene. All cells were green fluorescent protein (GFP)positive as judged by FACS analysis, and firefly luciferaseexpression was confirmed in vitro (data not shown). Thetumorigenicity of the three patients' eGFP.FFLuc-expres-sing GBM cell lines was confirmed by injecting 5 × 104

cells stereotactically into the right frontal cortex of SCID



mice. Bioluminescence imaging of tumor xenograftsshowed progressive and exponential growth of tumors inall experimental animals (Supplementary Fig. S3). Un-treated animals had a median survival of 17 days (range,14-22).To test the effector function of autologous HER2-specif-

ic T cells, 5 × 104 eGFP.FFLuc-expressing GBM cells fromall three patients were injected stereotactically into theright frontal cortex of SCID mice. On day 6 after tumor cellinjection, mice received an intratumoral injection of 2 ×106 autologous HER2-specific T cells or nontransduced Tcells. A subset of animals was not treated. We quantifiedtumor growth by serial bioluminescence. In untreated an-imals, and in animals treated with autologous nontrans-duced T cells (Fig. 5A), tumors grew exponentially overtime. In contrast, photon emission decreased significantlyin all eight mice after autologous HER2-specific T-cell in-jection, indicating tumor regression (Fig. 5B). GBM xeno-grafts from patients 2 and 3 regressed completely, whereasthe xenografts from patient 5 had a partial response. Thelight signal continued to be undetectable after 6 months infour of eight animals, indicating tumor eradication. Thiswas confirmed by histologic examination.Kaplan-Meier survival studies 60 days after tumor injec-

tion showed that untreated mice and mice receiving autol-ogous nontransduced T cells had a median survival of 14and 15 days, respectively. In contrast, mice treated withautologous HER2-specific T cells had a median survivalof 90 days, with 50% of the mice surviving >100 days(P < 0.001; Fig. 5C).To provide direct evidence that HER2-specific T cells al-

so kill autologous CD133-positive GBM stem cells in vivo,CD133-positive cells from the eGFP.FFLuc-expressingGBM cell lines of patient 2 were sorted and injected stereo-tactically into the right frontal cortex of SCID mice (1 ×104 cells; n = 8). Mice were imaged daily until the biolu-minescence signal was comparable to that of xenograftsobtained from unsorted cells. On day 8, mice were in-jected with autologous HER2-specific T cells or nontrans-duced T cells. Although tumors in animals treated withnontransduced T cells continued to grow exponentially,all animals treated with HER2-specific T cells had a mea-surable response, including two complete remissions, onbioluminescence imaging (Fig. 5D). This result furthersupports the conclusions of our in vitro experiments(Fig. 4) that HER2-specific T cells have antitumor activityagainst CD133-positive glioma cells.

Discussion

In this study, we investigated HER2-targeted T cells as apotential therapeutic agent for GBM and showed that Tcells from GBM patients can be readily genetically engi-neered to be rendered HER2 specific. These effector cellsrecognized autologous HER2-positive GBMs includingtheir CD133-positive stem cells in vitro and had potentantitumor activity in an orthotopic xenograft model.






It has proved difficult to generate tumor-specific T cellsin cancer patients using dendritic cell vaccines, most likelybecause tumor-specific T cells are present at low frequencyand in an anergic state due to the immunosuppressive en-vironment induced by malignancies such as GBMs (10,22). Adoptive T-cell transfer of ex vivo expanded tumor-specific T cells may overcome these limitations, and forcedexpression of antigen-specific CARs or transgenic α/βT-cell receptors (TCR) has been used to generate such ef-fector T lymphocytes (23–26). CARs combine the antigen-binding property of monoclonal antibodies with the lytic



and self-renewal capacities of T cells and have several ad-vantages over transgenic α/β TCR receptors (11, 12). CAR-expressing T cells recognize and kill tumor cells in anMHC-nonrestricted fashion so that target cell recognitionby CAR T cells is unaffected by some of the major mechan-isms by which tumors avoid MHC-restricted T-cell (α/βTCR) recognition, such as downregulation of HLA class Imolecules and defective antigen processing (11, 12). Inaddition, most tumors do not express costimulatory mo-lecules so that α/β TCR engagement is followed by incom-plete T-cell activation. We (27) and others (28, 29) have

Fig. 4. HER2-specific T cells target primary GBM stem cells. A, primary GBM cells from three patients (GBM patients 2, 3, and 5) were stained for CD133and isolated using high-speed sorting. Approximately 3% to 5% of the total primary GBM cell population was CD133 positive. B, this CD133-positivecell compartment was uniformly HER2 positive. Moreover, in all three tumors analyzed, the CD133-positive GBM stem cells expressed higher levelsof HER2 in comparison with the CD133-negative tumor cell population. C, in a 4-h 51Cr release assay, HER2-specific T cells from these three patients killedautologous CD133-positive cells as well as their CD133-negative counterparts. Autologous nontransduced T cells induced no appreciable killing.




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Fig. 5. Adoptively transferred HER2-specific T cells induce regression of autologous GBM xenografts in vivo. Primary GBM cells (5 × 104) from patient 2(two mice per group), patient 3 (three mice per group), and patient 5 (three mice per group) were injected stereotactically into the caudate nucleus of9- to 12-wk-old SCID mice followed by intratumoral injection of 2 × 106 autologous HER2-specific or nontransduced T cells 6 d after tumor inoculation.A, tumors grew progressively in untreated mice as shown for two representative animals (top row) and in mice receiving nontransduced T cells (middle row),whereas tumors regressed over a period of 2 to 5 d in response to a single injection of autologous HER2-specific T cells generated from the samepatient (bottom row). B, quantitative bioluminescence imaging. Autologous HER2-specific T cells induced tumor regression when compared withnontransduced T cells (two-tailed P = 0.002, Mann-Whitney U test). Solid arrows, time of T-cell injection; open arrows, background luminescence (mean,∼105 photons/s/cm2/sr); n, number of animals tested in each group. C, Kaplan-Meier survival curve. Survival analysis done 60 d after tumor establishment.Mice treated with autologous HER2-specific T cells had a significantly longer survival probability (P < 0.001) in comparison with untreated mice and micethat received nontransduced T cells.D, 1 × 104CD133-positive GBMcells from patient 2 were injected as described above followed by intratumoral injection of2 × 106 autologous HER2-specific or nontransduced T cells 8 d after tumor inoculation. Whereas tumors in animals treated with nontransduced T cells(n= 4) continued to grow exponentially, all of the animals treatedwith autologous HER2 T cells (n= 4) regressed, with two of these animals having no detectabletumors within 6 d after T-cell injection.

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shown that CARs can overcome this limitation if they in-corporate costimulatory endodomains within the chimericreceptor sequence or if they are expressed in virus-specificCTLs. Our current HER2-specific CAR with a CD28.ζ sig-naling domain induces immunostimulatory cytokine re-lease, including IL-2, from transduced cells of eight of nineHER2-positive GBM patients following CAR engagement.Several cell surface antigens have been identified as po-

tential targets for GBM-directed CAR T-cell therapies, in-cluding HER2, IL-13Rα2, EPH receptor A2, and EGFRvIII(14, 30–32). We chose HER2 because this antigen is ex-pressed by a high percentage of tumors (>70%), and signal-ing through HER2 deregulates cell proliferation, inhibitsapoptosis, and increases the metastatic potential of cancercells (33, 34). Moreover, HER2 expression increases withthe degree of anaplasia in glial tumors, and mutations inthe HER2 signaling pathway have been identified in glio-mas (35). Our own results confirmed a high frequency ofHER2 expression, showing the antigen on 9 of 10 GBM tu-mors by two independent methods (immunohistochemis-try and FACS analysis). As with other tumor-associatedantigens expressed by GBMs, there was moderate heteroge-neity in the expression pattern of HER2 within individualtumors (36, 37), and optimum killing in a clinical settingmay require targeting more than one tumor-associated an-tigen or the induction of epitope spreading beyond the orig-inal targets to prevent immune escape (20, 38).Despite the biological significance of HER2 signaling,

the expression of HER2 on GBMs is low in comparisonwith breast cancer, rendering HER2 monoclonal antibo-dies ineffective (19, 30). HER2-specific T cells allow target-ing of the relatively low levels of HER2 expressed by GBMsbecause the overall avidity of receptors arrayed on a T cellis greater than the avidity of a bivalent antibody, and en-gagement of even a limited number of TCR molecules issufficient to trigger a cytotoxic effector response (39, 40).One concern of targeting HER2-expressing malignancieswith T cells is “off-target” effects because the administra-tion of HER2 monoclonal antibodies has been associatedwith side effects, the most concerning of which is the poor-ly understood cardiac toxicity. If HER2-specific T cells arelong lived, this problem could be accentuated in severityand persistence. Potential cardiac toxicities are difficult tomodel in mice because T cells that are specific for humanHER2 do not recognize murine HER2. Nonetheless, wehave shown that HER2-negative cells are not killed byHER2-specific T cells. In addition, primary endothelialand epithelial cells (Supplementary Fig. S4) do not acti-vate HER2-specific T cells and five patients who receivedHER2-specific T cells had no dose-limiting toxicities (41,42). Finally, HER2 vaccines are well tolerated, and no car-diac toxicities were observed in patients who developedHER2-specific T-cell responses (43).CD133 expression has identified a population with

stem cell–like properties in normal and cancerous tissuesof the central nervous system (4, 5). In GBMs, these cellsare chemotherapy and radiotherapy resistant and mostlikely contribute to the ineffectiveness of conventional



therapies. For example, although temozolomide was re-ported to preferentially deplete CD133-positive stem cellsin primary GBMs in vitro, this effect was absent in O6-methylguanine-DNA methyltransferase–nonmethylatedtumors, which represent 50% to 70% of primary GBMsand carry a worse prognosis (44). The use of temozolo-mide in a randomized prospective clinical trial in patientswith GBM has thus only resulted in a marginal survivaladvantage (14.6 versus 12.1 months; ref. 3). We show herethat CD133-positive GBM stem cells express HER2 andare killed by autologous HER2-specific T cells in vitroand in vivo similar to CD133-negative GBM cells. Hence,immune-targeted therapies may eradicate malignant stemcells that are resistant to conventional therapy.Several studies have reported the infusion of activated T

lymphocytes systemically or into resection cavities of re-current or progressive malignant gliomas (45–47). Theseinfusions were safe and resulted in disease stabilizationand, in some instances, in partial regression. We injectedHER2-specific T cells directly into autologous GBM xeno-grafts in tumor-bearing mice. Tumors from patients 2 and3 regressed completely without the need for exogenous cy-tokines, whereas untreated tumors and tumors treatedwith nontransduced T cells continued to grow. Tumorsfrom patient 5 only had a partial response. Initial analysisrevealed no significant differences in the level of HER2expression as well as the in vitro effector function of HER2-specific T cells from these three patients. We are now inves-tigating other biological differences between the tumorsthat may account for variability in effector cell sensitivity.Tumors recurred in several animals most likely due to

limited T-cell persistence in vivo. Kahlon et al. (48) re-ported complete regression and no recurrence of U87 gli-omas in an orthotopic xenogeneic SCID model afterintratumoral injection of T cells expressing an IL-13Rα2–specific CAR. In their model, the U87 glioma cell line wasgenetically modified to secrete IL-2, a cytokine critical forT-cell survival and expansion in vivo. Tumor recurrence inour model may be due to inadequate persistence of hu-man HER2-specific T cells in the xenograft, a known lim-itation of SCID mouse models.In summary, this study shows that HER2-specific T cell

can be readily generated from GBM patients by gene trans-fer with HER2-specific CARs. HER2-specific T cells recog-nized and killed autologous HER2-positive GBM,including CD133-positive stem cells, ex vivo and inducedregression of experimental GBMs in vivo. Hence, the adop-tive transfer of HER2-redirected T cells may be an attractiveimmunotherapeutic approach for GBM.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Dr. Malcolm K. Brenner for the helpfuldiscussion and advice, Awateef Akrabi for assistance with




Ahmed et al.

484


FACS analysis, Christopher Threeton for assistance withflow sorting, and Drs. Richard L. Harper and James Rose(The Methodist Hospital neurosurgeons).

Grant Support

Clayton Foundation for Research, Dana Foundation,and American Brain Tumor Association. H.E. Heslop



is the recipient of a Doris Duke Distinguished ClinicalScientist Award.The costs of publication of this article were defrayed in

part by the payment of page charges. This article musttherefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.Received 5/29/09; revised 10/23/09; accepted 10/27/09;

published OnlineFirst 1/12/10.

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2010;16:474-485. Published OnlineFirst January 12, 2010.Clin Cancer Res Nabil Ahmed, Vita S. Salsman, Yvonne Kew, et al. and Induce Regression of Autologous Experimental TumorsHER2-Specific T Cells Target Primary Glioblastoma Stem Cells

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