research successful eradication of established peritoneal … · cancer therapy: preclinical...

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Published OnlineFirst July 13, 2010; DOI: 10.1158/1078-0432.CCR-10-0192

Cancer Therapy: Preclinical Clinical
Cancer
Research

Successful Eradication of Established Peritoneal OvarianTumors in SCID-Beige Mice following Adoptive Transfer ofT Cells Genetically Targeted to the MUC16 Antigen

Alena A. Chekmasova1, Thapi D. Rao1, Yan Nikhamin1, Kay J. Park2, Douglas A. Levine3,David R. Spriggs1, and Renier J. Brentjens1,4

Abstract

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Purpose: Most patients diagnosed with ovarian cancer will ultimately die from their disease. For thisreason, novel approaches to the treatment of this malignancy are needed. Adoptive transfer of a patient'sown T cells, genetically modified ex vivo through the introduction of a gene encoding a chimeric antigenreceptor (CAR) targeted to a tumor-associated antigen, is a novel approach to the treatment of ovariancancer.Experimental Design:We have generated several CARs targeted to the retained extracellular domain of

MUC16, termed MUC-CD, an antigen expressed on most ovarian carcinomas. We investigate the in vitrobiology of human T cells retrovirally transduced to express these CARs by coculture assays on artificialantigen-presenting cells as well as by cytotoxicity and cytokine release assays using the human MUC-CD+

ovarian tumor cell lines and primary patient tumor cells. Further, we assess the in vivo antitumor efficacyof MUC-CD–targeted T cells in SCID-Beige mice bearing peritoneal human MUC-CD+ tumor cell lines.Results: CAR-modified, MUC-CD–targeted T cells exhibited efficient MUC-CD–specific cytolytic acti-

vity against both human ovarian cell and primary ovarian carcinoma cells in vitro. Furthermore, expandedMUC-CD–targeted T cells infused through either i.p. injection or i.v. infusion into SCID-Beige mice bear-ing orthotopic human MUC-CD+ ovarian carcinoma tumors either delayed progression or fully eradicateddisease.Conclusion: These promising preclinical studies justify further investigation of MUC-CD–targeted

T cells as a potential therapeutic approach for patients with high-risk MUC16+ ovarian carcinomas.Clin Cancer Res; 16(14); 3594–606. ©2010 AACR.

Ovarian cancer is the sixth most common cancer world-wide and the seventh leading cause of cancer-related deathsin women (1, 2). Despite multimodality therapy with sur-gery and chemotherapy, most patients with ovarian carci-nomas have a poor prognosis. For this reason, alternativeapproaches to treating this disease are urgently needed.Infusion of a patient's own T cells genetically targeted

ex vivo to antigens expressed on the surface of tumor cellsis a promising novel approach to the adoptive immu-notherapy of cancer, and one that has only recently beenexplored in earnest in the clinical setting. T cells may begenetically modified to target tumor-associated antigens

ffiliations: Departments of 1Medicine, 2Pathology, 3Surgery,er for Cell Engineering, Memorial Sloan-Kettering Cancerw York, New York

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

ding Author: Renier J. Brentjens, Memorial Sloan-Ketteringnter, Box 242, 1275 York Avenue, New York, NY 10065.-639-7053; Fax: 212-772-8441; E-mail: [email protected].

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(TAA) through the retroviral introduction of genes encod-ing artificial T-cell receptors (TCR) termed chimericantigen receptors (CAR). CARs are most commonly com-posed of a single-chain fragment length antibody (scFv),derived from a murine monoclonal antibody (mAb) tar-geting a given TAA, fused to a transmembrane with orwithout an additional cytoplasmic signaling domain (typ-ically derived from CD8, CD28, OX-40, or 4-1BB), fusedto the TCRζ chain cytoplasmic signaling domain (3–13).When expressed by the T cells, the resulting construct, onengagement with the targeted antigen, induces T-cell acti-vation, proliferation, and lysis of targeted cells. To date,preclinical studies using CAR-modified T cells have shownpromising results in a wide variety of malignancies (3, 4,11, 14–18). More recently, this approach has been inves-tigated in phase I clinical trials (6, 19–21).Ovarian carcinomas seem to be relatively immunogenic

tumors capable of inducing an endogenous immune re-sponse because long-term prognosis of patients is marked-ly influenced by the degree and quality of the endogenousimmune response to the tumor. Specifically, it has beenwell documented that the presence of endogenous effectorT cells within the ovarian cancer tumor microenvironment

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Translational Relevance

Ovarian carcinomas seem to be immunogenic tu-mors because increased numbers of tumor-infiltratingT cells present in the pretreatment tumor are associatedwith an enhanced survival following surgery and che-motherapy. Patient T cells may be genetically modifiedto recognize tumor-associated antigens through the in-troduction of chimeric antigen receptors (CAR) specif-ically targeted to these antigens. We have generated aCAR, 4H11-28z, specific to the retained extracellularportion (MUC-CD) of MUC16, a glycoprotein overex-pressed on most ovarian carcinomas. We show that Tcells, when modified to express the 4H11-28z CAR,specifically lyse human ovarian cancer cells in vitro,and further show efficient antitumor efficacy in ortho-topic xenotransplant tumor models. Collectively, thesedata support the translation of these preclinical find-ings to the clinical setting in a planned phase I trialtreating patients with relapsed or refractory ovariancancer.

Eradication of Ovarian Carcinomas with Targeted T Cells


directly correlates to prolonged patient survival (22–25).In contrast, increasing numbers of immunosuppressiveCD4+ CD25hi regulatory T cells (Tregs) within the tumor,which in turn presumably abrogate the antitumor activityof infiltrating effector T cells, correlate with shorter patientsurvival (26–29). In fact, it seems that it is the ratio ofTregs to effector T cells within the tumor microenviron-ment that ultimately dictates whether the endogenous im-mune response to the cancer is of benefit or detriment tothe patient (24, 28). In this setting, the ability to generateand subsequently expand a population of tumor-targetedeffector T cells ex vivo, which are subsequently infused backinto the patient, may in turn skew the Treg to effector T-cellratio to one more favorable to eradicating the disease.Mucins are important biomolecules for cellular homeo-

stasis and protection of epithelial surfaces (1). MUC16 isone such mucin that is overexpressed on most ovarian car-cinomas and is an established surrogate serum marker(CA-125) for the detection and progression of ovariancancers (30–33). MUC16 is a glycosylated mucin com-posed of a large cleaved and released domain, termedCA-125, consisting of multiple repeat sequences, and aretained domain (MUC-CD), which includes a residualnonrepeating extracellular fragment, a transmembranedomain, and a cytoplasmic tail (34). Because the antigenis otherwise only expressed at low levels in the uterus,endometrium, fallopian tubes, ovaries, and serosa of theabdominal and thoracic cavities, MUC16 is a potentiallyattractive target for immune-based therapies.The fact that most of the extracellular domain of MUC16

is cleaved and secreted limits the utility of MUC16 as a tar-get antigen on ovarian carcinomas. To date, all reportedmAbs to MUC16 bind to epitopes present on the large

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secreted CA-125 fraction of the glycoprotein, with noneknown to bind to the retained extracellular fraction(MUC-CD) of the antigen (35–37). Because the MUC-CD fraction of the antigen is retained on the cell surface,generating T cells specific to this portion of MUC16 maylargely overcome the limitation of MUC16 as a target foradoptive cellular immunotherapy. To this end, we havepreviously generated a series of murine mAbs specific tothe retained MUC-CD extracellular domain (38). Using ahybridoma that expresses one such mAb, 4H11, we havesuccessfully constructed several CARs specific to theMUC-CD antigen.In this report, we show highly efficient retroviral

transduction of these MUC-CD–targeted CARs into hu-man T cells, with resulting T cells able to specifically tar-get and lyse MUC-CD+ tumor cell lines in vitro. Inaddition, we show significant cytotoxicity mediated by4H11-28z+ patient T cells and healthy donor T cellstargeting primary ascites-derived ovarian carcinoma cells.In vivo studies in immunocompromised SCID-Beige micebearing established peritoneal orthotopic MUC-CD+

human ovarian tumors show marked antitumor efficacyof MUC-CD–targeted T cells, following either i.p. or i.v.injection. These data serve as a rationale for future clin-ical trials using this approach in patients with high-riskovarian carcinomas.

Materials and Methods

Cell lines and T cellsTheOV-CAR3 and SK-OV3 tumor cell lines were cultured

in RPMI 1640 (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS), nonessential aminoacids, HEPES buffer, pyruvate, and 2-Mercaptoethanol (In-vitrogen). T80 cells originated from normal ovarian surfaceepithelium, which were immortalized by transfection withSV40 large T antigen, were kindly provided by Dr. Robert C.Bast (The University of Texas M. D. Anderson Cancer Cen-ter, Houston, TX) (39). T80 cells were cultured in 1:1 ratioof MCDB 105 medium (Sigma-Aldrich Co.) and Medium199 (Invitrogen) supplemented with 10% heat-inactivatedFBS. The PG13, HeLa, and gpg29 retroviral producer celllines were cultured in DMEM (Invitrogen) supplementedwith 10% FCS. NIH-3T3 artificial antigen-presenting cells(AAPC), described previously (3), were cultured in DMEMsupplemented with 10%heat-inactivated donor calf serum.Human T cells were isolated from peripheral blood ofhealthy donors under Institutional Review Board–ap-proved protocol 95-054 using BD Vacutainer CPT tubes(Becton Dickinson) as per the manufacturer's instructions.T cells were cultured in RPMI 1640 supplemented with 20IU/mL interleukin (IL)-2 (Novartis Pharmaceuticals), andwhere indicated, medium was supplemented with 10 ng/mL IL-15 (R&D Systems). Primary ovarian cancer cells de-rived from ascites specimens were cultured in RPMI 1640supplemented with 10% FBS. All media were supplemen-ted with 2 mmol/L L-glutamine (Invitrogen), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen).

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Isolation of patient ascites-derived ovariancancer cellsPrimary human ascites-derived cancer cells were ob-

tained from ovarian cancer patients undergoing surgeryfor newly diagnosed ovarian carcinomas under Institu-tional Review Board–approved protocol 97-134. The tu-mor cells were isolated from ascites fluid of patients bycentrifugation at 600 × g for 10 minutes at room temper-ature. Cells were washed once with 1× PBS and cultured inFBS-supplemented RPMI 1640 in tissue culture flasks. After5 days in culture, nonadherent cellswere removed, retainingthe tumor cell–enriched adherent fraction for further study.

Generation of the MUC-CD–targeted 4H11z and4H11-28z CARsThe heavy and light chain variable regions of the 4H11

mAb were derived from the hybridoma cell line 4H11.Using cDNA generated from 4H11 RNA, we isolatedthe VH coding region by RACE (rapid amplification ofcDNA ends) PCR using modified primers as describedelsewhere (40, 41). The VL chain variable region wascloned by standard PCR using modified primers as de-scribed by Orlandi et al. (42, 43). The resulting VH andVL fragments were subcloned into the TopoTA PCR 2.1cloning vector (Invitrogen) and sequenced. The VH andVL fragments were subsequently ligated to a (Gly4Ser)3spacer domain, generating the 4H11 scFv, and fused tothe human CD8 leader peptide (CD8L) by overlappingPCR (9, 42). To construct the MUC-CD–targeted 4H11CARs, the coding region of the CD8L-4H11 scFv wasfused to the human CD8 hinge and transmembrane do-mains (to generate the 4H11z CAR) or, alternatively, tothe CD28 transmembrane and cytoplasmic signaling do-mains (to generate the 4H11-28z CAR), fused to the TCRCD3ζ signaling domain (3, 9, 44). The resulting CAR con-structs were subsequently subcloned into themodifiedMo-loney murine leukemia virus retroviral vector SFG (45).VSV-G–pseudotyped retroviral supernatants derived fromtransduced gpg29 fibroblasts were used to construct stablePG13 gibbon ape leukemia virus envelope-pseudotypedretroviral-producing cell lines (42).

Retroviral gene transferIsolated healthy donor peripheral blood mononuclear

cells were activated with phytohemagglutinin at 2 μg/mL(Sigma), whereas patient T cells derived from fresh ascitessamples were isolated, activated, and expanded withDynabeads ClinExVivo CD3/CD28 beads (Invitrogen) fol-lowing the manufacturer's recommendations. ActivatedT cells were retrovirally transduced on retronectin-coatednontissue culture plates as described previously (46). Genetransfer was assessed on day 7 by fluorescence-activatedcell sorting (FACS).To generate the relevant NIH-3T3 murine fibroblast

AAPCs, a MUC-CD construct encoding the retained extra-cellular, transmembrane, and cytoplasmic domains of theMUC16 antigen was initially subcloned into SFG retrovi-ral vector, SFG(MUC-CD). 3T3(MUC-CD) AAPCs were

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generated by retroviral transduction of SFG(MUC-CD) in-to wild-type NIH-3T3 fibroblasts, whereas 3T3(MUC-CD/B7.1) AAPCs were generated by retroviral transduction ofpreviously established 3T3(B7.1) fibroblasts (42, 47). En-riched cell lines were isolated by FACS.OV-CAR3(MUC-CD), SK-OV3(MUC-CD), HeLa(MUC-

CD), OV-CAR3(MUC-CD/GFP-FFLuc), and SK-OV3(MUC-CD/GFP-FFLuc) cell lines were generated by retro-viral transduction with SFG(GFP-FFLuc) (48) and/or SFG(MUC-CD) VSV-G–pseudotyped retroviral supernatantsderived from gpg29 fibroblasts as described elsewhere(45). T80(MUC-CD) cells were generated by transfectionof the MUC-CD gene using the Vitality phrGFP II C vectorexpression system (Stratagene). Resulting tumor cells weresorted by FACS for MUC-CD expression.

In vitro analyses of CAR+ human T cellsTo assess in vitro expansion and cytokine release on

stimulation, 3 × 106 transduced T cells were coculturedfor 7 days after retroviral transduction in six-well tissueculture plates (BD Biosciences) on confluent NIH-3T3AAPCs in the absence of supplemented cytokines. Forin vivo studies, transduced T cells were expanded by cocul-ture on 3T3(MUC-CD/B7.1) AAPCs in RPMI 1640 supple-mented with 20 IU/mL IL-2 and 10 ng/mL IL-15 asdescribed previously (3, 44).

Western blot analysis of CAR expressionWestern blot analysis of T-cell lysates under reducing

conditions with 0.1 mol/L DTT (Sigma) was done as pre-viously described (47).

Cytotoxicity assaysIn vitro–modified T-cell cytotoxicity was assessed using

the DELFIA EuTDA assay (Perkin-Elmer LAS, Inc.) follow-ing the manufacturer's recommendations. Cytotoxicitywas assessed at 2 hours at serially diluted E:T ratios. Effec-tor T cells in these assays represent the CD8+ CAR+ T-cellfraction.

Cytokine detection assaysCytokine assays were done as per the manufacturer's

specifications using a multiplex Human Cytokine Detec-tion assay to detect IL-2 and IFN-γ (Millipore Corp.) usingthe Luminex IS100 system. Cytokine concentrations wereassessed using IS 2.3 software (Luminex Corp.).

In vivo SCID-Beige mouse tumor modelsIn all in vivo studies, 8- to 12-week-old Fox Chase C.B.-17

(SCID-Beige mice; Taconic) were injected i.p. with 3 × 106

tumor cells. Subsequently, CAR+ T cells, at indicated doses,were injected either i.p. or i.v. on either day 1 or 7 follow-ing tumor cell injection. Mice were monitored for distressas assessed by increasing abdominal girth, ruffled fur, anddecreased response to stimuli. Distressed mice were eutha-nized. All murine studies were done in the context of anInstitutional Animal Care and Use Committee–approvedprotocol (00-05-065).

Clinical Cancer Research





Bioluminescent imaging of GFP-FFLuc+ humanovarian tumor cells in SCID-Beige miceBioluminescent imaging (BLI) was done using Xenogen

IVIS imaging systemwith Living Image software (Xenogen).Briefly, GFP-FFLuc+ tumor-bearing mice were injected byi.p. with D-luciferin (150 mg/kg; Xenogen) suspended in200 μL PBS and imaged under 2% isoflurane anesthesiaafter 10 minutes. Image acquisition was done on a 25-cmfield of view at medium binning level for 0.5-minute expo-sure time (3, 44).

Flow cytometryAll flow cytometric analyses of T cells and tumor cells

were done using a FACScan cytometer with CellQuestsoftware (BD Biosciences). T cells were analyzed usingCAR-specific polyclonal goat Alexa Fluor 647 antibody(Molecular Probes); phycoerythrin-labeled anti-humanCD4, CD8, B7.1, and CD45RO (Caltag Laboratories);B7.2 (Invitrogen); phycoerythrin-conjugated anti-humanCCR7; and FITC-conjugated anti-human CD62L(eBioscience). MUC-CD expression was measured byFACS with Alexa Fluor 647–labeled 4H11 antibody(generated and labeled in the Memorial Sloan-KetteringCancer Center mAb core facility).

Carboxyfluorescein diacetate succinimidyl esterlabeling of CAR+ T cellsCAR+ T cells were stained with carboxyfluorescein dia-

cetate succinimidyl ester (CFSE) using the CellTraceCFSE cell proliferation kit following the manufacturer'srecommendations (Molecular Probes).

Assessment of in vivo T-cell persistenceSCID-Beige mice were infused i.p. with OV-CAR3

(MUC-CD) tumor cells and, 7 days later, treated by i.p.infusion with CAR+ T cells. Subsequently, mice were sac-rificed at days 2, 14, 21, and 28 following T-cell infusion.Peritoneal washes were collected using 10 mL PBS, RBCswere lysed with ACK lysing buffer (Lonza), and remain-ing cells were washed with 2% FBS/PBS and analyzed byFACS for persistence of human CD3+ T cells.

StatisticsSurvival data assessed by log-rank analysis using Prism

5.0 (GraphPad Software) and cytokine secretion data as-sessed by Student's two-tailed t test. In the comparisonbetween survival of i.p. versus i.v. modified T-cell infu-sion, two experiments were done with different follow-up times. For combined analysis, the stratified log-rankstatistic (stratified by experiment) was used to testwhether the survival rates differed between the i.p. andi.v. treatment groups.

Results

We have constructed SFG retroviral vectors encodingfirst-generation (4H11z) and second-generation costimu-latory (4H11-28z) CARs targeted to the MUC-CD antigen



using the 4H11 hybridoma, which generates a mAb spe-cific to the MUC-CD antigen (Fig. 1A). We confirmed ex-pression of appropriately sized CAR proteins by Westernblot analysis of resulting PG-13 retroviral producer cells(SFG-4H11z and SFG-4H11-28z) probed with a ζ chain–specific antibody (data not shown).To assess the function of the first-generation 4H11z

CAR, healthy donor T cells isolated from peripheral bloodwere retrovirally transduced to express the 4H11z CAR andcontrol T cells modified to express the irrelevant CD19-targeted 19z1 CAR (Fig. 1B). The function of the 4H11zCAR was assessed by proliferation of 4H11z-transduced Tcells following coculture on 3T3(MUC-CD/B7.1) AAPCs.Results show specific proliferation of 4H11z-transducedT cells when compared with 19z1-modified T cells (Fig.1C). These data are consistent with 4H11z CAR–mediatedspecific binding to the MUC-CD antigen and subsequentT-cell activation.Because most malignancies fail to express costimulatory

ligands, we further modified the 4H11z CAR to express theCD28 transmembrane and cytoplasmic costimulatory sig-naling domains, generating the 4H11-28z CAR (Fig. 1A).To assess whether the 4H11-28z CAR, when expressed byhuman T cells, was capable of generating both a primaryactivating signal (termed “signal 1”) through the ζ chainand a costimulatory signal (termed “signal 2”) throughthe CD28 cytoplasmic domain, which in turn allows forefficient T-cell proliferation in the absence of exogenouscostimulatory ligands, we compared T-cell proliferationfollowing coculture on either 3T3(MUC-CD) or 3T3(MUC-CD/B7.1) AAPCs in the absence of exogenous cyto-kines. As expected, the second-generation 4H11-28z+ T cellsmarkedly expandedwhen comparedwith 4H11z+ T cells oncoculture with 3T3(MUC-CD) AAPCs (P = 0.0047). In con-trast, both 4H11z+ and 4H11-28z+ T cells expanded equallywell on 3T3(MUC-CD/B7.1) AAPCs (P = 0.18; Fig. 2A).Costimulation mediated by the 4H11-28z CAR was furtherverified by analysis of day 2 tissue culture supernatantsfrom coculture experiments on 3T3(MUC-CD) AAPCs,showing significantly enhanced IL-2, a cytokine typicallysecreted in the context of T-cell costimulation, and IFN-γsecretion when compared with control 19-28z+ T cellsand first-generation 4H11z+ T cells (Fig. 2B).We next assessed the ability of 4H11-28z+ T cells to ex-

pand following weekly restimulations on 3T3(MUC-CD/B7.1) AAPC monolayers in the context of exogenous IL-2and IL-15 (3). In this setting, 4H11-28z+ T cells expandedgreater than two logs over 3 weeks compared with no ex-pansion of control 19-28z+ T cells (Fig. 2C). T cells trans-duced with 4H11-28z were further analyzed by FACS forCAR expression 7 days after initial transduction and follow-ing two subsequent costimulations on AAPCs, showing anexpected enrichment of the CAR+ T-cell fraction (Fig. 2D).

In vitro MUC-CD–specific activation of 4H11-28z+

T cellsTo assess MUC-CD specificity of 4H11-28z+ T cells, we

initially conducted a series of cytotoxicity assays using




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the MUC-CD− OV-CAR3 and SK-OV3 cell lines with orwithout further genetic modification with MUC-CD. Asexpected, 4H11-28z+ T cells efficiently lysed MUC-CD+

but not unmodified tumor cell lines, consistent withMUC-CD specificity mediated through the 4H11-28zCAR (Fig. 3A). To further verify these findings, we con-ducted similar studies on nonovarian carcinoma cell lines,HeLa, and an immortalized normal ovarian surface epithe-lial cell line, T80, showing similar results (SupplementaryFig. S1). We next assessed the activation of CAR-modified Tcells, as measured by proliferation and cytokine secretion,by coculture with these same cell lines. Consistent with4H11-28z CAR specificity for MUC-CD, T-cell proliferationas well as IL-2 and IFN-γ secretion only occurred in the set-ting of MUC-CD+ cell lines cocultured with 4H11-28z+ Tcells (Fig. 3B and C; Supplementary Fig. S1).Using more clinically relevant MUC-CD+ ovarian carci-

noma cells enriched from fresh ascites samples, healthydonor T cells modified to express the 4H11-28z CARsimilarly exhibited lysis of primary ovarian carcinoma



cells when compared with 19-28z–transduced T cells(Fig. 3D). Moreover, in three of three cases, we foundthat patient ascites-derived T cells modified to expressthe 4H11-28z CAR similarly lysed matched autologousprimary ovarian carcinoma cells (Fig. 3D).

In vivo antitumor activity of MUC-CD–targeted T cellsin SCID-Beige miceTo assess the in vivo antitumor activity of 4H11z+ and

4H11-28z+ T cells, we next generated an orthotopic xeno-transplant ovarian cancer tumor model by i.p. injection ofOV-CAR3(MUC-CD) tumor cells into SCID-Beige mice. Ifleft untreated, these mice developed marked ascites andmultiple nodular peritoneal tumors by 5 weeks followingtumor cell injection (Fig. 4A). All untreated tumor-bearingmice had to be euthanized by 7 weeks following tumorcell injection due to abdominal distention and evidenceof distress.For in vivo studies, cohorts of SCID-Beige mice, injected

i.p. with 3 × 106 OV-CAR3(MUC-CD/GFP-FFLuc) tumor

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Fig. 1. Design and in vitro analysisof MUC-CD–targeted CARs.A, schematic diagram of the first-generation 4H11z and second-generation 4H11-28z retroviralvectors. 4H11scFv: MUC16-specific scFv derived from theheavy (VH) and light (VL)chain variable regions of the mAb4H11; CD8: CD8 hinge andtransmembrane domains; CD28:CD28 transmembrane andcytoplasmic signaling domains;ζ chain: TCRζ chain cytoplasmicsignaling domain; LTR: longterminal repeat; black box:CD8 leader sequence; gray box:(Gly4Ser)3 linker; arrows indicatestart of transcription. B, FACSanalysis of human T cellsretrovirally transduced to expresseither the 4H11z or the 19z1 CAR.C, 4H11z+ but not 19z1+ T cellsexpand on 3T3(MUC-CD/B7.1)AAPC. CAR+ T cells werecocultured on 3T3(MUC-CD/B7.1)AAPC monolayers at 3 × 106 perwell of a six-well plate. Proliferationof CAR+ T cells was assessed byFACS in combination with viableT-cell counts as assessed bytrypan blue exclusion assays.


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cells on day 1, were initially treated with dose-escalatinglevels of CAR-modified T cells by i.p. injection on day 2.FACS analysis of modified in vitro–expanded T cells usedin our in vivo studies showed most infused T cells with aretained central memory phenotype as assessed by CD62L,CCR7, CD28, and CD45RO expression (SupplementaryFig. S2; ref. 49). These studies showed a dose-dependentin vivo antitumor response to MUC-CD–targeted T-celltherapy at a treatment dose of 1 × 107 4H11-28z+ T cellsfound to be the minimum dose required to achieve ameaningful long-term survival at day 70, with all micetreated at lower dose levels showing 100% mortality (Sup-plementary Fig. S3). Based on these findings, we chose thehighest tested dose level of 3 × 107 CAR+ T cells for furtherstudies. We subsequently repeated these studies with largercohorts of mice treated at this dose level of 4H11z+ or4H11-28z+ T cells. For negative controls, tumor-bearingmice were either untreated or treated with T cells modifiedto express the irrelevant CD19-targeted 19z1 CAR. Both



MUC-CD–targeted T-cell–treated cohorts showed statisti-cally significant enhanced survival when compared withuntreated or 19z1+ T-cell–treated control cohorts, withno statistically significant difference in survival when com-paring the 4H11z+ and 4H11-28z+ T-cell–treated cohorts(Fig. 4B; Supplementary Fig. S4A).As a further in vivo control for 4H11-28z MUC-CD spec-

ificity, we repeated these studies using SCID-Beige micebearing i.p. MUC-CD− SK-OV3(GFP-FFLuc) or SK-OV3(MUC-CD/GFP-FFLuc) human ovarian carcinoma tumorssimilarly treated with 3 × 107 4H11-28z+ T cells 1 day aftertumor cell injection. As expected, treatment with 4H11-28z+ T cells failed to result in any long-term survival ofSK-OV3(GFP-FFLuc) tumor-bearing mice, whereas 40%of mice bearing SK-OV3(MUC-CD/GFP-FFLuc) tumorsremain alive at day 70 (Supplementary Fig. S4B) with noevidence of disease as assessed by BLI (data not shown).To determine whether systemically infused MUC-CD–

targeted T cells successfully traffic to peritoneal tumors,

Fig. 2. In vitro comparison of T cells modified to express the first-generation 4H11z CAR to T cells modified to express the second-generation costimulatory4H11-28z CAR. A, CAR+ T cells were cocultured on MUC-CD monolayers with (right) or without (left) B7.1. CAR+ T cells (1 × 106 per well) were cocultured onAAPC monolayers in 12-well tissue culture plates in cytokine-free medium. Total viable T-cell counts were assessed on days 2, 4, and 7 by trypanblue exclusion assays. B, antigen-specific cytokine secretion by MUC-CD+ targeted T cells. C, expansion of CAR+ T cells following three cycles ofstimulation on 3T3(MUC-CD/B7.1). Arrows indicate first and second cycles of restimulation on AAPCs. D, FACS analysis of the CAR+ T-cell fraction of4H11-28z+ T cells increased following each weekly cycle of stimulation. I, FACS following initial transduction; II, FACS at 7 d following first stimulation onAAPCs; III, FACS at 7 d following second stimulation on AAPCs.




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we next compared i.p. to i.v. infusion of 4H11-28z+ T cellsin SCID-Beige mice 1 day after i.p. infusion of OV-CAR3(MUC-CD/GFP-FFLuc) tumors. Both i.p. and i.v. 4H11-28z+ T-cell–treated mice exhibited statistically enhancedsurvival when compared with untreated or 19-28z+ T-cell–treated control cohorts as assessed by BLI imaging(Fig. 5A) as well as overall survival (Fig. 5B; Supplemen-tary Fig. S5). Stratified log-rank analysis of merged datafrom two independent experiments showed statisticallyequivalent antitumor efficacy between i.p. and i.v. MUC-CD–targeted T-cell–treated cohorts (P = 0.092).We further confirmed trafficking of i.v. infused CFSE-

labeled 4H11-28z+ T cells to the peritoneum by FACS



analysis of single-cell suspensions of macerated OV-CAR3(MUC-CD) peritoneal tumor deposits (Fig. 5C).The presence of i.v. injected CFSE-labeled 19-28z+ controlT cells and 4H11-28z+ T cells 1 day following infusion intoSCID-Beige mice with advanced OV-CAR(MUC-CD) tu-mors (injected 7 days earlier) reveals a marked populationof human CD3/CFSE+ T cells within peritoneal OV-CAR3(MUC-CD) tumor deposits of 4H11-28z+ but not of con-trol 19-28z+ T-cell–treated mice.We next treated SCID-Beige mice bearing peritoneal OV-

CAR3(MUC-CD/GFP-FFLuc) tumor injected 7 days beforeadoptive T-cell therapy, a time point at which the micehad evidence of overt disease as assessed by BLI. A subset

Fig. 3. On coculture, 4H11-28z+ T cells specifically expand and lyse MUC-CD+ tumor cells. A, cytotoxicity assays of 4H11-28z+ T cells targetingOV-CAR3(MUC-CD) and SK-OV3(MUC-CD) tumor cells show efficient cytotoxicity mediated by 4H11-28z+ T cells from healthy donors compared withcontrol 19-28z+ T cells. B, antigen-specific proliferation of MUC-CD–targeted T cells after coculture with OV-CAR3(MUC-CD) or SK-OV3(MUC-CD)tumor cells. CAR+ T cells were cocultured with wild-type or MUC-CD–expressing tumor cells at 1:1 ratio for 7 d. Absolute T-cell numbers assessed on days2, 4, and 7 following coculture with ovarian tumor cells. The T-cell seeding density for T-cell proliferation assay was 1 × 106/mL. C, antigen-specific IL-2 andIFN-γ secretion by 4H11-28z–transduced T cells after 2 d of coculture on ovarian cancer cells. D, healthy donor T cells or autologous T cells isolatedfrom ascites, modified to express the 4H11-28z CAR, lyse primary patient ascites-derived MUC-CD+ tumor cells when compared with T cells modified toexpress the control 19-28z CAR.






of 4H11-28z+ T-cell–treated mice assessed to be tumor-freeby BLI on day 60 after tumor cell infusion was sacrificed,and peritoneal washes were analyzed for the presence ofMUC-CD/GFP-FFLuc+ tumor cells by FACS. At this timepoint, in contrast to control-treated mice sacrificed and an-alyzed at day 30, peritoneal washings from treated miceshowed <0.01% of analyzed cells to be MUC-CD/GFP+

(Supplementary Fig. S6A). Once more, we found that ther-apy withMUC-CD–targeted T cells initially eradicatedmostBLI evident disease in all treatedmice (Fig. 6A), with 75%ofmice ultimately developing relapsed disease at later timepoints, whereas 25% of treated mice survive at 120 days af-ter tumor cell infusion with no evidence of disease as as-sessed by BLI (Fig. 6B). Significantly, FACS analyses oftumor cell suspensions obtained from all 4H11-28z+ T-cell–treated mice with relapsed disease showed persistentexpression of the MUC-CD antigen (data not shown).To test for the persistence of modified T cells over time

in these studies, additional mice were infused i.p. withOV-CAR3(MUC-CD) tumors and treated on day 7 withmodified T cells and sacrificed serially following therapy.We analyzed for the presence of CAR+ T cells in peritonealwashes of 4H11-28z and 19-28z T-cell–treated tumor-bearing mice on days 2, 14, 21, and 28 after T-cell infusionby FACS, showing a decreasing but persistent population



of modified T cells out to 28 days after T-cell infusion (thelatest time point studied; Supplementary Fig. S6B).

Discussion

Based on analyses of patient tumor samples, ovariancarcinomas seem to be relatively immunogenic tumors.Specifically, researchers have found a direct correlation be-tween prognosis following surgery and chemotherapy andthe quantity of tumor-infiltrating T cells (TIL) in pretreat-ment tumor samples (25, 50, 51). Furthermore, othershave described an inverse correlation between prognosisfollowing therapy and pretreatment levels of Tregs withinthe tumor, which in turn presumably inhibit the antitu-mor function of tumor-specific effector TILs (26, 28, 52).Both of these findings imply a role for an endogenouseffector T-cell response to tumor in controlling diseaseprogression both before and following initial therapyand strongly support the contention that ovarian carcino-mas may be susceptible to killing by adoptive infusion ofautologous T cells targeted to ovarian tumor cell antigens.Although endogenous effector TILs are one source for

presumably tumor-specific T cells, an alternative approachto adoptive T-cell therapy is to isolate autologous periph-eral blood T cells, which in turn may be genetically

Fig. 4. Eradication of OV-CAR3(MUC-CD)tumors after i.p. treatment with first and secondgeneration of MUC-CD–targeted T cells.A, i.p. injection of OV-CAR3(MUC-CD) tumorsin untreated SCID-Beige mice results inabdominal distension and nodular peritonealtumors. Left, at 5 wk after i.p. injection ofOV-CAR3(MUC-CD) tumor cells, micedeveloped ascites as evidenced by a distendedabdomen (middle) when compared with atumor-free mouse; right, postmortemvisualization of the peritoneum shows nodulartumor masses (arrows) within the abdominalcavity. B, i.p. injection of 4H11z+ and4H11-28z+ T cells either delay tumorprogression or fully eradicate disease.Kaplan-Meier survival curve of SCID-Beigemice treated with first or second generation ofMUC-CD–targeted T cells.




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modified ex vivo to target tumor cell antigens. One suchgenetic approach is to retrovirally transduce patient T cellswith CARs targeted to surface-exposed antigens eitherunique to or overexpressed by the tumor. To this end,promising preclinical studies using this approach in othermalignancies have recently been translated to the clinicalsetting (6, 16, 19, 53). Application of this approach toovarian carcinomas requires the identification of suitabletarget antigens expressed on the tumor cell surface. To thisend, other investigators have studied this approach in vivoin the preclinical setting using CAR+ T cells targeted to dis-parate antigens overexpressed on ovarian carcinomas, in-cluding the α-folate receptor, the Lewis-Y antigen, Her2/neu, NKG2D ligands (MICA, MICB, and UL-16 bindingproteins), mesothelin, and MUC-1 (4, 11, 54–61). Specif-ically, in an elegant series of studies using a syngeneicorthotopic 1D8 tumor model of ovarian carcinoma inC57BL6 mice, Barber and Sentman (54) show efficienteradication of well-established i.p. tumors when treatedwith i.p. injections of syngeneic murine T cells modifiedto express the chNKG2D CAR. Furthermore, Hwu et al.(4) showed significant delays in tumor progression in im-munocompromised nude mice bearing orthotopic humanIGROV tumors following a single infusion of murineT cells modified to express an α-folate receptor–targeted



CAR. In the xenogeneic setting, several groups haveshown delayed tumor progression or complete antitumorresponses of subcutaneous human ovarian carcinoma celllines in immunocompromised mice following intratu-moral and/or i.v. infusion of human T cells expressingCARs specific to the Her2/neu and Lewis-Y antigens(59, 61, 62). Similarly, Wilkie et al. (60) and Carpenitoet al. (57) have recently published reports showing effi-cient antitumor efficacy in xenotransplant models of hu-man breast and mesothelioma tumors treated withhuman T cells modified, respectively, with CARs targetedto the MUC-1 and mesothelin antigens, antigens alsooverexpressed on ovarian carcinomas.In the clinical setting, Kershaw et al. (6) recently pub-

lished the results of a phase I dose escalation trial treatingpatients with relapsed ovarian carcinomas with autolo-gous T cells modified to express a first-generation CAR spe-cific to the α-folate receptor. The authors of this studyfound that therapy with targeted T cells was well tolerated,but noted a lack of antitumor response in these studiesrelated to poor persistence of modified T cells over timeas well as a yet undefined T-cell inhibitory factor in theserum of several treated patients.In our studies, we have chosen to target the MUC16

glycoprotein, which is overexpressed on most ovarian

Fig. 5. MUC-CD–targeted 4H11-28z+ T cells traffic to peritoneal OV-CAR3(MUC-CD/GFP-FFLuc) tumors following systemic i.v. infusion resulting inefficient antitumor efficacy. A, BLI of tumor progression of representative i.p. and i.v. 4H11-28z+ T-cell–treated mice with ultimately progressive diseasefollowing treatment compared with BLI of tumor progression in a representative control 19-28z+ T-cell–treated mouse. B, Kaplan-Meier survival curve ofSCID-Beige mice treated i.p. or i.v. with 4H11-28z+ T cells. Tumor eradication is enhanced after either i.p. or i.v. infusion of 4H11-28z+ T cells whencompared with control-treated mice. Both i.p. and i.v. 4H11-28z+ T-cell–treated mice exhibited statistically enhanced survival when compared with 19-28z+

T-cell–treated control cohorts, whereas survival between the i.p. and i.v. treated 4H11-28z+ T-cell cohorts was not statistically significant (P = 0.22).C, systemically injected CFSE-stained 4H11-28z+ T cells traffic to advanced i.p. OV-CAR(MUC-CD) tumors. Presence of i.v. injected CFSE-labeled 19-28z+

control T cells (left) and 4H11-28z+ T cells (right) 1 d following infusion into SCID-Beige mice with OV-CAR(MUC-CD) tumors injected 7 d earlier as assessedby FACS analysis of single-cell OV-CAR3(MUC-CD) tumor suspensions.






carcinomas (1, 30, 32, 33). The utility of MUC16 as atarget antigen for adoptive T-cell therapy is compromisedby the fact that most of the extracellular portion of thismolecule is cleaved by the tumor cell, secreted, and maybe detected in the serum as the CA-125 tumor marker.However, following cleavage of this secreted fraction ofMUC16, there remains a residual extracellular fractionof the glycoprotein, termed MUC-CD, which is retainedon the tumor cell surface and is therefore an attractivetarget for immune-based therapies. To this end, we useda murine hybridoma, 4H11, generated to the MUC-CDantigen (38) to construct a first-generation (4H11z) aswell as a second-generation costimulatory CAR (4H11-28z) specific to MUC-CD. Significantly, the antigen tothe 4H11 antibody is highly expressed on most pre-treatment ovarian carcinoma tumor samples obtainedfrom patients treated at our institution as assessed byimmunohistochemistry (38, 63).Consistent with previous studies, we found that T cells

transduced to express the second-generation 4H11-28zCAR, but not the first-generation 4H11z CAR, efficiently



expanded on coculture with 3T3(MUC-CD) fibroblastsin the absence of exogenous costimulation. This conclu-sion is further supported by the finding that 4H11-28z+

T cells secreted significantly higher levels of IL-2, a cyto-kine indicative of T-cell costimulation, and IFN-γ oncoculture on 3T3(MUC-CD) fibroblasts when comparedwith T cells transduced to express the first-generation4H11z CAR.Specificity of the 4H11-28z CAR to the MUC-CD anti-

gen was subsequently verified in vitro by comparing4H11-28z+ T-cell cytotoxicity and proliferation on a seriesof MUC-CD− ovarian carcinoma cell lines, as well asHeLA cells and the T80 immortalized normal ovariansurface epithelial cell line, to the same cell lines furthergenetically modified to express the MUC-CD antigen.To additionally validate the clinical relevance of our find-ings, we subsequently showed specific in vitro lysis of pri-mary ascites-derived tumor cells isolated from untreatedovarian carcinoma patients by both healthy donor alloge-neic 4H11-28z+ T cells and, more significantly, autolo-gous 4H11-28z+ patient ascites-derived T cells. These

Fig. 6. Eradication of BLI evident OV-CAR3(MUC-CD) tumors in SCID-Beige mice byi.p. infusion of 4H11-28z+ T cells. A, BLI of4H11-28z+ T-cell–treated mice with eitherrelapsed disease (middle row) or eradicateddisease (bottom row) compared with arepresentative 19-28z+ T-cell–treatedcontrol mouse. B, Kaplan-Meier survival curveof SCID-Beige mice with advanced OV-CAR3(MUC-CD/GFP-FFLuc) tumors treated i.p.with 4H11-28z+ T cells.




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data strongly support the contention that treatment withautologous 4H11-based CAR+ T cells has promise in fu-ture clinical applications.To assess the in vivo relevance of our in vitro findings, we

next generated several orthotopic human ovarian cancertumor models in SCID-Beige mice. These studies showederadication of early as well as more established BLI evidentperitoneal tumors following i.p. injection of healthy do-nor 4H11-28z+ T cells. In the setting of delayed therapy,tumor imaging by BLI could show initial marked eradica-tion of disease. However, loss of bioluminescent signal inthese studies did not preclude future relapse of disease be-cause most apparently tumor-free mice, as assessed by BLIat earlier time points of follow-up, developed relapsed dis-ease within the peritoneum over time, consistent with thenotion that BLI lacks the sensitivity to measure in vivomin-imal residual disease in demonstrated persistent expres-sion of the targeted MUC-CD antigen, consistent withthe notion that loss of target antigen expression by the tu-mor or immune selection of MUC-CD− tumors was notthe cause of tumor relapse. Although the source of re-lapsed disease in these mice remains speculative, studiesof i.p. infused 4H11-28z+ T-cell persistence show rapidlydeclining numbers of T cells over time. While modifiedT cells were still detectable in peritoneal washes out to28 days after T-cell infusion, these numbers were declin-ing, suggesting a loss of modified T-cell persistence as apotential source of disease relapse, which typically oc-curred at later time points.We further show trafficking of i.v. injected MUC-CD–

targeted T cells to peritoneal tumors by FACS. Significant-ly, tumor-bearing mice treated with i.v. infused 4H11-28z+ T cells exhibited similar antitumor efficacy when com-pared with i.p. treated mice as assessed by combined sur-vival data from two separate experiments using stratifiedlog-rank analysis.Although insightful, these xenotransplant murine tu-

mor models have significant limitations. Specifically,the biology of human T cells in immunocompromisedmice may significantly differ from autologous modifiedpatient T cells in the clinical setting, wherein these T cellsencounter an intact immune system, which may elicit animmune response to the CAR, and enter into a hostiletumor microenvironment containing immunosuppressiveTregs, immune-inhibitory cytokines (including IL-10 andtransforming growth factor-β), and myeloid-derived sup-pressor cells (64–66). To address these limitations, we arecurrently generating a more clinically relevant syngeneicimmunocompetent murine tumor model of ovarian carci-noma to further study the in vivo biology, immunogenicity,and antitumor efficacy of MUC-CD–targeted T cells.A further limitation of xenotransplant models is the in-

ability of these studies to address potential unforeseen off-target toxicities that may occur in the clinical setting,wherein infused CAR-modified T cells recognize antigennot only on tumor cells but also on normal tissues. Basedon previously published adverse events in recent clinicaltrials using CAR-modified T cells, this is a very real concern



with respect to the clinical feasibility of this adoptive T-cellapproach to cancer therapy. Specifically, three patientswith metastatic renal carcinoma treated with autologousT cells transduced to express the G250 CAR specific tothe TAA carboxyanhydrase IX developed significant livertoxicity (Common Toxicity Criteria grade 4 in patient 1,grade 2 in patient 2, and grade 3 in patient 3) due to anoff-target modified T-cell response to carboxyanhydrase IXexpressed by bile duct epithelial cells (20, 53). More re-cently, Morgan et al. reported the death of a patient withmetastatic colon cancer treated with ERBB2-targeted auto-logous 4D5-CD8-28BBz+ T cells. Following an extensivepostmortem analysis, the investigators of this study postu-late that the patient's death resulted from off-target recog-nition of the targeted ERBB2 antigen expressed by normallung tissue resulting in marked modified T-cell release ofinflammatory cytokines, including tumor necrosis factor-αand IFN-γ, leading to pulmonary toxicity, edema, and asubsequent cascading cytokine storm resulting in multior-gan failure and death (67).Although similar concerns may be raised in the setting

of treating patients with autologous T cells targeted to theMUC-CD antigen, we have conducted extensive immuno-histochemical studies using the 4H11 antibody to assessfor off-target binding to normal human tissues. Signifi-cantly, these studies showed no binding of the 4H11mAb in normal adult colon, rectum, small intestine, ecto-cervix, ovary, liver, pancreatic ducts, spleen, kidney, brain,and skin tissues. However, 4H11 did weakly bind cyto-plasm of endocervical gland cells and thymic corpuscles,the luminal surface of esophageal glands, as well as intra-cytoplasmic granules of bronchial epithelium and gastricglands (38). Significantly, these studies failed to showmembrane-bound antigen on the vascular surfaces ofany normal human tissues. Nevertheless, the potentialfor off-target toxicity in the clinical setting is not precludedby these studies. To this end, we acknowledge the possiblerequirement that further safety measures be added in theform of additional T-cell modification with a suicide genevector to enhance safety in the clinical setting.In conclusion, herein, we present the first published

data showing the feasibility of targeting MUC16, an anti-gen overexpressed on most ovarian carcinomas, throughadoptive therapy with genetically modified T cells targetedto the retained MUC-CD portion of the MUC16 antigen.Further, this report is the first to show efficient targeting ofT cells in an orthotopic murine model of ovarian cancer,showing efficacy of a single T-cell infusion of modifiedT cells in the absence of exogenous IL-2 cytokine support.Collectively, these data support the further planned trans-lation of this approach to the clinical setting in the form ofa phase I clinical trial in patients with persistent or re-lapsed ovarian carcinomas following initial therapy withsurgery and chemotherapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.






Grant Support

CA138738-01, PO1 CA052477 (D.R. Spriggs), Damon Runyon ClinicalInvestigator Award (R.J. Brentjens), Translational and Integrative MedicineFund Research Grant (Memorial Sloan-Kettering Cancer Center), and AnnualTerry Fox Run for Cancer Research (New York, NY) organized by the CanadaClubofNewYork, Kate's Team,WilliamH.Goodwin andAliceGoodwin, andthe Commonwealth Cancer Foundation for Research and the Experimental



Therapeutics Center of Memorial Sloan-Kettering Cancer Center, the GeoffreyBeene Cancer Foundation, and the Bocina Cancer Research Fund.

The costs of publicationof this articlewere defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 01/22/2010; revised 05/17/2010; accepted 05/18/2010;published OnlineFirst 07/13/2010.

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2010;16:3594-3606. Published OnlineFirst July 13, 2010.Clin Cancer Res Alena A. Chekmasova, Thapi D. Rao, Yan Nikhamin, et al. Cells Genetically Targeted to the MUC16 AntigenTumors in SCID-Beige Mice following Adoptive Transfer of T Successful Eradication of Established Peritoneal Ovarian

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