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Therapeutics, Targets, and Chemical Biology

In Vivo Persistence, Tumor Localization, and AntitumorActivity of CAR-Engineered T Cells Is Enhanced byCostimulatory Signaling through CD137 (4-1BB)

De-Gang Song1,3, Qunrui Ye1, Carmine Carpenito2, Mathilde Poussin1, Li-Ping Wang2,Chunyan Ji3, Mariangela Figini4, Carl H. June2, George Coukos1,2, and Daniel J. Powell Jr.1,2

AbstractHuman T cells engineered to express a chimeric antigen receptor (CAR) specific for folate receptor-a (FRa)

have shown robust antitumor activity against epithelial cancers in vitro but not in the clinic because of theirinability to persist and home to tumor in vivo. In this study, CARs were constructed containing a FRa-specificscFv (MOv19) coupled to the T-cell receptor CD3z chain signaling module alone (MOv19-z) or in combinationwith the CD137 (4-1BB) costimulatory motif in tandem (MOv19-BBz). Primary human T cells transduced toexpress conventional MOv19-z or costimulated MOv19-BBz CARs secreted various proinflammatory cytokines,and exerted cytotoxic function when cocultured with FRaþ tumor cells in vitro. However, only transfer of humanT cells expressing the costimulated MOv19-BBz CAR mediated tumor regression in immunodeficient micebearing large, established FRaþ human cancer. MOv19-BBz CAR T-cell infusion mediated tumor regression inmodels of metastatic intraperitoneal, subcutaneous, and lung-involved human ovarian cancer. Importantly,tumor response was associated with the selective survival and tumor localization of human T cells in vivo andwas only observed in mice receiving costimulated MOv19-BBz CAR T cells. T-cell persistence and antitumoractivity were primarily antigen-driven; however, antigen-independent CD137 signaling by CAR improved T-cellpersistence but not antitumor activity in vivo. Our results show that anti-FRa CAR outfitted with CD137costimulatory signaling in tandem overcome issues of T-cell persistence and tumor localization that limitthe conventional FRa T-cell targeting strategy to provide potent antitumor activity in vivo. Cancer Res; 71(13);4617–27. �2011 AACR.

Introduction

Immune targeting of tumor antigens that are overexpressedby cancer cells in numerous cancer types with limited expres-sion in normal tissues holds significant promise for wide-spread clinical application. Folate receptor-a (FRa) is aglycosylphosphatidylinositol-anchored protein that is over-expressed on the surface of cancer cells in a spectrum ofepithelial malignancies, including ovarian, breast, renal, colo-rectal, lung, and other solid cancers but limited in normaltissues (1–8). FRa overexpression is associated with high-

grade tumor progression, poor prognosis in ovarian cancer (9),and poor survival in breast cancer (10), and therefore repre-sents an attractive candidate for targeted biological therapy ofepithelial-derived cancers, particularly for epithelial ovariancarcinomas (EOC) where approximately 90% of cancersexpress FRa.

Chimeric antigen receptors (CAR) or "T-bodies" couple thehigh affinity binding of antibodies with the signaling domainsof the T-cell receptor (TCR) CD3z chain for specified trigger-ing of T-cell activation similar to the endogenous TCR (11).Despite promising results in neuroblastoma (12, 13) andlymphoma (14, 15), transfer of T cells genetically redirectedwith CAR to FRa in a phase I clinical trial for the treatment ofmetastatic ovarian cancer did not induce tumor regressiondue to the poor persistence of the gene-modified T cells in vivo,a lack of T-cell localization to tumor, and the induction of anundefined T-cell inhibitory factor in the serum of severalpatients (16). Persistence of tumor antigen–specific T cellsafter adoptive transfer correlates with tumor regression inpatients with advanced metastatic cancer (17), where trans-ferred T cells can localize to regressing lesions (18). Trans-ferred T cells persisting in the blood of responding patientsexpress high levels of costimulatory receptors (19, 20) andresponse to therapy is associated with the transfer of highnumbers of T cells expressing costimulatory receptors (21),

Authors' Affiliations: 1Ovarian Cancer Research Center, Department ofObstetrics and Gynecology and 2Abramson Cancer Center, Department ofPathology and Laboratory Medicine, University of Pennsylvania, Philadel-phia, Pennsylvania; 3Department of Hematology, Qilu Hospital, ShandongUniversity, Jinan, Shandong, P.R. China; and 4Department of ExperimentalOncology and Molecular Medicine, Istituto Nazionale dei Tumori, Milan,Italy

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

Corresponding Author: Daniel J. Powell, Jr., University of Pennsylvania,Rm. 1313 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104. Phone: 215-573-4783; Fax: 215-573-7627; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-0422

�2011 American Association for Cancer Research.

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suggesting that provision of costimulatory signals is necessaryto facilitate improved T-cell survival and antitumor responsein vivo. The addition of costimulatory domains, including theintracellular domain of CD28, and TNF receptor family mem-bers, CD134 (OX-40) and CD137 (4-1BB) into CARs can sig-nificantly augment the ability of these receptors to stimulatecytokine secretion and enhance antitumor efficacy in precli-nical animal models of solid tumors and leukemia that lackcognate costimulatory ligands (13, 22–24), and thus rationa-lizes the incorporation of costimulatory modules in the crea-tion of anti-FRa CAR therapy.

Here, we addressed the issue of limited FRa-specific T-cellpersistence and tumor activity in vivo through the introduc-tion of the CD137 costimulatory signaling domain into a FRa-specific CAR and studied the role of CD137 signaling in FRa-directed CAR T-cell therapy of human cancer. Compared with"first-generation" CAR that provide CD3z signaling to T cellsbut lack cis costimulatory signaling capacity, T cells expres-sing FRa-specific CAR with a CD137 signaling domain intandem showed minimally improved antitumor activity invitro, but markedly superior tumor regression capacity inestablished human ovarian cancer xenograft models, whichwas associated with enhanced T-cell persistence and tumorlocalization in vivo. Tumor regression and T-cell persistencewere both attainable by various routes of T-cell infusion, andintravenous (i.v.) cell infusion mediates the regression ofhuman cancer in xenograft models of advanced intraperito-neal (i.p.), subcutaneous (s.c.), and lung-involved metastaticdisease. T-cell persistence and tumor activity in vivo werelargely antigen-driven; however, provision of CD137 signalingin the absence of specific antigen recognition by CAR couldimprove T-cell persistence but not antitumor activity in vivo.Incorporation of the CD137 signaling domain in FRa-specificCARs thus overcomes the limitation of past CAR approachesby improving the persistence of transferred T cells in vivo, andbolstering their accumulation in tumor and antitumorpotency.

Materials and Methods

Anti-FRa chimeric immune receptor constructionThe MOv19 scFv-based (2, 25, 26) chimeric immune recep-

tor was constructed by using pCLPS lentiviral vector backboneconstructs previously described (22). CAR construction andlentivirus production are detailed in Supplementary Materialsand Methods.

Human T cellsPrimary human CD4þ and CD8þ T cells, which were

purchased from the Human Immunology Core at Universityof Pennsylvania, were isolated from healthy volunteer donorsfollowing leukapheresis by negative selection. All specimenswere collected under a protocol approved by a UniversityInstitutional Review Board, and written informed consent wasobtained from each donor. T cells were cultured in completemedia (RPMI 1640 supplemented with 10% heat inactivatedFBS, 100 U/mL penicillin, 100 mg/mL streptomycin sulfate, 10mmol/L HEPES), and stimulated with anti-CD3 and anti-CD28

monoclonal antibodies (mAb)-coated beads (Invitrogen) asdescribed (27). Twelve to twenty-four hours after activation, Tcells were transduced with lentiviral vectors at multiplicity ofinfection of approximately 5 to 10. CD4þ and CD8þ T cellsused for in vivo experiments were mixed at 1:1 ratio, activated,and transduced. Human recombinant interleukin-2 (IL-2;Novartis) was added every other day to a 50 IU/mL finalconcentration and a cell density of 0.5 � 106 to 1 � 106 cells/mL was maintained. Once T cells seemed to rest down, asdetermined by both decreased growth kinetics and cell sizingby using the Multisizer 3 Coulter Counter (Beckman Coulter),engineered T-cell cultures were adjusted to equalize thefrequency of transgene expressing cells prior to functionalassays.

Functional assaysCytokine release assays were carried out by an IFN-g ELISA

Kit (Biolegend) or by Cytometric Bead Array, according tomanufacturer's instructions (BD Biosciences) as described inSupplementary Materials and Methods. Cell-based biolumi-nescence and 51Cr release assays of cytolysis were carried outas previously described (28, 29).

Xenograft model of ovarian cancerMouse studies were carried out as previously described (22,

30) with modifications detailed in Supplementary Materialsand Methods.

ImmunohistochemistryFresh frozen tumor samples were sectioned for immuno-

histochemical analysis as described in Supplementary Materi-als and Methods.

Statistical analysisStatistical analysis was carried out by 2-way repeated

measures ANOVA for the tumor burden (tumor volume,photon counts). Student's t test was used to evaluate differ-ences in absolute number of transferred T cells, cytokinesecretion, and specific cytolysis. Kaplan–Meier survival curveswere compared by using the log-rank test. GraphPad Prism 4.0(GraphPad Software) was used for the statistical calculations.P < 0.05 was considered significant.

Results

CAR constructionThe mouse anti-human FRa-specific scFv MOv19 was

selected on the basis of its high binding affinity for FRa(108–109 M�1; refs. 2, 25, 26). FRa CAR constructs werecomprised of the MOv19 scFv linked to a CD8a hinge andtransmembrane region, followed by a CD3z signaling moietyalone (MOv19-z) or in tandem with the CD137 intracellularsignaling motif (MOv19-BBz; Fig. 1A). A signaling deficientFRa-specific CAR containing a truncated CD3z intracellulardomain (MOv19-Dz) was designed to assess the contributionof CD3z signaling. An anti-CD19 CAR containing CD3z andCD137 signaling motifs in tandem (anti–CD19-BBz) was usedas an antigen specificity control (30). CAR constructs were

Song et al.

Cancer Res; 71(13) July 1, 2011 Cancer Research4618




subcloned into the pCLPS lentiviral vector where transgeneexpression is driven off the cytomegalovirus promoter. Usinggene transfer technology established for clinical application,lentiviral vectors efficiently transduced primary human T cellsto express the anti-FRa CAR (Fig. 1B). T-cell transductionefficiency, as assessed by flow cytometry, was equilibrated forall constructs at approximately 50% in all assays.

Primary human FRa CAR T cells exert antigen-specificfunction in vitroBecause ovarian cancer frequently express FRa (2), a

panel of established human ovarian cancer cell lines thatexpress surface FRa at varying levels (SKOV3, A1847, andOVCAR3) was selected for assays (Fig. 1C). Two ovariancancer lines, C30 and PEO-1, were negative for FRa. Trans-

duced T cells expressing MOv19-BBz or MOv19-z CARsrecognized FRaþ tumor lines and secreted high levels ofIFN-g , but not when stimulated with FRa� lines (Fig. 1D).FRa-specific CAR T cells also secreted high levels of IL-2 andTNF-a when stimulated with FRaþ cancer cells and low butdetectable levels of IL-4 and IL-10 (Supplementary Fig. S1).MOv19 CARs functioned in both primary human CD4þ andCD8þ T cells. In all cases, MOv19-BBz T cells secreted moreIFN-g than MOv19-z T cells after specific stimulation. CD19-BBz CAR did not produce IFN-g , except when coincubatedwith K562 cells engineered to express surface CD19 antigen,and human T cells expressing MOv19-Dz CAR did notsecrete cytokine when stimulated with FRaþ cancer cells(Fig. 1D), showing that antigen specificity and CD3z signal-ing are required for CAR activity in T cells.

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Figure 1. Generation and specific immune recognition by FRa CAR–transduced human T cells in vitro. A, schematic representation of MOv19-based CARconstructs containing the CD3z cytosolic domain alone (MOv19-z) or in combination with CD137 costimulatory module (MOv19-BBz). FRa-specificCAR with a truncated CD3z domain (MOv19-Dz) and anti–CD19-BBz CAR are shown. VL, variable L chain; L, linker; VH, variable H chain; TM, transmembraneregion. B, MOv19 CAR expression (solid black line) on human CD3-gated cells after transduction with lentivirus compared with parallel untransducedT cells (filled gray histograms). Percent transduction is indicated. C, surface FRa expression (solid black line) by various human ovarian cancer cell lines by flowcytometry; isotype antibody control (filled gray histograms). D, antigen-specific IFN-g secretion by MOv19-z and MOv19-BBz CAR-transduced T cells but notMOv19-Dz anti–CD19-BBz T cells, following overnight incubation with FRaþ cancer cell lines. Mean IFN-g concentration � SEM (pg/mL) from triplicatecultures is shown. E, antigen-specific killing of FRaþ tumor cells by FRa CARþ CD8þ T cells in 18-hour bioluminescence assay at the indicated E/T ratio.Untransduced T cells (UNT) or gfp-transduced human CD8þ T cells served as controls.

Costimulated FRa-Specific CAR Therapy of Cancer

www.aacrjournals.org Cancer Res; 71(13) July 1, 2011 4619




To interrogate antigen-specific cytolytic potential, anti-FRaCAR CD8þ T cells were cocultured with FRa� AE17 (31), amouse malignant mesothelioma cell line, or AE17.FRa (anAE17 line derivative transduced to express high surface levelsof human FRa). In standard 4-hour chromium release and 24-hour bioluminescence assays, FRa-specific CAR T cells(MOv19-z and MOv19-BBz) specifically lysed AE17.FRa cellsbut not the parental AE17 line (Supplementary Fig. S2). T cellsexpressing anti–CD19-BBz, MOv19-Dz, or green fluorescentprotein (gfp) did not lyse AE17.FRa or AE17 cells. Consistentwith cytokine production results, primary human CD8þ T cellsexpressing MOv19-z or MOv19-BBz CAR directly and effi-ciently lysed FRaþ human ovarian cancer cell lines SKOV3and A1847, but not FRa� lines C30 or 624mel, a melanoma cellline (Fig. 1E). MOv19-BBz CAR T cells exhibited increasedcytotoxicity compared with MOv19-z CAR T cells, but not at alevel of statistical significance. Thus, human T cells trans-duced with FRa-specific CAR specifically recognize FRaþ

human and mouse cancer cells and exert MHC-unrestrictedcytotoxic activity in vitro.

Antitumor activity of primary human FRa CAR T cellsin vivo

CAR functional activity in vitro cannot adequately predictthe antitumor potential of transduced human T cells in vivo.The antitumor efficacy of FRa CAR constructs were evaluatedin a xenograft model of large, established cancer. Immunode-ficient NOD/SCID/IL-2Rgcnull (NSG) mice were inoculated s.c.with firefly luciferase (fLuc)-transfected FRaþ SKOV3 humanovarian cancer cells on the flank and received intratumoral(i.t.) injections of CARþ T cells on days 40 and 45 post-tumorinoculation (p.i.), when tumors were 250 mm3 or more in size.Tumors in mice receiving saline, MOv19-Dz CAR T cells, or gfpT cells progressed beyond the time of T cell transfer asmeasured by caliper-based sizing and bioluminescence ima-ging (BLI; Fig. 2A and B). Tumor growth was modestly delayedin mice receiving MOv19-z T cells (P ¼ 0.027), compared withall 3 control groups at the latest evaluated time point (38 daysafter first T-cell dose). In contrast, mice receiving i.t. injectionof MOv19-BBz T cells experienced rapid tumor regression,which was significantly better thanMOv19-z T cells (P < 0.001),indicating that incorporation of CD137 signals enhances over-all antitumor activity in vivo. Tumor-bearing mice treated withMOv19-BBz–transduced T cells delivered via i.v., i.p. injection,or i.t. routes experienced tumor regression (Fig. 2C). Followingi.v. or i.p. infusion of MOv19-BBz T cells, antitumor activitywas again observed, though delayed in regression by approxi-mately 7 days relative to i.t. delivery, indicating that althoughlocal injection is optimal, systemically infused CAR T cells canmarginalize upon adoptive transfer to mediate potent anti-tumor effects in vivo.

Persistence of primary human FRa CAR T cells in vivo isincreased by 4-1BB signals

The persistence of transferred tumor-reactive T cells fol-lowing adoptive T-cell therapy is highly correlated with tumorregression (17). In the experiments described earlier, periph-eral blood was collected from tumor-bearing mice 3 weeks

after the last T-cell dose and quantified for persistent humanCD4þ and CD8þ T cells (Fig. 2D). CD4þ and CD8þ T-cellcounts were highest in mice receiving MOv19-BBz CAR T cells,whether delivered by i.t., i.p., or i.v. routes of administration,compared with gfp, MOv19-Dz, and MOv19-z treatmentgroups. Notably, human T-cell counts in mice receivingMOv19-BBz CAR T cells by i.v. injection was significantlyhigher than those in the parallel MOv19-z CAR group (P <0.01), indicating a role for CD137 in T-cell survival in vivo.There was no significant difference in level of T-cell persis-tence amongmice receiving MOv19-BBz CAR T cells by i.v., i.t.,or i.p. injection (P ¼ 0.2), despite a trend toward less cells inthe i.v. injection group. Total T-cell counts in the MOv19-ztreatment group was statistically similar to other controlgroups including mice receiving saline in the absence ofhuman T-cell injection (Supplementary Fig. S3; P > 0.05),suggesting that antigen specificity alone is not sufficient forT-cell maintenance in vivo. This was primarily attributed topoor CD4þ T-cell persistence because circulating MOv19-zCAR CD8þ T cells persisted at greater numbers than MOv19-Dz CAR (P ¼ 0.026) or gfp (P ¼ 0.013) cells. Four weeks afterlast MOv19-BBz CAR T-cell dose, the absolute number ofhuman T cells persisting in the blood was inversely correlatedwith tumor burden of each group (Supplementary Fig. S3; r ¼–0.78). Tumor BLI results were consistent with the size ofresected residual tumors (Supplementary Fig. S4). Mechan-istically, enhanced persistence of MOv19-BBz CAR T cells,compared withMOv19-z, seemed to be attributed in part to anincreased upregulation of antiapoptotic Bcl-XL protein expres-sion after antigen stimulation (Supplementary Fig. S3). Thus,tumor regression was associated with the stable persistence ofengineered human T cells in vivo and supported by provisionof CD137 costimulation.

Tumor regression and T-cell persistence are antigen-driven in vivo

To determine whether MOv19-BBz CAR antitumor activityis antigen-specific, a comparative study was conducted withan anti-CD19–specific CAR also containing the CD137 signal-ing domain (30). NSG mice with established s.c. SKOV3 fLucþ

tumor receiving 2 i.t. T-cell injections experienced rapid tumorregression, whereas tumor grew progressively in mice treatedwith T cells expressing gfp or CD19-BBz CAR (Fig. 3A),excluding alloreactivity as a mechanism of tumor regression.Mice receiving MOv19-BBz T cells had significantly higherhuman CD4þ and CD8þ T cell counts than mice in anti-CD19CAR or gfp groups (Fig 3B; P ¼ 0.009), indicating that tumorantigen recognition drives the survival of the adoptivelytransferred T cells in vivo. Interestingly, T-cell persistencewas reproducibly higher in mice receiving anti–CD19-BBzCAR T cells than gfp T cells (P ¼ 0.012), suggesting thatpersistence of CAR T cells can be promoted in part through aCD137-driven process that does not require scFv engagementwith antigen. Nevertheless, there was no statistical differencein tumor control between anti–CD19-BBz CAR and gfp groups(P ¼ 0.065) even at the latest time point studied (day 73),showing that persistence in the absence of antigen specificityis insufficient to mediate tumor response. In this line, CAR

Song et al.





expressing T-cell frequency in the blood of tumor-bearingmice administered MOv19-BBz T cells was higher than thatobserved in mice receiving CD19-BBz CAR T cells, though notat statistical significance (Fig. 3C; P¼ 0.08). However, coupledwith increased T-cell counts, the total number of circulatingCARþ T cells persisting 1 month after infusion were signifi-cantly higher in mice receiving MOv19-BBz T cells (76 � 13cells/mL; P ¼ 0.013); mice in CD19-BBz CAR and gfp groupshad little to no detectable persistence of CARþ T cells withcounts of 12 � 4 cells/mL and 0 � 0 cells/ml, respectively(Fig. 3D). Consistent with the increased persistence of MOv19-BBz T cells in the blood of treated animals, immunohisto-chemical analysis revealed robust accumulation of humanCD3þ T cells in regressing SKOV3 lesions 6 weeks after i.v.T-cell administration (Fig. 4). Few CD3þ T cells were detected

in tumors resected at the same time from mice that receivedanti–CD19-BBz CAR or gfp-transduced T cells.

Tumor regression in the metastatic disease settingAdvanced ovarian cancer is a disease usually confined to the

peritoneal cavity with occasional metastatic spread to thepleural compartment. A xenogeneic model of advanced i.p.metastatic cancer was established to evaluate the functionalactivity of FRa-specific T cells against tumor localized to amore physiologically relevant compartment. NSG mice thatwere inoculated i.p. with SKOV3 fLucþ cells efficiently devel-oped peritoneal carcinomatosis which was readily evident30 days p.i., when MOv19-BBz or control anti–CD19-BBzCAR T-cell therapy was administered (Fig. 5A). Within 3 weeksof T-cell transfer, all mice that received control anti–CD19-BBz

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Figure 2. Human MOv19-BBz CAR T cells eradicate large preestablished tumors in vivo: effect of CD137 costimulatory signaling domains and route ofadministration. A, NSGmice bearing established s.c. tumor were treated with intratumoral injections of 8� 106 CARþ T cells on days 0 and 5 and imaged every2 weeks. Tumor growth was assessed by caliper measurement [V ¼ 1/2(length � width2)]. B, SKOV3 fLucþ bioluminescence signal was decreased inMOv19-BBz CAR treated mice compared with the MOv19-z and the control treatment groups 2 weeks and 4 weeks after last T-cell dose. C, SKOV3fLuc-bearing NSG mice were treated with 8 � 106 MOv19-BBz T cells via i.t., i.p., or i.v. routes. Tumor growth was assessed by caliper measurement.D, CD137 signaling enhances the survival of human CD4þ and CD8þ T cells in vivo on day 73 (4 weeks following last T-cell dose) in the peripheralblood. CD4 and CD8 T cells were quantitated from blood by using the TruCount method. Mean cell concentration (cells/mL) � SD for all evaluable micein each treatment group is shown.






CAR T cells developed distended abdomens, marked bloodyascites of approximately 5 to 8 mL volume and multiplenodular peritoneal tumors, and had to be euthanized due totumor-associated, abdominal distention (Fig. 5B and C). Bycomparison, mice treatedwithMOv19-BBzCAR T cells did notdevelop distended abdomens or ascites, and exhibited a pro-found enhancement in tumor-related survival (P¼ 0.0002)withno cases of tumor-related mortality in the MOv19-BBz CARgroup (Fig. 5C). At the time of euthanasia of mice treated withMOv19-BBz, tumor burden was minimal to none, but micerequired euthanizing due to signs of distress compatible withGVHD that develops in NSG mice following xenogeneic trans-fer of activated human lymphocytes (32). Still, median survivaltimes of 52 days after last T-cell infusion by i.v. injection and68 days by the i.p. route were observed in mice treated withMOv19-BBz CAR, compared with 9 and 12 days in the anti–CD19-BBz CAR T-cell groups, respectively (MOv19-BBz i.p. vs.anti–CD19-BBz i.p., P¼ 0.0023; MOv19-BBz i.v. vs. anti–CD19-BBz i.v., P¼ 0.0025; Fig. 5D). Two months after treatment withMOv19-BBzCAR cells via i.p. or i.v. routes, 60% (3 of 5) and 40%(2 of 5) of tumor-inoculated mice remained alive, respectively.

Occasionally, ovarian cancer patients develop lung metas-tases and pleural ascites formation requiring thoracentesis orother supportive management procedures during diseaseprogression (33). A model of metastatic ovarian cancer oflung was generated by inoculation of NSG mice with SKOV3fLucþ cells via tail-vein injection resulting in progressive lungmetastases in 100% of mice 3 days p.i. (Fig. 6). Two i.v.injections of MOv19-BBz T cells resulted in rapid regression

of lung metastasis in all treated animals 14 days p.i. and 80% (4of 5) of mice had no evidence of recurrence after 1 month. Bycontrast, disease progression occurred in all mice receivinganti–CD19-BBz T cells.

Discussion

CARs combine the high affinity and specificity of antigen-specific antibody, which binds cell surface determinants in anon–MHC-restricted manner, with the potent effector func-tions of T lymphocytes (11). Genetically retargeting of primaryhuman lymphocytes with CARs recognizing tumor-associatedantigens offers a robust and rapid avenue toward the genera-tion of tumor-reactive T cells for therapy. To date, CAR-basedtherapy has shown promising but often limited clinical activ-ity, despite the reproducible demonstration of strong effectoractivity in vitro (12–16). Effective adoptive T-cell therapy notonly requires antitumor activity, but also in vivo expansionand persistence of the infused tumor-reactive T cells (17). Inour study, we have addressed the central issue of limited CART-cell persistence and tumor activity in vivo (16) through theintroduction of the CD137 (4-1BB) costimulatory signalingdomain into a Mov19 scFv-based CAR.

CD137 is a TNF receptor family member that plays animportant role in T-cell proliferation and survival, particularlyfor T cells within the memory T-cell pool (34–36). We selectedCD137 on the basis of its demonstrated capacity to support ofCD8 T-cell expansion (36), and upregulate important anti-apoptotic protein Bcl-XL expression (37), and results showing

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m3 ) Figure 3. Tumor eradication by

CAR T cells is antigen-specific. A,NSG mice with s.c. SKOV3 fLucþ

tumor were treated with 8 � 106 Tcells (40% transduction efficiency)expressing MOv19-BBz, anti–CD19-BBz, or gfp via i.t. infusionon days 0 and 5 and measured fortumor volume by calipers every 2to 3 days. B, peripheral blood wascollected 3 weeks following lastT-cell infusion and quantified forthe absolute number of humanCD4þ and CD8þ T cells/ml ofblood. Mean cell count � SD isshown. C, FRa- and CD19-specific CAR expression onhuman CD3þ T cells fromperipheral blood of treated micemeasured by flow cytometry byusing goat anti-mouse IgG F(ab0)2.Mean CARþ expression frequency� SD per group is shown. D,absolute CARþ T-cell count wascalculated as number of humanCD3þ T cells/mL of blood timespercent CARþ. Mean count � SDwas determined.

Song et al.





that adoptive transfer of tumor-specific T cells costimulatedex vivo with 4-1BBL supports persistence and antitumoractivity in vivo (38). This work builds upon our previous studywhich showed high costimulatory receptor expression ontumor antigen–specific T cells persisting in the circulationin patients responding to adoptive immunotherapy (19), andcorrelations between costimulatory receptor expression ontransferred T cells and tumor regression (20;21). Like the "first-generation" Mov19-z CAR expressing CD3z signaling alone, Tcells engineered to express a "second-generation" Mov19-BBzCAR containing CD3z signaling and a CD137 signaling domainin tandem preferentially secrete high levels of Th1 cytokinesincluding IFN-g , TNF-a, and IL-2 upon tumor encounter andexert strong antitumor activity in vitro. Here, IFN-g cytokineproduction levels were generally associated with the level ofFRa expressed by tumor cell targets, and cytolysis of tumorcells by Mov19-z CAR and Mov19-BBz CAR T cells wasefficient even at a 3:1 effector to target cell (E/T) ratioin vitro. In all in vitro antitumor assays, engineered T cellsexpressing Mov19-BBz CAR outperformed Mov19-z CART cells, albeit not always to the level of statistical significance.Interestingly, the single exception was in the level of Th2cytokine secretion induced by tumor stimulation, where FRaengagement by Mov19-z CAR T cells induced greater IL-4 andIL-10 production, suggesting that combined CD3z and CD137signaling enforces a Th1 skewed response.The dichotomy between first- and second-generation CAR

vectors was most evident in in vivo studies where CD137

bearing Mov19-BBz CAR T cells facilitated superior regres-sion of large vascularized tumors in an established humanovarian cancer xenograft model, whereas tumor progressionwas almost unabated with Mov19-z CAR T cells. Transfer of16 � 106 total Mov19-BBz CAR T cells eliminated an esti-mated 2.5 � 108 tumor cells (assuming that a 250 mm3

tumor mass contains approximately 2.5 � 108 cells); ineffect, an approximately 1:15 E/T ratio. Consistent withprevious clinical observations (18, 39), tumor responsewas associated with enhanced T-cell persistence and tumorlocalization of Mov19-BBz CAR T cells in vivo, which seemedto be attributed in part to upregulated expression of Bcl-XL

following stimulation with tumor. Tumor regression wasantigen-specific, as transfer of anti–CD19-BBz T cells had noimpact on tumor progression. Tumor regression and T-cellpersistence were attainable via systemic or local T-celldelivery, showing the capacity of transferred T cells tocirculate, home to tumor and perform antitumor functions.Although i.v. injections are favorable in clinical applicationdue to the ease of administration and effective in our model,our data suggest that local administration of T cells mayprovide optimal therapeutic effect, which may be in part dueto increased T-cell trafficking to tumor and provision offavorable E/T ratios. However, such delivery may not beapplicable for tumors with multiple gross metastatic sites ormicrometastases.

Although Mov19-BBz and anti–CD19-BBz T cells could bedetected in the peripheral blood 3 weeks after T-cell infusion,

Figure 4. CAR T-cell localizationto tumor in vivo is antigen-specific. NSG mice with s.c.SKOV3 fLucþ tumors were treatedwith i.v. injections of 8 � 106 Tcells expressing MOv19-BBz(top), anti–CD19-BBz (middle), orgfp (bottom) on days 0 and 5.SKOV3 tumors grown forapproximately 40 additional dayswere collected from euthanizedmice and stained for human CD3expression (brown).Representative sections areshown at �100 magnifications.

MOv19-BBζ

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the accumulation of Mov19-BBz, but not anti–CD19-BBz Tcells, in FRaþ tumor lesions suggests that antigen-selectiveretention of CAR bearing T cells in tumor occurs and may berequisite in part for tumor regression (40). In a previous study,transferred TCR transgenic T cells migrated indiscriminatelyearly after adoptive transfer but experienced antigen-depen-dent activation exclusively in antigen-positive tumor resultingin tumor destruction (41). Transfer of chemokine receptorexpressing CAR T cells can enforce preferential migration totumor sites to boost antitumor activity in vivo (42). Our resultssupport the hypothesis that T-cell persistence, localization,and tumor activity in vivo are largely antigen-dependent, likelylinked, processes. Notably, the use of anti–CD19-BBz T cells asspecificity control in our assays, however, shows that provi-sion of CD137 signaling by CAR permitted improved T-cellpersistence but not antitumor activity in vivo through a

mechanism that is independent of scFv engagement withantigen, suggestive of low-level constitutive activity by theCD137 module, consistent with previous data (30). In thisscenario, it remains possible that persistence of nonspecificCD137-costimulated human T cells was driven by low-levelTCR recognition of xenoantigens in mice combined withconstitutive CD137 signaling by CAR, as shown by the occur-rence of graft-versus-host manifestations, which is an inherentlimitation of the xenogeneic NSG mouse model used.

T-cell–based targeting of FRa has been tested in patientswith advanced ovarian cancer with promising results. In anearlier clinical study, retargeted T cells were generated fortherapy by loading preactivated T cells with a bispecific mousemAb OC/TR, directed to the CD3 molecule on T lymphocytesand to FRa on EOC cells (43). Administration of FRa-redir-ected T cells to women with minimal residual ovarian cancer

MOv19-BBζ IP MOv19-BBζ IP

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Figure 5. Mov19-BBz T cells inhibit tumor growth and ascites formation in SKOV3 murine model of peritoneal carcinomatosis. A, NSG mice receivedi.p. injection of 5� 106 SKOV3 fLucþ tumor cells and were randomized into 4 groups before beginning therapy with 9� 106 T cells expressing MOv19-BBz oranti–CD19-BBz via i.p. or i.v. infusion on day 30 and 35 after tumor inoculation. B, representative NSG mice treated with MOv19-BBz T cells (left)via i.v. (top) or i.p (bottom) infusion. Mice treated with anti–CD19-BBz T cells (right) developed ascites as evidenced by a distended abdomen (middle).Postmortem visualization of the peritoneum shows nodular tumor masses (arrows; far right). C, Kaplan-Meier tumor-related survival curve of tumor-bearingNSG mice treated with either MOv19-BBz or anti–CD19-BBz T cells via i.v. or i.p. injection. D, Kaplan-Meier overall survival of tumor-bearing NSG mice.

Song et al.





resulted in antitumor responses in 27% of patients with mildto moderate immunotherapy-related toxicities; however, ther-apy was limited by the inability to generate stable anti-FRa–specific T-cell memory and the induction of human anti-mouse antibodies against the bispecific mAb in approximately90% of treated patients (44). In a phase I study of anti-FRaCAR therapy for cancer, Kershaw and colleagues (16) trans-ferred T cells that were retargeted to FRa by a first-generationMOv18 scFv-based CAR to immunocompetent patients withadvanced ovarian cancer. The parental MOv18 antibody has asimilar affinity for FRa (108�109 M�1) as MOv19 used in ourCAR construct (2, 26) though the relative affinities of their scFvproducts in CARs is not known. MOv18 and MOv19 also bindnon–cross-reactive epitopes (2), which may influence theirrelative ability to access surface antigen. Therapy usingMOv18-z CAR was safe and feasible; however, no patientexperienced a tumor response which was attributed to a lackof transferred T-cells persistence after infusion, poor tumorlocalization, and the development of a serum inhibitory factorthat reduced CAR T-cell activity in in vivo study (16). Ourstudies, combined with recent clinical strategies, addressthese issues. Similar to the study of Kershaw and colleagues(16), first-generation MOv19-z CAR, which redirected T-cellcytotoxicity in vitro, only delayed tumor progression in vivoand CARs did not persist long-term in vivo. We show thattumor response and T-cell persistence can be evoked byprovision of CD137 costimulatory signals to anti-FRa CART cells, which is facilitated principally by engagement of theirCAR with tumor antigen. Moreover, transfer of MOv19-BBz T

cells leads to increased accumulation of human T cells inregressing ovarian cancer lesions. Although the mouse anti-human MOv19 scFv used in the construction of the MOv19-BBz CAR is likely to elicit anti-mouse humoral responses inimmunocompetent recipients, as seen in past CAR studies andtrials using MOv18 scFv (16, 44, 45), nonmyeloablative immu-nosuppressive preconditioning can disable host endogenousimmunity to promote the in vivo persistence of T cellsexpressing CARs and TCRs of mouse origin, facilitating tumorregression (14, 46, 47). The use of immunodeficient NSG micemodels T-cell transfer in the setting of host lymphodepletion,albeit in the absence of human derivatives and endogenousimmune reconstitution. Still, the use of fully human anti-FRascFv candidates for the next generation of CAR-redirectedtherapy is worthy of investigation (26, 48). Our preclinicalresults support the notion that incorporation of the CD137signaling domain in FRa-specific CARs overcomes the limita-tions of past CAR approaches by improving the persistence oftransferred T cells in vivo, thereby increasing their retention intumor and bolstering antitumor potency. Careful considera-tions must be made when targeting of self/tumor antigenswith CARs or exogenous TCRs, which hold the potential formediating serious adverse events (47, 49); however, FRa,which is present on normal tissues, is localized primarily tothe apical surfaces of polarized epithelia, where it may beinaccessible to parenterally administered folate conjugatesand redirected T cells (50). Our results provide the rationalefor the clinical investigation of MOv19-BBz CAR T-cell therapyin combination with lymphodepleting preconditioning

Figure 6. Adoptive transfer ofFRa-specific T cells inducesregression of ovarian cancer lungmetastasis. A, NSG mice with3 day established SKOV3 fLucþ

tumor in the lungs received tail-vein injections of 6 � 106 T cellsexpressing either MOv19-BBz oranti–CD19-BBz on day 3 andday 8 and were monitored by BLI.B, photon emission from fLucþ

tumor cells was quantified and themean � SD bioluminescencesignal determined.

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regimens for the treatment of a wide spectrum of FRa-expressing epithelial malignancies.

Disclosure of Potential Conflict of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank Drs. Silvana Canevari, Gwenn Danet-Desnoyers, andMichael C. Milone for their suggestions and helpful discussions.

Grant Support

This work was supported by the Ovarian Cancer Research Fund, the SandyRollman Ovarian Cancer Foundation, the Joint Fox Chase Cancer Center, andUniversity of Pennsylvania Ovarian Cancer SPORE (P50 CA083638).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received February 8, 2011; revised April 7, 2011; accepted April 16, 2011;published OnlineFirst May 5, 2011.

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2011;71:4617-4627. Published OnlineFirst May 5, 2011.Cancer Res De-Gang Song, Qunrui Ye, Carmine Carpenito, et al. Costimulatory Signaling through CD137 (4-1BB)Activity of CAR-Engineered T Cells Is Enhanced by

Persistence, Tumor Localization, and AntitumorIn Vivo

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