Development of T Cells Redirected to Glypican-3 for the ......Cancer Therapy: Preclinical...

Cancer Therapy: Preclinical

Development of T Cells Redirected to Glypican-3 for theTreatment of Hepatocellular Carcinoma

Huiping Gao1,2, Kesang Li2, Hong Tu2, Xiaorong Pan2, Hua Jiang2, Bizhi Shi2, Juan Kong2,Hongyang Wang2,3,4, Shengli Yang2, Jianren Gu2, and Zonghai Li2

AbstractPurpose: The aim of our study is to elucidate whether T cells expressing GPC3-targeted chimeric antigen

receptor (CAR) can efficiently eliminate GPC3-positive HCC cells and their potential in the treatment of

HCC.

ExperimentalDesign: T cells expressing a first-generation and third-generationGPC3-targetedCARwere

prepared using lentiviral vector transduction. The in vitro and in vivo cytotoxic activities of the genetically

engineered CAR T cells were evaluated against various HCC cell lines.

Results: GPC3-targeted CAR T cells could efficiently kill GPC3-positive HCC cells but not GPC3-

negative cells in vitro. These cytotoxic activities seemed to be positively correlated with GPC3 expression

levels in the target cells. In addition, T cells expressing the third-generation GPC3-targeted CAR could

eradicate HCC xenografts with high level of GPC3 expression and efficiently suppress the growth of HCC

xenografts with low GPC3 expression level in vivo. The survival of the mice bearing established

orthotopic Huh-7 xenografts was significantly prolonged by the treatment with the third-generation

GPC3-targeted CAR T cells.

Conclusions: GPC3-targeted CAR T cells could potently eliminate GPC3-positive HCC cells, thereby

providing a promising therapeutic intervention for GPC3-positive HCC. Clin Cancer Res; 20(24); 6418–28.

�2014 AACR.

IntroductionHepatocellular carcinoma (HCC) is the fifth most com-

mon cancer and the third most common cause of cancermortality worldwide (1). Currently, surgery is the mosteffective treatment for HCC. However, tumor recurrenceafter a curative liver resection is very high, and the 5-yearsurvival rate remains at only 10% (2). Moreover, becausethe majority of patients with HCC are diagnosed at a latedisease stage, potentially curative therapies, including che-motherapy, chemoembolization, ablation, and proton

beam therapy, are frequently ineffective. Sorafenib (Nex-avar), the first clinically approved targeted drug therapy forHCC, could only extend the overall survival by 2 to 3months (3). Thus, it remains an urgent need for effective,life-prolonging strategies in the management of patientswith HCC.

Immunotherapy based on T cells modified with a chi-meric antigen receptor (CAR) has been demonstrated as apromising strategy for cancer treatment (4). CAR T cells canspecifically recognize tumor-associated antigen and elimi-nate tumor cells in a non–major histocompatibility com-plex-restricted manner. Several pilot clinical trials usingCAR T cells have recently been reported with promisingclinical outcomes (5).

GPC3 is a member of the glypican family of heparansulfate (HS) proteoglycans that are attached to the cellsurface by a glycosylphosphatidylinositol (GPI) anchor.GPC3 plays an important role in cellular growth, differ-entiation, and migration (6). Multiple studies have dem-onstrated that GPC3 is an attractive liver cancer–specifictarget because it is highly expressed in HCC but limitedexpressed in normal tissues (7). Currently, GPC3-targetedimmunotherapeutic strategies utilizing antibodies or apeptide vaccine have been explored for HCC treatment(8–12). The clinical data of the monoclonal antibodyGC33 indicated that it was well tolerated in advancedHCC, suggesting that GPC3 is a relatively safe target.

1Medical School of Fudan University, Shanghai, China. 2State Key Labo-ratory of Oncogenes & Related Genes, Shanghai Cancer Institute, RenjiHospital, Shanghai Jiaotong University School of Medicine, Shanghai,China. 3International Cooperation Laboratory on Signal Transduction,EasternHepatobiliary Surgery Institute, SecondMilitaryMedical University,Shanghai, China. 4National Center for Liver Cancer, Shanghai, China.

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

H. Gao and K. Li contributed equally to this article.

Corresponding Author: Zonghai Li, State Key Laboratory of Oncogenesand Related Genes, Shanghai Cancer Institute, Renji Hospital, MedicalSchool of Shanghai Jiaotong University, No. 25/Ln. 2200, Xie Tu Road,Shanghai 200032, China. Phone: 86-21-64436601; Fax: 86-21-64432027;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-1170

�2014 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 20(24) December 15, 20146418

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However, according to experimental knockdown results,GPC3 is not a lethal gene to HCC cells (13). Moreover,the naked antibody could not completely eliminatetumors in mouse models, and no partial or completeresponse was observed in the 15 patients evaluated duringthe phase I clinical trial of GC33, suggesting that thetherapeutic effects of a naked antibody may not be potentenough for curative treatment of HCC (14). In the phase Iclinical trial of a GPC3-derived peptide vaccine forpatients with HCC, the median overall survival seemedto positively correlate with the GPC3-specific CTL fre-quencies, suggesting that GPC3-targeted T cells could bepotential agents for HCC treatment (12). However,GPC3-based, CAR-engineered T cells have never beenreported. Therefore, in this study, we developed GPC3-specific CAR T cells and explored their potential for HCCtreatment.

Materials and MethodsConstruction of lentiviral vectors encoding CARsThe sequence encoding the anti-GPC3 scFv antibody in

the VL-VH orientation (Supplementary Fig. S1), based onthe sequence of GC33, which recognizes the C-terminalregion of GPC3 (8), was obtained by splicing PCR and theoverlap extension technique. As shown in Fig. 1A, theaGPC3-Z CAR (first generation) comprising the scFv GPC3linked to the human CD8a hinge and transmembranedomain (nucleotides 412–609, GenBank NM 001768.6)was fused to the intracellular signaling domain derivedfrom only the CD3x molecule (nucleotides 154–492,GenBank NM 198253.2). The aGPC3-28BBZ CAR (thirdgeneration) comprising the scFv GPC3 linked in-frame tothe hinge domain of the CD8a molecule (nucleotides412–546, GenBank NM 001768.6) was fused to the trans-membrane region of the human CD28 molecule (nucleo-tides 457–537, GenBank NM 006139.3), and the intracel-lular signaling domains (nucleotides 538–660, GenBankNM 006139.3), CD137 (nucleotides 640–765, GenBank

NM 001561.5), and CD3x molecules (nucleotides 154–492, GenBank NM 198253.2) in tandem. One CAR(aGPC3-DZ) carrying the truncated CD3x (nucleotides154–189, GenBank NM 198253.2), and another CAR(2D3-28BBZ) containing the irrelevant scFv 2D3 derivedfrom a hybridoma against the intracellular domain ofEGFRvA (15) were set as controls. All CAR constructs wereoverlapped with eGFP through a ribosomal skippingsequence (F2A) derived from the foot and mouth diseasevirus (16). Finally, all the products contained an MluI siteat the 50 end and a SalI site at the 30 end and were ligatedinto the third-generation nonself-inactivating EF-1a pro-moter-based lentiviral expression vector pWPT-eGFP (17).The primers are listed in Supplementary Table S1.

Cell linesHuman HCC cell lines (HepG2, Hep3B, PLC/PRF/5,

and SK-HEP-1) and 293T were obtained from the ATCC.Huh-7 cell line was obtained from the RIKEN Cell Bank.These cell lines were tested and authenticated by DNAprofiling for polymorphic short tandem repeat markers.For bioluminescence assays, Huh-7 cells with enforcedexpression of firefly luciferase (fLucþ) were established.293T and HCC cells were cultured in DMEM supplemen-ted with 10% FBS. All cells were routinely tested formycoplasma contamination.

Lentivirus productionRecombinant lentiviral particles were produced by a

calcium phosphate transfection system (17). Lentiviralparticles were concentrated 30-fold by ultracentrifugation(Beckman Optima XL-100 K, Beckman) for 2 hours at28,000 rpm.

Isolation, activation, transduction, and expansion ofhuman T cells

Peripheral blood mononuclear cells (PBMC) derivedfrom human donors were provided by the ShanghaiBlood Center. Primary human CD4þ and CD8þ T cellswere isolated from PBMCs by the negative selection usingRosetteSep kits (Stem Cells Technology). CD4þ andCD8þ T cells mixed at a 1:1 ratio were stimulated for24 hours with anti-CD3/anti-CD28 antibodies immobi-lized on tosyl-activated paramagnetic beads (Invitrogen)at a cell:bead ratio of 1:1 in accordance with the man-ufacturer’s instructions. T cells were then transducedwith the lentiviral vector at a multiplicity of infectionof 8 U/cell. The transduced T cells were cultured at aconcentration of 5 � 105 cells/mL in the presence ofrecombinant human IL2 (300 U/mL; Shanghai HuaxinHigh Biotech) every other day. Genetically modifiedT cells were used for functional assays when the lym-phocyte volume and proliferation showed a decrease.

Western blot analysisThe cell lysate was denatured and electrophoresed by

SDS-PAGE. The samples were then transferred to a nitro-cellulose membrane (Bio-Rad) and immunoblotted withthe targeted antibodies.

Translational RelevanceThere are limited strategies for the treatment of hepa-

tocelllar carcinomas (HCC). A majority of HCC cellsexpress Glypican-3 (GPC3), which has restricted expres-sion in normal tissues. In this study, we engineeredT cells with a first- and a third-generation (composedof costimulatory signaling domains from CD28 andCD137) chimeric antigen receptor (CAR) targetingGPC3. Our study indicates that the third-generationGPC3-targeted CAR T cells can efficiently destroyGPC3-positive human HCC cells in vitro and in vivo.Our preclinical study suggests that adoptive transfer of Tcells expressing a CAR-targeting GPC3 presents a prom-ising therapeutic strategy to treat HCC. Future clinicaltrials on this novel strategy are warranted.

GPC3-Targeted CAR T Cells for HCC Treatment

www.aacrjournals.org Clin Cancer Res; 20(24) December 15, 2014 6419




Cytotoxicity assays in vitroThe specific cytotoxicity of CAR-modified T cells toward

various HCC cells at the different effector:target ratios of3:1, 1:1, and 1:3 was measured after coculturing for 18hours by lactate dehydrogenase assay using the CytoTox96 nonradioactive cytotoxicity kit (Promega) according tothe manufacturer’s instructions.

Cytokine release assaysThe IFNg and IL2 cytokines secreted by the varying

genetically modified T cells were measured using an ELISAkit (MultiSciences Biotechnology).

Xenograft models of human hepatocellularcarcinoma

Six- to 8-week-old NOD/SCID mice were housed andtreated under specific pathogen-free conditions at theExperimental Animal Center of Shanghai Jiaotong Uni-

versity School of Medicine (Shanghai, China). All animalexperiments were carried out according to the protocolsapproved by the Shanghai Medical Experimental Ani-mal Care Commission. For the established subcutane-ous (s.c.) Huh-7 models, mice were inoculated subcuta-neously with 2 � 106 Huh-7 cells on the right flank onday 0. When the tumor burden was approximately 200 to300 mm3, mice were randomly allocated into fourgroups (n¼ 6) and assigned to receive one of the followinginjections: (i) genetically modified aGPC3-28BBZ CART cells dissolved in sterile saline (aGPC3-28BBZ);(ii) genetically modified 2D3-28BBZ CAR T cells in sterilesaline (2D3-28BBZ); (iii) genetically modified mockT cells in sterile saline (Mock); and (iv) saline onlywithout T cells. On day 13, to deplete of host lymphocytecompartments using cyclophosphamide and enhance thetumor treatment efficacy of the administered T cells (18),mice were injected intraperitoneally (i.p.) with 200 mg/

Figure 1. Characterization of GPC3-specific CAR T cells. A, schematic representation of a bicistronic lentiviral vector expressing CARs along witheGFP using the F2A ribosomal skipping sequence is shown. B, GPC3-specific CARs expression on human T cells transduced with lentiviralparticles were analyzed using flow cytometry by detection of GFP autofluorescence. Transduction efficiency was shown. C, in vitro expansionof T cells following activation with one cycle stimulation of aCD3/aCD28-coated magnetic beads on day 0 and transduction with the indicated CARson day 1. Genetically modified T cells were cultured in vitro for up to 2 weeks. On day 18, T cells were restimulated by aK562-64/86 withcoexpression of the extracellular domain of CD64 and CD86, in the presence of OKT3 (100 ng/mL) and recombinant human IL2 (300 U/mL). D, on day18, the general or antigen-specific reproliferation of aGPC3-28BBZ, and the negative control mock and 2D3-28BBZ–transduced T cellswere quantified.

Gao et al.

Clin Cancer Res; 20(24) December 15, 2014 Clinical Cancer Research6420




kg of cyclophosphamide. Then, 8 � 106 aGPC3-28BBZCAR T cells were intravenously (i.v.) injected on day 14.In established subcutaneous PLC/PRF/5 models, micewere inoculated with 5 � 106 PLC/PRF/5 cells in theright flank on day 0. On day 21, when the tumor burdenwas about 100 to 150 mm3, the mice received cyclophos-phamide (200 mg/kg i.p.), two doses of 8 � 106 aGPC3-28BBZ CAR T cells were intravenously injected on days 22and 30. Tumor dimensions were measured with calipers,and tumor volumes were calculated using the formula V¼ 1/2 (length � width2). Mice were euthanized when themean tumor burden in the control mice reached 1,500 to2,000 mm3. Xenografts were fixed with formalin, embed-ded in paraffin, and sections were cut and processed forIHC staining. For the established orthotopic HCC model,mice were inoculated with 2 � 106 Huh-7 (fLucþ) cells inthe liver on day 0. On day 13, mice were grouped (n ¼ 5)and received the injection of cyclophosphamide (200mg/kg i.p.) followed by the treatments with two doses ofintravenous injections of 5 � 106 genetically modified Tcells on days 14 and 21. The mice were imaged every weekfollowing T-cell administration to evaluate whetheraGPC3-28BBZ CAR T cells could suppress tumor growthor prolong the survival of the mice bearing orthotopicHCC xenografts. The transduction efficiencies of geneti-cally modified T cells used in experimental assays wereabout 50%.

Bioluminescence imagingIsoflurane-anesthetized animals were imaged using

the IVIS system (IVIS) followed by the intraperitonealinjection of 150 mg/kg D-luciferin (Promega). The datawere quantified using Living Image software (Caliper LifeSciences).

IHCThe HLiv-HCC150CS-01 tissue microarray (Outdo Bio-

tech) and sections of the normal kidney and gastric glandswere immunostained using an anti-GPC3 antibody (mAb1G12, BioMosaics Inc). To assess the persistence of theadministrated human T cells in xenografts, the sections offormalin-fixed, paraffin-embedded tumor tissues wereimmunostained using an anti-CD3 antibody (Thermo Sci-entific RM-9107-S0).

Statistical analysisStatistical analysis was performed by two-way repeated

measures ANOVA with Bonferroni post-tests for thetumor burden (tumor volume, tumor weight, and photoncounts). Differences in the absolute number of varioustransferred T cells were evaluated by Student’s t test. Theoverall survival statistics were calculated using the log-rank test (19). GraphPad Prism 5.0 was used for thestatistical calculations. P < 0.05 (�), P < 0.01 (��), andP < 0.001(��) were considered statistically significant.Additional information is described in Supplementary

Methods.

ResultsGeneration of CAR-GPC3 T cells by lentiviral vectortransduction

As shown in Fig. 1A, the lentiviral expression vectorsencoding the GPC3-targeted CARs including aGPC3-Z,aGPC3-28BBZ, or negative control CARs includingaGPC3-DZ and 2D3-28BBZ efficiently transduced humanT cells to coexpress the CAR protein and eGFP using the"self-cleaving" F2A peptide, respectively. The geneticallymodified T cells began to expand after activation. On day7, the expression of the different CARs in the transduced Tcells was demonstrated through eGFP expression. The trans-duction efficiencies were about 55% (Fig. 1B). According toviable cell counting assays (Trypan blue exclusion), theaCD3/aCD28-coated beads could induce 200- to 300-foldexpansions of CAR-modified T cells on day 18. In thepresence of OKT3 (100 ng/mL) and recombinant humanIL2 (300 U/mL), the expansion could be further increasedby 10- to 20-fold (Fig. 1C) after subsequent stimulation for7 days with cells of g-irradiated K562-based artificial anti-gen-presenting cells (referred to as aK562-64/86) whichexpress the membrane-bound extra-domains of CD64 andCD86 (Supplementary Fig. S2). To determine the antigen-specific expansion, the well-known GPC3-positive Huh-7cells and GPC3-negative SK-HEP-1 cells were used forspecific stimulations on day 18. The results indicated thatT cells expressing aGPC3-28BBZ were expanded by thestimulation of GPC3-positive Huh-7 cells but not byGPC3-negative SK-HEP-1 cells. In addition, both GPC3þ

and GPC3� HCC cell lines failed to elicit the expansion ofmock or 2D3-28BBZ transduced T cells (Fig. 1D).

Cytokines produced by GPC3-targeted CAR T cellsSurface expression of GPC3 on five HCC cell lines was

determined. The results of FACS and Western blot analysis(Fig. 2A) confirmed that SK-HEP-1 had no GPC3 expres-sion, and the other four HCC cell lines had varying levels ofGPC3 expression. In the cytokine production assay, greateramounts of IL2 (Fig. 2B) and IFNg (Fig. 2C) were producedby aGPC3-28BBZ CAR T cells than aGPC3-Z CAR T cells orthe control T cells including mock, aGPC3-DZ, or 2D3-28BBZ transduced T cells, when cocultured with GPC3-positvie cells. However, almost no induction of IL2 or IFNgwas observed when any of the engineered T cells werecocultured with SK-HEP-1 cells (Fig. 2B and C). Intriguing-ly, there was a positive correlation between IFNg secretionin GPC3-redirected CAR T cells transfected with eitheraGPC3-28BBZ or aGPC3-Z and the MFI of GPC3 expres-sion on the target cells (Fig. 2D).

In vitro cytotoxicity of T cells redirected to GPC3 onHCC cells

To determine whether T cells targeting GPC3 could spe-cifically recognize and kill GPC3-positive HCC cells, cyto-toxicity assays were performed by incubating the geneticallymodified T cells with each of the five HCC cell lines. Theresults indicated that both aGPC3-28BBZ and aGPC3-Z






CAR T cells could efficiently lyse the four GPC3-positiveHCC cell lines, but not the GPC3-negative SK-HEP-1 cellline (Fig. 3A), while the control effector cells (mock,aGPC3-DZ or 2D3-28BBZ) could not initiate specific lysison those HCC cells.

It was reported that two types of soluble GPC3 (sGPC3)exist in the blood serumof patients withHCC (20, 21). Oneis the N-terminal region of GPC3 (GPC3N, residues: S25-R358); the other is the full-length GPC3 without GPI(GPC3DGPI, residues: Q25-S560). To elucidate whethersGPC3 can prevent the activation and functional activityof the GPC3-targeted CAR T cells, the cytotoxic activities ofthe aGPC3-28BBZ–modified T cells toward Huh-7 cells inthe presence or absence of three types of recombinantsGPC3 were determined. The results indicated that theGC33-binding peptide fusion protein and GPC3DGPI, butnot GPC3N, could inhibit the cytotoxic activities of theaGPC3-28BBZ–modified T cells.However, this suppressionwas mild, as the highest inhibition ratio was approximately10% even at a concentration of 1,000 ng/mL GPC3DGPI(Fig. 3B).

CAR T cells redirected to GPC3 suppress thetumorigenesis of subcutaneous Huh-7 xenografts

NOD/SCID mice were coinjected subcutaneously withthe Huh-7 tumor cells and transduced T cells at an E:Tratio of 1:1 on the right flanks, with an additional groupreceiving the aGPC3-28BBZ T cells at an E:T ratio of 1:2.The results indicated that 2 out of the 6 mice were tumor

free in the group that received aGPC3-Z CAR T cells,while all mice were tumor free in the group that receivedaGPC3-28BBZ CAR T cells, even at an E:T ratio of 1:2(Supplementary Fig. S3A). In contrast, tumor outgrowthwas observed in all mice in the control groups (Supple-mentary Fig. S3B). The results indicated the aGPC3-28BBZ CAR T cells displayed a significantly stronger lyticactivity than the aGPC3-Z CAR T cells did.

aGPC3-28BBZ CAR T cells suppressed the growth ofestablished subcutaneous GPC3-positive HCCxenografts

To further explore the antitumor activities of aGPC3-28BBZ CAR T cells, NOD/SCID mice bearing establishedsubcutaneous Huh-7 xenografts were used. The potentantitumor effect was observed in the mice treated withaGPC3-28BBZ CAR T cells, whereas the other geneticallymodified T cells did not suppress tumor growth (Fig. 4A). Atthe experimental endpoint (day 28), 3 of the 5 mice treatedwith theaGPC3-28BBZCAR T cells were tumor free, where-as all mice in the control groups carried large tumors(Supplementary Fig. S4A). The antitumor effect of theaGPC3-28BBZ CAR T cells was very significant comparedwith the control groups, respectively (P < 0.001; Fig. 4A,Supplementary Fig. S4B). These results suggested thatGPC3-targeted CAR T cells could specifically eliminateHuh-7 cells in vivo.

To elucidate whether the in vivo cancer cell eliminatingability of aGPC3-28BBZ CAR T cells is dependent on the

Figure 2. IL2 and IFNg produced by GPC3-targeted CAR T cells cocultured with various HCC cell lines. A, surface GPC3 expression (black line) onvarious human HCC cell lines detected by flow cytometry and isotype antibody control (filled red histograms). Moreover, GPC3 expression invarious HCC cell lines was analyzed by Western blot analysis. IL2 (B) and IFNg (C) were secreted by the indicated genetically modified T cells coculturedwith various HCC cell lines for 24 hours. Results are representative of triplicates. D, correlation between the IFNg secretion from GPC3-targetedT cells and the MFI of GPC3 expression on the five HCC cell lines.

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expression level of GPC3, mice bearing PLC/PRF/5 xeno-grafts were also treatedwith the geneticallymodified T cells.Comparedwith the control groups, theaGPC3-28BBZCART cells could significantly suppress the growth of PLC/PRF/5tumors (Fig. 4B). At the end of the study (day 47), the micebearing PLC/PRF/5 tumors treated with aGPC3-28BBZCAR T cells still carried residual tumors (SupplementaryFig. S4C). The tumors treated with aGPC3-28BBZ CAR Tcells were significantly smaller in weight than those in thecontrol groups (aGPC3-28BBZ vs. saline, P ¼ 0.0332;aGPC3-28BBZ vs. mock, P ¼ 0.021; aGPC3-28BBZ vs.2D3-28BBZ, P ¼ 0.0367; Supplementary Fig. S4D). Theresults of these two models implicated that the cytotoxicactivities of GPC3-targeted CAR T cells were dependent onthe GPC3 expression level on the target cells.Previous studies have indicated that the persistence of

transferred T cells in vivo is highly correlated with tumorregression (22, 23). Therefore, we also detected the num-bers of human T cells in the peripheral blood of micebearing subcutaneous established Huh-7 or PLC/PRF/5xenografts 1 week after T-cell infusion. The results indi-cated that CD4þ and CD8þ T cell numbers were highestin the group treated with aGPC3-28BBZ CAR T cells in

both groups (P < 0.01; Fig. 4C and D). In addition, thepersisting T-cell number of the PLC/PRF/5 group was lessthan that of the Huh-7 group (CD4þ T cells: 123.5 � 16vs. 148 � 20; CD8þ T cells: 246.5 � 26 vs. 374.5 � 33).The persistence of human T cells was further certified byimmunostaining of the sections of Huh-7 tumors treatedwith aGPC3-28BBZ CAR T cells. The results revealed thathuman CD3þ T cells had accumulated in residual tumors2 weeks after intravenous T-cell administration (Fig. 5),while fewer T cells could be detected in the sections oftumors treated with 2D3-28BBZ or mock transduced Tcells. There was no specific staining in the sections treatedwith saline alone.

Human GPC3-targeted CAR T cells potentlysuppressed the growth of established orthotopicHuh-7 xenografts

To better evaluate the antitumor activities of GPC3-targeted CAR T cells, mice orthotopically transplantedwith Huh-7 (fLucþ) cells were used. After one week oftreatment, the group with aGPC3-28BBZ CAR T cells hadthe lowest tumor burden among the T-cell–transferredgroups (Fig. 6A). In the third week after the first dose of

Figure 3. In vitro cytotoxic activities of GPC3-targeted CAR T cells. A, primary human T cells transduced with the indicated lentiviral vectors werecoincubated with the five HCC cell lines at the varying effector: target ratios for 18 hours, respectively. Cell lysis was determined by a standardnonradioactive cytotoxic assay. Each data reflect the mean � SEM of triplicates. B, cytotoxicity of aGPC3-28BBZ CAR T cells on Huh-7 cellsin the absence or presence of mammalian cell-expressed GPC3DGPI and GPC3N, E.coli-expressed GST-fused GC33-binding peptide or BSA atthe effector:target ratio of 3:1 for 18 hours.






T-cell administration, the mice in the three controlgroups had swollen abdomens, whereas the mice of theaGPC3-28BBZ CAR T-cell–treated group had the normalabdomen (Supplementary Fig. S5). At the end of thisstudy, 3 out of 5 of the mice in the group receiving theaGPC3-28BBZ CAR T cells were liver tumor free, whereasall mice in the control groups carried large hepatomas(data not shown). One week after the last infusion, thetotal number of human CD4þ and CD8þ T cells andCAR-positive T cells from mice treated with aGPC3-28BBZ CAR T cells was significantly higher than that ofmice treated with 2D3-28BBZ CAR T cells or mock T cells(Fig. 6B), while no significant difference was observedbetween the 2D3-28BBZ and mock groups (P ¼ 0.197).The persistence of CARþ T cells was consistent with thetotal T cells (aGPC3-28BBZ vs. mock, P < 0.01; aGPC3-

28BBZ vs. 2D3-28BBZ, P < 0.01; 2D3-28BBZ vs. mock,P ¼ 0.22; Fig. 6C). These results suggested that tumorantigen recognition drove the survival of the infusedT cells in vivo. Previous studies indicated that the persis-tence of the CD137 signaling domain could upregulateBcl-XL expression and enhance the survival of T cells (24).We also observed increased expression of Bcl-XL inaGPC3-28BBZ CAR T cells driven by the GPC3 antigenin vitro (Supplementary Fig. S6), suggesting that theincreased number of CARþ T cells among the aGPC3-28BBZ CAR T cells might be ascribed to Bcl-XL upregula-tion. More importantly, all mice treated with the GPC3-specific CAR T cells survived for longer than 60 days,while the median survival durations of the saline, mock,and 2D3-28BBZ–treated mice were 33 days, 34 days, and39 days, respectively (Fig. 6D).

Figure 4. In vivo antitumor activities of aGPC3-28BBZ CAR T cells on established subcutaneous HCC tumor xenografts. A, growth curve of Huh-7xenografts treated with the indicated T cells or saline. At the endpoint, the residual tumors treated with aGPC3-28BBZ CAR T cells wassignificantly smaller than those in the control groups, respectively (��, P < 0.001). B, growth curve of PLC/PRF/5 xenografts treated with the indicatedT cells or saline. On day 47, the tumor in aGPC3-28BBZ group was significantly smaller than that in each control group (�, P < 0.05). The timepoint of T cells infusion or saline in figure A and B was shown with arrow. C and D, the quantities of circulating human CD4þ and CD8þ T cells from micebearing Huh-7 xenografts or PLC/PRF/5 treated with the indicated genetically modified T cells. Mean cell concentration (cells/mL) �SEM for micein genetically modified T cell treatment group and P values are shown.

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Discussion

In this study, we developed GPC3-targeted CAR T cellswith or without the costimulatory signaling domains ofCD28 and CD137. Both types of GPC3-targeted CAR T cellscould specifically destroy the HCC cells in an antigen-dependent manner. Similar to previous reports (25), theaGPC3-28BBZ CAR T cells that had the costimulatorysignaling domains displayed a significantly stronger cyto-toxic activity against theGPC3þHCC cells than theaGPC3-ZCART cells did, suggesting that the intracellular CD28 andCD137 costimulatory domains contributed to the increasedcytotoxic activities.Our data on established Huh-7 subcutaneous and ortho-

topic xenografts indicated that aGPC3-28BBZ CAR T cellscould eradicate liver cancer cells possessing a high level ofGPC3 expression. Compared with Huh-7 cells, PLC/PRF/5cells, which had a significantly lower level of GPC3 expres-sion, displayed less sensitivity to GPC3-targeted CAR T cellsboth in vitro and in vivo. In addition, there is a positiverelationship between IFNg secretion from GPC3-targetedCAR T cells and the GPC3 expression level in HCC cells.Together, these data suggested that GPC3 expression levelon the cell surface of HCC cell lines might be used as anindicator of the antitumor efficacy of GPC3-targeted CAR Tcells.Our previous study revealed that the levels of sGPC3

were 99.94 � 267.20 ng/mL with a median level of15.11 ng/mL (26). Our in vitro data in this study indicatedthat even a high concentration (approximately 1,000 ng/mL) of GPC3DGPI could only mildly inhibit the cytotoxicactivity of GPC3-targeted CAR T cells against GPC3-pos-itive HCC cells. We also observed that the GPC3DGPI(1,000 ng/mL) had no significant effect on IFNgand IL2secreted by the GPC3-targeted CAR T cells as well as theexpansion of GPC3-redirected T cells (data not shown).

Therefore, the persistence of sGPC3 in most of thepatients with HCC might not significantly inhibit theclinical efficacy of GPC3-targeted CAR T cells. However,the actual effect of the sGPC3 on the antitumor effect ofCAR T cells in vivo needs further study.

A major concern about CAR T cells, especially when thecostimulatory signaling domain of CD28 or CD137 isincluded, is the on-target off-tumor effect. Previously, onepatient with breast cancer died after the treatment withCAR T cells redirected to Erbb2 due to the expression ofthis receptor in lung tissues (27). Therefore, the specificityof the cancer-associated antigen targeted by the CAR Tcells is vitally important. Although several studies haveindicated that GPC3 is absent in normal tissues, a studyby Baumhoer and colleagues revealed that although mostnormal tissues stained negatively for GPC3, gastric glands[3/7 (43%)], kidney tubules [9/17 (53%)], and testiculargerm cells [2/16 (13%)] stained positively (7). Therefore,we examined the GPC3 expression in the kidney andgastric glands. Fortunately, our study revealed that thereare no obvious GPC3 expression in normal kidney tissues(10/10) and gastric glands (10/10; Supplementary Fig.S7). The contradictory results may be ascribed to thedifferent samples used. Another possibility is the falsepositivity of immunostaining. Anyway, considering theindividual difference, we thought it should be cautiouswhen the GPC3-redirected CAR T cells were applied inclinical trial. Several strategies such as the incorporationof suicide switches (28, 29), a "tumor sensing" approachby combining two types of antigen recognition withbalanced signaling (30) or iCARs technology (31) mightbe used to minimize the potential off-target toxicity of theCAR T cells.

As we know, one of the major challenges for cancertreatment lies in its heterogeneity. Several testis-antigenshave been targeted by T-cell–based immunotherapy.

Figure 5. aGPC3-28BBZ CAR T cells could be located in Huh-7 tumors. Tumors were collected from mice bearing Huh-7 subcutaneous xenograftstreated with aGPC3-28BBZ CAR T cells, 2D3-28BBZ, Mock T cells, or saline. Formalin-fixed, paraffin-embedded tumor sections were consecutively cutand stained for human CD3 expression (brown). [The images were taken with the microscope (BX41, Olympus, PA) and camera (DP70) under � 200magnifications. Scale bar was 200 mm].






However, most of the testis antigens are heterogeneouslyexpressed in the tumor tissue (32). In the tumor tissuemicroarray of 75 primary HCC patients, the differentlevels of GPC3 expression were evaluated by two experi-enced pathologists using a 4-point scale according to thestaining intensity (Supplementary Fig. S8A). Similar toprevious reports (7, 33), our data indicated that GPC3was expressed in 70.7% (53 out of 75) of patients withHCC (Supplementary Fig. S8B). In GPC3-positivepatients, 77% (41 out of 53) of patients carried highlevels of GPC3 expression (score � 2). Importantly,unlike testis-antigen, in patients carrying a high level ofGPC3 expression, GPC3 seemed to be homogenouslyexpressed in HCC tissues with a mean rate of GPC3-positive hepatoma cells equal to 88% (SupplementaryFig. S8C). This will be an important factor in the long-term clinical efficacy of GPC3-directed CAR T cells.

Taken together, the results of this study constitute thefirst report concerning CAR T cells redirected to GPC3 andthe first report about HCC treatment using CAR T cells.With their potent cancer cell-eliminating capacity, GPC3-targeted CAR T cells may be a promising therapeuticoption for HCC treatment.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: H. Wang, Z. LiDevelopment of methodology: X. Pan, J. Kong, H. WangAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): H. Gao, K. Li, H. Tu, Z. LiAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): H. Gao, H. Jiang, Z. LiWriting, review, and/or revision of the manuscript: H. Gao, Z. Li

Figure 6. aGPC3-28BBZ CAR T cells potently suppressed the established orthotopic Huh-7 xenografts in vivo. A, NOD/SCID mice bearing Huh-7 (fLucþ)orthotopic tumor were intravenous injected with 5 � 106 genetically-modified T cells on day 14 and 21, respectively. Mice were imaged weekly.Tumor growth was assessed by total bioluminescence signals. The growth of tumors treated with aGPC3-28BBZ CAR T cells was potently suppressedwhen compared with the control groups. B, the quantitative analysis of human CD4þ and CD8þ T cells by TruCount tubes. C, GFP-positiveperipheral blood cells were also counted. Mean cell concentration (cells/mL) �SEM for all evaluable mice in each treatment group are shown.D, the overall survival of mice treated with the indicated T cells or saline.

Gao et al.





Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. Tu, B. ShiStudy supervision: H. Wang, S. Yang, J. Gu

AcknowledgmentsThe authors thank Dr. Didier Trono (University of Geneva, Switzerland)

for the generous gift the pWPT-eGFP, psPAX2, and pMD2.G lentivirusplasmids.

Grant SupportThe study was funded by the Supporting Programs of the "Twelfth

Five-year Plan" for Science & Technology Research of China(2012ZX09103-301-005, 2012ZX10002), and the Research Fund of the

State Key Laboratory of Oncogenes and Related Genes (91-14-12),Shanghai Science and Technology Development Funds (Grant No.12JC1408300), Shanghai Rising-Star Program (A type; 13QA1403300),and One Hundred Talents Scientific Research Projects of Health Systemin Shanghai (XBR2013123).

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 indicatethis fact.

Received May 12, 2014; revised August 25, 2014; accepted September 9,2014; published OnlineFirst October 15, 2014.

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2014;20:6418-6428. Published OnlineFirst October 15, 2014.Clin Cancer Res Huiping Gao, Kesang Li, Hong Tu, et al. of Hepatocellular CarcinomaDevelopment of T Cells Redirected to Glypican-3 for the Treatment

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