A Tet-On Inducible System for Controlling CD19 …...Research Article A Tet-On Inducible System for Controlling CD19-Chimeric Antigen Receptor Expression upon Drug Administration Reona

Research Article

A Tet-On Inducible System for ControllingCD19-Chimeric Antigen Receptor Expressionupon Drug AdministrationReona Sakemura1, Seitaro Terakura1, Keisuke Watanabe1, Jakrawadee Julamanee1,2,Erina Takagi1, Kotaro Miyao1, Daisuke Koyama1, Tatsunori Goto1, Ryo Hanajiri1,Tetsuya Nishida1, Makoto Murata1, and Hitoshi Kiyoi1

Abstract

T cells genetically modified with a CD19 chimeric antigenreceptor (CD19CAR) are remarkably effective against B-cellmalignancies in clinical trials. However, major concerns remainregarding toxicities, such as hypogammaglobulinemia, due to B-cell aplasia or severe cytokine release syndrome after overactiva-tion of CAR T cells. To resolve these adverse events, we aimed todevelop an inducible CAR system by using a tetracycline regula-tion system that would be activated only in the presence ofdoxycycline (Dox). In this study, the second-generationCD19CAR was fused into the third-generation Tet-On vector(Tet-CD19CAR) and was retrovirally transduced into primaryCD8þ T cells. Tet-CD19CAR T cells were successfully generatedand had minimal background CD19CAR expression withoutDox. Tet-CD19CAR T cells in the presence of Dox were equiva-lently cytotoxic against CD19þ cell lines and had equivalent

cytokine production and proliferation upon CD19 stimulation,compared with conventional CD19CAR T cells. The Dox(þ)Tet-CD19CAR T cells also had significant antitumor activity in axenograft model. However, without Dox, Tet-CD19CAR T cellslost CAR expression and CAR T-cell functions in vitro and in vivo,clearly segregating the "On" and "Off" status of Tet-CD19CARcells by Dox administration. In addition to suicide-gene tech-nology, controlling the expression and the functions of CARwith an inducible vector is a potential solution for CAR T-celltherapy–related toxicities, and may improve the safety profileof CAR T-cell therapy. This strategy might also open the wayto treat other malignancies in combination with other CARor TCR gene–modified T cells. Cancer Immunol Res; 4(8); 658–68.�2016 AACR.

See related Spotlight by June, p. 643.

IntroductionT cells genetically modified with a CD19-chimeric antigen

receptor (CD19CAR) have emerged as a new therapy with aston-ishing treatment outcomes for relapsed or refractory B-cell malig-nancies, such as acute lymphoblastic leukemia (ALL; refs. 1–4),non-Hodgkin lymphoma (5, 6), and chronic lymphocytic lym-phoma (CLL; ref. 7). Previously published preclinical studies orclinical trials strongly suggest the possibility that this noveltechnology may also be applicable to patients with other typesof hematologic malignancies such as myeloid leukemia (8),multiple myeloma (9), or certain other types of solid tumors(10–12). However, because almost no antigens are truly tumorspecific, it is necessary to prepare for somedegree of adverse eventsrelated to CAR T-cell therapy. Upon engagement with target

antigen, CAR T cells become activated and proliferate until theantigen-bearing cells are eliminated. Although suchpowerfulCART-cell activity is the desired response, if the target antigen isexpressed on normal tissues, even in low amounts, severe adverseevents could occur. This activity has been described as the "on-target/off-tumor" effect. Indeed, CAR T-cell therapy targetingERBB2 and CAIX reportedly has involved lethal toxic accidentsin clinical trials that were due to cytokine release syndrometriggered by the off-target recognition of low amounts of antigen(13–17). Clinical trials of CD19CAR T-cell therapy also revealedtoxicities such as hypogammaglobulinemia, due to B-cellaplasia (18).

To address such problems, suicide gene systems have beendeveloped, and clinical application of suicide gene technologysuch as herpes simplex virus-1/thymidine kinase (HSV/TK;refs. 19–21) or inducible caspase-9 (iCasp9; refs. 22, 23) couldimprove the safety profile of engineered T-cell therapy. In partic-ular, iCasp9-transduced T cells showed effective CD20-specificcytotoxicity as well as efficient apoptotic removal of transducedT cells both in vitro and in vivo by administration of the homo-dimer agent AP1903 (24). The iCasp9 safety switch has attractedmuch attention and has nowbeen incorporated into CD30CART-cell (Clinical Trials.gov Identifiers NCT02274584) and mesothe-lin CAR T-cell (NCT02414269) clinical trials. Although suchsuicide gene systems have proved to be effective for eliminationof gene-modified T cells, the switching-on of the suicide geneleads to immediate induction of apoptosis in most of the gene-modified T cells, which may result in disease progression after

1Department of Hematology and Oncology, Nagoya University Grad-uate School of Medicine, Nagoya, Japan. 2Division of Clinical Hema-tology, Faculty of Medicine, Prince of Songkla University, Songkhla,Thailand.

Note: Supplementary data for this article are available at Cancer ImmunologyResearch Online (http://cancerimmunolres.aacrjournals.org/).

Corresponding Author: Seitaro Terakura, Department of Hematology andOncology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho,Showa-ku, Nagoya, Aichi 466-8560, Japan. Phone: 81-52-744-2145; Fax: 81-52-744-2161; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-16-0043

�2016 American Association for Cancer Research.

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elimination of these T cells. It is therefore desirable to develop asystem that can control transgene expression of genes such as CARand the T-cell receptor at will.

The tetracycline (Tet)-On system is an inducible gene expres-sion system for mammalian cells, in which the reverse Tet trans-activator (rtTA) fusion protein (25–27), which is composed of thedoxycycline-binding Tet-repressor mutant protein and the C-terminal activator domain from the herpes simplex virus VP16protein, was engineered to control gene expression with doxycy-cline (Dox). In the presence of Dox, rtTA activates the minimalpromoters that are fused downstream of an array of seven repeat-ed Tet-operator sequences (28). Until recently, all Tet-On systemshad required two separate vectors, one to introduce rtTA andanother with the inducible promoter to control the gene ofinterest. However, a one-vector system has recently been devel-oped (29), which has enabled transduction of a gene of interestinto primary immune cells. By utilizing this one-vector system,wetested whether it was possible to control CAR expression andfunctions using the Tet-On inducible system. We used CD19CARas the transgene model because CD19CAR T cells have shownclear antitumor efficacy.

In the present study, we aimed to develop not only safe but alsoefficient T-cell therapy by using the Tet-On inducible gene system.We describe herein our use of inducible CD19CAR with the Tet-On 3G inducible vector (Tet-CD19CAR), which has low back-ground expression and an equivalent functional profile to that ofthe original-CD19CAR (CD19CAR-OR).

Materials and MethodsHuman subjects

The research protocols of this study were approved by theInstitutional Review Board ofNagoyaUniversity Graduate Schoolof Medicine. Peripheral blood mononuclear cells (PBMC) wereobtained from healthy volunteer donors after written informedconsent and were obtained from each donor in accordance withthe Declaration of Helsinki.

Retroviral vector constructionThe CD19-binding scFv domain was constructed based on

the reported sequences of the mAb to CD19 (30, 31). Heavy-chain and light-chain variable region segments were linkedwith an 18-aa linker. scFv was then fused to a human IgG4hinge, a CD3z chain, a CD28 costimulatory domain, and atruncated version of the epidermal growth factor receptor(tEGFR) that lacked epidermal growth factor binding andintracellular signaling domains downstream of the self-cleavingT2A sequence (32–36). By inserting the T2A sequence betweenCD19CAR and tEGFR, the two proteins were coexpressed atequimolar levels from a single transcript. Cell-surface tEGFRwas detected using the biotinylated Erbitux mAb to EGFR(Bristol-Myers Squibb), and surface Fc was stained by goatantibody to human IgG Fc (Invitrogen). The pRetroX-TetOne3G vector (Takara Bio Inc.) was first digested with EcoRI. TheCD19CAR-tEGFR insert was then fused into the pRetroX-TetOne 3G vector in reverse orientation at the marked EcoRIsite, as shown in the sequence in Supplementary Fig. S1. TheTet-CD19CAR-encoding retrovirus was produced using thePhoenix-Ampho system (Orbigen). Finally, single-cell cloningwas performed to clone the virus-packaging cell that mostefficiently produced the retrovirus.

Cell linesThe SUP-T1 tumor cell line was obtained from the American

Type Culture Collection in 2013. K562, Raji, and SU-DHL6 celllinesweremaintained inour laboratory. These cell lineswere testedfor surface phenotype by flow cytometry to confirm their compat-ibility with reported phenotype as an authentication assay. All celllineswere cultured inRPMI1640mediumcontaining10%FBS,0.8mmol/L L-glutamine, and1%penicillin–streptomycin.Dr.MichaelC. Jensen (Seattle Children's Research Institute, Seattle,WA) kindlyprovided the lentivirus vector encoding green fluorescent protein(GFP)-firefly luciferase (ffluc). Raji-ffluc were derived by lentiviraltransduction with the GFP-ffluc gene and were then sorted forexpression of GFP. CD19-transduced K562 (CD19-K562) cellswere generated by retroviral transduction with the truncated CD19molecule as described elsewhere (31).

Generation, expansion, and selection of Tet-CD19CAR–transduced T cells

The PBMCs of a normal donor were isolated by centrifugationof whole blood using Ficoll-Paque (GE Healthcare). CD8þ lym-phocytes were then purified with immunomagnetic beads (Mil-tenyi Biotec), activated with anti-CD3/CD28 beads (Invitrogen),and transduced on days 3 and 4 after activation with a recombi-nant human fibronectin fragment (RetroNectin, Takara Bio Inc.)by centrifugation at 950�g for 45minutes at 32 �C together withthe retroviral supernatant. T cells were expanded in RPMI 1640medium containing 10% Tet system–approved fetal bovineserum (Takara Bio), 0.8 mmol/L L-glutamine, 1% penicillin–streptomycin, and 0.5 mmol/L 2-mercaptoethanol that was sup-plemented with recombinant human IL2 to a final concentrationof 50 IU/mL. Twenty-four hours before selection of CAR-positivecells, 100 ng/mL of Dox was added, and CAR-positive cells wereselected using biotin-conjugated anti-EGFRmAb and streptavidinbeads (Miltenyi Biotec). After the EGFR selection step, the trans-duced T cells were expanded with anti-CD3/CD28 beads (secondstimulation) that were supplemented with IL2 (50 IU/mL). Thesemanufactured T cells were used for further downstream experi-ments on day 15. To assess the potential influence of Tet-CD19CAR T cells toward normal B cells, CD19þ cells wereisolated from the PBMCs of healthy subjects by using immuno-magnetic beads (Miltenyi Biotec).

Flow cytometryAll samples were analyzed using flow cytometry with the FACS

Aria (BD Biosciences) instrument, and data were analyzed usingFlowJo software (Tree Star). Biotinylated Erbitux and streptavi-din–PEwere used to identify T cells that expressed the tEGFR (33).

51Cr release and coculture assaysFor the [51Cr] release assay, target cells such as K562, CD19-

K562, Raji, or SU-DHL6 cells were labeled for 2 hours with [51Cr],washed twice, dispensed at a concentration of 2� 103 cells per wellinto triplicate cultures in 96-well round-bottom plates, and incu-bated for 4hours at 37�CwithT cells at various effector-to-target (E:T) ratios. The percentage of specific lysis was calculated using astandard formula [(experimental � spontaneous release)/(maxi-mum load� spontaneous release)� 100 (%)] and is expressed asthe mean of triplicate samples. For the coculture assay, target cellswere labeledwith0.1mmol/L carboxyfluorescein succinimidyl ester(CFSE, Invitrogen), platedwith CAR T cells at a ratio of 1:1 without

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IL2, and the percentages of T cells and target cellswithin the live cellgates were assessed.

CAR T-cell proliferation assayTet-CD19CAR T cells or CD19CAR-OR T cells were stimulated

with either g-irradiated K562 or g-irradiated CD19-K562 cells at a1:1 ratio without IL2 supplementation. The numbers of CAR Tcells were counted 72, 96, and 120 hours after stimulation.

Intracellular cytokine staining and cytokine secretion assayTet-CD19CART cells or CD19CAR-OR T cells andK562, CD19-

K562, Raji, or SU-DHL6 cells were mixed at a 1:1 ratio in thepresence of brefeldin A (Sigma-Aldrich) and were then fixed and

permeabilizedwithCell Fixation/Permeabilization Kits (BDBios-ciences) for intracellular cytokine assays. After fixation, the T cellswere stained with mAbs to IFNg and CD8-allophycocyanin (BDBiosciences) and were analyzed using flow cytometry. Addition-ally, the concentrations of IFNg , TNFa, and IL2 in the supernatantwere measured using ELISA (BD Biosciences) after a 16-hourincubation.

Xenograftmodel of a CD19-positive tumor and bioluminescentimaging

The murine experimental procedures were approved by theInstitutional Animal Care and Use Committee of Nagoya Uni-versity Graduate School ofMedicine. Six- to 8-week-oldNOD/Shi

Before sorting

A

B CAfter sorting

tEGFR tEGFR

Figure 1.

Construction of the Tet-CD19CAR vector and surface CAR expression of Tet-CD19CAR–transduced SUP-T1 cells. A, schematic representation of the Tet-CD19CARconstruct. CD19CAR consisted of anti-CD19 scFv linked to CD3z, a CD28 costimulatory domain, and a truncated EGFR (tEGFR), which was used as atransduction or selection marker, via the T2A sequence. The Tet-On 3G transactivator is oriented in the forward direction downstream of the humanphosphoglycerate kinase 1 promoter (p-hPGK), and CD19CAR is transcribed in the reverse orientation under the pTRE3G promoter, which contains the Tet-responseelement. LTR, long terminal repeat; WPRE, Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element. B, SUP-T1 cells were transduced withthe Tet-CD19CAR–encoding retroviral supernatant. tEGFRþ cells were then purified using flow cytometry with a cell sorter. C, after sorting, the tEGFR wasstained with biotinylated erbitux, used as a transduction marker (left), and surface CD19CAR was stained with an anti-Fc Ab in the presence (solid line) or absence(dashed line) of Dox. Gray histograms show staining of unmodified SUP-T1 cells (B and C).

A B

C D

Figure 2.

Dox titration and kinetic analyses of CD19CAR expression upon Doxadministration and discontinuation. A and B, Dox titration. Theindicated concentrations of Dox were added to the medium ofTet-CD19CARþ SUP-T1 cells, following which the SUP-T1 cellswere stained with biotinylated erbitux (A) and the anti-Fc Ab (B)and analyzed using flow cytometry to determine cell-surfacetEGFR and CD19CAR expression, respectively. C and D, kineticanalyses of CD19CAR expression upon Dox administration (C)and discontinuation (D). For C, 100 ng/mL of Dox was added to themedium of Tet-CD19CARþ SUP-T1 cells and expression of thetEGFR, which reflects CAR expression, was then analyzed at varioussubsequent time points using flow cytometry. For D, 100 ng/mLof Dox was added to the medium of Tet-CD19CARþ SUP-T1 cells for16 hours. The medium containing Dox was then washed out, anddisappearance of CAR from the cell surface was then analyzedat various subsequent time points by analysis of expression ofthe tEGFR as in C. The means � SD of three independentexperiments are shown (A–D).

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Cancer Immunol Res; 4(8) August 2016 Cancer Immunology Research660




scid IL2Rg knockout (NOG)mice were inoculated with 0.5� 106

Raji-ffluc cells by intravenous (i.v.) injection. Seven days later, themice were i.v. injected with 5 � 106 untransduced CD8þ, Tet-CD19CAR, or CD19CAR-OR T cells. For luminescence-basedmeasurement of tumor growth, animals were injected intraper-itoneallywith 10mL/gbodyweight of 15mg/mLfireflyD-luciferinin PBS. Tumor progression was monitored using the IVIS Spec-trum System (Caliper Life Science) on days 7, 14, 21, 28, and 35after inoculation of the Raji-ffluc cells.

Statistical analysisDifferences between results were evaluated by a one-way or

two-way ANOVA analysis and the Bonferroni test, where appro-priate. Differences were considered statistically significant whenP < 0.05. Statistical analysis was performed usingGraphPad PrismVersion 5 software. Survival curves were constructed using the

Kaplan–Meier method, and statistical analyses of survival wereperformed using log-rank tests.

ResultsDesign and development of the Tet-CD19CAR retroviral vector

To develop inducible CD19CAR T cells, the all-in-one, third-generation (3G) tetracycline-inducible vector (29) was selected asthe backbone vector. Construction of this inducible Tet-OnCD19CAR vector is outlined in Fig. 1A. The Tet-On 3G protein(37), which is an advanced form of the Tet-On Advanced transac-tivator protein, is transcribed by a human PGK promoter. Thetranscribed Tet-On 3G protein binds to a 3G tetracycline responseelement promoter (PTRE3G; ref. 28) in the presence of Dox, andactivates transcription of the downstream gene. To reduce back-ground transcription in the absence of induction, the target gene

Figure 3.

Experimental scheme and surfaceexpression of CD19CAR aftertransduction of Tet-CD19CAR into CD8þ

T cells. A, schematic outline of thegeneration and assay of untransduced,CD19CAR-OR, and Tet-CD19CARtransduced CD8þ T cells. Afterimmunomagnetic bead selection ofCD8þ T cells, the cells were stimulatedwith CD3/28 beads and weretransduced twice, once on day 3 andonce onday 4.Onday 7, 24 hours beforeselection of CAR-positive cells, Dox (100ng/mL) was added. On day 8, EGFRselection and a second bead stimulationwere performed and the transducedcells were split into Dox(þ) and Dox(�)cultures. Untransduced T cells werecultured in parallel with transducedT cells, and all procedures used for theuntransduced T cells were exactly thesame as those used for thetransduced T cells except for virusaddition. Downstream assays wereperformed on day 15. stim, stimulation;Tdx, transduction. B, purity of Tet-CD19CAR and CD19CAR-OR T cellsbefore and after tEGFR selection asmeasured using flow cytometry.CD19CARþ cellswereenrichedby tEGFRselection.C, surfaceexpressionof tEGFRbefore and after tEGFR selection of Tet-CD19CAR cells. Each line represents aresult fromone donor. Datawerepooledfrom three independent experimentsfrom three donors. (�� , P < 0.01, paired ttest). D, surface expression of CD19CARprior to functional analysis on day 15 asmeasured using flow cytometry. E,CD19CAR surface expression on Tet-CD19CAR T cells in the presence orabsence of Dox. Data pooled from threeindependent experiments from threedonors are shown (mean and SEM;�� , P < 0.01, one-way ANOVA).Representative flow plots of threeindependent experiments from threedonors (B and D). NS, not statisticallysignificant.






(CD19CAR-T2A-tEGFR gene) was inserted into the vector in the 30

to 50 direction. A Tet-OnCD19CARplasmid vector was prepared inthe abovemanner and introduced into cells of the Phoenix-Amphoretrovirus packaging cell line. The resulting culture supernatantwascollected in order to obtain the retroviral vector.

Transduction of the Tet-CD19CAR geneTo determine whether Tet-CD19CAR gene introduction by

the prepared retroviral vector and its induced expression byDox administration were possible, the Tet-CD19CAR gene wastransduced into the SUP-T1 (38) human T-cell leukemia tumorcell line. Flow cytometric analysis confirmed tEGFR expressionafter the gene transduction, and the positive fraction was sortedto obtain tEGFRþ cells at >95% purity (Fig. 1B). Tet-CD19CARþ

SUP-T1 cells were positive for both tEGFR and Fc in thepresence of Dox, which showed that Tet-CD19CAR could beinduced by the addition of Dox (Fig. 1C). However, Tet-

CD19CARþ SUP-T1 cells apparently exhibited higher surfaceexpression of Fc and the tEGFR without Dox compared withthat of the untransduced control (Fig. 1C, dashed lines and grayhistograms, respectively).

Effect of Dox concentration on CAR expression dynamicsTet-CD19CARþ SUP-T1 cells were further analyzed to deter-

mine the Dox concentration required for induction of CARexpression. Flow cytometric analysis of both EGFR and Fc stainingdemonstrated that administration of Dox at a concentration of100 ng/mL or more was sufficient to obtainmaximal CAR expres-sion (Fig. 2A and B). CAR expression kinetics at initiation anddiscontinuationofDox administration to Tet-CD19CARþ SUP-T1cells were then examined. Flow cytometric measurements usingboth antibodies indicated that CAR expressionpeaked at 12 hoursafter Dox administration (Fig. 2C) and reached background-level24 hours after Dox removal from the culture medium (Fig. 2D).

CFSE (Target cell)

Untransduced

A

C

B

Figure 4.

Tet-CD19CAR T cells exhibited CD19-specific cytotoxicity only in thepresence ofDox.A andB,Cytotoxicitiesof the indicated CAR T cells against (A)CD19-K562 or (B) K562 cells wereassessed at the E:T ratio of 10:1 ina 51Cr release assay. Themeans and SEMare shown (�� , P < 0.001, one-wayANOVA analysis). NS, not statisticallysignificant. Each dot indicates mean oftriplicate results of independent T-celllines. C, the indicated CAR T cells andCFSE-labeled CD19-K562, Raji, orSU-DHL6 cells were cultured at a 1:1ratio without IL2 supplementation for96 hours. The percentages of survivingCAR T cells and various residual celllineswithin the live cell gates are shown.Data are representative of threeindependent experiments using threeindependent Tet-CD19CAR T-cell lines.

Sakemura et al.





Generation and purification of Tet-CD19CAR–transducedT cells

To investigate whether Tet-CD19CAR T-cell function could becontrolled by switching Dox on and off, we transduced primary Tlymphocytes from PBMCs obtained from healthy donors. Asdescribed in Materials and Methods, the transduced and untrans-duced CD8þ lymphocytes were prepared. After day 11, the culturewasdivided intoaDox(þ) groupandaDox(�) group, and the cellswere harvested on day 15 for various functional assays (Fig. 3A).Although the transduction efficiency of Tet-CD19CAR was onlyapproximately5%to10%, followinga tEGFRpurificationprocess apurity of more than 90% of tEGFRþ cells was obtained for bothCD19CAR-OR and Tet-CD19CAR T cells (Fig. 3B and C). After anadditional course of expansion culture, both CD19CAR-OR andDox(þ)Tet-CD19CARTcellshadmaintainedapurityofmore than

80%,whereasDox(�) Tet-CD19CART cellswere almost complete-ly negative for CD19CAR expression (Fig. 3D). Dox(�) Tet-CD19CAR T cells were "leaky" in their expression of CAR, com-pared with untransduced T cells as well as with Tet-CD19CARþ

SUP-T1 cells [Fc-mean fluorescence intensity (MFI; mean� SEM);untransduced, 89 � 8; Dox(�), 1,706 � 536; Fig. 3D and E].However, the Tet-CD19CAR expression induced by Dox wasapproximately 20-fold higher than that of the noninduced state[Fc-MFI (mean � SEM); Dox(þ), 37,423 � 5,617; Fig. 3E].

Cytotoxicity of Tet-CD19CART cells against CD19-K562 cells inthe presence of Dox

Because the Tet-CD19CAR was expressed weakly even in thenoninduced state, therewas a concern aboutwhetherDox(�) Tet-CD19CAR T cells might function as CD19CAR T cells in amanner

Untdx

Untdx

Untdx

Untdx

A

B C D E

Po

siti

ve

Figure 5.

Tet-CD19CAR T cells produce andsecrete various cytokines uponstimulation with CD19-positive cell linesonly in the presence of Dox. A, IFNgproduction after CD19 stimulation.CD19CAR-OR T cells or Tet-CD19CART cells cultured with or without Doxwere stimulated with various CD19þ

tumor cells at a 1:1 ratio for 4 hours, andwere then permeabilized and stainedfor intracellular IFNg . Representativeflow plots of three independentexperiments from three donors areshown.B, thepercentages of T cells thatstained positive for IFNg are shown.CD19CAR-OR T cells or Tet-CD19CART cells cultured with or without Doxwere stimulated with CD19-K562 cellsand stained for intracellular IFNg as inA. Data were pooled from threeindependent experiments withTet-CD19CAR T cells from three donors(mean and SEM; �� , P < 0.01, one-wayANOVA analysis). C–E, the secretion of(C) IL2, (D) IFNg , and (E) TNFa into theculture supernatant upon stimulation ofthe indicated CD19CAR T cells withCD19-K562 cells was analyzed usingELISA. CD19CART cellswere stimulatedwith g-irradiated CD19-K562 at a ratioof 1:1, and culture supernatants wereharvested after 16 hours (mean andSEM; �� , P < 0.0001; �� , P < 0.01, one-way ANOVA analysis). NS, notstatistically significant; Untdx,untransduced.






similar to CD19CAR-OR cells. We investigated the cytolytic activ-ity of Tet-CD19CAR T cells in the noninduced state with standard51Cr-release assays. The cytolytic activity of CD19CAR-OR T cellsand Dox(þ) Tet-CD19CAR T cells against both CD19-K562 andK562 cellswas equivalent. ComparedwithDox(þ) Tet-CD19CART cells, the cytolytic activity of Dox(–) Tet-CD19CAR T cells wassignificantly lower against CD19-K562 cells, but was comparabletoward K562 cells. Compared with untransduced cells, Dox(�)Tet-CD19CAR T cells were slightly more cytotoxic against CD19-K562, but the difference was not statistically significant (Fig. 4Aand B). To evaluate the integrated activity of Tet-CD19CARcytotoxicity and expansion of CD19þ target cells, coculture assayswith CD19-K562, Raji, and SU-DHL6 cells were performed.CD19-MFI was 2,809, 2,505, and 2,166 for CD19-K562, Raji,and SU-DHL6, respectively. Untransduced or Dox(�) Tet-CD19CART cells didnot showa significant cytotoxic effect againstCD19þ target cells, whereas Dox(þ) Tet-CD19CAR andCD19CAR-OR T cells eradicated the target cells (Fig. 4C). Thesedata showed that the weak CAR expression observed on Dox(�)Tet-CD19CAR T cells is below the range at which it exercisessignificant cytotoxic activity.

IFNg from Tet-CD19CAR T cells upon exposure to CD19-expressing tumor or B cells

We then investigated whether Dox administration could reg-ulate Tet-CD19CAR expression so that it could influence thecytokine-producing and cytokine-secreting ability of the trans-duced cells. We first determined intracellular IFNg production ofthe various CD19CAR T cells after stimulation with variousCD19þ cell lines and K562 cells by staining for intracellular IFNg .Although untransduced and Dox(�) Tet-CD19CAR T cellsshowed almost no intracellular IFNg production, Dox(þ) Tet-CD19CAR and CD19CAR-OR T cells showed robust and compa-rable intracellular IFNgþ fractions (Fig. 5A and B). We thentitrated the dose of Dox to assess whether it was possible toregulate the degree of IFNg production. Dox was administered 24hours prior to the assay using various concentrations of Dox. CARexpression on Tet-CD19CAR T cells was precisely regulated byDox in a dose-dependent manner (Supplementary Fig. S2A).Intracellular IFNg measurement showed that at least 75 ng/mLof Dox was required for Tet-CD19CAR T cells to produce themaximum amount of IFNg after stimulation with CD19-K562cells (Supplementary Fig. S2B).

We next examined whether Tet-CD19CAR T cells retained asafety profile against normal B cells in the absence of Dox. Byusing immunomagnetic beads, CD19þ cells with a purity ofmorethan 90% were obtained from donor PBMCs. The CD19 MFI ofnormal B cells was lower than that of Raji and of other CD19þ

tumor cell lines (Supplementary Fig. S3A). CD19CAR-OR T cellsand Tet-CD19CAR T cells were stimulated with normal B cells,and intracellular IFNg production was assessed. WhereasCD19CAR-OR T cells and Dox(þ) Tet-CD19CAR T cells showedsimilar IFNg production, Dox(�) Tet-CD19CAR T cells did notproduce IFNg (Supplementary Fig. S3B and S3C). We furtherassessed the secretion of various cytokines by the various CAR Tcells using ELISA. CAR T cells were cocultured with K562/CD19-K562 cells, and the concentration of IL2, IFNg , and TNFa in theculture supernatants was measured after 16 hours. No significantcytokine production was observed in any coculture with K562cells (IFNg , less than 0.3 ng/mL; IL2, less than 0.06 ng/mL). Dox(�) Tet-CD19CAR T cells secreted only low amounts of each

cytokine, which were similar to those of the untransduced T cells,whereas the Dox(þ) Tet-CD19CAR T cells produced amounts ofeach cytokine similar to those produced by CD19CAR-OR T cells(Fig. 5C–E).

Expansion of Tet-CD19CAR T cells upon CD19 stimulation inthe presence of Dox

Robust proliferation after exposure to target cells is one of themost important functions of CAR T cells. To investigate possibledifferences in the ability of Dox(þ) and Dox(�) Tet-CD19CAR Tcells to proliferate upon CD19 stimulation, CAR T cells werecocultured with CD19-K562 cells in a 1:1 ratio without IL2supplementation, and CAR T-cell expansion was measured bycounting viable cells. No growth was observed in the untrans-duced T cells, and only minimal expansion was observed in theDox(�) Tet-CD19CAR T cells (Fig. 6A). On the other hand, theexpansion of Dox(þ) Tet-CD19CAR T cells was similar to that ofCD19CAR-OR T cells. The difference in cell growth between Dox(þ) and Dox(�) Tet-CD19CAR T cells was statistically significant96 hours after stimulation with CD19-K562 cells (Fig. 6A).

Expansion upon stimulation with other CD19þ cells, such asRaji and SU-DHL6 cells, was also examined. In the presenceof Dox, Tet-CD19CAR T cells showed statistically significant

Untransduced

A

CB

Untdx

Untdx

Figure 6.

Tet-CD19CAR T cells exhibited proliferative potential only in the presence ofDox. A–C, the indicated CD19CAR T cells were incubated with g-irradiated (A)CD19-K562, (B) Raji, or (C) SU-DHL6 cells at a ratio of 1:1, without exogenous IL2.CAR T-cell expansion was measured by counting viable cells. Dox(þ) Tet-CD19CAR T cells showed significantly higher cell expansion after 4 dayscompared with Dox(�) Tet-CD19CAR T cells (mean and SEM; �� , P < 0.001,two-way ANOVA analysis). Data were pooled from three independentexperiments using three independent Tet-CD19CAR T-cell lines. Data are shownas fold expansion over a time course in (A), and as fold expansions at 120 hours inB and C. NS, not statistically significant.

Sakemura et al.





expansion, compared with Dox(�) Tet-CD19CAR T cells after 5days of culture with either of these cell lines (Fig. 6B and C).

Dox administration functionally segregated Tet-CD19CAR Tcells in an in vivo model

To further assess the therapeutic effect of Tet-CD19CAR T cellsand their functional segregation byDox administration, we used aRaji-xenografted NOG mouse model. Raji-ffluc cells (5 � 105)were inoculated by tail-vein injection, and, 7 days later, a singledose of 5� 106 Tet-CD19CAR, CD19CAR-OR, or untransduced Tcells was administered i.v. Before CAR T-cell injection, tumor flux

was similar in all the treatment groups. Dox was given in drinkingwater, and Raji-ffluc tumor growth was monitored every 7 daysusing bioluminescence imaging (Fig. 7A). Tumor flux was calcu-lated as the sum of tumor signal intensity measured in the entirebody of each mouse. Tumor growth was suppressed in mice thatreceived Tet-CD19CAR T cells in the presence of Dox. Thissuppression was similar to that seen in mice that receivedCD19CAR-OR T cells. In contrast, tumor flux inmice that receivedTet-CD19CAR T cells in the absence of Dox increased similarly tothat ofmice that receiveduntransduced T cells (Fig. 7B andC). Theobserved tumor flux in the Tet-CD19CAR T cells in the presence of

A

B

C D

Figure 7.

Dox administration was required for Tet-CD19CAR T cells to show a suppressive function against a CD19þ tumor in vivo. A, schematic representation ofthe in vivo experimental procedure. NOG mice were inoculated with 5 � 105 Raji-ffluc cells via tail-vein injection on day 0. On day 7, mice were treated with eitheruntransduced CD8þ T cells, Tet-CD19CAR T cells with/without Dox, or CD19CAR-OR T cells. The tumor burden of the mice was then assessed weekly usingbioluminescence imaging (BLI).B,BLI ofmice bearing Raji-ffluc tumors. Mice treatedwith Tet-CD19CAR T cells in the presence of Dox showed statistically significanttumor regression. On the other hand, Tet-CD19CAR T cells without Dox could not suppress tumor growth. Data are representative of three independent experimentsusing three independent Tet-CD19CAR T-cell lines. C, tumor flux in individual mice on days 7, 14, 21, and 28 of inoculation of the Raji-ffluc cells. Tumorflux was calculated as the sum of the tumor signal intensity of the entire body following a 30-second exposure time. Mice treated with Tet-CD19CAR T cellsin the presence of Dox showed statistically significant tumor regression compared with mice treated with Tet-CD19CAR T cells without Dox (�� , P < 0.01, one-wayANOVA). The statistically significant difference was observed on days 21 and 28. Data are plotted as means � SEM. D, Kaplan–Meier curves of the survivalof mice treated with Dox(þ) Tet-CD19CAR T cells or Dox(�) Tet-CD19CAR T cells. Mice treated with Dox(þ) Tet-CD19CAR T cells showed significantlyprolonged survival compared with mice treated with Dox(�) Tet-CD19CAR T cells (�� , P < 0.0001, log-rank test). Data were pooled from three independentexperiments with Tet-CD19CAR T cells from three donors, including 9 to 12 mice per group in C and D.






Dox was significantly lower than that in the Tet-CD19CAR T cellsin the absence of Dox (P < 0.01).

Additionally, the mice that received Tet-CD19CAR T cells withDox showed prolonged survival compared with the mice thatreceived Tet-CD19CAR T cells without Dox (P < 0.0001, log-ranktest; Fig. 7D). Despite the transient tumor suppression in themicethat received Tet-CD19CAR T cells in the presence of Dox, allmiceeventually died with tumor growth.

DiscussionIn the present study, we fused anti-CD19CAR into an all-in-

one, 3G tetracycline-inducible vector. Tet-CD19CAR retrovirusvector was prepared, and SUP-T1 and primary T cells weresuccessfully transduced. With 75 ng/mL or more of Doxsupplementation, Tet-CD19CAR expressed CD19CAR in bothSUP-T1 cells and in human primary CD8þ cells. In functionalassays, Dox(þ) Tet-CD19CAR T cells effectively lysed a varietyof CD19þ target cells at various E:T ratios, secreted cytokinessuch as IFNg , IL2, and TNFa, and proliferated upon CD19þ cellstimulation in vitro. Furthermore, Dox(þ) Tet-CD19CART cells also showed significant antitumor activity in a mousexenograft model. On the other hand, Dox(�) Tet-CD19CAR Tcells could not behave as CAR T cells either in vitro or in vivo. Tothe best of our knowledge, this is the first report of the controlof CAR expression and function by the Tet-On induciblesystem.

High response rates have been reported in clinical trials ofCD19CAR T-cell therapies for B-cell malignancies, even inpatients with relapsed or refractory-phase diseases. In addition,other clinical trials of CAR T-cell therapy or T-cell receptor (TCR)gene–modified therapies that have targeted solid tumors havealso shown favorable outcomes (39–42). However, some casesresulted in unpredictable or uncontrollable adverse events due toon-target/off-tumor toxicity, such as pulmonary events resultingfromHER2-specific CART-cell therapy (13), lethal cardiac toxicitydue to MAGE-A3 TCR T-cell therapy (43), or a severe neurologictoxicity derived from MAGE-A3 TCR T-cell therapy (44). Theseunfavorable events were sometimes life threatening and associ-ated with lethal outcomes.

To preclude such problems, combining suicide gene technol-ogies, such as HSV/TK (19–21) or iCasp9 (22–24), with adoptiveT-cell therapy has been investigated. In particular, iCasp9, whichconsists of the human FK506-binding protein with an F36Vmutation linked tohuman caspase-9, induces rapid andprofounddeath of gene-modified cells and can effectively remove trans-ferred T cells, as reported in some preclinical and clinical trials(24). These suicide gene strategies were developed to strengthenthe safety profiles of adoptive T-cell therapies, but inducingapoptosis of gene-modified T cells disables them and could welllead to cancer progression. This possibility led us to developinducible CAR T cells.

Instead of inducing permanent deletion of CAR T cells in asuicidal manner, we sought to regulate CAR expression in aninducible fashion. In clinical settings, this strategy may allowcontrol of the transgene expression by administration of a drug toa patient on demand. Conversely, when shutdown of the trans-gene expression is required, drug administration can be stopped.Several drug-inducible transgene expression systems have beendescribed to date, and the representative inducible vectors are theTet-On system and the Cre/LoxP system. The Cre-recombinase

efficiently targets the LoxP sequence and catalyzes the insertion orexcision of DNA between two LoxP sites (45). However, a majorlimitation of the Cre/LoxP system is that it permanently eitheractivates or inactivates the gene of interest, whereas the Tet-Oninducible systems allow reversible regulation of the gene ofinterest. We therefore considered the Tet-On system to be moresuitable for CAR T-cell therapy. Other systems for small-moleculeregulation of CAR-T function have also been developed, in whichsignaling from the CAR, rather than CAR expression, is regulatedby a small molecule (46). The CAR function is reportedly fine-tuned by the "ON-switch" CAR systems, showing that the strategyis promising.

The Tet-On inducible vector that we used in the presentstudy has a high signal-to-noise ratio of up to 14,000- to25,000-fold in human cell lines (29). Although this is a highlyefficient inducible vector, some background expression wasstill seen, such as weak surface CAR expression with subse-quent low cytotoxicity toward CD19þ cell lines, and lowproliferation upon CD19 stimulation. However, vector leak-iness was limited to situations where a target cell physicallyencountered CAR T cells over a short period, such as over the4-hour incubation time in the 51Cr release assay or in acoculture assay of less than 72 hours. Moreover, Tet-CD19CART cells did not secrete various cytokines without Dox and didnot proliferate even over a short period of time. Thus, incoculture assays that were performed with an incubation timeof 96-hour Dox(�) Tet-CD19CAR T cells lost their ability tokill the target cells. A similar effect was seen in the xenograftmodel in which Dox(�) Tet-CD19CAR T cells were unable tokill tumor cells in vivo. The combined observations indicatedthat the functions of Tet-CD19CAR T cells could be success-fully controlled by Dox administration, even though this maynot occur immediately.

Another possible drawback of the Tet-On inducible system isthe potential for immunogenicity. Because this system uses a partof bacterial protein fused to a viral protein, this could lead torejection of the gene-modified cells (47), as has been describedwith the HSV/TK suicide system or even with the murine derivedscFv protein (4, 48).

To achieve immediate removal of T cells, other groups havedemonstrated an alternative safety system that involves elimina-tion of T cells by targeting either the tEGFR (33, 49) or CD20 (50),which are coexpressed with CD19CAR on the T-cell surface.According to these reports, effective removal of the target cellscould be achieved by specific antibody administration. Becausewe adopted the strategy of coexpressing CAR and the tEGFR, ourconstruct has a built-in alternative for immediate shutdown ofgene-modified T cells for added safety.

As we have seen in this study, one of the advantages of thesystem that we used is the safety profile of Tet-CD19CAR Tcells against autologous B cells. Without Dox treatment,incubation of CD19þ cell lines with Tet-CD19CAR T cellsinduced little intracellular IFNg and very low levels of secretedcytokines. This result therefore indicates that adoption of theTet-On inducible system may bring a major benefit in terms ofavoiding on-target/off-tumor effects. Our observation of thedifferent level of recognition between CD19lo cells (normal Bcells) and CD19hi tumor cells by Dox (þ) Tet-CD19CART cells suggests that we could adjust the CAR or TCR expres-sion to recognize only the target antigen expression of thetumor by applying inducible technology.

Sakemura et al.





The major problem regarding CAR T-cell therapy is the risk ofthe on-target/off-tumor effect, as we discussed above. Tumor-associated antigens (TAA) are commonly expressed in normaltissues, resulting in activation of TAA-specific T cells in thesetissues. Thus, adoptive T-cell therapy targeting TAA may resultin normal tissues being attacked, and this is amajor concernwhendesigning a new TAA-specific adoptive T-cell therapy. The appli-cation of an "inducible switch" technology may allow us to fine-tune CAR T-cell activity to discriminate between tumor versusnormal tissue, and to extend the safety profile of such therapy. Byapplying this technology, it might be possible to expand newpotential therapies targeting myeloid leukemia or solid tumors,which have been considered difficult to deal with because of on-target/off tumor effects.

In conclusion, an inducible CAR expression approach mayprovide a safe and efficient treatment strategy against CD19þ

malignancies. This strategy might also open the way to treat othermalignancies in combination with other CAR or TCR gene–modified T cells.

Disclosure of Potential Conflicts of InterestH. Kiyoi reports receiving commercial research support from Bristol-Myers

Squibb, Chugai Pharmaceutical Co. Ltd., Kyowa Hakko Kirin Co. Ltd., Dai-nippon Sumitomo Pharma, Zenyaku Kogyo, and Fujifilm Corporation. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: R. Sakemura, S. Terakura, K. WatanabeDevelopment of methodology: R. Sakemura, S. Terakura, K. Watanabe

Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Sakemura, S. Terakura, E. Takagi, K. Miyao,T. Goto, R. HanajiriAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):R. Sakemura, S. Terakura, E. Takagi, K.Miyao, T.Goto,R. HanajiriWriting, review, and/or revision of the manuscript: R. Sakemura, S. Terakura,J. Julamanee, M. Murata, H. KiyoiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Julamanee, D. Koyama, M. Murata, H. KiyoiStudy supervision: S. Terakura, M. Murata, H. Kiyoi

AcknowledgmentsWe are very grateful to Yoko Matsuyama and Chika Wakamatsu for their

technical assistance.

Grant SupportThis work was supported by grants from the Foundation for Promotion of

Cancer Research (Tokyo, Japan; to S. Terakura), the Japan Society for thePromotion of Science KAKENHI (24790969 and 15k09497 to S. Terakura),Practical Research for Innovative Cancer Control (15ck0106067h0002 to S.Terakura), and Practical Research Project for Allergic Diseases and Immunology(15ek0510010h0003 to M. Murata). J. Julamanee was supported by a grantfrom the Research Foundation of Prince of Songkla University (grant MOE.0521.1.0601(2)/6058).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received February 26, 2016; revised April 21, 2016; accepted May 3, 2016;published OnlineFirst June 21, 2016.

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2016;4:658-668. Published OnlineFirst June 21, 2016.Cancer Immunol Res Reona Sakemura, Seitaro Terakura, Keisuke Watanabe, et al. Receptor Expression upon Drug AdministrationA Tet-On Inducible System for Controlling CD19-Chimeric Antigen

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