ChimericAntigenReceptorTCellswithDissociatedSignaling ...redirected activityin vitro and in vivo (ref. 13; Fig. 1A and B). The costimulatory anti-FRa CAR (F-28) construct comprised

Research Article

ChimericAntigenReceptorTCellswithDissociatedSignalingDomains Exhibit Focused Antitumor Activity with ReducedPotential for Toxicity In Vivo

Evripidis Lanitis1,3, Mathilde Poussin1, Alex W. Klattenhoff2, Degang Song1, Raphael Sandaltzopoulos3,Carl H. June2, and Daniel J. Powell Jr1,2

AbstractAdoptive immunotherapy using T lymphocytes genetically modified to express a chimeric antigen receptor

(CAR-T) holds considerable promise for the treatment of cancer. However, CAR-based therapies may involve on-target toxicity against normal tissues expressing low amounts of the targeted tumor-associated antigen (TAA). Tospecify T cells for robust effector function that is selective for tumor but not normal tissue, we developed a trans-signaling CAR strategy, whereby T-cell activation signal 1 (CD3z) is physically dissociated from costimulatorysignal 2 (CD28) in two CARs of differing antigen specificity: mesothelin and a-folate receptor (FRa). HumanT cellswere geneticallymodified to coexpress signal 1 (anti-Meso scFv-CD3z) and signal 2 (anti-FRa scFv-CD28) CARs intrans. Trans-signaling CAR-T cells showedweak cytokine secretion against target cells expressing only one TAA invitro, similar to first-generation CAR-T cells bearing CD3z only, but showed enhanced cytokine secretion uponencountering natural or engineered tumor cells coexpressing both antigens, equivalent to that of second-generation CAR-T cells with dual signaling in cis. CAR-T cells with dual specificity also showed potent anticanceractivity and persistence in vivo, which was superior to first-generation CAR-T cells and equivalent to second-generation CARs. Importantly, second-generation CAR-T cells exhibited potent activity against cells expressingmesothelin alone, recapitulating normal tissue, whereas trans-signaling CAR-T cells did not. Thus, a dualspecificity, trans-signaling CAR approach can potentiate the therapeutic efficacy of CAR-T cells againstcancer while minimizing parallel reactivity against normal tissues bearing single antigen. Cancer Immunol Res;1(1); 43–53. �2013 AACR.

IntroductionGenetic redirection of T cells with chimeric antigen recep-

tors (CAR) that link an antigen-specific single-chain antibodyfragment (scFv) to intracellular signaling domains is at theforefront of cancer immunotherapy (1, 2). CARs functionallyredirect T cells with high specificity to various surface antigenson tumor cells independent of MHC restriction and antigenprocessing, and therefore bypass major mechanisms by whichtumors escape immune recognition. T cells bearing a first-generation CAR having only the T-cell CD3z intracellularsignaling domain either fail to persist or become anergic, astumor cells frequently lack requisite ligands for costimulation(3). This incomplete activation of CAR-T cells in vivo seems to

limit their persistence, and has thus hampered their efficacy inclinical trials for lymphoma (4), neuroblastoma (5), ovariancancer (6), or renal cell cancer (7). To overcome these limit-ations, second-generation CAR-T cells were developed thatincorporate the intracellular domain of various costimulatorymolecules such as CD28, 4-1BB, OX-40, and CD27 leading toimproved expansion, persistence, and activity of the CAR-Tcells in preclinical mouse models (8, 9) and in clinical studies(2, 10, 11). Still, the enhanced potency of these CARs can beassociated with autoimmunity due to on-target toxicitiesagainst normal tissues expressing lower levels of the tumor-associated antigen (TAA). For instance, administration of highnumbers of T cells bearing an anti-ErbB2 CAR comprising theCD28 and 4-1BB costimulatory domains to a lymphodepletedpatient with metastatic colon cancer resulted in rapid onset ofpulmonary toxicity with lung infiltrates and a "cytokine storm"followed by cardiac arrest and death (12). Clearly, the devel-opment of strategies limiting potential early- or late-phasetoxicity is of importance. We have previously generated a fullyhuman anti-mesothelin CAR capable of conferring potent invitro and in vivo effector functions to primary T cells againstmesothelin-expressing tumors (13). Mesothelin-redirectedCAR-T cells also hold the potential to inflict damage againstnormal mesothelial cells lining the pleura, peritoneum as wellas epithelial cells of the trachea, tonsils, fallopian tube, and the

43

Authors' Affiliations: 1Department ofObstetrics andGynecology,OvarianCancer Research Center; 2Department of Pathology and Laboratory Med-icine, Abramson Cancer Center, University of Pennsylvania, Philadelphia,Pennsylvania; and 3Department ofMolecular Biology andGenetics, Demo-critus University of Thrace, Alexandroupolis, Greece

Corresponding Author: Daniel J. Powell Jr, University of Pennsylvania,3400 Civic Center Blvd., Bldg. 421, Smilow Center for TranslationalResearch, Rm 8-103, Philadelphia, PA 19104. Phone: 215-573-4783; Fax:215-573-7627; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-13-0008

�2013 American Association for Cancer Research.

CancerImmunology

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rete testis, which express low levels of mesothelin (14, 15). Tolimit "on-target" toxicity and improve tumor-focused targetingand attack, we have developed and tested the concept of atrans-signaling CAR strategy where the T-cell activation signal1 (CD3z module) is physically dissociated from the costimu-latory signal 2 (CD28module). Becausemesothelin and a-folatereceptor (Fra) are TAAs coexpressed in the majority of epi-thelial ovarian cancers, but expressed differentially and at lowlevels in normal tissues (14, 16–19), 2 independent CARs ofdistinct specificitywere used: a signal 1CAR (meso-CD3zonly),and a signal 2 CAR (FRa-CD28 only) using prevalidated scFvs(13, 20). In this fashion, T cells transduced to coexpress bothCARs exhibit potent in vitro and in vivo effector functions thatare driven by tumor encounter and coupled with diminisheddamage to normal tissues.

Materials and MethodsCAR constructs

The F-28 CAR was constructed by using lentiviral vectorbackbone constructs previously described (20). CAR construc-tion and lentivirus production are detailed in SupplementaryMaterials and Methods.

Recombinant lentivirus productionHigh-titer replication-defective lentiviral vectors were pro-

duced and concentrated as previously described (13).

Human T-cell transductionPrimary human T cells, purchased from the Human Immu-

nology Core at University of Pennsylvania (Philadelphia, PA),were isolated from healthy volunteer donors following leuka-pheresis by negative selection. All specimens were collectedunder a University Institutional Review Board–approved pro-tocol, and written informed consent was obtained from eachdonor. T-cell activation and lentiviral transduction was con-ducted as previously described (13).

Functional assaysCytokine release assays were carried out using an IFN-g

ELISA Kit (Biolegend). 51Cr-release and CD107 degranulationassays of cytolysis were conducted as previously described(13, 21). The Cytometric Bead Array and apoptosis assay wereconducted according to manufacturer's instructions (BD Bios-ciences). Functional assays are further detailed in the Supple-mentary Materials and Methods.

Xenograft model of ovarian cancerMouse studies were carried out as detailed in the Supple-

mentary Materials and Methods.

ImmunohistochemistryFresh frozen tumor samples were sectioned for immuno-

histochemical analysis as described in the SupplementaryMaterials and Methods.

Statistical analysisStatistical evaluation was conducted using two-tailed Stu-

dent t test. GraphPad Prism 4.0 (GraphPad Software) was used

for the statistical calculations. P values less than 0.05 wereconsidered significant.

ResultsCAR construction

Anti-mesothelin CAR constructs comprised the P4 scFvlinked to a CD8a hinge and transmembrane region, followedby a CD3z signaling moiety alone (referred to as M-z) or intandem with the CD28 intracellular signaling motif (M-28z),which were previously shown to confer specific mesothelin-redirected activity in vitro and in vivo (ref. 13; Fig. 1A and B).The costimulatory anti-FRa CAR (F-28) construct comprisedthe MOv-19 scFv linked to a CD8a hinge and the CD28transmembrane region and intracellular signaling motif; thesignaling-deficient F-Dz construct lacks a functional signal-ing domain. F-28 CAR expression was monitored throughthe GFP transgene, which is included in the CAR lentiviralconstruct with its expression driven from the same EF1apromoter, which regulates CAR expression. GFP signal washighly correlated with protein-L binding to the mouse scFvof the CAR highlighting the reliability of this reporter geneexpression (Supplementary Fig. S1). Primary human T cellswere efficiently transduced with the 2 CAR-encoding lenti-viral vectors with more than 40% dual-transduced T cellsreproducibly expressing both CARs (Fig. 1C). CAR-T cellpopulations were adjusted to equivalent frequencies of anti-mesothelin CAR-T cells (60%–70%) by adding untransducedT cells for all functional assays.

Trans-signaling CAR-T cells exert superior antigen-specific cytokine secretion in vitro

To evaluate the in vitro effector functions of CAR-T cells inresponse to cells that express mesothelin alone versus tumorcells that coexpress mesothelin and FRa, TAA-negative C30cancer cell lines, which lack endogenous CD80/86 costimu-latory ligands were engineered to overexpress either one orboth of these antigens (Fig. 2A). T cells engineered to expressthe M-z CAR recognized and secreted similar levels of IFN-gwhen cocultured with C30 cells expressing either mesothelin(C30M) or mesothelin and FRa (C30M/F). In comparison,second-generation M-28z CAR-T cells exerted superior IFN-g secretion against all C30 cell variants expressing mesothe-lin. In contrast, trans-signaling M-z/F-28 CAR-T cells pro-duced low levels of IFN-g against C30M, similar to M-z CAR-T cells, but exerted enhanced IFN-g production whenexposed to C30M/F tumor cells (Fig. 2B). M-z/F-28 CARactivity against C30M/F cells surpassed that of M-z CAR-Tcells showing that expression of both antigens by tumor cellspromotes the costimulation of M-z/F-28 CAR-T cellsthrough the F-28 CAR (Fig. 2B). Consistent with this notion,coexpression of the M-z and the signaling-deficient F-DzCAR in T cells did not enhance their response againstC30M/F cells. M-28z CAR-T cells produced more IFN-g thanM-z/F-28 CAR-T cells using this artificial target cell systemwhere cells are engineered to express antigens at supra-physiologic levels. T cells expressing the F-28 CAR alone didnot produce any IFN-g , consistent with a lack of CD3-z

Lanitis et al.

Cancer Immunol Res; 1(1) July 2013 Cancer Immunology Research44




signaling. Moreover, control T cells transduced to expressGFP or an anti-CD19 CAR containing CD3z with CD28signaling motifs in tandem (CD19-28z; ref. 22) did not pro-duce cytokines after stimulation with CD19-negative C30cells, illustrating the need for antigen-specificity (Fig. 2B).We next tested trans-signaling CAR-T cell activity against

an ovarian cancer cell line that naturally expresses bothmesothelin and FRa, A1847 (Fig. 2C). M-z/F-28 CAR-T cellssecreted IFN-g in response to A1847 that was significantlyhigher than that secreted by M-z CAR-T cells but similar tocis-signaling M-28z CAR-T cells, showing that the naturalexpression of both antigens on tumor cells is capable ofinducing costimulatory effects in trans-signaling CAR-T cells(Fig. 2D). Interleukin (IL)-2 secretion from the dual trans-duced M-z/F-28 CAR-T cells was also similar to second-generation M-28z CAR-T cells and higher than that of M-zCAR-T cells indicating that integrated delivery of signal 1and 2 in trans or cis can enhance production of IL-2 cytokine(Fig. 2E).

Trans-signaling CAR-T cells show in vitro cytolyticpotency

Degranulation is a quantitative indicator of lytic functionby T cells (21). M-z and M-z/F-28 CAR-T cell degranula-tion was accompanied by upregulation of surface coexpres-sion of mobilized CD107 (lysosomal-associated membraneprotein 1) and the activation-associated marker CD69 inresponse to C30M but not when stimulated with either C30or C30F cells (Fig. 3A). Consistent with active costimulation,M-28z CAR-T cells displayed a superior cytolytic phenotypeagainst C30M compared with M-z and M-z/F-28 CAR-T cells.However, exposure to A1847 (Mþ/Fþ) tumor cells with bothantigens led to enhanced degranulation by both M-28zand M-z/F-28 CAR-T cells, relative to M-z CAR-T cells(Fig. 3A). Anti-CD19 CAR-T cells did not degranulate inresponse to C30 or A1847 cells (Supplementary Fig. S2). Inshort-term chromium release assays, all of the variousmesothelin-directed CAR-T cell populations (M-28z, M-z,and M-z/F-28) showed cytolytic activity against single

Figure 1. Generation and expression of mesothelin and Fra-specific CARs. A, schemata of the anti-mesothelin (P4)–based CAR constructs containing theCD3z endodomain alone (M-z) or in combination with the CD28 costimulatory module (M-28z). B, schemata of the anti-FRa (F)–based CAR constructscontaining the scFv (MOV-19) fused through the CD8a hinge and CD28 TM to the CD28 costimulatory module (F-28). A truncated anti-FRa CAR with nosignaling domains is shown (F-Dz). P4, anti-mesothelin scFv; VL, variable L chain; L, linker; VH, variable H chain; TM, transmembrane region. C, P4 CARexpression detected on humanCD3-gated cells via recombinantmesothelin protein staining after transduction with lentivirus comparedwith untransduced Tcells. T cells bearing the F-28 CAR were detected via GFP transgene expression. Transduction efficiencies are indicated with the percentage of CAR-expressing cells within the transduced populations.

Trans-Signaling CAR-T Cells for Focused Tumor Targeting

www.aacrjournals.org Cancer Immunol Res; 1(1) July 2013 45




antigen-expressing C30-M target cells with M-28z CAR-Tcells exhibiting greater cytolytic activity than M-z/F-28 andM-z cells at all ratios tested. A1847 target cells were alsolysed by all anti-mesothelin CAR-T groups, yet M-28z and M-z CAR-T showed statistically comparable and higher lysisthan M-z/F-28 CAR-T cells at effector to target (E:T) ratios�10:1 (Fig. 3B). At lower E:T ratios, M-28z CAR-T cells lyseddual antigen A1847 cells more efficiently than both M-z andM-z/F-28 groups suggesting that in tandem costimulatorysignaling may permit more efficient killing in the short term.Control GFP and F-28 CAR-T cells did not exhibit cytolyticfunction against either cell target.

Trans-signaling CAR-T cells resist AICDIncorporation of costimulatory domains into CARs can

increase resistance of CAR-T cells to apoptosis upon activationby tumors (23). To investigate if provision of CD28 costimula-tion in trans protects T cells from antigen-induced cell death(AICD), single or dual CAR-bearing T cells were measured for

their rate of apoptosis following coculturewith A1847 (Mþ/Fþ)tumor cells. Apoptosis [7-aminoactinomycin D (7-AAD)þ An-nexin Vþ] was elevated in M-z T cells exposed to A1847 (34%)for 3 days but reduced in trans-signaling M-z/F-28 CAR-T cells(0.5%–1%; Fig. 4A). Consistent with past studies (24), cis-signaling M-28z CAR-T cells were also resistant to AICD.Control F-28 or CD19-28z CAR-T cells displayed no AICD inthe absence of specific antigenic stimulation (Fig. 4B).

Dual-specific CAR-T cells possess enhanced antitumorpotency and persistence in vivo

The capability of trans-signaling CAR-T cells to inhibithuman tumor outgrowth was evaluated in vivo in immuno-deficient NOD/SCID/IL-2R-gcnull (NSG) mice inoculated sub-cutaneously with 1 � 106 firefly luciferase-expressing A1847cells. Mice with established tumors (150–200 mm3) receivedintravenous injections of CAR-T cells. Four weeks after thefirst T-cell dose, tumor growth was inhibited modestly in micereceiving M-z CAR-T cells (P ¼ 0.043), compared with saline,

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Figure 2. Trans-signaling CAR-T cells exert superior antigen-specific cytokine secretion in vitro compared with first-generation CAR-T cells. A, detectionof surface mesothelin and/or FRa protein expression in genetically modified C30 tumor cells by flow cytometry. B, primary human T cells transducedwith M-z and F-28 CARs exert superior IFN-g secretion compared with M-z CAR-T cells. Mean IFN-g concentration� SEM (pg/mL) is shown. ��, P < 0.01comparing M-z and M-z/F-28 CAR-T cells against C30-M/F cancer cells. C, coexpression of mesothelin and FRa by A1847 ovarian cancer cell line. D,trans-signaling CAR-T cells secrete higher levels of IFN-g in response to A1847. Mean IFN-g concentration � SEM (pg/mL) is shown. �, P < 0.05comparing M-z and M-z/F-28 CAR-T cells against A1847 cancer cells. E, trans-signaling CAR-T cells secrete more IL-2 (pg/mL) in response to A1847than M-z CAR-T. Values represent the mean � SEM concentration of triplicate wells.

Lanitis et al.





CD19-28z CAR-T cells, F-28 CAR-T cells, or GFP-T cell controlgroups (Fig. 5A). Compared with M-z CAR-T cells, transfer ofM-28z or M-z/F-28 CAR-T cells mediated even more potentinhibition of tumor outgrowth (P ¼ 0.028) indicating thatincorporation of CD28 signaling domain in cis or in transenhances antitumor activity in vivo against tumors coexpres-sing both TAAs (Fig. 5A). Bioluminescence imaging resultsfrom treated mice confirmed the lower tumor burden in thosetreated with the trans-signaling CAR-T cells compared withfirst-generation CAR-T cells. Tumors exhibited similar lowlevels of bioluminescence regardless of whether the mice weretreated with trans- or cis-signaling CAR-T cells (Fig. 5B). Twoweeks after first T-cell dose, peripheral blood CD8þ T andCD4þ T cell counts frommice injected withM-28z orM-z/F-28CAR-T cells were similar and higher than in theM-z group (P <0.05; Fig. 5C). No substantial human T-cell persistence wasobserved inmice treatedwith CD19-28z CAR-T, F-28 CAR-T, orGFP-T cells.

Trans-, but not cis-, signaling CAR-T cells exhibit morelimited activity against cells bearing single antigen invivoFRa-deficient A1847 cells (A1847Mþ/F�) were generated

via transduction with lentiviral particles encoding a FRa-specific short hairpin RNA (shRNA), as a surrogate for normalhuman mesothelial cells expressing only mesothelin for usein modeling in vivo. Fluorescence-activated cell sorting result-ed in an enriched cancer cell population (�98%), which

lacked surface FRa expression (Supplementary Fig. S3A). FRaexpression was unaltered after engineering cells with controlshRNA (A1847Mþ/Fþ). Besides the lack of FRa expression,the 2 lines seemed identical by all measures. No differencein in vitro growth kinetic or viability of A1847Mþ/Fþ andA1847Mþ/F�cells was observed (Supplementary Fig. S4) andcontrol anti-FRa (F-28z) CAR-T cells produced minimal IFN-gin response to stimulation with A1847 cells after FRa silencing;similar to levels seen in response to PEO-1 ovarian cancer cells,which have undetectable surface FRa expression via flowcytometry (ref. 9; Supplementary Fig. S5A and S5B). In cocul-ture assays, IFN-g secretion by trans-M-z/F-28 CAR-T cells wassignificantly reduced in response to A1847Mþ/F� comparedwith A1847Mþ/Fþ, a confirmation of potent effector functiononly upon engagement of both antigens (Supplementary Fig.S6). In addition, M-z/F-28 and M-z CAR-T cells reacted equi-valently against A1847Mþ/F� cells. As expected, cis-M-28zCAR-T cells secreted significantly higher amounts of IFN-gagainst A1847Mþ/F�, similar to the IFN-g levels achievedagainst A1847Mþ/Fþ.

To evaluate the in vivo potency of trans- or cis-signalingT cells against A1847 cells coexpressing or lacking FRa,A1847Mþ/Fþ and A1847Mþ/F� cells were inoculated sub-cutaneously separately in the same NSG mice on oppositehind flanks. A1847Mþ/Fþ and A1847Mþ/F� tumors wereestablished comparably and grew similarly in vivo (Fig. 1A andB). Mice bearing 2 established A1847 (�330 mm3) tumorsreceived tail vein injections of CAR-T cells and tumor

Figure 3. Trans-signaling CAR-Tcells show in vitro cytolytic potency.A, dual CAR-T cells exert superiordegranulation and express T-cellactivation markers in response tospecific stimulation. DetectableCD107a, b and CD69 expression inCAR-T cells during coculturewith theindicated tumor target or control cellline by flow cytometry. B, cytolyticfunction of dual CAR-T cells. Primaryhuman T cells transduced to expressM-z, M-z/F-28, M-28z, F-28, or GFPwere cocultured with 51Cr-labelednative C30-F, C30-M, or A1847cancer cell lines for 4 hours at theindicated E:T ratio. Error barsindicateSD. ��,P<0.001 comparingM-z/F-28 and M-28z CAR-T cellsagainst C30-M cancer cells.






outgrowth was monitored. The efficiency of A1847Mþ/Fþ

tumor outgrowth inhibition was identical between trans-M-z/F-28 CAR-T and cis-M-28z CAR-T cell groups (Fig. 6A).However, inhibition of A1847Mþ/F� outgrowth was signifi-cantly attenuated in the trans-M-z/F-28 CAR-T cell groupcompared with the cis-M-28z group (P ¼ 0.0045; Fig. 6B).Notably, trans-signaling CAR-T cells were less effective ininhibiting the outgrowth of A1847Mþ/F�, compared with theiractivity against the A1847Mþ/Fþ tumor in the same mice (P¼0.0001; Fig. 6C). Bioluminescence imaging of the tumors con-firmed these results (Fig. 6D).

Preferential accumulation of trans-signaling CAR-T cellsin tumor in vivo

The accumulation of trans- and cis-signaling CAR-T cells inregressing tumors from treated and euthanized mice withestablished masses in both flanks was measured via immuno-histochemical analysis for the detection of human CD3þ T cells(Fig. 7). The abundance of T cells was equally high in dual(A1847Mþ/Fþ) or single antigen-expressing tumors (A1847Mþ/F�) frommice treatedwith cis-M-28zCAR-T cells. In contrast, inmice treated with trans-M-z/F-28 CAR-T cells, a significantincrease in T-cell accumulation was observed in tumors expres-sing both antigens with lower numbers observed in singleantigen tumors, illustrating selectivity. Few CD3þ T cells weredetected in tumors resected from mice that received CD19-28zCAR-T cells. Thus, the survival and accumulation of trans-signaling CAR-T cells into tumor sites is dependent on theengagement of the FRa antigen to the FRa specific-CAR, aninteraction that triggers optimal costimulation.

DiscussionAdoptive immunotherapy involving genetic modification

of T cells with antigen-specific, chimeric, single-chain recep-tors is a promising approach for the treatment of cancer(25). However, the therapeutic value of adoptively trans-ferred, gene-engineered T cells may be compromised byextensive autoimmune damage to normal tissues expressingthe target antigen (26). This adverse side effect has beenencountered in clinical trials using T cells genetically mod-ified to express a tumor antigen-specific CAR (27). Mild tosevere autoimmune toxicity has been reported followingtransfer of tumor-reactive T cells including liver toxicityupon adoptive transfer of anti-CAIX CAR-T cells (28) due toCAIX expression on bile duct epithelium. Serious adverseevents (SAE) involving death of a patient has been observedin 2 distinct clinical trials following adoptive transfer ofCAR-T cells redirected against ErbB2 (12) or CD19 (29),though the role for modified T cells in the latter SAE remainsunclear. Collectively, studies in preclinical mouse modelsand in patients have indicated that the number of T cellsadministered, the expression levels and localizationof antigen in normal tissues, the type of signaling domainsincorporated into chimeric receptors and the level ofimmune preconditioning used are parameters that must becarefully considered in the design of safe engineered T-celltherapeutic strategies (26, 30–35).

CARs with one or more costimulatory signals confer a newpotential to respond to target antigen with sustained prolif-eration and cytotoxicity, and resistance to AICD and regulatoryT-cell suppression (20, 36). However, powerful costimulationraises a potential danger for cross-reactivity against normal

Figure 4. Trans-signaling CAR-Tcells resist AICD upon antigen-specific stimulation. A, apoptosis inmixed CAR-T cell populationcontaining dual CAR-transducedT cells (M-z/F-28), single CAR-transduced T cells (M-z or F-28),and untransduced T cells that werestimulated with A1847(Mþ/Fþ)cancer cells for 3 days. Cis-M-28zCAR-T cells were cocultured inparallel with A1847 for the sametimeperiod. After 3 days, apoptosisby AICD was quantified usingannexin V and 7-AAD staining. B,AICD is not observed in CAR-Tcells stimulated with antigen-negative cancer cells (C30; M�/F�)for 3 days.

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host tissues expressing low levels of the target TAA. Oneapproach to forestall this problem involves the physical sep-aration of the signal 1module (CD3z) from the signal 2module(costimulation) through their incorporation into 2 distinctCARs specific for 2 different antigens, to recapitulate naturalT-cell biology and function. In this way, dual CAR-T cells mayselectively traffic, survive, and exert sustained proliferationwithin the tumor microenvironment, as synergistic signalswould be delivered to T-cells preferentially at that location.Hence, the potential for "on-target" toxicity should be reducedcommensurately. Indeed, CARs can be engineered to providecostimulation alone (37). For example, Jurkat cells engineeredto coexpress hapten-specific CD3z- and CD28-based CARs,triggered complementary signaling leading to IL-2 production(38). So far, early attempts to preferentially redirect CAR-T cellsagainst tumors expressing multiple antigens to limit potentialtoxicity were not successful enough to hold promise for clinicalapplication. In one study, CAR-T were outfitted with 2 first-generation CARs specific for ErbB2 and FRa to increasespecificity for tumor. These dual-transduced CAR-T cellsexpressed similar amounts of total surface CARs as mono-transduced T cells, that is each individual CAR was expressedat a lower level (39). Thus, the dual-transduced T cells cross-reacted considerably less avidly with target cells expressingonly a single antigen than with tumor cells expressing both

antigens. Because these CAR-T cells exhibited no costimula-tion activity due to the lack of any costimulatory domains ineither of the CARs, they were unlikely to survive, persist, andclear tumors in vivo. Another recent study proposed dualtargeting of ErbB2 and MUC1 in breast cancer by generatingdual CAR-T cells transduced with both a CD28 containingMUC1 CAR and a CD3z containing ErbB2 CAR (40). Thesedual CAR-T cells were even less efficient than first-generationCAR-T cells and failed to secrete IL-2 in response to trans-signaled costimulation upon encounter with a second antigen,thus rendering the system unsuitable for further preclinicalinvestigation.

Here, we used 2 distinct CARs, redirected against meso-thelin and FRa, to test the concept of combinatorial CARsignaling for highly selective antitumor activity. The anti-mesothelin CAR contained the CD3z signaling motif alone,whereas the anti-FRa CAR included only the intracellulardomain of the CD28 costimulatory molecule. Proper costimu-lation of these engineered T cells relies upon the coexpressionof both FRa and mesothelin on the tumor cell surface. Incomparisons between trans-signaling CAR-T cells with con-ventional first- and second-generation anti-mesothelin CAR-Tcells, trans-signaling CAR-T cells showed similar in vitropotency to cis-signaling CAR-T cells and were capable of pro-ducing increased levels of T-helper cell (TH1) cytokines

Figure 5. Trans-signaling CAR-T cells exert superior antitumor effector functions in vivo compared with first-generation CAR-T cells. A, in vivo inhibitionof large preestablished tumors by M-z/F-28 CAR-T cells: effect of the CD28 costimulatory signaling domain in trans. NSG mice bearing establishedsubcutaneous tumors were treated with 2 intravenous injections of 7.5 � 106 M-z, M-z/F-28, or M-28z CAR-T cells or control anti-CD19-28z and GFPT cells or saline on days 55 and 59 posttumor inoculation (arrows). Tumor growth was assessed by caliper measurement. Mean tumor volume(mm3 � SEM) is shown with n ¼ 5 for all groups. �, P < 0.05 comparing M-z CAR-T–treated mice with M-z/F-28 or M-28z CAR-T–treated mice. B, A1847fLucþ bioluminescence signal is similar in M-z/F-28 and M-28z CAR-T-treated mice and less than in M-z–treated mice 4 weeks after first T-cell dose.Control groups show no decrement in the bioluminescence signal. C, stable persistence of CD28 cis- or trans-costimulated CAR-T T cells in vivo.Peripheral blood was collected 2 weeks after the first T-cell infusion and quantified for the absolute number of human CD4þ and CD8þ T cells/mL ofblood. Mean cell count � SEM is shown with n ¼ 5 for all groups. �, P < 0.05 comparing M-z CAR-T–treated mice with M-z/F-28 CAR-T–treated mice.






compared with first-generation CAR-T cells. Unlike the find-ings of Wilkie and colleagues (40), tumor-induced costim-ulation through the anti-FRa-28 CAR triggered enhancedsecretion of IL-2, a cytokine known to promote T-cell prolife-ration and persistence in vivo (41, 42). Furthermore, costim-ulation delivered in trans was sufficient to protect thoseCAR-T cells from AICD, similar to the effects seen in cis-signaling CAR-T cells (9, 24). The cytolytic potential oftrans-signaling CAR-T cells in vitro was similar to first andsecond-generation CAR-T cells. This is consistent with pre-vious studies showing no statistically significant differencein specific tumor lysis by CAR-T cells that include or lackincorporated costimulatory domains (11, 20, 43).

The significance of a trans-signaling CAR approach is besttested in preclinical models where the targeting of bothhuman tumor cells and representative normal tissue cells byCAR-T cells can be evaluated simultaneously. Ovariantumors generally overexpress both FRa (90%) and mesothe-lin (70%), rendering ovarian cancer an attractive studyparadigm (17, 44). Moreover, the pattern of mesothelin and

FRa expression on normal tissues is largely nonoverlapping.Mesothelin is expressed by mesothelial cells lining the pleuraand peritoneum and at low levels by epithelial cells of thetrachea, tonsils, fallopian tube, and the testis (14, 15). FRaexpression is limited to the apical surface of the kidneyproximal tubules, choroid plexus, lung epithelium, thyroid,and intestinal brush border epithelial cells (45, 46). In ourstudy, in vivo antitumor activity of trans-signaling CAR-Tcells was initially tested by adoptively transferring thedifferent CAR-T cell populations into immunodeficient micewith established ovarian cancer, using the A1847 humanovarian cancer cell line expressing both mesothelin and FRa.Trans-signaling CAR-T cells exhibited potent antitumoractivity against ovarian cancer in vivo, equivalent to thatachieved using conventional cis-signaling CAR-T cells. Moreimportantly, the finding that trans-signaling CAR-T cells aremore selective, potent, and localized within cancers furn-ished with both antigens, but spare normal cells bearing asingle antigen, may have important translational ramifica-tions. Indeed, tumor localization seems to be a key factor for

Figure 6. Cis-, but not trans-, signaling CAR-T cells exhibit enhanced in vivo activity against cells bearing a single antigen. In vivo antitumor efficacy oftrans- or cis-signaling T cells against A1847 cells expressing or lacking FRa. A total of 7.5 � 106 A1847(Mþ/Fþ) and A1847(Mþ/F�) cells were inoculatedsubcutaneously separately in the same NSG mice on opposite hind flanks. Mice with 2 established A1847 tumors (�330 mm3) received tail veininjections of CAR-T cells on days 45 and 49 posttumor inoculation (arrows). Tumor growth was assessed by caliper measurement. A, the efficiency ofA1847(Mþ/Fþ) tumor outgrowth inhibition was identical between trans-M-z/F-28 CAR-T and cis-signaling M-28z CAR-T cell groups. B, inhibition ofA1847(Mþ/F�) outgrowth in the trans-M-z/F-28 CAR-T group was reduced compared with the cis M-28z group (��, P < 0.01 in M-z/F-28 vs. M-28z).C, trans-signaling CAR-T cells were not as effective in inhibiting the outgrowth of A1847(Mþ/F�) as in inhibiting the A1847(Mþ/Fþ) tumor in thesame mice. Mean tumor volume (mm3 � SEM) is reported with n ¼ 5 for all groups in A–C. ��, P < 0.01 comparing tumor growth of A1847(MþFþ) versusA1847(MþF�) in mice treated with M-z/F-28 CAR-T. D, FLucþ bioluminescence signal from A1847(Mþ/Fþ) tumors is dramatically lower compared withthe signal derived from A1847(Mþ/F�) masses in the samemice treated with M-z/F-28 CAR-T cells 4 weeks after first T-cell dose. In the cis M-28z group,MþFþ and MþF� A1847 tumors exhibit similarly low levels of FLucþ bioluminescence signal. The CD19-28z CAR-T treatment group showed nodecrement in bioluminescence signal.

Lanitis et al.





the success of CAR-T cell therapy (6). For example, mesothe-lin redirected T cells coexpressing an anti-mesothelin CARand a chemokine receptor (CCR2) showed increased local-ization in malignant pleural mesotheliomas secreting thechemokine CCL2, and thus improved the therapeutic out-come in preclinical mouse models (47).Upon trafficking to the normal tissues expressing mesothe-

lin alone, trans-signaling CAR-T cells receive only signal 1 uponantigen contact. This may render the designer T cells suscep-tible to AICD or drive them into anergy (24, 36, 48) andconsequently restrict their potential for long-term persistenceand tumor elimination when applied to patients. Alternatively,recognition of FRa alone and singular transmission of CD28signal, in the absence of mesothelin-directed CD3z-signals,does not activate trans-signaling CAR-T cells. Importantly,equal regression of tumors was observed in our model usingcis- or trans-signaling CAR-T cells, illustrating the idea that adual CAR-T cell approach can modestly improve safety forclinical application without significantly diminishing the anti-tumor potential of the second-generation CAR approach. DualCAR-T cells which receive combined CD3z and costimulatorysignals upon antigen-specific interactions with tumor did notseem to be rendered costimulation-independent and endowedwith the capacity to react strongly and systemically against thetissue expressing single antigen. This was apparent in ourpreclinical model where there was a significant difference inthe control of tumor bearing 2 antigens versus the more rapidgrowth of cells with only 1 antigen following treatment withM-z/F-28 CAR-T cells. This was further supported by the statis-tically higher infiltration and accumulation of trans-signalingCAR-T cells into the tumor sites where both antigens wereexpressed.Our preclinical findings extend beyond the in vitro study of

Wilkie and colleagues (40) and were rationalized by the finding

that our dual CAR-T cells are more favorable effector cells foradoptive cell transfer than first-generation CAR-T cells due totheir resistance to AICD and ability to secrete elevated levels ofTH1 cytokine including IL-2 in response to trans-signaledcostimulation upon encounter with a second antigen. Sincethe submission of our article, an additional study has nowconfirmed these enhanced effector properties of dual signalingCAR-T cells upon secondary antigen encounter in vitro (49).Using CARs specific for prostate-specific membrane antigenand prostate stem cell antigen, this report confirmed thepreferential killing of tumor cells bearing both antigens invivo, compared with single antigen target cells, thus reducingthe potential for harmful side effects (49). The factor(s)accounting for the reduced effector cell efficiency and IL-2production by dual CAR-T cells stimulated with both antigensin vitro observed in the Wilkie and colleagues' study (40)remain unclear. Differences in specificity, affinity, and stoichi-ometry of CARs, as well as different model systems may beinfluencing factors, however, the observation of high level IL-2production by T cells bearing a ErbB2-specific CAR with nocostimulatory endodomain in this study (40) is unusual, notobserved in multiple other studies (8, 13, 43) and suggestsuniqueness of this particular first-generation CAR.

Trans-signaling CARs represent a novel approach to focusCAR-T cells to tumor cells and reduce their impact on singleantigen-expressing cells; however, this method is highlydependent upon identification of 2 antigens, which arenearly uniformly expressed in a particular cancer type, withrelatively low and nonoverlapping expression in normaltissues. Accordingly, the identification and application ofalternative approaches to limit CAR-T-mediated auto-immune effects remains warranted. One approach to limitsuch toxicity is the careful design of a dose-escalationstrategy to better define the optimal T-cell dose (26). Some

Figure 7. Trans-signaling CAR-T cells preferentially localize to dual antigen-expressing tumors in vivo. A, NSG mice with subcutaneouslyestablished A1847 tumors expressing or not FRa in opposite flanks received intravenous injections of 7.5� 106 CAR-T cells expressing CD19-28z (top),M-z/F-28 (middle), or M-28z (bottom) on days 45 and 49 posttumor inoculation. Mice were euthanized after 4 weeks, and tumors were collected andstained for human CD3 expression (brown) or isotype control. Representative sections for MþFþ and MþF� A1847 tumor derivatives are shown(�10 magnifications). B, quantification of CD3þ T cells within A1847(Mþ/Fþ) and A1847(Mþ/F�) tumors in the differentially treated groups. CD3þ T cellswere counted in 10 randomly selected intratumoral fields of each slide at 20X magnification. Data are expressed as the means � SE with n ¼ 5 for allgroups. �, P < 0.05 comparing the tumor T-cell infiltration in A1847(Mþ/Fþ) versus A1847(Mþ/F�) in M-z/F-28 CAR-T-treated mice.






of the potential side effects of nontumor cell recognitionby CAR-T cells can be overcome by the coexpression ofconditional suicide genes such as incorporation of HSV-TK,the cytoplasmic domain of Fas, or an inducible caspase intogene-engineered T cells to abort aberrant T-cell responses(30–33). Indeed, the iCasp9 cell-suicide system has beenshown to increase the safety of cellular therapies in pati-ents that received T cells depleted of alloreactive pro-genitor cells (34). Alternatively, electroporation of T cellswith optimized RNAs for transient CAR expression hasbeen effective in preclinical mouse models and might bypassthe associated safety concerns of integrating gene vectors(50). These approaches toward cell product safety are notmutually exclusive and future application of dual CAR-Tcells engineered in combination with suicide switches asdescribed earlier may better permit preferential CAR-T cellaccumulation and activity within the tumor microenvironmentand in a safe manner.

Disclosure of Potential Conflicts of InterestC.H. June has ownership interest (including patents) in Novartis, which has

licensed inventions related to this research, and he may receive royalties frominventions licensed to Novartis. No potential conflicts of interest were disclosedby the other authors.

Authors' ContributionsConception and design: E. Lanitis, C.H. June, D.J. Powell JrDevelopment of methodology: E. Lanitis, C.H. June, D.J. Powell JrAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): E. Lanitis, M. Poussin, A.W. Klattenhoff, D.J. Powell JrAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): E. Lanitis, M. Poussin, D.J. Powell JrWriting, review, and/or revisionof themanuscript:E. Lanitis,M. Poussin, A.W. Klattenhoff, R. Sandaltzopoulos, C.H. June, D.J. Powell JrAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): E. Lanitis, M. Poussin, D. Song, D.J.Powell JrStudy supervision: D.J. Powell Jr

AcknowledgmentsThe authors thank Drs. Steven Albelda, Gwenn Danet-Desnoyers, Michael

Kalos, Michael Milone, Edmund Moon, Richard Kronish, Nathalie Scholler, andDenarda Dangaj for helpful suggestions, discussions, and reagents.

Grant SupportThisworkwas supported by grants from theW.W. Smith Charitable Trust, the

Kronish Research Foundation for the Treatment of Ovarian Cancer, the SandyRollman Ovarian Cancer Foundation, the Ovarian Cancer Research Fund (PPD-Penn-01.12), NIH RO1-CA168900 and 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 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 December 5, 2012; revised February 11, 2013; accepted February 12,2013; published OnlineFirst April 7, 2013.

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2013;1:43-53. Published OnlineFirst April 7, 2013.Cancer Immunol Res Evripidis Lanitis, Mathilde Poussin, Alex W. Klattenhoff, et al.

In VivoPotential for Toxicity Domains Exhibit Focused Antitumor Activity with Reduced Chimeric Antigen Receptor T Cells with Dissociated Signaling

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ChimericAntigenReceptorTCellswithDissociatedSignaling ...redirected activityin vitro and in vivo (ref. 13; Fig. 1A and B). The costimulatory anti-FRa CAR (F-28) construct comprised