Reversible Transgene Expression Reduces ...(Caliper Life Sciences) after injecting D-Luciferin (150 mg/kg i.p.). Mice were euthanized after the tumor burden reached a luminescence

Research Article

Reversible Transgene Expression ReducesFratricideandPermits4-1BBCostimulationofCART Cells Directed to T-cell MalignanciesMaksim Mamonkin1,2, Malini Mukherjee1,3,4, Madhuwanti Srinivasan1,Sandhya Sharma1,5, Diogo Gomes-Silva1,6, Feiyan Mo1,5, Giedre Krenciute1,3,Jordan S. Orange2,3,4,5, and Malcolm K. Brenner1,3,5

Abstract

T cells expressing second-generation chimeric antigen recep-tors (CARs) specific for CD5, a T-cell surface marker present onnormal and malignant T cells, can selectively kill tumor cells.We aimed to improve this killing by substituting the CD28costimulatory endodomain (28.z) with 4-1BB (BB.z), as 28.zCD5 CAR T cells rapidly differentiated into short-lived effectorcells. In contrast, 4-1BB costimulation is known to promotedevelopment of the central memory subpopulation. Here, wefound BB.z CD5 CAR T cells had impaired growth comparedwith 28.z CD5.CAR T cells, due to increased T-cell–T-cellfratricide. We demonstrate that TRAF signaling from the 4-1BBendodomain upregulated the intercellular adhesion molecule

1, which stabilized the fratricidal immunologic synapsebetween CD5 CAR T cells. As the surviving BB.z CD5 CART cells retained the desired central memory phenotype, weaimed to circumvent the 4-1BB–mediated toxicity using aregulated expression system that reversibly inhibits CAR expres-sion. This system minimized CAR signaling and T-cell fratricideduring in vitro expansion in the presence of a small-moleculeinhibitor, and restored CAR expression and antitumor functionof transduced T cells in vivo. These studies reveal a mechanismby which 4-1BB costimulation impairs expansion of CD5CAR T cells and offer a solution to mitigate this toxicity.Cancer Immunol Res; 6(1); 47–58. �2017 AACR.

IntroductionIncorporation of one or more costimulatory endodomains in

chimeric antigen receptors (CARs) increases the expansion andpersistence of CAR T cells after tumor challenge (1–3). Clinicallyused endodomains are commonly derived from either the Igsuperfamily gene CD28 or TNFR superfamily members 4-1BB(CD137) and OX40 (CD134; ref. 4), coupled to CD3z. T cellsexpressing CD19 CARs harboring either CD28 or 4-1BB endodo-mains have induced complete remissions in patients withadvanced B-cell malignancies, though each endodomain mayproduce distinct biological effects (5–8). Incorporation ofCD28.z in CD19 CAR T cells results in their rapid expansion andimmediate antitumor activity, whereas 4-1BB.z CD19 CAR T cells

may persist longer and establish immune control (5–9). Thesedifferences could be attributed, at least in part, to the role ofCD28-derived PI3K signaling in enhancing T-cell activation andspurring differentiation to cytotoxic but short-lived effector cells(10–12). In contrast, 4-1BB signaling induces more limited T-celldifferentiation, favoring the development of central memoryT cellswith longer-termpersistence (13).Despite these differencesin biological activity, whether coupling CD19CARs toCD28 or to4-1BB signaling domains produces superior clinical outcomesremains unknown. Thus, continued exploration of both classes ofsignaling endodomains is necessary for future studies of CART-cell activity in humans.

Some potentially targetable tumor-associated antigens, partic-ularly those associated with lymphoid malignancies, are alsoexpressed by T cells (14–17). Expression of a CAR specific forthese antigens in T cellsmay promote their fratricide and thereforecompromise expansion. This problem is particularly relevant forthe development of CAR T-cell–based therapies for T-lineagemalignancies. The pan–T-cell marker CD5 is being investigatedfor the treatment of T-cell malignancies such as T-cell acutelymphoblastic leukemia (T-ALL) and peripheral lymphoma(17). This approach is feasible because CD5 is downregulatedfrom the T-cell surface upon ligation with its ligand or antibody,allowing CD5þ T-cell tumors to be targeted even by CD5 CAR Tcells that are themselves CD5þ (17). Thus, expression of a CD28.zCD5 CAR in activated T cells resulted in transient and limitedfratricide followed by robust expansion of CD5 CAR T cells.CD28.z CD5 CAR T cells had high cytotoxicity against malignantT cells but limited in vivo persistence. Although the short life spanof effector-enriched 28.z CD5 CAR T cells may reduce the extent

1Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children'sHospital, Houston Methodist Hospital, Houston, Texas. 2Department of Pathol-ogy and Immunology, Baylor College of Medicine, Houston, Texas. 3Departmentof Pediatrics, Baylor College of Medicine, Houston, Texas. 4Center for HumanImmunobiology, Texas Children's Hospital, Houston, Texas. 5InterdepartmentalGraduate Program in Translational Biology and Molecular Medicine, BaylorCollege of Medicine, Houston, Texas. 6Instituto Superior T�ecnico, University ofLisbon, Lisbon, Portugal.

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

Corresponding Author: M. Mamonkin, Baylor College of Medicine, 1102 BatesStreet, Suite FC1770.02, Houston, TX 77030. Phone: 832-824-4683; Fax: 832-825-4732; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-17-0126

�2017 American Association for Cancer Research.

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and duration of potential off-tumor toxicities in patients (e.g., T-cell aplasia), it may also limit the durability of antitumorresponses. Therefore, we hypothesized that replacing CD28 withthe 4-1BB costimulatory endodomain in CD5 CARs wouldrestrain effector differentiation of CD5 CAR T cells and increasetheir in vivo persistence.

We found that incorporation of 4-1BB in the CD5 CAR indeedaugmented the formation of central memory T cells. We observedthat 4-1BB costimulation also enhanced fratricide of CD5 CAR Tcells and impaired their expansion, an adverse effect also pro-duced by other TNFR superfamily–derived CAR endodomains.Nonetheless, by developing a CAR expression system that revers-ibly disrupts this deleterious CAR signaling and prevents CART-cell fratricide in vitro, we were able to explore the potentialbenefits of 4-1BB signaling without promoting fratricide.

Materials and MethodsCAR constructs and vectors

The second-generation CD5 CAR containing a CH3 IgG Fcspacer and CD28-derived transmembrane region and endodo-main (28.z) was described previously (17). Other CD5 CARconstructs used in this study were created by replacing the CD28endodomain with cytoplasmic regions from ICOS, 4-1BB, CD27,CD30, OX40, or HVEM genes. OX40 and 4-1BB endodomainswere amplified fromGD2- and CD19-specific CARs that are beingevaluated clinically at Baylor College of Medicine (BCM). ICOS,CD27, CD30, and HVEM genes were subcloned from a cDNAlibrary of genes expressed in activated T cells using standardmolecular cloning techniques. In ICOS.z andHVEM.z CD5CARs,the CH3 spacer was replaced with the CD8a stalk due to unstableexpression in the original configuration. All CAR constructs con-tained the transmembrane region from CD28 except ICOS.zwhere the transmembrane region from ICOS was used. TRAF-binding sites in the 4-1BB endodomain were created using site-directed mutagenesis with the InFusion cloning Kit (Clontech).CAR constructs were subcloned into SFG gammaretroviral vectorand sequence-verified by Sanger DNA sequencing. Gammaretro-viral transductionof T cellswas performed as previously described(17). Transduced T cells were expanded in the presence of IL7(10 ng/mL) and IL15 (10 ng/mL) in RPMI-1640/Click's (CTL)media supplemented with 10%FBS and 1% L-glutamine. Primaryhuman peripheral blood mononuclear cells were isolated fromhealthy donors after informed consent using the protocolapproved by the BCM Institutional Review Board and in accor-dance with the Declaration of Helsinki.

Flow cytometryAntibodies were purchased from Beckman Coulter [CD3

(UCHT1), CD4 (SK3), CD5 (UCHT2), CD8 (SK1), CD45RA(2H4)], BD Biosciences [CCR7 (150503)], or Biolegend[intercellular adhesion molecule 1 (ICAM-1; HA-58)]. AnnexinV and/or 7-AAD (BD Biosciences) was used to detect apoptoticcells. Goat anti-mouse Fc-specific or Fab-specific polyclonal anti-bodies (Jackson Immunoresearch) were used to detect CARexpression on the cell surface. Samples were acquired on BDGallios and analyzed with FlowJo v.9.

Cell cultureJurkat andCCRF T-ALL cell linesmodified to express GFP-FFluc

were described previously (17). CD5 CAR T cells were plated with

tumor cells at a 1:4 initial effector-to-target ratio and cultured for72 hours in the absence of exogenous cytokines. Cells wereacquired by flow cytometry and quantified at the end of coculturein the presence of 7-AAD and Countbright counting beads(Invitrogen).

For ICAM-1 blockade, CD5 CAR T cells were cultured in thepresence of 2 mg/mL anti–ICAM-1-blocking antibodies (84H10;Bio-Rad) or vehicle control for 72 hours. Cell death and countswere measured by flow cytometry as indicated above.

Western blot analysisWestern blot analysis was performed as previously described

(18). Briefly, cells were dissociated using lysis buffer with phos-phatase and protease inhibitors (Thermo Scientific). Proteinconcentrations were determined using a Bio-Rad protein assay(BioRad). Samples were denatured in Laemmli buffer containing2-Mercaptoethanol andboiled at 95�C for 10minutes. Cell lysateswere run on a premade 10% SDS polyacrylamide gel (BioRad)and transferred to nitrocellulose membranes (BioRad). Mem-branes were then blocked with 5% BSA or milk and probedwith primary antibodies followed by horseradish peroxidase–conjugated secondary antibodies. Blots were developed usingSuperSignal West Dura Extended Duration Substrate (ThermoScientific) and exposed to GeneMate Blue Basic AutoradiographyFilm (BioExpress). Antibodies used in the study are as follows:anti-CD3.z (sc-1239; Santa Cruz Biotechnology, Inc.), anti-CD247 (pY142) (558402; BDPharmigen), pIKKa/b and IKKa(#9936; Cell Signaling Technology), and GAPDH (sc-47724;Santa Cruz Biotechnology, Inc.). Secondary antibodies used inthe study are as follows: goat anti-mouse (sc-2005; Santa CruzBiotechnology, Inc.) and goat anti-rabbit (111-035-003; Jack-son ImmunoResearch Laboratories, Inc.). ImageJ (NationalInstitutes of Health, Bethesda, MD) was used for Western blotquantification. Protein levels were normalized to loading con-trols (GAPDH).

Immunofluorescence analysisFor confocal microscopy, conjugates between CAR T cells and

target cells were incubated for 30 minutes at 37�C and then fixedand stained for F-actin (Phalloidin 568), Pericentrin (rabbit anti-Pericentrin; Abcam), and ICAM-1 (mouse anti–ICAM-1; BDBiosciences). Pericentrin was detected using Alexa Fluor PacificBlue secondary antibody to rabbit IgG, and ICAM-1 was detectedusing 488 Alexa Fluor secondary antibody to mouse IgG. Con-jugates were imaged as Z stacks of 0.2 mm thickness to cover theentire volume of the immune synapse (IS), determined individu-ally for each conjugate on a Leica SP8 confocal microscope using a100�objective.Densityof F-actinand ICAM-1accumulationat theimmune synapse of CAR T cells was calculated using the formula[volume � mean fluorescence intensity (MFI)] for an equal num-ber of 1 mm3 boxes selected to cover the IS (19). Microtubule-organizing center (MTOC) to synapse distance was measured asdescribed before (20).

Tet-OFF expression systemGammaretroviral backbone from pRetroX-Tight-Pur (Clon-

tech) was digested with EcoRI–EcoRV. An expression cassettecomprised of the EF1a/LTR hybrid promoter (pCDH-CMV-MCS-EF1-copGFP; System Biosciences), and tTA-Advanced(pRetroX-TetOFF; Clontech) was assembled and inserted intothe gammaretroviral backbone using the InFusion Cloning Kit

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(Clontech). CD5 CAR constructs were subcloned downstreamof the Tet-responsive elements and upstream of the EF1a/LTRpromoter. Gammaretroviral transduction of T cells was per-formed as previously described (17). To suppress transgeneexpression, transduced T cells were cultured in the presence of10 to 50 ng/mL doxycycline (Sigma Aldrich). Doxycycline wasreplaced every 48 to 72 hours.

Mouse xenograft modelEight- to 10-week-old male or female NOD.Cg-Prkdcscid

Il2rgtm1Wjl/SzJ (NSG) mice (The Jackson Laboratory) were inoc-ulated intravenously with 1 � 106 Jurkat-FFluc cells. Mice werethen ear-tagged and randomly assigned to experimental groups.Mice received a single intravenous injection of 1.0 � 106 CART cells 3 or 7 days after tumor engraftment. Tumor burden wasmonitored by in vivo imaging with an IVIS Imaging system(Caliper Life Sciences) after injecting D-Luciferin (150 mg/kgi.p.). Mice were euthanized after the tumor burden reached aluminescence level of 108 photons/sec or after displaying signsof distress associated with graft-versus-host disease (GVHD) orhigh tumor burden. Peripheral blood was collected by tail veinbleeding. All animal experiments were conducted in compliancewith the Institutional Animal Care and Use Committee of BCM.

Statistical analysisUnpaired two-tailed Student t test was used to determine statis-

tical significance for 2-sample comparison, and one-way ANOVAwith Bonferroni posttest correction was used for multiple compar-isons. P values below 0.05 were considered statistically significant.All statistical analyses were performed in GraphPad Prism 6.

Results4-1BB costimulation abrogates the expansion of CD5 CAR Tcells

We previously reported that T cells expressing a second-generation CD5 CAR with the CD28 costimulatory endodomain(28.z) have antitumor activity (17). To examine the role of 4-1BBcostimulation in CD5 CARs, we substituted 28.z with the 4-1BBendodomain (BB.z), leaving the rest of the CAR backbone intact(Fig. 1A). Both 28.z and BB.z CD5 CARs were expressed on the cellsurfaceof transducedTcells, and their expressioncorrelatedwith thedownregulation of CD5 (Fig. 1A), reflecting the rapid internaliza-tion of CD5 upon binding to the CAR. Expression of the BB.z CD5CAR resulted inenrichment forCCR7þCD45RA� centralmemoryTcells (Fig. 1B); however, the BB.z CD5 CAR T cells failed to expandcompared with control or 28.z CD5 CAR T cells (Fig. 1C). Theimpaired growth of BB.z CD5 CAR T cells correlated with signifi-cantly enhanced apoptosis (Fig. 1D), indicating that the expressionofBB.zCD5CARaugmentedT-cell death. The increasednumbersof28.z CD5 CAR T cells could not be attributed to an associatedfunctional exhaustion and loss of cytotoxicity or fratricide as thesecells retained high cytotoxic activity even 21 days after transduction(Supplementary Fig. S1). To determine whether the increasedfratricide was a result of an elevated expression of BB.z CD5 CARin T cells (Fig. 1A), we increased the expression of 28.z CD5CAR byreplacing the CH3 Fc spacer with a short IgG Fc–derived hinge andevaluated T-cell expansion (Supplementary Fig. S2A and S2B).Elevated 28.z CD5 CAR expression did not abrogate T-cell growth(Supplementary Fig. S2C), indicating that the inability of BB.z CART cells to expand is not due to increased CAR expression.

TNFR superfamily costimulatory endodomains impairexpansion of CD5 CAR T cells

To investigate whether only 4-1BB impaired expansion of CD5CAR T cells, or whether this was a more general phenomenonobserved with other TNFR superfamily genes (OX40, CD27,CD30, and HVEM), we investigated the role of each gene inregulating CD5 CAR T-cell expansion. We incorporated thesecostimulatory endodomains in a panel of second-generationCD5CARs and then measured T-cell expansion following transduc-tion. We found that T cells expressing CD5 CAR with immuno-globulin superfamily endodomains CD28 or ICOS (28.z andICOS.z) expanded similarly to those expressing the first-genera-tion CD5 CAR (z), whereas CD5 CAR T cells incorporating theTNFR superfamily costimulatory domains OX40, CD27, CD30,and HVEM showed reduced expansion, resembling the defectobserved in BB.z CD5 CAR T cells (Fig. 2A). Although all CD5CARswere expressed normally on T cells 3 days after transduction(Fig. 2B),weobserved complete or partial downregulationofCD5CARs with TNFR costimulatory endodomains in T cells a weeklater (Fig. 2B). We detected higher frequencies of the centralmemory subpopulation in all T cells expressing TNFR CD5 CARscomparedwithCD5CARswith IgSF superfamily endodomains orno costimulation (Supplementary Fig. S3), indicating all TNFRendodomains can promote central memory formation in CAR-transduced T cells. The poor expansion of all TNFR domain-harboring CD5 CAR T-cell populations was not associated withincreased expression of CD5 on the cell surface, as cell-surfaceexpression of CD5 remained equally low in all CD5 CAR T cells(Fig. 2C). Thus, signaling from other TNFR endodomains pro-duced the same effects onphenotype and toxicity as 4-1BB in theseCD5CART cells. T cells expressing a third-generation28.BB.zCD5CAR behaved like BB.z CAR T cells, and they failed to expand(Fig. 2D). Because the phenotype of BB.z CD5 CAR T cells cannotbe reversed by additional CD28 costimulation, 4-1BB signalingmay play a dominant role in impairing expansion and survival ofCD5 CAR T cells.

TRAF2 signaling from the 4-1BB endodomain enhancedapoptosis in CD5 CAR T cells

Although CD5 CAR T cells downregulate surface expression ofCD5, CD5 gene transcription remains intact (17). This observa-tion suggests that CD5 CAR can constantly interact with residualligand and therefore produce tonic CAR signaling, an interpreta-tion supported by the presence of comparable levels of sponta-neous phosphorylation of the CAR-derived z chain in both 28.zand BB.z CD5CAR T cells (Fig. 3A).Hence, the selective toxicity ofBB.z CD5 CAR is not associated with the differences in themagnitude of tonic signaling. However, unlike CD28, whichdirectly activates PI3K signaling, 4-1BB signals through TRAFadapter proteins leading to the activation of the IkB kinasecomplex (IKKa/b), which subsequently disinhibits the transcrip-tion factor NF-kB prompting its nuclear translocation and tran-scriptional activity (21–23). We detected robust steady-statephosphorylation of IKKa/b in BB.z CAR T cells compared withthe 28.z controls (Fig. 3B). Because 4-1BB and CD28 costimula-tion elicits different signaling pathways, we determined whetherthe toxicity of BB.z CD5 CAR in T cells is driven by 4-1BB–derivedTRAF signaling. We disrupted the N-terminal (mut1QEED!QAAA)and the C-terminal (mut2PEEEE!PEAAA) motifs in the 4-1BB endo-domain to prevent binding of TRAF2 (mut1) or TRAF1, TRAF2,and TRAF3 (mut2; ref. 21). Alterations in TRAF-binding motifs

Regulated CAR Expression Minimizes Tonic Signaling

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did not compromise cell surface expression of CD5 CAR (Fig. 3C)but reduced the rate of apoptosis in BB.z CAR T cells (Fig. 3D) anddecreased IKKa/b phosphorylation (Supplementary Fig. S4),resulting in normal expansion of T cells expressing either of themutant BB.z CARs (Fig. 3E). Despite expanding normally, BB.zCD5 CAR T cells with mutated TRAF-binding motifs were unableto sequentially kill CD5þ tumor cells (CCRF); their killing capac-ity was similar to first-generation CD5 CAR T cells lackingcostimulation (Fig. 3F). Moreover, disruption of TRAF2 signalingreduced the frequency of central memory T cells (SupplementaryFig. S5). These results indicate that although tonic 4-1BB–derivedTRAF signaling promotes apoptosis in BB.z CAR T cells, acuteantigen-driven TRAF2 signaling is essential for the costimulatoryproperties of the 4-1BB endodomain.

BB.z CD5 CAR T cells form fratricidal immunologic synapsesBecause CD5 expression in 28.z CD5 CAR T cells produces

transient and limited fratricide, we investigated whether constantTRAF signaling in BB.z CD5 CAR T cells enhanced their fratricide.Consistent with this hypothesis, we observed formation of immu-nologic synapses between BB.z CD5 CAR T cells characterized by

polymerization of actin (refs. 24, 25; Fig. 4A and B). A similareffectwas observed in T cells expressing a third-generation 28.BB.zCD5 CAR (Fig. 4C).We also detected increased recruitment of theMTOC to the immunologic synapse in BB.z but not 28.z ormutated BB.z CD5 CAR T cells (Fig. 4D and E), suggesting self-directed cytotoxicity of BB.z CAR T cells (24, 25).

Engagement of adhesionmolecules such as leukocyte function-associated antigen-1 (LFA-1) on T cells and its ICAM ligands ontarget cells is central to the formation of a stable immunologicsynapse (25–27). Because NF-kB can directly activate ICAM-1gene transcription (28, 29),we investigatedwhether chronic TRAFsignaling can similarly enhance ICAM-1 expression in BB.z CD5CAR T cells. In fact, surface ICAM-1 expression was significantlyelevated in BB.z CD5 CAR T cells compared with 28.z CD5 CAR Tcells (Fig. 5A), and blunting TRAF signaling as above in BB.z CD5CAR prevented this upregulation (Fig. 5A). We also detectedsignificant ICAM-1 upregulation in T cells expressing 28.BB.zCD5 CAR (Fig. 5B) or CD5 CARs with other TNFR endodomains(Fig. 5C), which correlatedwith the formation of large cell clustersduring in vitro expansion (Supplementary Fig. S6). ICAM-1 wasrecruited to the synapse interface (Fig. 5D and E), potentially

Figure 1.

Expression of BB.z CD5 CAR abrogates T-cell expansion. A, Schematic representation of CD5 CAR constructs and their expression in T cells 4 days aftertransduction. B, Frequency of CCR7þ CD45RAþ (na€�ve-like) and CCR7þ CD45RA� (central memory) cells among T cells 13 days after transduction with 28.zor BB.z CD5 CAR, compared with nontransduced control T cells. The rest of the cells were comprised by terminally differentiated effector and effectormemory T cells. Data are shown as mean � SD (P ¼ 0.0331 by unpaired Student t test, n ¼ 3). C, Expansion of T cells transduced with 28.z or BB.z CD5 CARand mock-transduced cells (Ctrl). Data are shown as mean � SD (n ¼ 3). D, Representative histograms showing Annexin V staining of CD5 CAR T cells.Bar graphs show summarized data from days 8 and 13 after transduction (P ¼ 0.019 and 0.0044 by unpaired Student t test, respectively). Data representfour to six independent experiments.

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strengthening the fratricidal synapse. Blocking ICAM-1 withmonoclonal antibodies in BB.z CD5 CAR T cells for 48 hourssignificantly reduced apoptotic cells (Fig. 5F) and enhanced T-cellexpansion (Fig. 5G). These results indicate that constant TRAF2signaling promoted BB.z CD5 CAR T-cell apoptosis by enhancingICAM-1 expression and stabilizing the fratricidal immunologicsynapse.

Conditional CAR expression system eliminates fratricide ofBB.z CD5 CAR T cells

Because enhanced apoptosis in T cells was a direct consequenceof tonic BB.z CD5 CAR activation, we determined whether reduc-ing CAR expression could reverse the toxicity. We attenuated toxictonic signaling by expressing the BB.z CD5 CAR from a self-inactivating lentiviral vector, which reduced surface expressionof the CAR (30). This improved cell viability and decreased tonicNF-kB activation, as reflected by normal ICAM-1 expression(Supplementary Fig. S7). Unfortunately, reduced CAR expressionalso significantly impaired the cytotoxicity of BB.z CD5 CAR Tcells (Supplementary Fig. S7), indicating that the T-cell fratricideand the antitumor activity of BB.z CD5 CAR expression cannotreadily be separated. Because such permanent reduction of CAR

expressionwould compromise the antitumor activity of T cells, weevaluated an alternative approach that minimized fratricide dur-ing expansionwhile enabling subsequent antitumor function.Wedesigned a conditional retroviral Tet-OFF expression system inwhich CAR transcription is driven by a transactivator (rTA)encoded downstream of the CAR under the control of a consti-tutive synthetic promoter (Fig. 6A). Addition of the small-molecule doxycycline (Dox) during in vitro culture prevented rTAfrom binding the CAR promoter, resulting in minimal CARexpression on the cell surface (Fig. 6B). We observed normalexpansion of BB.z CD5CAR T cells cultured in the presence ofDox(Fig. 6C), indicating that inhibitingCARexpressionduring cultureprevents CAR T-cell fratricide. The residual CD5 CAR expressionobserved in Tet-OFF–transduced T cells didnot trigger cytotoxicityby CD5 CAR T cells against CD5þ target cells (Supplementary Fig.S8). The phenotype of BB.z Tet-OFF cells was different from thephenotype of 28.z CD5 CAR T cells and mirrored that of thenontransduced T cells (Fig. 6D). Removing Dox from the mediaresulted in restoration of CAR expression over 4 to 5 days (Fig. 6E)and concomitant downregulation of CD5 (Supplementary Fig.S9). Conversely, adding Dox to CD5 CAR T cells minimized CARexpression within 24 hours (Fig. 6F). AfterDoxwithdrawal, T cells

Figure 2.

CD5 CARs containing TNFR superfamily endodomains preclude T-cell expansion. A, Expansion of T cells transduced with CD5 CARs containing theendodomains indicated. Nontransduced cells were used as control. Data are shown as mean � SD (n ¼ 3). B, Representative histograms show expressionof CD5 CARs on T cells at the indicated time points after transduction using anti-mouse F(ab)2-specific antibodies. Vertical line denotes mean 28.z CD5CAR expression. C, Expression of CD5 in CD5 CAR T cells 7 days after transduction. D, Expansion of T cells expressing second- (28.z) or third-generation(28.BB.z) CD5 CARs. Data are shown as mean � SD (P ¼ 0.0006 by unpaired Student t test, n ¼ 3). Data represent two to three independent experiments.






with freshly acquired CAR expression were able to control tumorgrowth in vitro upon coculture with CD5þ cell lines Jurkat andCCRF-CEM(Fig. 6G)with similar effectiveness as the 28.z control.As in T cells with constitutively expressed BB.z CARs, we observedenriched central memory in Tet-OFF BB.z CD5 CAR T cells 7 daysafter Dox withdrawal (Fig. 6H). We observed similar increases inthe expansion and frequency of undifferentiated T-cell subsetsand restoration of cytotoxicity with a Tet-OFF 28.z CD5 CAR(Supplementary Fig. S10). These results indicate that the Tet-OFFCAR expression system produces functional CD5 CAR T cells inthe absence of Dox.

BB.z Tet-OFF CD5 CAR T cells expand in vivo and exhibitsuperior antitumor activity

We tested the ability of the BB.z Tet-OFF CAR T cells to protectagainst systemic T-cell leukemia progression in amouse xenograft

modelwhereNSGmice received a single dose ofCART cells 3 daysafter engraftment with Jurkat-FFluc cells (Fig. 7A). We removedDox from BB.z Tet-OFF CD5 CAR T cells immediately prior tointravenous injection, resulting inminimal CAR expression at thetime of injection (Fig. 7B). Despite delayed expression of the BB.zCAR in the Tet-OFF system, these T cells produced superiormousesurvival (Fig. 7C) and suppression of leukemia (Fig. 7D) com-pared with control 28.z CD5 CAR T cells. Restoration of BB.z CARexpression in T cells prior to in vivo administration significantlyreduced the ability of CD5 CAR T cells to control the tumor(Supplementary Fig. S11), indicating the need to delay CARexpression following in vivo administration. We observed a sig-nificant expansion of BB.z CAR T cells in peripheral blood oftumor-bearing mice (Fig. 7E and F), which correlated with theupregulation of CAR and concomitant downregulation of CD5(Fig. 7G). These data demonstrate the ability of the Tet-OFF

Figure 3.

Disrupting TRAF-binding motifs in the 4-1BB endodomain restores T-cell expansion but ablates costimulation. A, Western blot analysis showingphosphorylation of the CAR-embedded z chain in CD5 CAR T cells 7 days after transduction. Nontransduced T cells were used as controls. B, Phosphorylationof IKKa/IKKb in CD5 CAR T cells 7 days after transduction. C, Expression of CD5 CARs with mutated TRAF-binding sites in T cells 7 days after transduction.D, Frequency of apoptotic T cells expressing normal or mutated CD5 CARs 7 days after transduction. P ¼ 0.002 calculated by one-way ANOVA withBonferroni posttest correction (n ¼ 3). E, Expansion of T cells transduced with normal or mutated CD5 CARs. P ¼ 0.0015 calculated at day 14 by one-wayANOVA with Bonferroni posttest correction (n ¼ 3). F, CD5 CAR T cells were cocultured with GFPþ CCRF cells at an effector-to-target ratio of 1:4 for two3-day rounds. Fresh CCRF cells were added to CD5 CAR T cells at the end of round 1 (R1) to restore the initial effector-to-target ratio. Representative dotplots show frequency of tumor cells at the end of each round. Data represent two to four independent experiments.

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expression system to overcome fratricide and generate functionalBB.z CAR T cells in vitro and in vivo.

Discussion4-1BB costimulation can enhance the in vivo survival and

persistence of CAR T cells and may contribute to their antitumorfunction. In this study, however, we show that when a T-cellmalignancy is targeted, the incorporation of 4-1BB or otherTNFR-related endodomains into a self-reactive CD5 CAR can alsostabilize fratricidal immunologic synapses in a TRAF2- and ICAM-1–dependent manner, decreasing survival of CAR-expressing Tcells. We found that a Tet-OFF–inducible retroviral expressionsystem overcomes fratricide by controllably repressing BB.z CARexpression in the presence of doxycycline in vitro while allowingCAR expression and antitumor activity to be restored once trans-duced T cells are introduced in vivo.

Although both CD28 and 4-1BB endodomains have been usedto deliver costimulation in CAR T cells, they have different roles inT-cell antitumor activity and effects on T-cell phenotype. CD28 isexpressed by both na€�ve and effector/memory T cells and is activeduring the initial priming of na€�ve T cells (31, 32). CD28 signalingdirectly activates PI3K signaling, which augments activation ofT cells and promotes their differentiation to short-lived cytotoxic

effector cells (11, 12). As a result, 28.z CD5 CAR T cells are highlycytotoxic but have limited persistence inmouse xenograft models(17). In contrast to CD28, 4-1BB is upregulated in T cells afterinitial stimulation, and therefore physiological 4-1BB ligationdelivers late costimulatory signals to the activated T cells (31–33).4-1BB signaling protects T cells against activation-induced celldeath (34) and facilitates fatty acid oxidation and increasedrespiration, enhancing development of central memory T cells(13). We found, however, that constant CAR activationupregulates ICAM-1, which in turn sustains cell contactbetween CAR T cells and enhances fratricide, thus decreasingexpansion of CD5 CAR T cells.

The interaction between ICAM-1 and its receptor LFA-1 pro-motes T-cell adhesion and intercellular contact. LFA-1 is anintegrin expressed by all T cells, and TCR stimulation triggers aconformational change in LFA-1, promoting ligand binding andproviding additional costimulation for T cells (35, 36). In con-trast, ICAM-1 ismainly present onother cells: expressionof ICAM-1 on antigen-presenting cells is critical for efficient T-cell primingand memory differentiation (37–39), whereas upregulation ofICAM-1 on the endothelium facilitates T-cell migration (26, 40).ICAM-1 levels in T cells are usually low, although IL2, IL12, andIFNg can upregulate its expression (41, 42). Expression of ICAM-1promotes homotypic T-cell–T-cell interactions, leading to the

Figure 4.

4-1BB endodomain in CD5 CAR enhances formation of immunologic synapses between T cells. A, Confocal representative images (original magnification,�100) of 28.z and BB.z CD5 CAR T cells. Shown are bright field (BF) and single-color anti–F-actin (red) images. Arrows indicate cell–cell contacts. B andC, CD5 CAR T cells were assessed for accumulation of F-actin at synapses formed with each other. P < 0.01 by unpaired two-tailed Student t test. D, Representativeimages showing recruitment of MTOC to the immunological synapse between 28.z and BB.z CD5 CAR T cells. E, Distance between MTOC and immunologicalsynapse formed between two CD5 CAR T cells (P ¼ 0.0002 by one-way ANOVA with Bonferroni posttest correction). Cells were imaged as a z stack on aLeica SP8 confocal microscope. Data are representative of 3 independent experiments with n ¼ 3 donors in each.






formation of T-cell clusters that may facilitate paracrine IL2signaling and regulate T-cell effector functions (42). In our study,we found that expression of BB.z CD5CARs similarly upregulatedICAM-1 in T cells, which correlated with increased clustering.Although CD5 is rapidly internalized upon ligation (43, 44), ourresults indicate that the increased intercellular adhesion andcontinuous CAR signaling promoted formation of immunologicsynapses between CD5 CAR T cells manifested in polymerizationof actin and recruitment of MTOC. Blocking ICAM-1 with amonoclonal antibody decreased fratricide and augmented sur-vival of BB.z CD5 CAR T cells, indicating increased intercellularadhesion promoted self-elimination.

In addition to 4-1BB, other TNFR superfamily membersincluding OX40 and CD27 have been explored as costimula-tory endodomains for CARs. Although 4-1BB costimulates both

CD4þ and CD8þ T cells, ligation of OX40 primarily boosts theproliferation and function of CD4þ T cells (33). Integration ofboth CD28 and OX40 in a GD2-specific CAR enhanced effectorfunction and antitumor activity of GD2 CAR T cells in vitro (45).Similarly, incorporation of the CD27-derived costimulatoryendodomain into an FR-specific CAR increased CAR T-cellpersistence and antitumor function compared with controlT cells expressing 28.z or z-only CAR (46). The potentialcostimulatory function of HVEM and CD30 genes for CARshas not yet been explored. All these TNFR superfamily genescan activate both canonical and noncanonical NF-kB pathwaysvia TRAF2-mediated signaling, albeit by recruiting distinct andpartially nonoverlapping sets of TRAF proteins (32, 33). Wefound that in addition to 4-1BB, other TNFR endodomains alsoenriched the central memory subpopulation of CD5 CAR

Figure 5.

Upregulation of ICAM-1 in T cells expressing CD5 CARs with TNFR costimulatory endodomains promotes fratricide. A, Expression of surface ICAM-1 inCD5 CAR T cells 7 days after transduction. Total relative MFI of ICAM-1 is shown in the bar graph (P < 0.0001 by one-way ANOVA with Bonferroni posttestcorrection, n ¼ 3). B, Upregulation of ICAM-1 in 28.BB.z CD5 CAR T cells on day 7 after transduction (P ¼ 0.03 by unpaired two-tailed Student t test).C, ICAM-1 expression in CD5 CAR T cells. Vertical line denotes average expression level in 28.z CD5 CAR T cells. D, Confocal representative images (originalmagnification, �100) of CD5 CAR T cells showing F-actin (silver) and ICAM-1 (red). Arrow indicates cell–cell contact area. E, CD5 CAR T cells wereassessed for accumulation of ICAM-1 at synapses formed with each other. P < 0.0001 by one-way ANOVA with Bonferroni correction. F, BB.z CD5 CART cells were cultured in the presence of ICAM-1 blocking antibodies for 72 hours, and apoptosis was measured by Annexin V staining as shown on therepresentative histograms (P ¼ 0.043 calculated by paired two-tailed Student t test). G, Increased expansion of BB.z CD5 CAR T cells incubated in thepresence of ICAM-1 blocking antibodies for 72 hours (P ¼ 0.0098 by paired two-tailed Student t test). Data are representative of two to threeindependent experiments.

Mamonkin et al.





T cells, but that incorporation of any of these domains into theCD5 CAR led to similar defects in T-cell expansion associatedwith upregulation of ICAM-1, highlighting a general mecha-nism mediated by TRAF2 signaling. These results indicate that

4-1BB and other TNFR superfamily endodomains can be det-rimental in CARs specific for antigens that may be expressed inT cells even at a low level. Therefore, the optimal costimulatoryendodomain(s) in a CAR may need to be determined both by

Figure 6.

DOX-regulated expression system enables controllable CAR expression and prevents fratricide of BB.z CD5 CAR T cells during expansion. A, Schematicrepresentation of the Tet-OFF expression system. Both 50 and 30 LTR elements are mutated to eliminate endogenous promoter activity, and CD5 CAR isencoded downstream of the PTight promoter. Tetracycline transactivator (tTA) is expressed under the constitutive hybrid EF1a/LTR promoter and, in theabsence of DOX, activates the PTight promoter and drives CD5 CAR expression. Addition of DOX prevents tTA from activating the promoter and minimizesCD5 CAR expression. B, Activated T cells were transduced with Tet-OFF BB.z CD5 CAR and cultured with or without DOX. Representative histograms showCAR expression in T cells 4 days after transduction. C, Expansion of T cells transduced with Tet-OFF BB.z CD5 CARs with or without DOX, compared with 28.z CD5CAR T cells or control nontransduced cells. Data are shown as mean � SD (P¼ 0.0006 by one-way ANOVA with Bonferroni posttest correction, n¼ 3). D, Surfacephenotype of T cells transduced with 28.z, BB.z, or Tet-OFF BB.z CD5 CAR and expanded in the presence of DOX for 11 days, compared with nontransducedcontrol T cells. Numbers denote the frequency of na€�ve-like (TN) and central memory (TCM) T cells, and bar graphs show summarized data for CD4þ and CD8þ T cells(mean � SD, n ¼ 3). E, Tet-OFF BB.z CD5 CAR T cells expanded in the presence of DOX were washed and transferred into cell culture media without DOX.Histogram shows the kinetics of CAR re-expression in T cells after 96 hours of culture. F,Addition of DOX to Tet-OFF BB.z CD5 CAR T cells represses CAR expressionafter 24 hours of culture. G, Tet-OFF BB.z CD5 CAR T cells were expanded in the presence of DOX for 11 days followed by DOX withdrawal and CAR re-expression.CD5 CAR T cells were then cocultured with CD5þ tumor cell lines Jurkat and CCRF at an effector-to-target ratio of 1:4 for 3 days. Dot plots show representativefrequency of GFPþ tumor cells at the end of coculture. Bar graphs show absolute counts of viable tumor cells at the end of coculture (n ¼ 3). Dashed line denotesinitial number of tumor cells upon plating.H,Surface phenotypeof Tet-OFFBB.z CD5CART cells 7 days after DOXwithdrawal, comparedwith nontransduced and28.z CAR T cells. Numbers denote the frequency of na€�ve-like (TN) and central memory (TCM) T cells, similar to D. Data represent three independent experiments.






which population of CAR T cells should be expanded, and theexpression pattern of the target antigen.

Several studies have demonstrated the tonic CAR signalingsignificantly affects T-cell differentiation and antitumor function.Tonic ligand-independent signaling in 28.z GD2 CAR resulted in

accelerated terminal differentiation of T cells in vitro and acqui-sition of an exhausted phenotype (47). In a mouse model,continuous activation through the CD28.z CD19 CAR impairedthe ability of T cells to adequately respond to simultaneous TCRstimulation leading to functional deletion of dual-reactive T cells

Figure 7.

Tet-OFF BB.z CD5 CAR T cells expanded in vivo and protected mice against systemic leukemia. A, Ffluc-expressing GFPþ Jurkat cells were systemicallyengrafted into NSG mice 3 days before a single intravenous injection of 28.z or Tet-OFF BB.z CD5 CAR T cells. B, Expression of CD5 CAR on T cells before injection.C, Overall survival of animals receiving 28.z or BB.z Tet-OFF CD5 CAR T cells (28.z vs. BB.z, P < 0.0001 by Mantel–Cox test, n ¼ 6–9). D, Development ofsystemic leukemia in control and CD5 CAR–treated animals. E, T cells transduced with 28.z or BB.z Tet-OFF CD5 CAR were injected into mice 7 days afterJurkat engraftment. Dot plots show expansion of adoptively transferred T cells in peripheral blood at indicated time points after CAR T-cell injection.Numbers indicate relative frequency of human T cells. F, Overall expansion of T cells in peripheral blood of tumor-bearing mice at the indicated time points(P ¼ 0.0046 by one-way ANOVA with Bonferroni posttest correction, n ¼ 3). N.D., not detectable. G, Representative plot showing expression of CD5 andCD5 CAR in Tet-OFF BB.z CD5 CAR T cells 27 days after injection. Cells with upregulated CD5 CAR and downregulated CD5 are gated. Data representthree independent experiments with 3 to 9 animals in each individual experiment.

Mamonkin et al.





in vivo (48). Although 28.z CD5 CAR T cells also demonstratedenhanced effector differentiation, their cytotoxicity was sustainedfor at least 3 weeks after stimulation, indicating those cells werenot functionally exhausted by the continuous CAR signaling.Therefore, the differential survival of 28.z and BB.z CD5 CART cells after CAR transduction was not a result of impairedcytotoxicity of the former cells but rather reflected enhancedfratricide by the latter.

One way to attenuate tonic CAR signaling is to decrease itsexpression on T cells. We found that a reduction in BB.z CARexpression indeed decreased fratricide of transduced T cells butalso compromised their tumor-directed cytotoxicity. There-fore, we created the single-vector gammaretroviral Tet-OFFsystem to temporally control CAR expression and thus limitboth CAR T-cell fratricide and antigen-driven exhaustion.Using this system, CAR expression is suppressed in the pres-ence of the small-molecule doxycycline in vitro but recoversupon introduction of the cells into a doxycycline-free in vivoenvironment. The Tet-OFF system restores normal expressionand function of BB.z CD5 CAR after doxycycline withdrawal,allowing CAR-mediated cytotoxicity and central memory dif-ferentiation to be restored to the transduced T cells. Injectionof these cells in leukemia-bearing mice protected againstsystemic leukemia progression resulting in greater T-cellexpansion and prolonged suppression of leukemia comparedwith 28.z CD5 CAR T cells. Re-expression of the BB.z CD5 CARin vivo did not result in immediate fratricide of CAR T cells,likely due to the excessive dilution of these cells in mousetissues and competition with malignant blasts for targetcells. In addition to enabling normal expansion of CD5CAR-transduced T cells, this system has several advantagesover "always-on" expression systems. Unlike Tet-OFF systemsthat require cotransduction with two separate vectors (oneplasmid containing the transgene and another encoding thetransactivator), ours utilizes only one gammaretroviral vector,thus simplifying the manufacturing process and increasingtransduction efficiency. Although expression of the synthetictransactivator may promote an immune response againstTet-OFF T cells in humans, it remains to be seen whether themagnitude of this response would limit the expansion or long-term persistence of transgenic T cells.

Taken together, these studies show that TNFR costimulationenhances fratricide of CAR T cells specific for T-cell malignancies.We identified the mechanism of toxicity and described a solutionto circumvent this limitation. Our results have direct implicationson the design of CARs targeting T-cell antigens for optimal clinicalperformance.

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

Authors' ContributionsConception and design: M. Mamonkin, J.S. Orange, M.K. BrennerDevelopment of methodology: M. Mamonkin, M. Mukherjee, M. Srinivasan,D. Gomes-Silva, J.S. OrangeAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Mamonkin, M. Srinivasan, S. Sharma, F. MoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Mamonkin, M. Mukherjee, M. Srinivasan,G. Krenciute, J.S. OrangeWriting, review, and/or revision of the manuscript: M. Mamonkin,M.K. BrennerAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Mamonkin, G. Krenciute, M.K. BrennerStudy supervision: M. Mamonkin, M.K. BrennerOther (acquisition and analysis of high-resolution microscopic images):M. Mukherjee

AcknowledgmentsThis study was supported by a grant from the NIH, NCI (P50 CA126752), by

the ASH Scholar Award (to M. Mamonkin), and by a Stand Up To Cancer – St.Baldrick's Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113; to M.K. Brenner). Stand Up To Cancer is a program of the Entertain-ment Industry Foundation. Research grants are administered by the AmericanAssociation for Cancer Research, the scientific partner of SU2C. The authors alsoappreciate the support of shared resources in the Cancer Center (support grantNCIP30CA125123). The imaging work was supported by R01AI067946(to J.S. Orange).

The authors thank Catherine Gillespie for editing the article.

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.

ReceivedMarch 14, 2017; revised August 7, 2017; acceptedOctober 23, 2017;published OnlineFirst October 27, 2017.

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Mamonkin et al.




2018;6:47-58. Published OnlineFirst October 27, 2017.Cancer Immunol Res Maksim Mamonkin, Malini Mukherjee, Madhuwanti Srinivasan, et al.

Malignancies4-1BB Costimulation of CAR T Cells Directed to T-cell Reversible Transgene Expression Reduces Fratricide and Permits

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