Functional Tuning of CARs Reveals Signaling Threshold...

Research Article

Functional Tuning of CARs Reveals SignalingThreshold above Which CD8þ CTLAntitumor Potency Is Attenuated due toCell Fas–FasL-Dependent AICDAnnette K€unkele1, Adam J. Johnson1, Lisa S. Rolczynski1, Cindy A. Chang1,Virginia Hoglund1, Karen S. Kelly-Spratt1, and Michael C. Jensen1,2,3

Abstract

Chimeric antigen receptor (CAR) development is biasedtoward selecting constructs that elicit the highest magnitudeof T-cell functional outputs. Here, we show that componentsof CAR extracellular spacer and cytoplasmic signalingdomain modulate, in a cooperative manner, the magnitude ofCD8þCTL activation for tumor-cell cytolysis and cytokinesecretion. Unexpectedly, CAR constructs that generate thehighest in vitro activity, either by extracellular spacer lengthtuning or by the addition of cytoplasmic signaling modules,

exhibit attenuated antitumor potency in vivo, whereas CARstuned for moderate signaling outputs mediate tumor eradica-tion. Recursive CAR triggering renders CTLs expressing hyper-active CARs highly susceptible to activation-induced cell death(AICD) as a result of augmented FasL expression. CAR tuningusing combinations of extracellular spacers and cytoplasmicsignaling modules, which limit AICD of CD8þCTLs, may be acritical parameter for achieving clinical activity against solidtumors. Cancer Immunol Res; 3(4); 368–79. �2015 AACR.

IntroductionApproaches to cancer immunotherapy, whereby T cells are

genetically modified to express chimeric antigen receptors (CAR),are the subject of considerable early-phase clinical trials (1).Whereas dramatic antitumor potency is observed in patientstreated with CD19-specific CAR T cells for B-cell malignancies,such as acute lymphoblastic leukemia and non–Hodgkin lym-phomas, challenges to achieve similar responses in patientsharboring solid tumors are considerable (2–4). At present, thedevelopment and clinical testing of CAR-redirected T-cell adop-tive therapy in cancer patients is largely empiric and constrainedby a variety of technical parameters that affect feasibility ofexecuting clinical phase I trials. Two parameters related to cellproducts that can be defined with greater precision are thecomposition of T-lymphocyte subset(s) and the tuning of CARsignaling for functional outputs that maximize their antitumoractivity. Our group has studied the therapeutic activity of CAR-expressing central memory T cells, a stable antigen-experiencedcomponent of the T-cell repertoire having stem cell–like features

and capacity to repopulate long-lived functional memory nichesfollowing adoptive transfer (5–9).

Moving beyond the targeting of CD19-expressing B-cell malig-nancies, a significant challenge for the field is the identificationand vetting of cell-surface target molecules that are amenable toCAR T-cell recognition with tolerable "on" target "off" tumorreactivity (10, 11). Once identified, however, approaches to tunenew CARs for signaling outputs are presently rudimentary. Para-meters that are generally perceived as central to CAR developmentare the affinity of the target molecule CAR antigen-bindingdomain and the signaling modules of the cytoplasmic domain.We and others have described the significant impact of theextracellular spacer in contributing to CAR T-cell performanceand the growing appreciation that CAR spacers need to adjust thebiophysical synapse distance between a T cell and a tumor cell toone that is compatible for T-cell activation (12–14). Given thateach new scFv and target molecule define a unique distance fromthe tumor cell plasma membrane, the adjustment of CAR spacersis unique to each construct and derives via empiric testing oflibraries of spacer length variants.

The present study evaluates the contribution of both extracel-lular spacer length and cytoplasmic signalingmodule selection onthe performance of a CAR specific for a tumor-selective epitope onCD171 (L1-CAM) that is recognized by monoclonal antibodyCE7 and was previously tested as a first-generation CAR in aclinical pilot study (15–17). Using in vitro functional assays forCAR-redirected effector potency, we observed a quantitative hier-archy of effector outputs based on spacer dimension in the contextof second- and third-generation cytoplasmic signaling domains.We observed a striking discordance in CAR T-cell performancein vitro versus in vivodue to fratricidal activation-induced cell death(AICD) of the most functionally potent CAR formats. These datareveal new and potentially clinically relevant parameters for

1BTCCCR, Seattle Children's Research Institute, Seattle, Washington.2Department of Pediatrics, University of Washington, Seattle,Washington. 3Department of Bioengineering, University of Washing-ton, Seattle,Washington.

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

Corresponding Author: Michael C. Jensen, Ben Towne Center for ChildhoodCancer Research, Seattle Children's Research Institute, 1100 Olive Way, Suite100, Seattle, WA 98101. Phone: 206-884-4100; Fax: 206-987-1241; E-mail:[email protected]

doi: 10.1158/2326-6066.CIR-14-0200

�2015 American Association for Cancer Research.

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inspection in the development of CAR T-cell immunotherapy forsolid tumors.

Materials and MethodsCAR construction and lentiviral production

CD171-specific CARs were constructed using (G4S)3 peptide–linked VL and VH segments of the CE7-IgG2 monoclonal anti-body (18). The scFv was codon optimized and subsequentlylinked to variable spacer length domains based on 12AA [shortspacer (SS)/"hinge-only"], 119AA [medium spacer (MS)/"hinge-CH3"] or 229AA [long spacer (LS)/"hinge-CH2-CH3"] derivedfrom human IgG4-Fc. All spacers were linked to the transmem-brane domain of human CD28 and to signaling modules com-prising either the cytoplasmic domain (i) of 4-1BB alone (2GCAR) or (ii) of CD28 (mutant) and 4-1BB (3G CAR), with eachsignaling module fused to the human CD3-z endodomain (19).The cDNA clones encoding CAR variants were linked to a down-stream T2A ribosomal skip element and truncated EGF receptor(EGFRt), cloned into the epHIV7 lentiviral vector, and CD171-CAR lentiviruses were produced in 293T cells (20).

Real-time PCRTotal RNA was extracted from T cells using the RNeasy Mini Kit

(Qiagen). cDNA was synthesized by reverse transcription usingthe First Strand Kit (Life Technologies). RNA quantification wasperformed using FasL primers (IDT) and the CFX96 real-timedetection system (Bio-Rad). Housekeeping gene actin was used asa control. Data were analyzed using CFX Manager Softwareversion 3.0.

Protein expressionWestern blot analysis. T cells were harvested, washed in PBS, andlysed in protease inhibitor (Millipore). Proteins were analyzedusing SDS–PAGE followed byWestern blotting using anti-CD247(CD3-z; BD Biosciences). Signals were detected using an OdysseyInfrared Imager, and band intensities were quantified usingOdyssey v2.0 software (LI-COR).

Flow cytometry. Immunophenotyping was conducted using fluor-ophore-conjugated mAbs: CD4, CD8, CD27, CD28, CD45RA,CD45RO, CD62L, CCR7 (Biolegend). Cell-surface expression ofL1-CAM was analyzed using a fluorophore-conjugated mAb(Clone 014; Sino Biological). EGFRt expression was analyzedusing biotinylated cetuximab (Bristol-Myers Squibb) and a fluor-ophore-conjugated streptavidin secondary reagent. To assess acti-vation and AICD fluorophore-conjugatedmAbs for CD25, CD69,CD137, CD178 (Fas Ligand) and CD95 (Fas, all Biolegend) wereused. Caspase-3 activity was measured using CaspGlow(eBioscience) following the manufacturer's protocol. Flow anal-yses were performed on an LSRFortessa (BD Biosciences), anddata were analyzed using FlowJo software (Treestar).

Generation of T-cell lines expressing CD171-CARsSamples of heparinized blood were obtained from healthy

donors after written informed consent following a research pro-tocol approved by the Institutional Review Board of SeattleChildren's Research Institute (SCRI IRB #13795). Peripheralblood mononuclear cells (PBMC) were isolated using ficoll (GEHealthcare), andCD8þCD45ROþCD62Lþ centralmemory T cells(TCM) were isolated using immunomagnetic microbeads (Milte-

nyi). First, CD8þCD45ROþ cells were obtained by negative selec-tion using a CD8 T-cell isolation kit and CD45RA beads, thenenriched for CD62L, activated with anti-CD3/CD28 beads at abead-to-cell ratio of 3:1 (Life Technologies) and transduced onday 3 by centrifugation at 800� g for 30 minutes at 32�C withlentiviral supernatant (multiplicity of infection ¼ 5) supplemen-ted with 1 mg/mL protamine sulfate (APP Pharmaceuticals).T cells were expanded in RPMI (Cellgro) containing 10% heat-inactivated FCS (Atlas), 2 mmol/L L-glutamine (Cellgro), supple-mented with a final concentration of 50 U/mL recombinanthuman IL2 (Chiron Corporation), and 1 ng/mL IL15 (Miltenyi).The EGFRtþ subset of each T-cell line was enriched by immuno-magnetic selection with biotin-conjugated Erbitux (Bristol-MyersSquibb) and streptavidinmicrobeads (Miltenyi; 21). CD171-CARand mock control T cells were expanded using a rapid expansionprotocol (9). T cells used for in vivo assays were derived bystimulation with CD3/CD28 beads (S1) followed by 2 rapidexpansions (R2) and cryopreserved 14 days (D14) after the secondrapid expansion stimulation.

Cell linesThe neuroblastoma cell lines Be2 and SK-N-DZ were

obtained from the ATCC. Be2-GFP-ffLuc_epHIV7 and SK-N-DZ-GFP-ffLuc_epHIV7 were derived by lentiviral transductionwith the firefly luciferase (ffLuc) gene and purified by sorting onGFP. Both cell lines were further transduced with CD19t-2A-IL2_pHIV7 to generate IL2-secreting cell lines purified by sort-ing on CD19t. All neuroblastoma cell lines were culturedin DMEM (Cellgro) supplemented with 10% heat-inactivatedFCS and 2 mmol/L of L-glutamine. EBV-transformed lympho-blastoid cell lines (TMLCL) and TMLCLs that expressed mem-brane-tethered CD3epsilon-specific scFvFc derived from OKT3mAb (TMLCL-OKT3; ref. 6) were cultured in RPMI 1640supplemented with 10% heat-inactivated FCS and 2 mmol/Lof L-glutamine.

CAR T-cell receptor signalingAfter coculturing 1 � 106 effector and target cells for 4 to 8

minutes, cells were processed to measure Erk/MAP kinase 1/2activity according to the 7-Plex T-cell Receptor Signaling Kit(Millipore). Protein concentration was measured using the PierceBCA Protein Assay Kit (Thermo Scientific).

In vitro T-cell assaysCytotoxicitymeasured by chromium release assay (CRA).Target cellswere labeled with 51Cr (Perkin Elmer), washed, and incubated intriplicate at 5� 103 cells/well with T cells (S1R2D12-14) at variouseffector-to-target (E:T) ratios. Supernatants were harvested after a4-hour incubation for g-counting using Top Count NTX (PerkinElmer), and specific lysis was calculated (16).

Cytotoxicity measured by the biophotonic luciferase assay. Neuro-blastoma cell lines containing GFP-ffLuc_epHIV7 were cocul-tured with effector cells at a 5:1 E:T ratio. The effector cells wereon their first, second, or third round of tumor cell encounter asdescribed above. To assess the amount of viable tumor cells leftafter T-cell encounter, D-luciferin was added, and after 5 min-utes the biophotonic signal from the neuroblastoma cells wasmeasured using an IVIS Spectrum Imaging System (PerkinElmer).

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Cytokine release. A total of 5 � 105 T cells (S1R2D12-14) wereplated with stimulator cells at a 2:1 E:T ratio for 24 hours. IFNg ,TNFa, and IL2 in the supernatant were measured using Bioplexcytokine assay and Bioplex-200 system (Bio-Rad).

Stress test. To mimic recursive antigen encounters, we started acoculture of adherent target cells and freshly thawed nonad-herent effector cells at a 1:1 E:T ratio. After 24 (round I) and 48(round II) hours, T-cell viability was assessed using the GuavaViaCount Assay (Millipore), and nonadherent effector cellswere moved to a new set of adherent target cells at a 1:1 E:Tratio. After rounds I, II, and III (72 hours), T cells wereharvested and treated with a dead cell removal kit (Miltenyi)before further analysis.

ImmunohistochemistryTumor samples were obtained from patients diagnosed with

neuroblastoma and treated in the Department of Pediatric Hema-tology-Oncology of Seattle Children's Hospital (SCRI IRB#13740).

Human neuroblastomas and mouse brains were harvested,fixed, processed, paraffin embedded, and cut into 5-mm sec-tions. Antigen retrieval was performed using Diva decloakerRTU (Biocare Medical). Primary antibodies were incubatedwith sections overnight at 4�C and diluted in blocking bufferas follows: rat monoclonal anti-human CD3 (Clone CD3-12;Bio-Rad) 1:100, mouse monoclonal anti-human Ki67 (CloneMIB-1; Dako) 1:200, rabbit polyclonal anti-human cleavedcaspase-3 (Biocare Medical) 1:100, rabbit polyclonal anti-human Granzyme B (Covance) 1:200, and mouse monoclonalanti-human CD171 (clone 14.10; Covance) 1:200. Secondaryantibodies (Life Technologies) were incubated with sections for2 hours at room temperature and diluted at 1:500 in PBS with0.2% BSA.

Slides were imaged on an Eclipse Ci upright epifluorescencemicroscope (Nikon) equippedwith aNuancemultispectral imag-ing system and analyzed with InForm analysis software (PerkinElmer).

Experiments in NOD/SCID/gc�/� miceNSGmouse tumormodelswere conductedunder SCRI IACUC-

approved protocols.

Intracranial NSG mouse neuroblastoma xenograft model. Adultmale NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ [NODscid gamma (NSG)]micewere obtained from the Jackson Laboratory or bred in house.Mice were injected intracranially (i.c.) on day 0 with 2� 105 IL2-secreting, ffLuc-expressing Be2 or SK-N-DZ tumor cells 2 mmlateral, 0.5mmanterior to the bregma, and 2.5mmdeep from thedura. Mice received a subsequent intratumoral injection of 2 �106 CAR-modified CD8þ TE(CM) either 7 (therapy response mod-el) or 14 (stress test model) days later. In the stress test model,micewere euthanized 3 days after T-cell injection, and brainswereharvested for immunohistochemistry (IHC) analysis.

For bioluminescent imaging of tumor growth, mice receivedintraperitoneal (i.p.) injections of D-luciferin (4.29 mg/mouse).Mice were anesthetized with isoflurane and imaged using an IVISSpectrum Imaging System 15 minutes after D-luciferin injection.Luciferase activity was analyzed using Living Image SoftwareVersion 4.3 and the photon flux analyzed within regions ofinterest (all Perkin Elmer).

Statistical analysesStatistical analyses were conducted using Prism software

(GraphPad). Data are presented as means� SD or SEM, as statedin the figure legends. The Student t test was conducted as a two-sided unpaired test with a confidence interval of 95%. Statisticalanalyses of survival were conducted by the log-rank test. Resultswith a P value of less than 0.05 were considered statisticallysignificant.

ResultsMagnitude of CAR-triggered cytolytic and cytokine functionaloutputs can be incrementally modulated based on CARextracellular spacer size

The biophysical synapse between a CAR-expressing T cell and atumor cell is influenced by the epitope location on the tumor cell-surface target molecule relative to the distance from the tumorcell's plasma membrane. We hypothesized that CAR extracellularspacer size tuning to accommodate a functional signaling synapseis a key attribute to engineering bioactive CARs. To assess theimpact of CD171-specific CAR extracellular spacer size, we assem-bled a set of spacers using modular domains of human IgG4 asfollows: "long spacer" (LS) IgG4 hinge-CH2-CH3, "mediumspacer" (MS) IgG4 hinge-CH3 fusion, and "short spacer" (SS)IgG4 hinge. Each spacer variant was fused to a CD28-transmem-brane domain followed by a second-generation (2G) 4-1BB:zeta-endodomain thatwas linked to the cell-surface EGFRt tag (Fig. 1A;ref. 21). We generated sets of spacer variant 2G-CARþ/EGFRtþ

human CD8þ central memory–derived effector T-cell lines (TE(CM)) from purified CD8þCD45ROþCD62Lþ central memoryprecursors by immunomagnetic selection (Supplementary Fig.S1A and S1B). Following expansion, lentivirally transducedTE(CM) cell lines were further enriched for homogeneous levelsof EGFRt expression by cetuximab immunomagnetic positiveselection (21). We confirmed the similar surface expression levelsof each of the CAR spacer variants by anti-murine F(ab)-specificand EGFR-specific flow-cytometric staining and protein expres-sion quantified by Western blot for CD3z of each T-cell line(Fig. 1B and C).

We first sought to determine whether the magnitude of 2G-CAR–triggered in vitro activation of CD8þ TE(CM) is influenced byspacer domain size. The human tumor cell lines Be2 and SK-N-DZwere selected based on L1-CAM expression levels that are similarto those of clinical tumor specimens (Supplementary Fig. S2).Following activation by CD171þ human neuroblastoma cells,CD171-specific 2G-CAR(LS)-CD8þ TE(CM) exhibited 3.1-foldhigher levels of phospho-ERK (P ¼ 0.003), and 5.7-fold higherpercentage of cells expressing the activation marker CD137 (P ¼0.015), as compared with their CD171-CAR(SS) counterparts(Fig. 1D and E). 2G-CAR(MS) exhibited intermediate levels ofphospho-ERK and CD137 induction as compared with LS and SS2G-CARs. Next, we sought to determine whether spacer size alsomodulated the magnitude of antitumor cytolytic activity in anLS>MS>SS pattern. Using a 4-hour CRA, we observed that lysis ofCD171þ neuroblastoma target cells followed the same potencygradient of LS>MS>SS against both CD171high Be2 and CD171low

SK-N-DZ cell lines (Fig. 1F and Supplementary Fig. S3A). Fur-thermore, activation for cytokine secretion followed the sameincremental output hierarchy such that 2G-CAR(LS) produced8.4-fold higher amount of IFNg (P ¼ 0.003), 6.3-fold more IL2(P < 0.0001) and 6.1-fold higher levels of TNFa (P ¼

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Figure 1.CARextracellular spacer tunes antitumor effector outputs ofCD8þCTLs. A, schematic of CD171-specific 2G-CARextracellular domain spacer variants. B, humanCD8þ

TE(CM) cell-surface expression of 2G SS, MS, or LS spacer variants and EGFRt detectedwith antimurine F(ab) and cetuximab. C, expression levels of 2G-CAR detectedby CD3-z–specific Western blot. D, 2G-CAR–induced levels of phospho-ERK upon coculture with CD171þ Be2 neuroblastoma cells at a 1:1 E:T ratio (n � 3 percondition). E, 2G-CAR activation–induced CD137 surface expression upon tumor coculture as in D. F, antitumor lytic activity of spacer variant 2G-CAR-CTLsdetermined by CRA. Fold-specific lysis of LS andMS spacers relative to SS 2G-CAR-CTLs at a 10:1 E:T ratio. G, stimulation of cytokine secretion inmixed 2G-CAR-CTLtumor cultures (n � 5 per condition). Fold cytokine production comparison is relative to SS 2G-CAR as in F. � , P < 0.05.






0.005; Fig. 1G) as compared with those elicited by 2G-CAR(SS),with the induction by 2G-CAR(MS) falling between these twoextremes. These data demonstrate that the biophysical synapsecreated by CARs can be tuned by spacer size such that incrementallevels of activation and functional outputs are achieved. Based onstandard CAR development criteria typically utilized in the field,the 2G-CAR(LS) spacer variant would be a lead candidate forfurther development for clinical applications.

Inverse correlation of spacer-modulated CAR-redirected CTLfunctional activity in vitro with in vivo antitumor potency

To delineate the relationship of the observed potency of CARsignaling based on in vitro assays to therapeutic activity in vivo,we performed adoptive transfer experiments in NSG mice withestablished human neuroblastoma xenografts stereotacticallyimplanted in the cerebral hemisphere (Fig. 2A). Surprisingly,Be2 tumor–engrafted mice treated with intratumoral injectionof 2G-CAR(LS) exhibited no therapeutic activity, necessitatinganimal euthanasia approximately 3 weeks after tumor inocu-lation (Fig. 2B and C). In comparison, biophotonic tumorsignal was reduced and survival enhanced in mice treated with2G-CAR(SS)-CD8þ TE(CM) and to an intermediate extent inmice treated with the 2G-CAR(MS) variant (P ¼ 0.001; mediansurvival of the different groups: LS ¼ 20 days, mock ¼ 21 days,MS ¼ 59.5 days, SS ¼ 76 days; Fig. 2D). SK-N-DZ tumor–engrafted mice exhibited an SS>MS>>LS hierarchy of tumorresponses with uniform tumor clearance and 100% survival ofanimals treated with 2G-CAR(SS)-CD8þ TE(CM) (Supplemen-tary Fig. S3C and S3D). Failure of 2G-CAR(LS)–redirected CTLs,which we inject directly into engrafted tumors, could not beattributed to their failure to survive adoptive transfer and beactivated in situ, as equivalent intratumor densities of trans-ferred T cells that expressed Granzyme B and Ki67 wereobserved by IHC early after adoptive transfer (day 3; Fig.2E–H). While not statistically significant (P ¼ 0.34), 2G-CAR(LS)-CD8þ TE(CM), in which activated caspase-3 was detected,were 12.1-fold more frequent than their 2G-CAR(SS) counter-parts (Fig. 2H). These data reveal an unexpected discordancebetween the in vitro antitumor potency of CAR-redirected T cellsdictated by extracellular spacer size and their in vivo antitumortherapeutic activity.

Augmented activation-induced cell death accompanieshyperactive signaling outputs of long-spacer formatted second-generation CAR upon recursive antigen exposure

We hypothesized that whereas in vitro activation for cytolysisin a 4-hour CRA is the consequence of a limited duration ofCAR signaling, the in vivo tumor model requires recursiverounds of activation to achieve tumor eradication. Thus, thesignaling performance of a particular CAR format that dictatessuperior metrics in vitro may fail to reveal the consequences ofthe signaling amplitude in vivo. To reproduce recursive serialstimulation in vitro, we devised a CAR T-cell:tumor cell cocul-ture "stress test" assay whereby every 24 hours, CAR T cells areharvested and recursively transferred to culture dishes seededwith tumor cells adjusting for a constant viable 1:1 T-cell:tumorcell ratio (Fig. 3A). We utilized Be2 cells modified to expressfirefly luciferase to concurrently track tumor-cell killing uponeach of three rounds of serial transfer. The recursive activationof the 2G-CAR spacer variant lines resulted in equivalent loss ofantitumor activity by round III (Fig. 3B). In addition, analysis

of each spacer variant-expressing effector cells after each roundby flow-cytometric measurement revealed that 2G-CAR(LS)-CD8þ TE(CM) displayed higher frequencies of cells expressingactivation markers CD25 and CD69, as compared with their2G-CAR(SS) counterparts (round I, 79.4% vs. 46.8%, P ¼0.007; round II, 74.0% vs. 47.6%; round III, 65.7% vs.42.1%, P ¼ 0.037; Fig. 3C).

In contrast with the LS>MS>SS pattern of upregulation ofactivation markers in round I that mimicked our earlier in vitroanalysis, we observed an LS/MS>SS loss of T-cell viability that wasmost substantial in round III (round III percent dead cells, LS58.7%,MS 62.6% vs. SS 21.1%, LS vs. SS P¼ 0.024 andMS vs SS P¼ 0.007; Fig. 3D). To substantiate that the asymmetric loss of T-cell viability by 2G-CAR(LS)-CTLs occurring with recursive acti-vation was the result of exaggerated AICD, we assessed themechanism of cell death focusing on FasL–Fas-mediated T-cellfratricide (22). We observed tumor-induced CAR activation-dependent upregulation of FasL followed an LS>MS>SS hierarchyas 2G-CAR(LS)-CD8þ TE(CM) displayed 4.8-fold and 2.5-foldhigher FasL surface expression, and 5.5-fold and 3.3-fold higherFasL mRNA abundance than the short or medium spacer CAR Tcells, respectively (long vs. short: P < 0.0001 and P ¼ 0.002; longvs.medium: P < 0.0001 and P¼ 0.016; Fig. 3E and F). To link FasLexpression with increased Fas-mediated apoptosis, we analyzedcaspase-3 activity and observed 13.2-fold higher levels of cleavedcaspase-3 in 2G-CAR(LS)-CD8þ TE(CM) as compared with their SScounterpart (P < 0.0001; Fig. 3G). Finally, we subjected 2G-CAR(LS)-CD8þ TE(CM) to siRNA knockdown of Fas or FasL, thenexposed them to tumor and observed a 1.4-fold (Fas; P ¼0.005) and 1.6-fold (FasL; P ¼ 0.0001) increase in T-cell viabilityafter round III, respectively (Fig. 3H). To verify that our siRNAknockdown led to a reduction of Fas/FasL, we assessed theirsurface expressionon the 2G-CAR(LS)-CD8þTE(CM) andobserveda 91.3% reduction in Fasþ (P < 0.0001) and 80.1% reduction inFasLþ CTLs (P < 0.0001) than 2G-CAR(LS)-CD8þ TE(CM) treatedwith scrambled siRNA (Supplementary Fig. S4A and S4B). Inaggregate, these data demonstrate that tuning of CAR spacer sizecanmodulate downstream signaling events that result in not onlydifferential magnitudes of antitumor functional outputs butcoordinated increases in susceptibility to AICD. The balancebetween these two processes for optimal in vivo antitumoractivity may not always be achieved by spacer tuning to achievethe highest levels of CAR-signaling outputs, as exemplified byour comparison of 2G-CAR(LS) and 2G-CAR(SS) structuralvariants.

Augmentation of CAR cytoplasmic endodomain compositionreverts short spacer CD171-CAR to AICD-prone variant uponrecursive tumor encounter

Third-generation CARs contain two costimulatory endodo-main modules in series with the CD3-z activation module andhave been reported to augment the magnitude of cytolysis andcytokine production levels over their second-generation coun-terparts (23). We assembled a CD171-specific 3G-CAR throughthe addition of a CD28 endodomain to the 2G 4-1BB:zeta-endodomain (Fig. 4A). CD8þ TE(CM) expressing comparablelevels of 2G-CAR(SS) and 3G-CAR(SS) were derived frompurified TCM precursors by immunomagnetic selection(Fig. 4B and C). 3G-CAR(SS)-CD8þ TE(CM) demonstrated an8.4-fold higher induction of CD137 expression upon tumorcontact than their second-generation counterparts (P <

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0.0001; Fig. 4D), a 1.3-fold increase in cytolytic activity againstBe2 targets (E:T of 1:10; P ¼ 0.0001; Fig. 4E) and 5.1-fold moreIL2 and 2.5-fold more TNFa secretion (P < 0.0001 and P ¼0.003; Fig. 4F).

Next, we assessed whether the 3G endodomain, in the contextof an extracellular short spacer, could selectively enhance antitu-mor activity in vivo without exacerbation of AICD. Surprisingly,the in vivo antitumor activity of 3G-CAR(SS)-CD8þ TE(CM) against

Figure 2.Inverse correlation between CAR spacer–dependent CTL functional potency in vitro and antitumor activity in vivo. A, schema of the intracranial (i.c.) NSGmouse neuroblastoma xenograft therapy model and biophotonic signal of ffLucþBe2 tumors at day þ6 following stereotactic implantation. B, biophotonic tumorsignal response to intratumorally infused 2G-CAR-CD8þTE(CM) spacer variants (n ¼ 6 mice per group). The LS 2G-CAR cohort was euthanized on day 20 dueto tumor-related animal distress. C, Kaplan–Meier survival plot of treated cohorts from B. D, quantitation of intratumoral 2G-CAR T cells at the time of symptomatictumor progression. T-cell density determined by counting human CD3þ cells and reported as total number per 40 hpf (data representative of individualtumor analysis). E, timeline of tumor retrieval fromNSGmicebearingBe2 i.c. xenografts and treatedwith 2G-CAR-CTLs for subsequent IHC/immunofluorescence (IF)inspection. F, representative tumor andcontralateral hemisphere immunofluorescence images forCD3, Ki67, and activated caspase-3 costainingof SS 2G-CAR-CTLs.G, immunofluorescence quantification of persisting 2G-CAR spacer variant CTLs 3 days after intratumoral implantation; n ¼ total human CD3þ cells per 40 hpfas in D. H, percentage of CD3þ T cells that coexpress Granzyme B, Ki67, and activated caspase-3 (n ¼ average cells/40 hpf from the analysis of two individualengrafted mice). � , P < 0.05; N.S., not statistically significant.






bothBe2 (Fig. 5A) and SK-N-DZ (Fig. 5B)was inferior, thoughnotto a statistically significant degree to their 2G-CAR(SS) counter-parts. These findings were not attributable to differences in short-term persistence of CAR T cells within tumors based on similardensities of human CD3þ T cells detected 3 days after adoptivetransfer (Fig. 5C). Despite the finding of higher frequencies ofGranzyme Bþ 3G-CAR(SS) T cells compared with 2G-CAR(SS)intratumoral T cells, we again observed augmented numbers ofthird-generation T cells with activated caspase-3, suggesting thatthe augmented costimulation through a combined effect of CD28and 4-1BB was capable of hyperstimulation resulting in height-ened AICD, despite the context of a short spacer extracellulardomain (Fig. 5D). This was confirmed by comparing their per-formance using the in vitro stress test assay. Following each roundof tumor stimulation, we observed higher frequencies of CD25þ

CD69þ T cells in the 3G-CAR(SS)-T-cell population (Fig. 6A)

accompanied by increased frequencies of dead T cells throughsuccessive rounds of activation (Fig. 6B). Augmented AICD wasagain associated with heightened levels of FasL expression bysurface staining andmRNA content, which in turn coincided withincreased levels of activated caspase-3 (Fig. 6C–E). These datademonstrate that overtuning of CAR-signaling outputs based onintracellular signaling domain composition negatively impactedon a tuned short spacer dimension in a combinatorial manner byenhancing FasL-mediated T-cell AICD.

DiscussionCARs are capable of mediating multiplexed signaling outputs

that trigger redirected antitumor T-cell effector function (24–26).It stands to reason that the tuning of CARs for effective T-cellantitumor activity will be more stringent in solid tumor

Figure 3.Recursive antigen exposure in vitroresults in differential FasL-mediatedAICDbased onCAR spacer dimension.A, schema of the in vitro stress testassay for the analysis of CAR-T-cellfunctional status and viability uponrepetitive stimulationwith tumor cells.B, quantification of residual viablefLucþ Be2 tumor cells after successiverounds of 2G-CAR transfer (% tumorviability ¼ average of threeindependent experiments). C, flowcytometric quantification of CD25 andCD69 surface expression followingsuccessive rounds of coculture withBe2 cells at an E:S ratio of 1:1 (% CD25þ

CD69þ values ¼ average of twoindependent experiments). D, 2G-CAR-T-cell viability determination bythe Guava Viacount assay after eachround. The percentage of dead T-cellvalues derived as in C. E, frequency ofFasLþ 2G-CAR-CTLs before and after8-hour coculturewith Be2 cells (E:S 1:1;each data point is derived from anindependent experiment). F, foldinduction of FasL mRNA transcriptionmeasured by RT-qPCR upon cocultureof MS and LS 2G-CAR spacer variantsrelative to SS 2G-CAR-CTLsnormalized to b-actin (average offive independent experiments).G, frequency of activated caspase-3þ

2G-CAR-CTLs following 16-hourcoculture with Be2 cells (E:S 1:1; values¼ average of four independentexperiments). H, effect of siRNAknockdown of Fas or FasL onapoptosis induction in LS 2G-CAR-CTLs after three rounds. Averageviability determination by the GuavaViacount assay performed in threeindependent experiments ("–"condition, mock-electroporated Tcells; "scr" condition, scrambledsiRNA). � , P < 0.05: N.S.,not statistically significant.

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applications, and that empiric designs of CARs based on limitedunderstanding of the impact of their composition on in vivoantitumor function will only hamper progress in human clinicalapplications. Here, we systematically interrogate CAR structurefunction in human central memory–derived CD8þ effector CTLsfocusing on the combinatorial effects of extracellular spacer

dimension in the context of cytoplasmic signaling module com-position. By surveyingCAR-signaling strength using in vitro assays,we have identified a potency hierarchy of CAR structural variants.These analyses have revealed a range of CAR-signaling outputspermissive for in vivo antitumor activity above which in vivopotency is attenuated by heightened AICD.

Figure 4.Augmented costimulation via a third-generation CD28:4-1BB:zeta cytoplasmic domain results in enhanced effector function outputs in vitro. A, schematicof 2G- versus 3G-CAR-composition. B, human CD8þ TE(CM) cell-surface expression of 2G- versus 3G-CAR(SS) and EGFRt detected with anti-murine F(ab) andcetuximab. C, 2G- and 3G-CAR(SS) expression levels detected by CD3-z-specific Western blot. D, 2G- versus 3G-CAR(SS) activation–induced CD137 surfaceexpression upon tumor coculture. E, antitumor lytic activity of 2G- versus 3G-CAR(SS)-CTLs determined by CRA. Fold-specific lysis of SS-3G- relative to SS-2G-CTLat a 10:1 E:T ratio (average of three independent experiments). F, stimulation of cytokine secretion in 2G- versus 3G-CAR(SS)-CTL tumor cocultures (n � 6 percondition). Fold cytokine production comparison is relative to 2G-CAR(SS) as in E. � , P < 0.05.






The evolution of CAR design has proceeded to date via a largelyempiric process, and has focused predominantly on the augmen-tation of signaling outputs through combinatorial modules ofcostimulatory receptor cytoplasmic domains fused in series toimmunoreceptor tyrosine-based activation motif (ITAM)-con-taining activationdomains (27–29).Comparisons of the functionof CTLs expressing first-, second-, or third-generation CARs havetypically beenmade in the context of a "stock" extracellular spacerdomain preferred by a particular laboratory, ranging from full-length IgGs to relatively short CD8a hinges or membrane-prox-imal portions of CD28 (30–32). Our group and others havestudied the impact of spacer dimension on CAR signaling andfunctional activity (14, 19). Unlike a T-cell receptor contact withpeptide-loaded HLA class I or II, which defines a scripted bio-physical gap between T-cell plasma membrane and target-cellplasma membrane that is permissive for assembly of a supramo-lecular activation complex, CARs do not conform to this dimen-sional relationship as a consequence of the target molecule'sstructural dimensions, the scFv's epitope location on the targetmolecule, and the CAR's spacer size (33). While the first twodimensions are unique to each selected antigen and antibody-binding domain, the CAR spacer is size tunable and can com-pensate to some extent in normalizing the orthogonal synapsedistance betweenCART cell and target cell. This topography of theimmunological synapse between a T cell and a target cell alsodefines a distance that cannot be functionally bridged by a CARdue to a membrane-distal epitope on a cell-surface target mole-cule that, even with a short spacer CAR, cannot bring the synapsedistance in to an approximation for signaling (13). Likewise,

membrane-proximal CAR target antigen epitopes have beendescribed for which signaling outputs are only observed in thecontext of a long spacer CAR (34).

Using a CD171-specific scFv-binding domain derived from theCE7 mAb, we first assessed the impact of extracellular spacer sizeon signaling outputs from a 4-1BB:zeta second-generation CAR.We observed incremental gains of function in signaling outputsbasedon in vitro assays as spacer size increased from the short IgG4hinge spacer, to an intermediate hinge:CH3, to the full-lengthIgG4-hinge:Fc spacer. Because prior studies have revealed reducedsurvival of LSCART cells due to interaction between FcgRþ cells inthe lung and the Fc portion of the CAR after intravenous injection(14, 35), we used for in vivo testing a direct intratumoral route ofCD8þ CTL administration to study the direct effect of spacerlength on CAR T cells within a solid tumor that provides IL2locally, as a surrogate for infusional IL2 and/or codelivery ofCD4þ Th1 T cells. Unexpectedly, the antitumor potency of intra-tumorally injected CAR-CD8þ CTLs was inversely correlated tospacer size (i.e., SS>MS>>LS) and in vitro functional potency.Given these findings, we hypothesized that commonly employedin vitro assays that assess CAR-T-cell function upon a singlelimited-duration tumor cell encounter fail to detect the subse-quent fate of CAR T cells upon recursive tumor exposure, aswouldbe predicted to occur within solid tumors in vivo. To better assessthis possibility, wedevised an in vitro assay inwhichCART cells arerecursively exposed to equal numbers of biophotonic reportergene-expressing tumor cells. Tumor-cell killing can thereby bequantified biophotonically and retrieved CAR T cells can beinterrogated for activation status, viability, and caspase activity

Figure 5.3G-CAR(SS)-CTLs do not exhibit enhanced antitumor activity in vivo. A, Kaplan–Meier survival curves of Be2 tumor cell–engrafted NSG mice treated with2G- versus 3G-CAR(SS)-CTLs (n ¼ 5–6 per group, sham-transduced CTL control in black). B, Kaplan–Meier survival curves for SK-N-DZ tumor cell–engraftedmice treated as in A. C, 2G- versus 3G-CAR(SS)-T-cell intratumoral persistence 3 days following adoptive transfer; n ¼ number of CD3þ cells per 40 hpf in 2independently derived tumor specimens. D, immunofluorescence detection of Granzyme Bþ and activated caspase-3þ CD3þ CTLs as described in C. � , P < 0.05.

K€unkele et al.





after each round of tumor coculture. We observed, upon threerecursive tumor encounters, disproportionate increases in thefrequency of T cells undergoing apoptosis among 2G-CAR(LS)T cells as compared with 2G-CAR(SS)-T-cells. The exaggeratedAICD correlated with heightened LS CAR-induced expression ofFasL and activated caspase-3 relative to SS CAR. AICD in LS CAR Tcells was reduced by siRNA knockdown of FAS or FasL beforeexposure to tumor cells. These in vitro findings reveal a T cell–intrinsic Fas–FasL-dependent mechanism of AICD that correlateswith limited intratumoral persistence of LS CAR T cells. In aggre-gate, these data demonstrate that the nonsignaling extracellularspacer is amajor tunable CAR design element that affects not onlysignaling activity but persistence ofCART cells in solid tumors in aT cell–intrinsicmanner, independent of interactionswith Fcþ cellsof the reticulo-endothelial system (14).

Given the relation between spacer dimension and in vivosurvival in the context of a 4-1BB:zeta CAR, we sought to under-stand whether the short spacer dimension would be genericallyoptimal in the context of the augmented signaling outputs of athird-generation CD28:4-1BB:zeta CAR endodomain format.Consistent with observations made by multiple other groups,the CD171-specific 3G-CAR(SS) stimulated heightened levels ofcytolytic activity and cytokine synthesis compared with the 2G-CAR(SS) upon in vitro tumor stimulation. However, the augment-ed signaling outputs of the 3G-CAR in the context of its shortspacer also increased FasL expression, exacerbated apoptosis asindicated by increased levels of activated caspase-3 and resulted in

higher frequencies of cell death. Correspondingly, we observedimpaired in vivo antitumor efficacy of the 3G-CAR(SS) T cells, ascompared with the 2G-CAR(SS) due to attenuated in vivo intra-tumoral survival. While CD28 costimulates T cells upon initialantigen activation and enhances T-cell viability by deflectingAICD through Nuclear Factor of Activated T Cells (NFAT)-regu-lated increases in cFLIPshort, published studies have also revealedthat recursive CD28 costimulation of previously activated T cellscan reduce their subsequent survival via augmented FasL expres-sion and, consequently, increased AICD (36). It is interesting,therefore, to speculate whether recursive CD28 signalingmediatedby anti-CD19 CD28:zeta CAR-T-cells is responsible for the rela-tively short persistence duration in treated ALL patients, as com-pared with the often prolonged persistence of anti-CD19 4-1BB:zeta-treated patients in reported clinical trials (2, 37). In aggregate,these data demonstrate that in vivo potency of CAR-redirected Tcells is dependent, in part, on identifying permissive combinationsof size-optimized extracellular spacer domains in the context of aparticular cytoplasmic signaling domain composition. Further-more, we describe an in vitro assay for assessing the proclivity ofa CAR construct to induce AICD in primary human CD8þ CTLsupon recursive activation events. These studies, using a solid tumormodel system, reveal that "overtuning" of CARs based on in vitrofunctional assays can lead to the selection of constructs that exhibitsuboptimal in vivo potency due to excessive AICD.

There is as yet no predictive structural model that can reliablydirect a priori how CARs should be built based on target molecule

Figure 6.Recursive antigen exposure in vitro results in differential FasL-mediated AICD based on CAR cytoplasmic signaling in the context of a short spacer extracellulardomain. A, flow-cytometric quantification of CD25 and CD69 surface expression by 2G- versus 3G-CAR(SS)-CTLs following successive rounds of coculturewith Be2 cells at an E:S of 1:1 (% CD25þCD69þ values derived from average of 2 independent experiments). B, 2G- versus 3G-CAR(SS)-T-cell viability determinationby the Guava Viacount assay after each round of tumor coculture. The percentage of dead T-cell values derived as previously described. C, frequency ofFasLþ 2G- versus 3G-CAR(SS)-CTLs before and after 8-hour coculturewith Be2 cells at an 1:1 E:S ratio (each data point of 5 per 2G-CAR spacer variant is derived froman independently conducted experiment). D, fold induction of FasL mRNA transcription measured by RT-qPCR upon coculture of 2G- versus 3G-CAR(SS)-CTLsnormalized to b-actin (average results from five independent experiments). E, frequency of cytosolic activated caspase-3þ 2G- versus 3G-CAR(SS)-CTLsfollowing 16-hour coculture with Be2 cells at an 1:1 E:S ratio (values derived from average of four independent experiments). � , P < 0.05.






epitope location relative to the plasma membrane of the tumorcell. Moreover, commonly used surrogate in vitro bioassays mayinstruct away from a definitive choice of CAR composition thatresults in the greatest differential between high-level functionalantitumor CAR-T-cell outputs and low-level AICD.Ourwork heredemonstrates that a CAR structural library screen technique usingthe in vitro stress test assaymay be a valuable additional parameterto integrate into CAR engineering. It is conceivable that geneticstrategies might limit the susceptibility of hyperactive CAR con-structs to undergo AICD, such as forced overexpression of cFLIP orToso, or, vector-directed synthesis of siRNAs that knock downFasL or Fas (38, 39). Additional secondary consequences of CARovertuning also require interrogation, such as the predilection ofhyperactive CARs to trigger expression of inhibitory receptors,such as PD-1, capable of enforcing an exhausted T-cell functionalstatus within PD-L1þ solid tumors (40, 41). Our data demon-strate in a solid tumor model that (i) CAR structure functionin vitro testing using commonly employed functional assays canmisdirect the selection of candidate constructs as common prac-tice is to focus on those constructs that display the highestfunctional activity, and (ii) potency tuning of CAR-redirectedeffector CTLs has an upper limit above which gains in themagnitude of effector outputs are negated by augmentation inAICD upon recursive triggering through the CAR. These resultshave guided our selection of a CD171-specific short spacerCAR for a phase I study in children with relapsed/refractoryneuroblastoma.

Disclosure of Potential Conflicts of InterestM.C. Jensen reports receiving commercial research support from, has own-

ership interest (including patents) in, and is a consultant/advisory boardmember for Juno Therapeutics, Inc. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: A. K€unkele, K.S. Kelly-Spratt, M.C. Jensen

Development of methodology: A. K€unkele, A.J. Johnson, L.S. Rolczynski,C.A. Chang, K.S. Kelly-Spratt, M.C. JensenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L.S. Rolczynski, C.A. Chang, V. Hoglund,K.S. Kelly-SprattAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):A. K€unkele, A.J. Johnson, L.S. Rolczynski, C.A. Chang,K.S. Kelly-Spratt, M.C. JensenWriting, review, and/or revision of the manuscript: A. K€unkele, A.J. Johnson,L.S. Rolczynski, C.A. Chang, K.S. Kelly-Spratt, M.C. JensenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. K€unkele, L.S. Rolczynski, C.A. Chang,K.S. Kelly-Spratt, M.C. JensenStudy supervision: A. K€unkele, K.S. Kelly-Spratt, M.C. Jensen

AcknowledgmentsThe authors thank A. DeWispelaere, A. Ravanpay, and C. Crane for helpful

advice. The Jensen laboratory at BTCCCR is the recipient of financial supportfrom the Ben Towne Pediatric Cancer Research Foundation, Make Some Noise:Cure Kids Cancer Foundation Incorporation, Seattle Children's Guild Associ-ation, Andrew McDonough Bþ Foundation, Journey4A Cure, Zulily, St.Baldrick's Foundation, and Life Sciences Discovery Fund.

Grant SupportA. K€unkele was supported by the German Research Foundation (DFG,

Deutsche Forschungsgemeinschaft). M. Jensen was supported by a Stand UpTo Cancer–St. Baldrick's Pediatric Dream Team Translational Research grant(SU2C-AACR-DT1113). Stand Up To Cancer is a program of the EntertainmentIndustry Foundation administered by the American Association for CancerResearch.

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

Received October 27, 2014; revised December 22, 2014; accepted December22, 2014; published OnlineFirst January 9, 2015.

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2015;3:368-379. Published OnlineFirst January 9, 2015.Cancer Immunol Res Annette Künkele, Adam J. Johnson, Lisa S. Rolczynski, et al. FasL-Dependent AICD−

CTL Antitumor Potency Is Attenuated due to Cell Fas+Which CD8Functional Tuning of CARs Reveals Signaling Threshold above

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