Functional Tuning of CARs Reveals Signaling Threshold ... · CAR-expressing central memory T cells, a stable antigen-experienced component of the T-cell repertoire having stem cell-like

1

Functional Tuning of CARs Reveals Signaling Threshold Above Which CD8+

CTL Antitumor Potency is Attenuated Due to Cell Fas-FasL-Dependent AICD

Annette Künkele1, Adam J. Johnson1, Lisa S. Rolczynski1, Cindy A. Chang1, Virginia

Hoglund1, Karen S. Kelly-Spratt1, Michael C. Jensen1-3

1 BTCCCR, Seattle Children’s Research Institute, Seattle, U.S.A.

2 University of Washington, Department of Pediatrics, Seattle, U.S.A.

3 University of Washington, Department of Bioengineering, Seattle, U.S.A.

Running Title: CAR Mediated Activation Induced Cell Death

Keywords: CAR, CD171, solid tumor, AICD, Fas/FasL pathway

Grant support: A. Künkele was supported by the German Research Foundation

(DFG, Deutsche Forschungsgemeinschaft). M. Jensen was supported by a Stand Up

To Cancer–St. Baldrick’s Pediatric Dream Team Translational Research Grant

(SU2C-AACR-DT1113). Stand Up To Cancer is a program of the Entertainment

Industry Foundation administered by the American Association for Cancer Research.

Corresponding author:

Michael C. Jensen, MD

Ben Towne Center for Childhood Cancer Research

Seattle Children’s Research Institute

1100 Olive Way, Suite 100

Seattle, WA 98101

Email: [email protected]

Phone: +1-206-987-1241 Fax: +1-206-884-4100

on January 9, 2020. © 2015 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 9, 2015; DOI: 10.1158/2326-6066.CIR-14-0200

http://cancerimmunolres.aacrjournals.org/

2

Conflict of interest: M.C.J. is a founder and equity holder in Juno Therapeutics, Inc.

Word Count: 5953

Number of Figures: 6

Number of Supplemental Figures: 4




3

Abstract:

Chimeric antigen receptor (CAR) development is biased towards selecting constructs

that elicit the highest magnitude of T-cell functional outputs. Here, we show that

components of CAR extracellular spacer and cytoplasmic signaling domain modulate,

in a cooperative manner, the magnitude of CD8+CTL activation for tumor cell

cytolysis and cytokine secretion. Unexpectedly, CAR constructs that generate the

highest in vitro activity, either by extracellular spacer length tuning or addition of

cytoplasmic signaling modules, exhibit attenuated antitumor potency in vivo, whereas

CARs tuned for moderate signaling outputs mediate tumor eradication. Recursive

CAR triggering renders CTLs expressing hyperactive CARs highly susceptible to

activation-induced cell death (AICD) as a result of augmented FasL expression. CAR

tuning using combinations of extracellular spacers and cytoplasmic signaling

modules, which limit AICD of CD8+CTLs may be a critical parameter for achieving

clinical activity against solid tumors.




4

Introduction

Approaches 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

patients treated with CD19-specific CAR-T-cells for B-cell malignancies, such as

acute lymphoblastic leukemia and non-Hodgkin lymphomas, challenges to achieve

similar responses in patients harboring solid tumors are considerable (2-4). At present,

the development and clinical testing of CAR-redirected T-cell adoptive therapy in

cancer patients is largely empiric and constrained by a variety of technical parameters

that impact feasibility of executing clinical phase I trials. Two parameters related to

cell products that can be defined with greater precision are the composition of T-

lymphocyte subset and the tuning of CAR-signaling for functional outputs that

maximize their antitumor activity. Our group has studied the therapeutic activity of

CAR-expressing central memory T cells, a stable antigen-experienced component of

the T-cell repertoire having stem cell-like features and capacity to repopulate long-

lived functional memory niches following adoptive transfer (5-9).

Moving beyond the targeting of CD19-expressing B-cell malignancies, a

significant challenge for the field is the identification and vetting of cell surface target

molecules that are amenable to CAR-T-cell recognition with tolerable “on” target

“off” tumor reactivity (10, 11). Once identified, however, approaches to tune new

CARs for signaling outputs are presently rudimentary. Parameters that are generally

perceived as central to CAR development are the affinity of the target molecule CAR

antigen-binding domain and the signaling modules of the cytoplasmic domain. We

and others have described the significant impact of the extracellular spacer in

contributing to CAR-T-cell performance and the growing appreciation that CAR




5

spacers need to adjust the biophysical synapse distance between a T cell and a tumor

cell to one that is compatible for T-cell activation (12-14). Given that each new scFv

and target molecule define a unique distance from the tumor cell plasma membrane,

the adjustment of CAR spacers are unique to each construct and derive via empiric

testing of libraries of spacer length variants.

The present study evaluates the contribution of both extracellular spacer

length and cytoplasmic signaling module selection on the performance of a CAR

specific for a tumor-selective epitope on CD171 (L1-CAM) that is recognized by

monoclonal antibody CE7 and was previously tested as a first generation CAR in a

clinical pilot study (15-17). Using in vitro functional assays for CAR-redirected

effector potency, we observed a quantitative hierarchy of effector outputs based on

spacer dimension in the context of second and third generation cytoplasmic signaling

domains. We observed a striking discordance in CAR-T-cell performance in vitro

versus in vivo due to fratricidal activation-induced cell death (AICD) of the most

functionally potent CAR formats. These data reveal new and potentially clinically

relevant parameters for inspection in the development of CAR-T-cell immunotherapy

for solid tumors.




6

Materials and Methods

CAR construction and lentiviral production

CD171-specific CARs were constructed using (G4S)3 peptide linked VL and

VH segments of the CE7-IgG2 monoclonal antibody (18). The scFv was codon-

optimized and subsequently linked to variable spacer length domains based on 12AA

(short spacer (SS)/“hinge-only”), 119AA (medium spacer (MS)/“hinge-CH3”) or

229AA (long spacer (LS)/“hinge-CH2-CH3”) derived from human IgG4-Fc. All

spacers were linked to the transmembrane domain of human CD28 and to signaling

modules comprising either the cytoplasmic domain (i) of 4-1BB alone (2G CAR) or

(ii) of CD28 (mutant) and 4-1BB (3G CAR), with each signaling module fused to the

human CD3-ζ endodomain (19). The cDNA clones encoding CAR-variants were

linked to a downstream 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 PCR

Total RNA was extracted from T cells using the RNeasy Minikit (Qiagen).

cDNA was synthesized by reverse transcription using the First Strand Kit (Life

Technologies). RNA quantification was performed using FasL primers (IDT) and the

CFX96 real-time detection system (Biorad). Housekeeping gene actin was used as a

control. Data were analyzed using CFX Manager Software version 3.0.

Protein expression

Western Blot: T cells were harvested, washed in PBS and lysed in protease

inhibitor (Millipore). Proteins were analyzed using SDS/PAGE followed by Western




7

blotting using anti-CD247 (CD3-ζ, BD Biosciences). Signals were detected using an

Odyssey Infrared Imager and band intensities were quantified using Odyssey v2.0

software (LI-COR).

Flow Cytometry: Immunophenotyping was conducted using fluorophore-

conjugated mAbs: CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CCR7

(Biolegend). Cell surface expression of L1-CAM was analyzed using a fluorophore-

conjugated mAb (Clone 014, Sino Biological). EGFRt expression was analyzed using

biotinylated cetuximab (Bristol-Myers-Squibb) and a fluorophore-conjugated

streptavidin secondary reagent. To assess activation and AICD fluorophore-

conjugated mAbs for CD25, CD69, CD137, CD178 (Fas Ligand) and CD95 (Fas, all

Biolegend) were used. Caspase 3 activity was measured using CaspGlow

(eBioscience) following the manufacturer’s protocol. Flow analyses were performed

on an LSRFortessa (BD Biosciences) and data were analyzed using FlowJo software

(Treestar).

Generation of T-cell lines expressing CD171-CARs

Samples of heparinized blood were obtained from healthy donors after written

informed consent following a research protocol approved by the Institutional Review

Board of Seattle Children’s Research Institute (SCRI IRB #13795). Peripheral blood

mononuclear cells (PBMC) were isolated using ficoll (GE Healthcare) and

CD8+CD45RO+CD62L+ central memory T cells (TCM) were isolated using

immunomagnetic microbeads (Miltenyi). First, CD8+CD45RO+ cells were obtained

by negative selection using a CD8 T-cell isolation kit and CD45RA beads, then

enriched for CD62L, activated with anti-CD3/CD28 beads at a bead to cell ratio of

3:1 (Life Technologies) and transduced on day 3 by centrifugation at 800g for 30




8

minutes at 32°C with lentiviral supernatant (multiplicity of infection [MOI]=5)

supplemented with 1mg/mL protamine sulfate (APP Pharmaceuticals). T cells were

expanded in RPMI (Cellgro) containing 10% heat-inactivated fetal calf serum (FCS,

Atlas), 2mmol/L L-glutamine (Cellgro), supplemented with a final concentration of

50U/ml recombinant human interleukin (IL)-2 (Chiron Corporation), and 1ng/ml IL15

(Miltenyi). The EGFRt+ subset of each T-cell line was enriched by immunomagnetic

selection with biotin-conjugated Erbitux (Bristol-Myers-Squibb) and streptavidin-

microbeads (Miltenyi) (21). CD171-CAR and mock control T cells were expanded

using a rapid expansion protocol (9). T cells used for in vivo assays were derived by

stimulation with CD3/CD28 beads (S1) followed by 2 rapid expansions (R2) and

cryopreserved 14 days (D14) after the second rapid expansion stimulation.

Cell lines

The neuroblastoma (NB) cell lines Be2 and SK-N-DZ were obtained from the

American Type Culture Collection. Be2-GFP-ffLuc_epHIV7 and SK-N-DZ-GFP-

ffLuc_epHIV7 were derived by lentiviral transduction with the firefly luciferase

(ffLuc) gene and purified by sorting on GFP. Both cell lines were further transduced

with CD19t-2A-IL2_pHIV7 to generate IL2-secreting cell lines purified by sorting on

CD19t. All NB cell lines were cultured in DMEM (Cellgro) supplemented with 10%

heat-inactivated FCS and 2mmol/L L-glutamine. EBV-transformed lymphoblastoid

cell lines (TMLCL) and TMLCL that expressed membrane-tethered CD3epsilon-

specific scFvFc derived from OKT3 mAb (TMLCL-OKT3) (6) were cultured in

RPMI 1640 supplemented with 10% heat-inactivated FCS and 2mmol/L L-glutamine.

CAR-T-cell receptor signaling




9

After co-culturing 1x106 effector and target cells for 4-8min, cells were

processed to measure Erk/MAP Kinase ½ activity according to the 7-Plex T-cell

receptor signaling kit (Millipore). Protein concentration was measured using the

Pierce BCA Protein Assay Kit (Thermo Scientific).

In vitro T-cell assays

Cytotoxicity measured by Chromium Release Assay (CRA): Target cells were

labeled with 51Cr (Perkin Elmer), washed and incubated in triplicate at 5x103

cells/well with T cells (S1R2D12-14) at various effector to target (E:T) ratios.

Supernatants were harvested after a 4-hour incubation for γ-counting using Top Count

NTX (Perkin Elmer) and specific lysis was calculated (16).

Cytotoxicity measured by Biophotonic Luciferase Assay: NB cell lines

containing GFP-ffLuc_epHIV7 were co-cultured with effector cells at a 5:1 E:T ratio.

The effector cells were on their first, second or third round of tumor cell encounter as

described above. To assess the amount of viable tumor cells left after T-cell

encounter, D-Luciferin was added and after 5minutes the biophotonic signal from the

NB cells was measured using an IVIS Spectrum Imaging System (Perkin Elmer).

Cytokine release: A total of 5x105 T cells (S1R2D12-14) were plated with

stimulator cells at a 2:1 E:T ratio for 24hours. IFNγ, TNF�, and IL2 in the

supernatant were measured using Bioplex cytokine assay and Bioplex-200 system

(Bio-rad).

Stress Test: To mimic recursive antigen encounters, we started a co-culture of

adherent target cells and freshly thawed non-adherent 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

Guava ViaCount Assay (Millipore) and non-adherent effector cells were moved to a




10

new set of adherent target cells at a 1:1 E:T ratio. After round I, II and III (72hours) T

cells were harvested and treated with a dead cell removal kit (Miltenyi) before further

analysis.

Immunohistochemistry (IHC)

Tumor samples were obtained from patients diagnosed with NB and treated in

the Department of Pediatric Hematology-Oncology of Seattle Children’s Hospital

(SCRI IRB #13740).

Human NBs and mouse brains were harvested, fixed, processed, paraffin

embedded and cut into 5μm sections. Antigen retrieval was performed using Diva

decloaker RTU (Biocare Medical). Primary antibodies were incubated with sections

overnight at 4°C and diluted in blocking buffer as follows: rat monoclonal anti-human

CD3 (Clone CD3-12, Bio-rad) 1:100, mouse monoclonal anti-human Ki67 (Clone

MIB-1, Dako) 1:200, rabbit polyclonal anti-human cleaved caspase 3 (Biocare

Medical) 1:100, rabbit polyclonal anti-human granzyme B (Covance) 1:200, mouse

monoclonal anti-human CD171 (Clone 14.10, Covance) 1:200. Secondary antibodies

(Life technologies) were incubated with sections for 2hours at room temperature and

diluted 1:500 in PBS with 0.2%BSA.

Slides were imaged on an Eclipse Ci upright epifluorescence microscope

(Nikon) equipped with a Nuance multispectral imaging system and analyzed with

InForm analysis software (Perkin Elmer).

Experiments in NOD/SCID/γc-/-mice

NSG mouse tumor models were conducted under SCRI IACUC approved

protocols.




11

Intracranial NSG Mouse Neuroblastoma Xenograft Model: Adult male

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ [NODscid gamma (NSG)] mice were obtained from

the Jackson Laboratory or bred in-house. Mice were injected intracranially (i.c.) on

day 0 with 2x105 IL2-secreting, ffLuc-expressing Be2 or SK-N-DZ tumor cells 2mm

lateral, 0.5mm anterior to the bregma and 2.5mm deep from the dura. Mice received a

subsequent intra tumoral injection of 2x106 CAR-modified CD8+ TE(CM) either seven

(therapy response model) or fourteen (stress test model) days later. In the stress test

model mice were euthanized 3 days after T-cell injection and brains were harvested

for IHC analysis.

For bioluminescent imaging of tumor growth, mice received intraperitoneal

(i.p.) injections of D-luciferin (4.29mg/mouse). Mice were anesthetized with

isoflurane and imaged using an IVIS Spectrum Imaging System 15 minutes after D-

luciferin injection. Luciferase activity was analyzed using Living Image Software

Version 4.3 and the photon flux analyzed within regions of interest (all Perkin Elmer).

Statistical analyses

Statistical analyses were conducted using Prism Software (GraphPad). Data

are presented as means±SD or SEM as stated in the figure legends. Student t test was

conducted as a two-sided unpaired test with a confidence interval of 95%. Statistical

analyses of survival were conducted by log-rank testing. Results with a P value less

than 0.05 were considered statistically significant.




12

Results

Magnitude of CAR-triggered cytolytic and cytokine functional outputs can be

incrementally modulated based on CAR extracellular spacer size.

The biophysical synapse between a CAR-expressing T cell and a tumor cell is

influenced by the epitope location on the tumor cell surface target molecule relative to

the distance from the tumor cell’s plasma membrane. We hypothesized that CAR

extracellular spacer size tuning to accommodate a functional signaling synapse is a

key attribute to engineering bioactive CARs. In order to assess the impact of CD171-

specific CAR extracellular spacer size, we assembled a set of spacers using modular

domains of human IgG4 as follows: “long spacer”(LS) IgG4 hinge-CH2-CH3,

“medium spacer”(MS) IgG4 hinge-CH3 fusion, and “short spacer”(SS) IgG4 hinge.

Each spacer variant was fused to a CD28-transmembrane domain followed by a

second generation (2G) 4-1BB:zeta-endodomain that was linked to the cell surface

EGFRt tag (Fig.1A) (21). We generated sets of spacer variant 2G-CAR+/EGFRt+

human CD8+ central memory-derived effector T-cell lines (TE(CM)) from purified

CD8+CD45RO+CD62L+ central memory precursors by immunomagnetic selection

(Supplemental Fig.1A+B). Following expansion, lentivirally transduced TE(CM) cell

lines were further enriched for homogeneous levels of EGFRt expression by

cetuximab immunomagnetic positive selection (21). We confirmed the similar surface

expression levels of each of the CAR spacer variants by anti-murine F(ab)-specific

and EGFR-specific flow cytometric staining and protein expression quantified by

western blot for CD3ζ of each T-cell line (Fig.1B+C).

We first sought to determine if the magnitude of 2G-CAR-triggered in vitro

activation of CD8+ TE(CM) is influenced by spacer domain size. The human tumor cell

lines Be2 and SK-N-DZ were selected based on L1-CAM expression levels that are




13

similar to those of clinical tumor specimens (Supplemental Fig.2). Following

activation by CD171+ human NB cells, CD171-specific 2G-CAR(LS)-CD8+ TE(CM)

exhibited 3.1-fold higher levels of phospho-ERK (p=0.003), and 5.7-fold higher

percentage of cells expressing the activation marker CD137 (p=0.015) as compared to

their CD171-CAR(SS) counterparts (Fig.1D+E). 2G-CAR(MS) exhibited

intermediate levels of phospho-ERK and CD137 induction as compared to LS and SS

2G-CARs. Next we sought to determine if spacer size also modulated the magnitude

of antitumor cytolytic activity in a LS>MS>SS pattern. Using a 4-hour CRA, we

observed that lysis of CD171+ NB target cells followed the same potency gradient of

LS>MS>SS against both CD171high Be2 and CD171low SK-N-DZ cell lines (Fig.1F

and Supplemental Fig.3A). Further, activation for cytokine secretion followed the

same incremental output hierarchy such that 2G-CAR(LS) produced 8.4-fold higher

amount of IFNγ (p=0.003), 6.3-fold more IL2 (p<0.0001) and 6.1-fold higher levels

of TNFα (p=0.005; Fig.1G) as compared to those elicited by 2G-CAR(SS), with the

induction by 2G-CAR(MS) falling between these two extremes. These data

demonstrate that the biophysical synapse created by CARs can be tuned by spacer

size such that incremental levels of activation and functional outputs are achieved.

Based on standard CAR development criteria typically utilized in the field, the 2G-

CAR(LS) spacer variant would be a lead candidate for further development for

clinical applications.

Inverse correlation of spacer-modulated CAR-redirected CTL functional activity

in vitro with in vivo antitumor potency

To delineate the relationship of the observed potency of CAR-signaling based

on in vitro assays to therapeutic activity in vivo, we performed adoptive transfer




14

experiments in NSG mice with established human NB xenografts stereotactically

implanted in the cerebral hemisphere (Fig.2A). Surprisingly, Be2 tumor-engrafted

mice treated with intratumoral injection of 2G-CAR(LS) exhibited no therapeutic

activity necessitating animal euthanasia approximately 3 weeks after tumor

inoculation (Fig.2B+C). In comparison, biophotonic tumor signal was reduced and

survival enhanced in mice treated with 2G-CAR(SS)-CD8+ TE(CM) and to an

intermediate extent in mice treated with the 2G-CAR(MS) variant (p=0.001; median

survival of the different groups: LS=20 days, mock=21 days, MS=59.5 days, SS=76

days) (Fig. 2D). SK-N-DZ tumor-engrafted mice exhibited a SS>MS>>LS hierarchy

of tumor responses with uniform tumor clearance and 100% survival of animals

treated with 2G-CAR(SS)-CD8+ TE(CM) (Supplemental Fig.3C+D). Failure of 2G-

CAR(LS)-redirected CTLs, which we inject directly into engrafted tumors, could not

be attributed to their failure to survive adoptive transfer and be activated in situ, as

equivalent intratumor densities of transferred T cells which expressed granzyme B

and Ki67 were observed by IHC early after adoptive transfer (day3) (Fig.2E-H).

While not statistically significant (p=0.34), 2G-CAR(LS)-CD8+ TE(CM) in which

activated caspase 3 was detected were 12.1-fold more frequent than their 2G-

CAR(SS) counterparts (Fig.2H). These data reveal an unexpected discordance

between the in vitro antitumor potency of CAR-redirected T cells dictated by

extracellular spacer size and their in vivo antitumor therapeutic activity.

Augmented Activation-Induced Cell Death Accompanies Hyperactive Signaling

Outputs of Long-Spacer Formatted Second Generation CAR Upon Recursive

Antigen Exposure




15

We hypothesized that whereas in vitro activation for cytolysis in a 4-hour

CRA is the consequence of a limited duration of CAR-signaling, the in vivo tumor

model requires recursive rounds of activation to achieve tumor eradication. Thus, the

signaling performance of a particular CAR format that dictates superior metrics in

vitro may fail to reveal the consequences of the signaling amplitude in vivo. In order

to reproduce recursive serial stimulation in vitro, we devised a CAR-T-cell:tumor cell

co-culture “stress test” assay whereby every 24 hours, CAR-T-cells are harvested and

recursively transferred to culture dishes seeded with tumor cells adjusting for a

constant viable 1:1 T-cell:tumor cell ratio (Fig.3A). We utilized Be2 cells modified to

express firefly luciferase to concurrently track tumor-cell killing upon each of three

rounds of serial transfer. The recursive activation of the 2G-CAR spacer variant lines

resulted in equivalent loss of antitumor activity by round III (Fig.3B). Additionally,

analysis of each spacer variant-expressing effector cells after each round by flow

cytometric measurement revealed that 2G-CAR(LS)-CD8+ TE(CM) displayed higher

frequencies of cells expressing activation markers CD25 and CD69, as compared to

their 2G-CAR(SS) counterparts (round I 79.4% vs 46.8%, p=0.007; round II 74.0% vs

47.6%; round III 65.7% vs 42.1%, p=0.037) (Fig.3C).

In contrast to the LS>MS>SS pattern of upregulation of activation markers in

round I that mimicked our earlier in vitro analysis, we observed a LS/MS>SS loss of

T-cell viability that was most substantial in round III (round III percent dead cells LS

58.7%, MS 62.6% vs SS 21.1%, LS vs SS p=0.024 and MS vs SS p=0.007) (Fig.3D).

To substantiate that the asymmetric loss of T-cell viability by 2G-CAR(LS)-CTLs

occurring with recursive activation was the result of exaggerated AICD, we assessed

the mechanism of cell death focusing on FasL-Fas-mediated T-cell fratricide (22). We

observed tumor-induced CAR activation-dependent upregulation of FasL followed a




16

LS>MS>SS hierarchy as 2G-CAR(LS)-CD8+ TE(CM) displayed 4.8-fold and 2.5-fold

higher FasL surface expression, and, 5.5-fold and 3.3-fold higher FasL mRNA

abundance than the short or medium spacer CAR-T-cells, respectively (long vs short:

p<0.0001 and p=0.002; long vs medium: p<0.0001 and p=0.016) (Fig.3E+F). To link

FasL expression with increased Fas-mediated apoptosis, we analyzed caspase 3

activity and observed 13.2-fold higher levels of cleaved caspase 3 in 2G-CAR(LS)-

CD8+ TE(CM) as compared to their SS counterpart (p<0.0001)(Fig.3G). Lastly, we

subjected 2G-CAR(LS)-CD8+ TE(CM) to siRNA knockdown of Fas or FasL, then

exposed them to tumor and observed a 1.4-fold (Fas)(p=0.005) and 1.6-fold

(FasL)(p=0.0001) increase in T-cell viability after round III, respectively (Fig.3H).

To verify that our siRNA knock-down led to a reduction of Fas/FasL, we assessed

their surface expression on the 2G-CAR(LS)-CD8+ TE(CM) and observed a 91.3%

reduction in Fas+ (p<0.0001) and 80.1% reduction in FasL+ CTLs (p<0.0001) than

2G-CAR(LS)-CD8+ TE(CM) treated with scrambled siRNA (Supplemental Fig.4A+B).

In aggregate, these data demonstrate that tuning of CAR spacer size can modulate

downstream signaling events that result not only in differential magnitudes of

antitumor functional outputs but coordinated increases in susceptibility to AICD. The

balance between these two processes for optimal in vivo antitumor activity may not

always be achieved by spacer tuning to achieve the highest levels of CAR-signaling

outputs as exemplified by our comparison of 2G-CAR(LS) and 2G-CAR(SS)

structural variants.

Augmentation of CAR Cytoplasmic Endodomain Composition Reverts Short

Spacer CD171-CAR to AICD-Prone Variant Upon Recursive Tumor Encounter




17

Third generation CARs contain two co-stimulatory endodomain modules in

series with the CD3-ζ activation module and have been reported to augment the

magnitude of cytolysis and cytokine production levels over their second generation

counterparts (23). We assembled a CD171-specific 3G-CAR through the addition of a

CD28-endodomain to the 2G 4-1BB:zeta-endodomain (Fig.4A). CD8+TE(CM)

expressing comparable levels of 2G-CAR(SS) and 3G-CAR(SS) were derived from

purified TCM precursors by immunomagnetic selection (Fig.4B+C). 3G-CAR(SS)-

CD8+ TE(CM) demonstrated an 8.4-fold higher induction of CD137 expression upon

tumor contact than their second generation counterparts (p<0.0001)(Fig.4D), a 1.3-

fold increase in cytolytic activity against Be2 targets (effector to target ratio 1:10,

p=0.0001)(Fig.4E) and 5.1-fold more IL2 and 2.5-fold more TNFα secretion

(p<0.0001 and p=0.003)(Fig.4F).

Next we assessed whether the 3G endodomain, in the context of an

extracellular short spacer, could selectively enhance antitumor activity in vivo without

exacerbation of AICD. Surprisingly, the in vivo antitumor activity of 3G-CAR(SS)-

CD8+ TE(CM) against both Be2 (Fig.5A) and SK-N-DZ (Fig.5B) was inferior, though

not to a statistically significant degree to their 2G-CAR(SS) counterparts. These

findings were not attributable to differences in short-term persistence of CAR-T-cells

within tumors based on similar densities of human CD3+ T cells detected 3 days after

adoptive transfer (Fig.5C). Despite the finding of higher frequencies of granzyme B+

3G-CAR(SS)-T-cells compared to 2G-CAR(SS) intratumoral T cells, we again

observed augmented numbers of third generation T cells with activated caspase 3,

suggesting that the augmented costimulation through a combined effect of CD28 and

4-1BB was capable of hyperstimulation resulting in heightened AICD, despite the

context of a short spacer extracellular domain (Fig.5D). This was confirmed by




18

comparing their performance using the in vitro stress test assay. Following each round

of tumor stimulation, we observed higher frequencies of CD25+CD69+ T cells in the

3G-CAR(SS)-T-cell population (Fig.6A) accompanied by increased frequencies of

dead T cells through successive rounds of activation (Fig.6B). Augmented AICD was

again associated with heightened levels of FasL expression by surface staining and

mRNA content, that in turn coincided with increased levels of activated caspase 3

(Fig.6C-E). These data demonstrate that over tuning of CAR-signaling outputs based

on intracellular signaling domain composition negatively impacted on a tuned short

spacer dimension in a combinatorial manner by enhancing FasL-mediated T-cell

AICD.




19

Discussion

CARs 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-cell antitumor activity will be more stringent in solid

tumor applications and that empiric designs of CARs based on limited understanding

of the impact of their composition on in vivo antitumor function will only hamper

progress in human clinical applications. Here, we systematically interrogate CAR

structure-function in human central memory-derived CD8+ effector CTLs focusing on

the combinatorial effect(s) of extracellular spacer dimension in the context of

cytoplasmic signaling module composition. By surveying CAR-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 outputs permissive

for in vivo antitumor activity above which in vivo potency is attenuated by heightened

AICD.

The evolution of CAR design has proceeded to date via a largely empiric

process, and has focused predominantly on the augmentation of signaling outputs

through combinatorial modules of costimulatory receptor cytoplasmic domains fused

in series to immunoreceptor tyrosine-based activation motif (ITAM)-containing

activation domains (27-29). Comparisons of the function of CTLs expressing first,

second, or third generation CARs have typically been made in the context of a “stock”

extracellular spacer domain preferred by a particular laboratory, ranging from full

length IgGs to relatively short CD8α-hinges or membrane proximal portions of CD28

(30-32). Our group and others have studied the impact of spacer dimension on CAR-

signaling and functional activity (14, 19). Unlike a T-cell receptor contact with

peptide loaded HLA Class I or II, which defines a scripted biophysical gap between




20

T-cell plasma membrane and target-cell plasma membrane that is permissive for

assembly of a supramolecular activation complex, CARs do not conform to this

dimensional relationship as a consequence of the target molecule’s structural

dimensions, the scFv’s epitope location on the target molecule, and the CAR’s spacer

size (33). While the first two dimensions are unique to each selected antigen and

antibody binding domain, the CAR spacer is size tunable and can compensate to some

extent in normalizing the orthogonal synapse distance between CAR-T-cell and target

cell. This topography of the immunological synapse between a T cell and a target cell

also defines a distance that cannot be functionally bridged by a CAR due to a

membrane distal epitope on a cell surface target molecule that, even with a short

spacer CAR, cannot bring the synapse distance in to an approximation for signaling

(13). Likewise, membrane proximal CAR target antigen epitopes have been described

for which signaling outputs are only observed in the context of a long spacer CAR

(34).

Using a CD171-specific scFv-binding domain derived from the CE7 mAb, we

first assessed the impact of extracellular spacer size on signaling outputs from a 4-

1BB:zeta second generation CAR. We observed incremental gains of function in

signaling outputs based on in vitro assays as spacer size increased from the short IgG4

hinge spacer, to an intermediate hinge:CH3, to the full length IgG4-hinge:Fc spacer.

Since prior studies have revealed reduced survival of LS CAR-T-cells due to

interaction between FcγR+ cells in the lung and the Fc-portion of the CAR after

intravenous injection (14, 35), we used for in vivo testing a direct intratumoral route

of CD8+ CTL administration to study the direct effect of spacer length on CAR-T-

cells within a solid tumor that provides IL2 locally, as a surrogate for infusional IL2

and/or co-delivery of CD4+ Th1 T cells. Unexpectedly, the antitumor potency of




21

intratumorally injected CAR-CD8+ CTLs was inversely correlated to spacer size (i.e.

SS>MS>>LS) and in vitro functional potency. Given these findings, we hypothesized

that commonly employed in vitro assays that assess CAR-T-cell function upon a

single limited duration tumor cell encounter fail to detect the subsequent fate of CAR-

T-cells upon recursive tumor exposure, as would be predicted to occur within solid

tumors in vivo. To better assess this possibility we devised an in vitro assay in which

CAR-T-cells are recursively exposed to equal numbers of biophotonic reporter gene-

expressing tumor cells. Tumor-cell killing can thereby be quantified biophotonically

and retrieved CAR-T-cells can be interrogated for activation status, viability, and

caspase activity after each round of tumor co-culture. We observed that upon three

recursive tumor encounters, disproportionate increases in the frequency of T cells

undergoing apoptosis among 2G-CAR(LS)-T-cells as compared to 2G-CAR(SS)-T-

cells. The exaggerated AICD correlated with heightened LS CAR-induced expression

of FasL and activated caspase 3 relative to SS CAR. AICD in LS CAR-T-cells was

reduced by siRNA knockdown of FAS or FasL prior to exposure to tumor cells. These

in vitro findings reveal a T-cell intrinsic Fas-FasL-dependent mechanism of AICD

that correlates with limited intratumoral persistence of LS CAR-T-cells. In aggregate

these data demonstrate that the non-signaling extracellular spacer is a major tunable

CAR design element that impacts not only on signaling activity but persistence of

CAR-T-cells in solid tumors in a T-cell intrinsic manner, independent of interactions

with Fc+ cells of the RE system (14).

Given the relation between spacer dimension and in vivo survival in the

context of a 4-1BB:zeta CAR, we sought to understand if the short spacer dimension

would be generically optimal in the context of the augmenting signaling outputs of a

third generation CD28:4-1BB:zeta CAR endodomain format. Consistent with




22

observations made by multiple other groups, the CD171-specific 3G-CAR(SS)

stimulated heightened levels of cytolytic activity and cytokine synthesis compared to

the 2G-CAR(SS) upon in vitro tumor stimulation. However, the augmented signaling

outputs of the 3G-CAR in the context of its short spacer also increased FasL

expression, exacerbated apoptosis as indicated by increased levels of activated

caspase 3 and resulted in higher frequencies of cell death. Correspondingly, we

observed impaired in vivo antitumor efficacy of the 3G-CAR(SS)-T-cells, as

compared to the 2G-CAR(SS) due to attenuated in vivo intratumoral survival. While

CD28 costimulates T-cells upon initial antigen activation and enhances T-cell

viability by deflecting AICD through NFAT-regulated increases in cFLIPshort,

published studies have also revealed that recursive CD28 costimulation of previously

activated T cells can reduce their subsequent survival via augmented FasL expression

and consequently, increased AICD (36). It is interesting therefore, to speculate if

recursive CD28 signaling mediated by anti-CD19 CD28:zeta CAR-T-cells is

responsible for the relatively short persistence duration in treated ALL patients, as

compared to 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 T cells is dependent, in part, on identifying permissive

combinations of size-optimized extracellular spacer domains in the context of a

particular cytoplasmic signaling domain composition. Further, we describe an in vitro

assay for assessing the proclivity of a CAR-construct to induce AICD in primary

human CD8+ CTLs upon recursive activation events. These studies, using a solid

tumor model system, reveal that “over tuning” of CARs based on in vitro functional

assays, can lead to selection of constructs that exhibit suboptimal in vivo potency due

to excessive AICD.




23

There is as yet no predictive structural model that can reliably direct a priori

how CARs should be built based on target molecule epitope location relative to the

plasma membrane of the tumor cell. Moreover, commonly used surrogate in vitro

bioassays may instruct away from a definitive choice of CAR composition that results

in the greatest differential between high-level functional antitumor CAR-T-cell

outputs and low-level AICD. Our work here demonstrates that a CAR structural

library screen technique using the in vitro stress test assay may be a valuable

additional parameter to integrate into CAR engineering. It is conceivable that genetic

strategies might limit susceptibility of hyperactive CAR constructs to undergo AICD,

such as forced over expression of cFLIP or Toso, or, vector-directed synthesis of

siRNAs that knock down FasL or Fas (38, 39). Additional secondary consequences of

CAR over tuning also require interrogation, such as the predilection of hyperactive

CARs to trigger expression of inhibitory receptors, such as PD-1, capable of

enforcing an exhausted T-cell functional status within PD-L1+ solid tumors (40, 41).

Our data demonstrate in a solid tumor model that, 1) CAR structure-function in vitro

testing using commonly employed functional assays can misdirect selection of

candidate constructs as common practice is to focus on those constructs that display

the highest functional activity, and, 2) potency tuning of CAR-redirected effector

CTLs has an upper limit above which gains in the magnitude of effector outputs are

negated by augmentation in AICD upon recursive triggering through the CAR. These

results have guided our selection of a CD171-specific short spacer CAR for a Phase I

study in children with relapsed/refractory neuroblastoma.




24

Acknowledgement

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

The Jensen Laboratory at BTCCCR is the recipient of financial support from the Ben

Towne Pediatric Cancer Research Foundation, Make Some Noise:Cure Kids Cancer

Foundation Incorporation, Seattle Children’s Guild Association, Andrew McDonough

B+ Foundation, Journey4A Cure, Zulily, St. Baldrick’s Foundation and Life Sciences

Discovery Fund.




25

References

1. Kershaw MH, Westwood JA, Slaney CY, Darcy PK. Clinical application of genetically modified T cells in cancer therapy. Clin Trans Immunol. 2014;3:e16. doi: 10.1038/cti.2014.7. 2. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. The New England journal of medicine. 2013;368(16):1509-18. doi: 10.1056/NEJMoa1215134. PubMed PMID: 23527958; PubMed Central PMCID: PMC4058440. 3. Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Science translational medicine. 2013;5(177):177ra38. doi: 10.1126/scitranslmed.3005930. PubMed PMID: 23515080; PubMed Central PMCID: PMC3742551. 4. Kochenderfer JN, Dudley ME, Kassim SH, Somerville RP, Carpenter RO, Stetler-Stevenson M, et al. Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma and Indolent B-Cell Malignancies Can Be Effectively Treated With Autologous T Cells Expressing an Anti-CD19 Chimeric Antigen Receptor. Journal of clinical oncology : official journal of the American Society of Clinical Oncology. 2014. doi: 10.1200/JCO.2014.56.2025. PubMed PMID: 25154820. 5. Berger C, Jensen MC, Lansdorp PM, Gough M, Elliott C, Riddell SR. Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates. The Journal of clinical investigation. 2008;118(1):294-305. doi: 10.1172/JCI32103. PubMed PMID: 18060041; PubMed Central PMCID: PMC2104476. 6. Wang X, Berger C, Wong CW, Forman SJ, Riddell SR, Jensen MC. Engraftment of human central memory-derived effector CD8+ T cells in immunodeficient mice. Blood. 2011;117(6):1888-98. doi: 10.1182/blood-2010-10-310599. PubMed PMID: 21123821; PubMed Central PMCID: PMC3056638. 7. Terakura S, Yamamoto TN, Gardner RA, Turtle CJ, Jensen MC, Riddell SR. Generation of CD19-chimeric antigen receptor modified CD8+ T cells derived from virus-specific central memory T cells. Blood. 2012;119(1):72-82. doi: 10.1182/blood-2011-07-366419. PubMed PMID: 22031866; PubMed Central PMCID: PMC3251238. 8. Graef P, Buchholz VR, Stemberger C, Flossdorf M, Henkel L, Schiemann M, et al. Serial transfer of single-cell-derived immunocompetence reveals stemness of CD8(+) central memory T cells. Immunity. 2014;41(1):116-26. doi: 10.1016/j.immuni.2014.05.018. PubMed PMID: 25035956. 9. Wang X, Naranjo A, Brown CE, Bautista C, Wong CW, Chang WC, et al. Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale. Journal of immunotherapy. 2012;35(9):689-701. doi: 10.1097/CJI.0b013e318270dec7. PubMed PMID: 23090078; PubMed Central PMCID: PMC3525345. 10. Leuci V, Mesiano G, Gammaitoni L, Aglietta M, Sangiolo D. Genetically redirected T lymphocytes for adoptive immunotherapy of solid tumors. Current gene therapy. 2014;14(1):52-62. PubMed PMID: 24365144. 11. Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunological reviews. 2014;257(1):107-26. doi: 10.1111/imr.12131. PubMed PMID: 24329793; PubMed Central PMCID: PMC3874724.




26

12. Moritz D, Groner B. A spacer region between the single chain antibody- and the CD3 zeta-chain domain of chimeric T cell receptor components is required for efficient ligand binding and signaling activity. Gene therapy. 1995;2(8):539-46. PubMed PMID: 8593604. 13. James SE, Greenberg PD, Jensen MC, Lin Y, Wang J, Till BG, et al. Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane. Journal of immunology. 2008;180(10):7028-38. PubMed PMID: 18453625; PubMed Central PMCID: PMC2585549. 14. Hudecek M, Sommermeyer D, Kosasih PL, Silva-Benedict A, Liu L, Rader C, et al. The non-signaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer immunology research. 2014. doi: 10.1158/2326-6066.CIR-14-0127. PubMed PMID: 25212991. 15. Schonmann SM, Iyer J, Laeng H, Gerber HA, Kaser H, Blaser K. Production and characterization of monoclonal antibodies against human neuroblastoma. International journal of cancer Journal international du cancer. 1986;37(2):255-62. PubMed PMID: 3943922. 16. Gonzalez S, Naranjo A, Serrano LM, Chang WC, Wright CL, Jensen MC. Genetic engineering of cytolytic T lymphocytes for adoptive T-cell therapy of neuroblastoma. The journal of gene medicine. 2004;6(6):704-11. doi: 10.1002/jgm.489. PubMed PMID: 15170741. 17. Park JR, Digiusto DL, Slovak M, Wright C, Naranjo A, Wagner J, et al. Adoptive transfer of chimeric antigen receptor re-directed cytolytic T lymphocyte clones in patients with neuroblastoma. Molecular therapy : the journal of the American Society of Gene Therapy. 2007;15(4):825-33. doi: 10.1038/sj.mt.6300104. PubMed PMID: 17299405. 18. d'Uscio C, Blaser K. Establishment of anti-human neuroblastoma-selective isotype-switch variants. Journal of immunological methods. 1992;146(1):63-70. PubMed PMID: 1735783. 19. Hudecek M, Lupo-Stanghellini MT, Kosasih PL, Sommermeyer D, Jensen MC, Rader C, et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ROR1-specific chimeric antigen receptor T cells. Clinical cancer research : an official journal of the American Association for Cancer Research. 2013;19(12):3153-64. doi: 10.1158/1078-0432.CCR-13-0330. PubMed PMID: 23620405; PubMed Central PMCID: PMC3804130. 20. Ausubel LJ, Hall C, Sharma A, Shakeley R, Lopez P, Quezada V, et al. Production of CGMP-Grade Lentiviral Vectors. BioProcess international. 2012;10(2):32-43. PubMed PMID: 22707919; PubMed Central PMCID: PMC3374843. 21. Wang X, Chang WC, Wong CW, Colcher D, Sherman M, Ostberg JR, et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood. 2011;118(5):1255-63. doi: 10.1182/blood-2011-02-337360. PubMed PMID: 21653320; PubMed Central PMCID: PMC3152493. 22. Arakaki R, Yamada A, Kudo Y, Hayashi Y, Ishimaru N. Mechanism of activation-induced cell death of T cells and regulation of FasL expression. Critical reviews in immunology. 2014;34(4):301-14. PubMed PMID: 24941158. 23. Wang J, Jensen M, Lin Y, Sui X, Chen E, Lindgren CG, et al. Optimizing adoptive polyclonal T cell immunotherapy of lymphomas, using a chimeric T cell receptor possessing CD28 and CD137 costimulatory domains. Human gene therapy. 2007;18(8):712-25. doi: 10.1089/hum.2007.028. PubMed PMID: 17685852.




27

24. Jensen MC, Riddell SR. Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells. Immunological reviews. 2014;257(1):127-44. doi: 10.1111/imr.12139. PubMed PMID: 24329794; PubMed Central PMCID: PMC3991306. 25. Cheadle EJ, Gornall H, Baldan V, Hanson V, Hawkins RE, Gilham DE. CAR T cells: driving the road from the laboratory to the clinic. Immunological reviews. 2014;257(1):91-106. doi: 10.1111/imr.12126. PubMed PMID: 24329792. 26. Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor design. Cancer discovery. 2013;3(4):388-98. doi: 10.1158/2159-8290.CD-12-0548. PubMed PMID: 23550147; PubMed Central PMCID: PMC3667586. 27. Maher J, Brentjens RJ, Gunset G, Riviere I, Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta /CD28 receptor. Nature biotechnology. 2002;20(1):70-5. doi: 10.1038/nbt0102-70. PubMed PMID: 11753365. 28. Campana D, Schwarz H, Imai C. 4-1BB chimeric antigen receptors. Cancer journal. 2014;20(2):134-40. doi: 10.1097/PPO.0000000000000028. PubMed PMID: 24667959. 29. Lanitis E, Poussin M, Klattenhoff AW, Song D, Sandaltzopoulos R, June CH, et al. Chimeric antigen receptor T Cells with dissociated signaling domains exhibit focused antitumor activity with reduced potential for toxicity in vivo. Cancer immunology research. 2013;1(1):43-53. doi: 10.1158/2326-6066.CIR-13-0008. PubMed PMID: 24409448; PubMed Central PMCID: PMC3881605. 30. Cooper LJ, Topp MS, Serrano LM, Gonzalez S, Chang WC, Naranjo A, et al. T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect. Blood. 2003;101(4):1637-44. doi: 10.1182/blood-2002-07-1989. PubMed PMID: 12393484. 31. Marin V, Kakuda H, Dander E, Imai C, Campana D, Biondi A, et al. Enhancement of the anti-leukemic activity of cytokine induced killer cells with an anti-CD19 chimeric receptor delivering a 4-1BB-zeta activating signal. Experimental hematology. 2007;35(9):1388-97. doi: 10.1016/j.exphem.2007.05.018. PubMed PMID: 17656004. 32. Brentjens RJ, Latouche JB, Santos E, Marti F, Gong MC, Lyddane C, et al. Eradication of systemic B-cell tumors by genetically targeted human T lymphocytes co-stimulated by CD80 and interleukin-15. Nature medicine. 2003;9(3):279-86. doi: 10.1038/nm827. PubMed PMID: 12579196. 33. James JR, Vale RD. Biophysical mechanism of T-cell receptor triggering in a reconstituted system. Nature. 2012;487(7405):64-9. doi: 10.1038/nature11220. PubMed PMID: 22763440; PubMed Central PMCID: PMC3393772. 34. Grada Z, Hegde M, Byrd T, Shaffer DR, Ghazi A, Brawley VS, et al. TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy. Molecular therapy Nucleic acids. 2013;2:e105. doi: 10.1038/mtna.2013.32. PubMed PMID: 23839099; PubMed Central PMCID: PMC3731887. 35. Hombach A, Hombach AA, Abken H. Adoptive immunotherapy with genetically engineered T cells: modification of the IgG1 Fc 'spacer' domain in the extracellular moiety of chimeric antigen receptors avoids 'off-target' activation and unintended initiation of an innate immune response. Gene therapy. 2010;17(10):1206-13. doi: 10.1038/gt.2010.91. PubMed PMID: 20555360. 36. Boussiotis VA, Lee BJ, Freeman GJ, Gribben JG, Nadler LM. Induction of T cell clonal anergy results in resistance, whereas CD28-mediated costimulation primes




28

for susceptibility to Fas- and Bax-mediated programmed cell death. Journal of immunology. 1997;159(7):3156-67. PubMed PMID: 9317113. 37. Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K, et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Science translational medicine. 2014;6(224):224ra25. doi: 10.1126/scitranslmed.3008226. PubMed PMID: 24553386. 38. Kreuz S, Siegmund D, Scheurich P, Wajant H. NF-kappaB inducers upregulate cFLIP, a cycloheximide-sensitive inhibitor of death receptor signaling. Molecular and cellular biology. 2001;21(12):3964-73. doi: 10.1128/MCB.21.12.3964-3973.2001. PubMed PMID: 11359904; PubMed Central PMCID: PMC87059. 39. Hitoshi Y, Lorens J, Kitada SI, Fisher J, LaBarge M, Ring HZ, et al. Toso, a cell surface, specific regulator of Fas-induced apoptosis in T cells. Immunity. 1998;8(4):461-71. PubMed PMID: 9586636. 40. Oestreich KJ, Yoon H, Ahmed R, Boss JM. NFATc1 regulates PD-1 expression upon T cell activation. Journal of immunology. 2008;181(7):4832-9. PubMed PMID: 18802087; PubMed Central PMCID: PMC2645436. 41. Greenwald RJ, Freeman GJ, Sharpe AH. The B7 family revisited. Annual review of immunology. 2005;23:515-48. doi: 10.1146/annurev.immunol.23.021704.115611. PubMed PMID: 15771580.




29

Figure Legends

Fig.1. CAR extracellular spacer tunes antitumor effector outputs of CD8+ CTLs.

(A) Schematic of CD171-specific 2G-CAR extracellular domain spacer variants. (B)

Human CD8+ TE(CM) cell surface expression of 2G SS, MS or LS spacer variants and

EGFRt detected with anti-murine F(ab) and cetuximab. (C) Expression levels of 2G-

CAR detected by CD3-ζ-specific Western Blot. (D) 2G-CAR-induced levels of

phospho-ERK upon co-culture with CD171+ Be2 NB cells at a 1:1 E:T ratio (n≥3 per

condition). (E) 2G-CAR activation-induced CD137 surface expression upon tumor

co-culture as in (D). (F) Antitumor lytic activity of spacer variant 2G-CAR-CTLs

determined by CRA. Fold-specific lysis of LS and MS spacers relative to SS 2G-

CAR-CTLs at a 10:1 E:T ratio. (G) Stimulation of cytokine secretion in mixed 2G-

CAR-CTL mixed tumor cultures (n ≥5 per condition). Fold-cytokine production

comparison is relative to SS 2G-CAR as in (F).

Fig.2. Inverse correlation between CAR spacer-dependent CTL functional potency in

vitro and antitumor activity in vivo.

(A) Schema of intracranial (i.c.) NSG mouse NB xenograft therapy model and

biophotonic signal of ffLuc+Be2 tumors at day +6 following stereotactic implantation.

(B) Biophotonic tumor signal response to intratumorally infused 2G-CAR-

CD8+TE(CM) spacer variants (n=6 mice per group). LS 2G-CAR cohort was euthanized

on day 20 due to 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 symptomatic tumor progression. T-cell density determined by counting human

CD3+ cells and reported as total number per 40hpf’s (data representative of individual

tumor analysis). (E) Timeline of tumor retrieval from NSG mice bearing Be2 i.c.




30

xenografts and treated with 2G-CAR-CTLs for subsequent IHC/IF inspection. (F)

Representative tumor and contralateral hemisphere immunofluorescence (IF) images

for CD3, Ki67 and activated caspase 3 co-staining of SS 2G-CAR-CTLs. (G) IF

quantification of persisting 2G-CAR spacer variant CTLs 3days after intratumoral

implantation. N=total human CD3+cells per 40hpf as in (D). (H) Percentage of CD3+

T cells that co-express granzyme B, Ki67, and activated caspase 3

(n=avg.#cells/40hpf from analysis of two individual engrafted mice).

Fig.3. Recursive antigen exposure in vitro results in differential FasL-mediated AICD

based on CAR spacer dimension.

(A) Schema of in vitro stress test assay for analysis of CAR-T-cell functional status

and viability upon repetitive stimulation with tumor cells. (B) Quantification of

residual viable fLuc+ Be2 tumor cells after successive rounds of 2G-CAR transfer

(%tumor viability=average of 3 independent experiments). (C) Flow cytometric

quantification of CD25 and CD69 surface expression following successive rounds of

co-culture with Be2 cells at an effector:stimulator (E:S) ratio of 1:1 (%CD25+CD69+

values=average of 2 independent experiments). (D) 2G-CAR-T-cell viability

determination by Guava Viacount assay after each round. %Dead T-cells values

derived as in (C). (E) Frequency of FasL+ 2G-CAR-CTLs before and after 8-hour co-

culture with Be2 cells (E:S 1:1; each data point is derived from an independent

experiment). (F) Fold-induction of FasL mRNA transcription measured by rt-qPCR

upon co-culture of MS and LS 2G-CAR spacer variants relative to SS 2G-CAR-CTLs

normalized to beta-actin (average of 5 independent experiments). (G) Frequency of

activated caspase 3+ 2G-CAR-CTLs following 16-hour co-culture with Be2 cells (E:S

1:1; values=average of 4 independent experiments). (H) Effect of siRNA knockdown




31

of Fas or FasL on apoptosis induction in LS 2G-CAR-CTLs after 3 rounds. Average

viability determination by Guava Viacount assay performed in 3 independent

experiments (“-“ condition is mock electroporated T-cells, “scr” condition scrambled

siRNA).

Fig.4. Augmented co-stimulation via a third generation CD28:4-1BB:zeta

cytoplasmic domain results in enhanced effector function outputs in vitro.

(A) Schematic of 2G-versus 3G-CAR-composition. (B) Human CD8+ TE(CM) cell

surface expression of 2G-versus 3G-CAR(SS) and EGFRt detected with anti-murine

F(ab) and cetuximab. (C) 2G- and 3G-CAR(SS) expression levels detected by CD3-ζ-

specific Western Blot. (D) 2G- versus 3G-CAR(SS) activation-induced CD137

surface expression upon tumor co-culture. (E) Antitumor lytic activity of 2G- versus

3G-CAR(SS)-CTLs determined by CRA. Fold-specific lysis of SS-3G- relative to SS-

2G-CTL at a 10:1 E:T ratio (average of 3 independent experiments). (F) Stimulation

of cytokine secretion in 2G- versus 3G-CAR(SS)-CTL tumor co-cultures (n≥6 per

condition). Fold-cytokine production comparison is relative to 2G-CAR(SS) as in (E).

Fig.5. 3G-CAR(SS)-CTLs do not exhibit enhanced antitumor activity in vivo.

(A) Kaplan-Meier survival curves of Be2 tumor-cells engrafted NSG mice treated

with 2G- versus 3G-CAR(SS)-CTLs (n=5-6 per group, sham-transduced CTL control

in black). (B) Kaplan-Meier survival curves for SK-N-DZ tumor-cells engrafted mice

treated as in (A). (C) 2G- versus 3G-CAR(SS)-T-cell intratumoral persistence 3days

following adoptive transfer. N= #of CD3+ cells per 40hpf in two independently

derived tumor specimens. (D) IF detection of granzyme B+ and activated caspase 3+

CD3+ CTLs as described in (C).




32

Fig.6. Recursive antigen exposure in vitro results in differential FasL-mediated AICD

based on CAR cytoplasmic signaling in the context of a short spacer extracellular

domain.

(A) Flow cytometric quantification of CD25 and CD69 surface expression by 2G-

versus 3G-CAR(SS)-CTLs following successive rounds of co-culture with Be2 cells

at an E:S of 1:1 (%CD25+CD69+ values derived from average of two independent

experiments). (B) 2G- versus 3G-CAR(SS)-T-cell viability determination by Guava

Viacount assay after each round of tumor co-culture. %Dead T-cells values derived as

previously described. (C) Frequency of FasL+ 2G- versus 3G-CAR(SS)-CTLs before

and after 8-hour co-culture with Be2 cells at an 1:1 E:S ratio (each data point of 5 per

2G-CAR spacer variant is derived from an independently conducted experiment). (D)

Fold-induction of FasL mRNA transcription measured by rt-qPCR upon co-culture of

2G- versus 3G-CAR(SS)-CTLs normalized to beta-actin (average results from 5

independent experiments). (E) Frequency of cytosolic activated caspase3+ 2G- versus

3G-CAR(SS)-CTLs following 16-hour co-culture with Be2 cells at an 1:1 E:S ratio

(values avg. of 4 independent experiments).






















Published OnlineFirst January 9, 2015.Cancer Immunol Res Annette Künkele, Adam J Johnson, Lisa S Rolczynski, et al. Fas-FasL Dependent AICDWhich CD8+ CTL Antitumor Potency is Attenuated Due to Cell Functional Tuning of CARs Reveals Signaling Threshold Above

Updated version

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

Access the most recent version of this article at:

Material

Supplementary

http://cancerimmunolres.aacrjournals.org/content/suppl/2015/01/09/2326-6066.CIR-14-0200.DC1

Access the most recent supplemental material at:

Manuscript

Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerimmunolres.aacrjournals.org/content/early/2015/01/09/2326-6066.CIR-14-0200To request permission to re-use all or part of this article, use this link



http://cancerimmunolres.aacrjournals.org/lookup/doi/10.1158/2326-6066.CIR-14-0200

http://cancerimmunolres.aacrjournals.org/content/suppl/2015/01/09/2326-6066.CIR-14-0200.DC1

http://cancerimmunolres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://cancerimmunolres.aacrjournals.org/content/early/2015/01/09/2326-6066.CIR-14-0200


Documents

Functional Tuning of CARs Reveals Signaling Threshold ... · CAR-expressing central memory T cells, a stable antigen-experienced component of the T-cell repertoire having stem cell-like