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Blood First Edition Paper, prepublished online June 5, 2015; DOI 10.1182/blood-2014-11-612903

Eradication of B-ALL Using Chimeric Antigen Receptor-Expressing T cells Targeting the TSLPR Oncoprotein

1

1

1

1

2,3, Htoo Zarni

Haiying Qin , Monica Cho , Waleed Haso , Ling Zhang , Sarah K. Tasian

4,6, Oo

5 Luca Negri ,

Gian

Maude

2,3,

Yongshun

David T. Teachey

4,6, Daugaard

Poul H.B.

2,3,

7 Lin ,

7 Jizhong Zou ,

David M. Barrett

5 Sorensen ,

8

Barbara S. Mallon , Shannon

2,3,

1

Rimas J. Orentas , Mads

2,3, Stephan A. Grupp

and Terry J. Fry

1

1 Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland; USA

2Division of

Oncology, Children's Hospital of Philadelphia, Philadelphia,

Pennsylvania; USA

3Department of Pediatrics,

University of Pennsylvania Perelman School of Medicine,

Philadelphia, Pennsylvania; USA

4Vancouver Prostate Centre, 5Department of Molecular

Vancouver, British Columbia; Canada

Oncology, BC Cancer Agency, Vancouver, British

Columbia; Canada

6Department of

Urologic Sciences, University of British Columbia, Vancouver, British

Columbia; Canada

7iPSC

Core, National Heart Lung and Blood Institute, National Institutes of Health,

Bethesda, Maryland; USA

8NIH

Stem Cell Unit, National Institute of Neurological Diseases and Stroke, National

Institutes of Health, Bethesda, Maryland; USA

Corresponding Author:

Terry J. Fry, MD Pediatric Oncology Branch, Center for Cancer Research NCI, NIH Bethesda, MD 20816 [email protected] ph: 301-402-0215 fax: 301-451-7052

Short title: Eradication of B-ALL by anti-TSLPR CAR T cells

Scientific category: LYMPHOID NEOPLASIA

Disclaimer The content of this publication does not necessarily reflect the views of policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

1

Copyright © 2015 American Society of Hematology

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Key Points

1) Adoptive transfer of T cells genetically modified to express anti-TSLPR chimeric antigen receptors can cure B-ALL in xenograft models

2) Anti-TSLPR CAR constructs containing a CH2CH3 spacer domain were inactive against TSLPR-overexpressing B-ALL

2

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Abstract

Adoptive transfer of T cells genetically modified to express chimeric antigen

receptors (CARs) targeting the CD19 B cell-associated protein have demonstrated

potent activity against relapsed/refractory B lineage acute lymphoblastic leukemia

(B-ALL). Not all patients respond, and CD19-negative relapses have been observed.

Overexpression of the thymic stromal lymphopoietin receptor (TSLPR; encoded by

CRLF2) occurs in a subset of adults and children with B-ALL and confers a high risk of relapse. Recent data suggest the TSLPR signaling axis is functionally important,

suggesting that TSLPR would be an ideal immunotherapeutic target. We

constructed short and long CARs targeting TSLPR and tested efficacy against

overexpressing B-ALL. Both CARs demonstrated activity

TSLPR CAR T cells mediated leukemia regression.

CRLF2-

in vitro, but only short

In vivo activity of the short CAR

was also associated with long-term persistence of CAR-expressing T cells. Short

TSLPR CAR treatment of mice engrafted with a TSLPR-expressing ALL cell line

induced leukemia cytotoxicity with comparable efficacy to that of CD19 CAR T cells.

Short TSLPR CAR T cells also eradicated leukemia in four xenograft models of

human

CRLF2-overexpressing ALL. Finally, TSLPR has limited surface expression on

normal tissues. TSLPR-targeted CAR T cells thus represent a potent oncoprotein-

targeted immunotherapy for high-risk ALL.

3

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Introduction

Acute lymphoblastic leukemia (ALL) is the most common oncologic diagnosis in

children. Substantial progress has been made in upfront therapy for pediatric ALL

such that >80% of patients will be cured

1-3.

Nonetheless, ALL remains a significant

cause of death from cancer in children due to initial refractoriness or relapse of

leukemia that no longer to responds to cytotoxic chemotherapy

4,5.

Furthermore,

long-term therapy-induced morbidity is a major issue, particularly in those patients

deemed to be at high risk for relapse who are thus treated with more intensive

6

regimens under current risk-adapted protocols . ALL occurs less frequently in

adults than in children, but the prognosis for adult ALL is much worse despite

7

treatment with multi-agent cytotoxic chemotherapy . Treatment of young adults on

pediatric-type regimens has improved outcome, but not to the level achieved in

8

children . Thus, for multiple reasons, there remains the need for new treatments for

ALL for all age groups that is not based on intensification of cytotoxic chemotherapy.

After more than a decade of study with modest clinical progress, immune-based

treatment for cancer has shown much promise in recent years. In particular, the

adoptive cell transfer of T cells genetically modified to express chimeric antigen

receptors (CARs) targeting antigens expressed on lymphoid cells have

demonstrated potent activity in B cell malignancies, including ALL, with reported

remission rates of 70-90% in multiply relapsed or chemotherapy-refractory

patients

9-14.

The surface protein targeted in the majority of these trials is the CD19

antigen that is expressed on both malignant and non-malignant B cells. However,

not all patients respond to CD19-redirected CAR T cells, and relapses have been

observed, in some cases due to loss of CD19 expression

14.

Loss of CD19 has also

been observed after treatment with a bispecific antibody-based reagent targeting

CD19 and CD3

15.

Alternative cell surface targets are thus needed. In principle, the

optimal target for immunotherapy is a cell surface protein that is functionally

important for the leukemic cell such that relapse due to antigen loss is less likely to

occur.

4

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Substantial progress in leukemia genomics has resulted in the identification of genes

3

and pathways that are dysregulated in ALL . One such category are those associated

with cytokine signaling, including IL-7 receptor pathways and in particular CD127

(encoded by

IL7RA)16.

Thymic stromal lymphopoietin (TSLP) is a cytokine that

binds to the TSLPR, a heterodimeric receptor complex comprised of a TSLPR

subunit (encoded by the

CRLF2 gene) and a CD127 subunit, and induces JAK/STAT

pathway signaling. Overexpression of the TSLPR has been identified in 5-15% of

children and adults with B-ALL, largely due to

resulting in alternative promoters

17-22.

CRLF2 translocations or deletions

Importantly, in a number of studies,

CRLF2

rearrangements have been associated with poor prognosis in both pediatric and

adult ALL

23-29,

and emerging studies have identified activation of the TSLPR

signaling pathway as biologically important for ALL blasts

30,31.

Recent

comprehensive genomic analyses of high risk B-ALL cases demonstrated that

CRLF2

rearrangements in ALL are frequently associated with a gene expression profile

highly similar to that of Philadelphia chromosome-positive ALL, but without the

BCR-ABL1 fusion. Patients with these “Philadelphia chromosome-like” (Ph-like) leukemias respond poorly to conventional chemotherapy and have high rates of

relapse

17.

Multiple groups have now demonstrated that

CRLF2 rearrangements

account for half of Ph-like ALL genomic alterations and are also highly associated

with concomitant

JAK1 and JAK2 point mutations17,18,20,21,32.

We maintain that the

TSLPR functions as an ALL oncoprotein given its cell surface expres sion and

association with poor clinical outcomes and thus may be an ideal

immunotherapeutic target. Furthermore, TSLPR expression in normal tissues

appears to be restricted. We demonstrate that a TSLPR CAR can completely

eradicate human

CRLF2-rearranged (TSLPR-overexpressing) ALL in multiple model

systems, including patient-derived xenografts (PDX).

Materials and Methods

Cells and culture conditions The following B cell acute lymphoblastic leukemia (ALL) cell lines were used:

MUTZ5 (DSMZ ACC 490), REH-TSLPR (native REH transduced with human TSLPR),

5

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and non-transduced REH as a TSLPR-negative control. Cell lines were cultured in

media supplemented with 10% heat-inactivated FBS (Gemini Bioproducts), 10mM

HEPES, 100 U/mL penicillin, 100 ug/mL streptomycin, and 2mM L-glutamine

(Invitrogen). The 293T retroviral vector packaging cell line (Clontech) was cultured

in DMEM (Invitrogen). In addition, PDX models of

CRLF2-rearranged Ph-like ALL (de

novo specimens JH331, JH352, and NH362 and relapse specimen ALL4364; Table 1) were used for

in vivo testing of TSLPR CAR T cells. PDX models were created as

previously described

33,34

using viably cryopreserved Ph-like ALL specimens banked

in the Children’s Oncology Group or Children’s Hospital of Philadelphia leukemia

biorepositories under Institutional Review Board (IRB)-approved research

protocols. For these studies, secondary and tertiary PDX models were used for

relapse and

de novo ALL specimens, respectively. Briefly, primary leukemia cells

from peripheral blood or bone marrow were intravenously injected into non-obese

diabetic/severe combined immunodeficient (NOD.Cg-

mice (10

6

Prkdcscid) Il2rgtm1wjl/SzJ (NSG)

cells/mouse). Splenocytes harvested from successfully engrafted

xenografts were reinjected (10

6 cells/mouse) to create secondary

and tertiary

xenografts for treatment trials. Human peripheral blood mononuclear cells (PBMCs)

from healthy donors were obtained from the Department of Transfusion Medicine at

the National Institutes of Health (NIH) Clinical Center under an IRB-approved

protocol. All research specimens from human subjects were obtained with informed

consent in accordance with the Declaration of Helsinki.

Construction of TSLPR chimeric antigen receptors TSLPR binding single chain fragment variable (scFv) sequences were determined

from the anti-TSLPR producing hybridoma 3G11 obtained from the MD Anderson

Cancer Center as described in Supplemental Methods. For construction of the long

CAR constructs, the CH2CH3 domains from IGHG1 (gb|AAC82527.1 aa 98-329) were

included. The leader sequence for the scFv coding for T-cell surface glycoprotein

CD8 alpha chain was included to facilitate membrane trafficking. The CAR-encoding

amino acid sequences were reverse translated, codon optimized, and synthesized as

single constructs (DNA 2.0). These constructs were then subcloned into a third

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generation lentiviral plasmid (pELNS-BBzeta) containing a CD8 transmembrane

domain, a 41BB (CD137) signaling domain and a CD3zeta domain (kindly provided

by Dr. Carl June at the University of Pennsylvania

35

and previously described

36).

Lentiviral vector production and T cell transduction TSLPR CAR-encoding lentiviral vectors were produced by transient transfection of

the 293T cell line as previously described.

35

Briefly, 293T cells were plated into

poly-D lysine coated 15 cm plates (BD Biosciences). The following day, 293T cells

were transfected using lipofectamine 2000 (Life Technologies) with plasmids

encoding the TSLPR CAR along with packaging and envelope vectors

(pMDLg/pRRE,

pMD-G, and pRSV-Rev kindly provided by Dr. Richard Morgan of the Surgery Branch,

Center for Cancer Research, NCI, NIH). Lentiviral supernatants were collected 48-72

hours post-transfection, centrifuged at 3000 RPM for 10 minutes to remove cell

debris, then stored at -80°C. Human PBMCs from normal donors were activated with

a 1:1 ratio of CD3/CD28 microbeads (Life Technologies) in AIM-V media containing

40 IU/mL recombinant IL-2 (teceleukin, rhIL-2; Roche) for 24 hours. Activated T

cells were resuspended at 2 million cells per 3 mL of lentiviral supernatant plus 1

mL of fresh AIM-V media with 10

μg/mL protamine sulfate and 40 IU/mL IL-2 and

cultured in 6-well plates. Plates were centrifuged at 1000 x g for 2 hours at 32°C and

then incubated at 37°C overnight. A second transduction was performed the

following day. On the third day following transduction, the CD3/CD28 beads were

removed, and the cells were cultured at 300,000 cells/mL in AIM-V containing 100

IU/mL IL-2 with fresh IL2-containing media added every 2-3 days until harvest at

day 8 or 9.

Flow cytometry analysis Surface expression of CAR-transduced T cells was determined by flow cytometry

using a TSLPR-Fc (R&D Systems) followed by incubation with PE-F(ab)

F(ab)

2

2

or APC-

specific for human IgG-Fc (Jackson ImmunoResearch Laboratories).

Alternatively, biotin-conjugated protein L (Thermo Scientific) was used to detect

7

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CAR expression after incubation with streptavidin-conjugated PE (BD Biosciences).

Expression of CD19, CD22, and TSLPR on B-ALL lines were detected using the

following anti-human antibodies: CD45-PerCP-Cy5.5 (eBioscience), CD19-Pacific

Blue, CD19-APC-Cy7, CD10-PE-Cy7, CD22-PE, and TSLPR-APC (Biolegend). T cells

were characterized with the following antibodies: CD3-APC-Cy7, CCR7-FITC

(CD197), CD45RA-APC, CD4-Pacific Blue (BioLegend), CD45-PerCP-Cy5.5

(eBioscience), and CD8-V500 (BD Biosciences). The binding of the 3G11 hybridoma

supernatant to the TSLPR-overexpressing ALL lines was detected with goat anti-

mouse IgG-PE (BD Biosciences). Dead cells were excluded by staining with Fixable

Viability Dye eFluor® 506 (eBioscience).

Cellular cytotoxicity and cytokine assays Target cells were labeled with 100 uCi

51Cr (PerkinElmer)

for 1 hour. After washing,

5,000 targets per well were coincubated for 4 to 6 hours with bead-purified (Pan T

Cell II isolation kit [Miltenyi Biotec]) transduced T cells at various effector to target

(E:T) ratios. Assay supernatants were counted for

51Cr

release using LumaPlates

(PerkinElmer) and a Top Count Reader (Packard). Specific lysis was calculated as

follows: % lysis = (experimental lysis - spontaneous lysis)/(maximum lysis -

spontaneous lysis) X 100. 1x10

5

CAR T cell or control T cells were washed three

times and then co-cultured with target cell at 1:1 ratio in 96-well plate. All samples

were run in triplicates. The overnight culture supernatants were measured for

cytokine levels with either IFNg ELISA kits (R&D) or multiplex assay (Meso Scale

Discovery) as per the manufacturer’s instructions.

In vivo studies Animal studies were carried out under protocols approved by the NCI Bethesda

Animal Care and Use Committee. B-ALL cell lines and previously xenografted human

B-ALL specimens were IV injected into NSG mice (NOD scid gamma, NOD.Cg-

Prkdcscid Il2rgtm1Wjl/SzJ; Jackson ImmunoResearch Laboratories) as previously described

33.

For non-luciferase-expressing PDXs models, animals were treated with

TSLPR T cells once ≥5% human ALL was detectable by flow cytometry in peripheral

8

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blood. For luciferase-expressing lines, leukemia was detected using the Xenogen

IVIS Lumina (Caliper Life Sciences). NSG were injected intraperitoneally with 3 mg

D-luciferin (Caliper Life Sciences) and were imaged 6 minutes later with an

exposure time of 3 minutes. Living Image Version 4.1 software (Caliper Life

Sciences) was used to analyze the bioluminescent signal flux for each mouse as

2

photons/s/cm /sr. Leukemia burden in non-luciferase expressing xenografts was

measured by flow cytometry of peripheral blood, bone marrow, and spleen.

Tissue Microarray Tissue microarray (TMA) is a screening tool that allows hundreds of tissue samples

to be analyzed quickly and conveniently. Formalin-fixed, paraffin-embedded (FFPE)

tissue blocks from 48 healthy patients were selected for use as normal controls.

H&E stained slides from these tissue blocks were annotated for desired areas, and

the slides were then used as guides for core selection. TMAs were constructed on a

Beecher Manual Tissue Arrayer I (MTA I). For this TMA, 1 mm cores of normal FFPE

tissue were punched from a donor block and arrayed into a recipient paraffin block.

Cores were placed into the recipient block following a coordinate grid map. Each

tissue type was represented by three cores when possible. A total of 138 cores were

used in each TMA block. Tissue types represented were aorta, adipose, bladder,

breast, cartilage, cervix, colon, connective tissue, diaphragm, epididymis, heart,

kidney, liver, lung, mesentery, ovary, pancreas, small intestine, skeletal muscle,

smooth muscle, spleen, testes, thymus, thyroid, tonsil, trachea, and skin.

Immunohistochemistry with the anti-CRLF2 clone 109626 (Abcam) antibody was

performed on the Ventana Discovery XT platform. The protocol included a CC1

standard retrieval process. The antibody was diluted using SignalStain enhancer

(Cell Signaling) to a final dilution of 1:1500. CRLF2 was further detected using

Ultramap anti Rabbit HRP and the Chromomap DAB detection kit. The TMA was

scanned with a SCN400 Leica scanner at 40X magnification.

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Results

Cloning and construction of TLSPR-targeted chimeric antigen receptors and generation of TSLPR CAR expressing T cells We first confirmed binding of an anti-TSLPR antibody produced by the 3G11

hybridoma to

CRLF2-rearranged (TSLPR-overexpressing) B-ALL cell lines and

xenografted primary specimens (TSLPRhi ALL, Figure 1A). 3G11 does not bind

murine TSLPR, thus precluding the ability to assess toxicity in murine models (not

shown). The sequences for the heavy and light chain variable regions (Fv) were then

determined (Supplemental Methods). Single chain Fv (scFv) sequences were

constructed using a glycine linker and inserted into a chimeric antigen receptor

lentiviral vector backbone encoding CD8α hinge and CD8 transmembrane regions

with CD3ζ and 41BB (CD137) intracellular domains (short CAR; Figure 1B).

Because distance of the scFv from the T cell surface may affect CAR function, a

construct containing an immunoglobulin CH2CH3 spacer domain between the scFv

and the transmembrane sequence was also generated (long CAR). Lentiviral vectors

encoding the TSLPR CARs were then used to transduce CD3/CD28 bead-activated

human T cells resulting in a high efficiency of gene transfer as detected by both

protein L and a TSLPR Fc fusion protein (Figure 1C). There were no differences in

cell expansion between TSLPR short CAR or cells CD19 CAR or CD22 CAR

transduced cells (Supplemental Figure 1). Importantly, TSLPR reportedly has

limited expression in normal tissues outside of the immune system where it has

been found on dendritic cells (with the highest expression on a subset of myeloid

DCs) and some T cells (a small subset of CD4+ T cells)

37,38.

To confirm this, we

performed immunohistochemistry on a normal TMA (Figure 1D). As expected,

there were scattered rare cells in lymphoid tissues and thymus demonstrating

TSLPR expression, possibly representing dendritic cells and developing T cells.

There was also some staining in renal tubular cells and colonic mucosa, although the

staining pattern in these tissues was not consistent with cell surface expression. In

the liver, there was cytoplasmic staining in cells likely representing Kupffer cells.

Finally, there were scattered cells in the basal layer of the skin with cytoplasmic

staining. There was no staining in the myocardium. There was also no evidence for

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in vivo reactivity of TSLPR CAR T cells against a panel of normal tissue cell lines including cardiomyocytes or neurocytes induced from pluripotent stem cells

(Supplemental Figure 2). Importantly, as has been shown in primary

rearranged B-ALL specimens

31,

CRLF2-

the TSLPRhi ALL cell line MUTZ5 and a human

TSLPRhi PDX (JH331) express TSLPR at comparably high levels to those of CD19

and CD22 (Figure 1E). REH expresses very low levels of TSLPR relative to the

TSLPRhi ALL and an REH line transduced with human TSLPR.

Both short and long TSLPR CAR T cells demonstrate in vitro activity against TSLPRhi ALL We next tested whether T cells transduced with the TSLPR CAR constructs

demonstrated activity

in vitro when incubated with the B-ALL cell line REH

transduced to express TSLPR (REH-TSLPR), as well as a naturally TSLPR-

overexpressing

CRLF2-rearranged ALL line (MUTZ5).

As shown in Figure 2A, both

short and long CAR T cells produce high levels of interferon gamma (IFN

tumor necrosis factor alpha (TNF

found that, in addition to IFN

γ) and

α) when incubated with REH-TSLPR. We also

γ and TNFα, substantial amounts of IL-2 and IL-8 were

produced (Supplemental Figure 3).

When the lytic capacity of TSLPR CAR T cells

was measured, the short and long constructs demonstrated equivalent activity

against REH-TSLPR (Figure 2B). However, the short TSLPR CAR T cells appeared to

have higher lytic capacity against MUTZ5 than the long CAR T cells despite

comparable levels of TSLPR surface expression on both REH-TSLPR and MUTZ5. As

an additional negative control we also assessed TLSPR CAR T cell reactivity against

the parent (TSLPRlo) cell line. Minimal cytokine production (Figure 2A) or cell lysis

(data not shown) was observed.

Only short TSLPR CAR T cells are active against B-ALL in vivo, correlating with greater in vivo persistence than long TSLPR CAR T cells We next tested the ability for TSLPR CAR T cells to eradicate ALL

in vivo when

injected into mice engrafted with TSLPR-expressing ALL. Four days after IV

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injection of luciferase-expressing REH-TSLPR, leukemia was detectable at low levels.

Injection of 15 x 10

6

short CAR T cells completely eradicated ALL in this cell line

model (Figure 2C). Interestingly, despite equivalent

in vitro activity against REH-

TSLPR, long CAR T cells had minimal impact on leukemia progression in mice. To

determine the reason for the disparate activity of the long and short CAR constructs,

we next looked at CAR T cell persistence by flow cytometry and found that short

CAR T cells were present in greater numbers in the peripheral blood compared to

long CAR T cells (Figures 2D and E). Interestingly, this was most notable at later

time points despite progression of TSLPR-expressing ALL in recipients of long CAR

T cells. Thus the marked increase in activity seen with the short TSLPR CAR

construct compared to the long construct was associated with greater persistence of

short CAR-expressing T cells.

Short CAR T cells eradicate TSLPR overexpressing ALL when infused at high leukemic burden To test the short TSLPR CAR T cells in higher leukemia burden models, CAR T cell

infusion was delayed until day 16 after REH-TSLPR injection in NSG mice (Figure

3A). Remarkably, 10x10

6

short TSLPR CAR T cells were still able to induce rapid

clearance of highly-engrafted TSLPRhi ALL with maintenance of leukemia clearance

in the majority of mice through day 40 (Figure 3A). Importantly, there was no

evidence for any alteration in the rapid progression of TLSPRlo ALL by TSLPR CAR T

cells, demonstrating that activity is dependent on expression levels of the CAR

target. CD8+ T cells generally exhibit greater

in vitro lytic function and are thought

to be the most important mediators of direct anti-tumor activity

compared to CD4+ T cells. Interestingly, although the

in vivo when

in vitro expansion protocol

utilized in these experiments resulted in a predominance of CD4+ T cells prior to

infusion, CD8+ T cells had expanded markedly by day 50 and represented the

largest T cell subset

in vivo (Figure 3B).

As expected, this expansion of CD8+ CAR-

expressing T cells was associated with expression of surface markers associated

with effector phenotypes by day 50 (Figures 3 C and D). There was also a

substantial percentage of CAR T cells with a CCR7+/CD45RA- phenotype, consistent

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with a central memory subset, thought to be important for persistence and

sustained anti-tumor activity.

Quantitative assessment of short CAR T cells defines curative dose in established leukemia We next performed an

in vivo short CAR T cell dose titration to define the range over

which activity is observed and to determine if the short TSLPR could cure animals

with high levels of engrafted leukemia. As shown in Figure 4, short TSLPR CAR cells

were curative at 15 x 10

6

cells per mouse and with clear activity at 5-10 x 10

mouse and some activity as low as 1 x 10

6

6

per

per mouse in well-engrafted animals,

particularly noted as improved survival (Figure 4B). Again, there was minimal

activity seen following infusion of the long CAR T cells with only slight decrease in

leukemic burden in the peripheral blood (Figure 4C) and luciferase activity at day 6

(Figure 4D) compared to GFP control T cells, but no difference between the two

groups at any of the later timepoints. These data are consistent with the failure of

the long CAR T cells to persist

in vivo.

Short TSLPR CAR T cells eradiate pre-B-ALL that naturally overexpresses TSLPR All of the results shown thus far utilized an engineered REH ALL cell line that was

transduced to overexpress TSLPR, but also allowed assessment of the target

specificity of the CAR by direct comparison to the non-TSLPR-overexpressing parent

REH cell line (TSLPRlo). We thus next tested the short TSLPR construct in 3 PDX

models of primary

de novo Ph-like ALL with CRLF2 rearrangements (Figure 5A).

As

shown in Figure 5B, the short TSLPR CAR eradicated leukemia in the luciferase-

expressing human TSLPRhi ALL PDX model JH331, as measured by bioluminescent

imaging.

These responses were durable in a repeat experiment in which mice were

imaged for 70 days (Supplemental Figure 4). The short TSLPR CAR also eradicated

leukemia in blood, bone marrow, and spleens of 2 additional

PDX models (Figures 5C and 5D and not shown).

13

de novo TSLPRhi ALL

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TSLPR overexpression has been associated with clinically high risk ALL and, in

many cases, an aggressive and chemotherapy-refractory phenotype. We thus tested

the short TSLPR CAR against an aggressive relapsed

CRLF2-rearranged ALL

specimen (ALL4364) that is lethal by 60 days after IV injection into NSG mice. As

shown in Figures 5E-H, short TSLPR CAR T cells demonstrated potent activity

against this aggressive TSLPRhi ALL, resulting in decreased splenomegaly and

reduction in blast counts in spleens and blood of xenografted animals as early as day

14 following CAR T cell injection. Interestingly, although activity in the bone

marrow was also observed, clearance of leukemia was less rapid and not

statistically significant at this early evaluation time. However, CAR T cell treatment

was associated with eventual clearance of aggressive TSLPRhi ALL and prolonged

survival (Figure 5H). Analysis of late relapses demonstrated retained expression of

TSLPR, indicating that failure was not due to loss of antigen (Figure 5I).

Comparable activity of TSLPR CAR to CD19 CAR and CD22 CAR We next compared the activity of the short TSLPR CAR to a CD19 CAR containing the

same scFv (FMC68) as those currently demonstrating potent clinical activity

CD22 CAR construct with potent activity in human xenograft models

39.

10

and a

Importantly,

all of the CAR constructs contained the same 41BB costimulatory domain (Figure

6A) and demonstrated comparable transduction efficiencies (Figure 6B). As shown

in Figures 6C and D, T cells transduced with the short TSLPR CAR, CD19 CAR, or

CD22 CAR T cells were curative in the JH331 PDX model at a dose of 3x10

cells with durable responses with 0.3x10

6

6

CAR T

TSLPR CAR T cells.

Discussion Much recent progress has been made in immunotherapeutic approaches to treat

cancer, particularly in the area of adoptive cell therapy

40.

For B-ALL, the use of T

cells modified with CARs that fuse T cell signaling constructs with antibody

recognition domains targeting the B cell-associated antigen CD19 has resulted in

remarkable complete remission rates in heavily-pretreated, chemotherapy-

refractory patients, including those who have relapsed after allogeneic

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hematopoietic stem cell transplantation

9-11.

These studies have provided important

proof-of-principle of the potency of adoptive cell therapy using CAR-expressing T

cells. However, leukemia relapses with loss of expression of the CD19 target has

been observed

14.

CD19 loss has also been observed following treatment of ALL

patients with blinatumomab, a CD19/CD3 bispecific T cell-engaging reagent

15.

These observations indicate that, while CD19 is an excellent target, it is likely not

required for B-ALL survival. Thus identification of additional ALL targets is needed,

particularly those less likely to be lost from the surface of the leukemic blasts and,

theoretically, more likely to result in durable responses when targeted.

Recent gene expression profiling studies have revealed overexpression of the

CRLF2

gene in 5-15% of children and adults with B-ALL, which results in high levels of

TSLPR cell surface expression that is easily measurable by flow cytometry

assays

17,18,31.

Detailed analyses of these leukemias indicated that TSLPR

overexpression was associated with translocations or upstream deletions resulting

in alternative promoter regions in the majority of patients resulting in

overexpression of a non-mutated receptor

20.

Importantly, although survival and

relapse data have been somewhat discrepant in standard-risk vs. high-risk

patients

23,26,41,42,

CRLF2 rearrangements and other Ph-like kinase alterations are

associated with a greater risk of relapse and inferior outcomes in high-risk

patients

18.

More recent genetic studies have demonstrated that Ph-like ALL

(including those with

CRLF2 rearrangements) are highly associated with IKZF1

deletions, which are also prevalent in Ph+ ALL

17,23,24,43.

TSLPR is thus an excellent

target for molecularly-targeted therapeutic approaches, as its overexpression

occurs in a sizable subset of B-ALL patients who have high risks of recurrence and

generally poor outcomes.

The CRLF2 chain forms the heterodimeric TSLPLR complex with the IL-7R

with inducible signaling by binding of the TSLP ligand

44.

α chain

Important intracellular

mediators of TSLP signaling include members of the JAK/STAT pathway, such as

15

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JAK1, JAK2, and STAT5. Physiologically, TSLP is important in allergic inflammation

and T cell development and can contribute to B cell development. Recently,

targeting of the TSLP/TSLPR axis has been shown to be therapeutically beneficial in

patients with allergic asthma

(encoding the IL7R

45.

In B-ALL, recurring mutations in the

IL7RA gene

α chain) have been identified that result in homodimerization

and autophosphorylation of the TSLPR with ligand-independent signaling in

contrast to the overexpression of non-mutated receptor in the majority of

overexpressing ALL

16.

CRLF2-

Nonetheless, these data provide further evidence that IL-

7/TSLP signaling pathways are functionally important for B-ALL. JAK2 mutations

have also been identified in B-ALL and are frequently associated with

rearrangements

17,20,46,

CRLF2

although the specific mutations are different than those

found in myeloproliferative neoplasms

47.

Although JAK inhibition is under study for

CRLF2-rearranged and/or JAK-mutant B-ALL and preclinical efficacy has been demonstrated in PDX models

33,

it remains unclear whether such agents will be

effective in patients with JAK-activated Ph-like ALL or whether multiple inhibitors

will be necessary given the diversity of mutations. Targeting the TSLPR protein on

the cell surface thus provides another universal option for these high-risk patients.

Collectively, these data indicate that B-ALL biology is firmly linked to this cytokine

receptor family and suggest that targeting of these pathways could provide a

therapeutic option.

In these studies, we demonstrate that T cells engineered with a CAR targeting the

TSLPR protein are potent therapeutically and can eradicate TSLPR-overexpressing

leukemia in murine xenograft models of human Ph-like ALL. Importantly, we

observed these phenomena in multiple TSLPR-overexpressing B-ALL models.

Interestingly, although other CAR constructs have successfully utilized a spacer

region between the cell membrane and the scFv, T cells transduced with the long

version of our anti-TSLPR CAR did not demonstrate

comparable activity

in vitro.

in vivo activity despite

We demonstrate that this lack of efficacy of the long

construct T cells is associated with lack of persistence

16

in vivo.

Similar observations

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have been recently reported for other CAR constructs

48,49.

Finally, at suboptimal

CAR T cell dosing, residual or recurrent B-ALL did not lose surface expression of

TSLPR.

In summary, we demonstrate that targeting of TSLPR using engineered chimeric

antigen receptor T cells is efficacious against

CRLF2-rearranged B-ALL and may be a

promising therapeutic option for patients with these high-risk leukemias. To our

knowledge, these data are the first demonstration that targeting of a cytokine

receptor and oncoprotein can induce robust leukemia cytotoxicity. Although CAR-

based immunotherapies have demonstrated potent activity in B cell ALL, such

approaches are currently under study only in patients with recurrent or

chemorefractory ALL. Our findings also create opportunities to utilize TSLPR

targeting with other treatment approaches, such as scFv toxin conjugates or

bispecific antibody reagents, for patients with

CRLF2-rearranged ALL as an

intervention earlier in therapy given the high risk of relapse in these patients.

Acknowledgements

These studies were supported in part by the Stand Up To Cancer - St. Baldrick’s

Pediatric Dream Team Translational Research Grant (SU2C-AACR-DT1113; S.K.T.,

M.D., D.M.B., R.J.O., P.H.B.S., S.A.G., and T.J.F.). Stand Up To Cancer is a program of the

Entertainment Industry Foundation administered by the American Association for

Cancer Research. The studies were also supported by a grant from the William

Lawrence and Blanche Hughes Foundation (T.J.F.). The TMA was constructed at the

Biopathology and Morphology Cores of The Research Institute at Nationwide

Children’s Hospital in Columbus OH, with tissues submitted by the Pediatric and

Southern Divisions of the NCI-supported CHTN (UMICA1837390). S.K.T. was also

supported by the Alex’s Lemonade Stand Foundation (ALSF), the University of

Pennsylvania Institute for Translational Medicine and Therapeutics (ITMAT)

Transdisciplinary Program in Translational Medicine and Therapeutics

(UL1RR024134 from the National Center For Research Resources), and the NCI

17

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(K08CA184418) and was an ALSF Scholar in Developmental Therapeutics. D.M.B. is

a St. Baldrick’s Foundation Scholar. S.A.G. is the Yetta Deitch Novotny Endowed

Chair in Pediatric Oncology

. We would also like to thank Crystal L. Mackall, M.D. for

helpful discussions.

Authorship

Contribution: H.Q performed experiments and wrote the manuscript. W.H, M.C., and

L.Z. performed experiments and analyzed data. S.K.T. provided key patient-derived

xenografts and edited the manuscript. H.Z., G.N., M.D, and P.H.B.S. performed and

interpreted the TSLPR tissue microarray. J.Z., Y.L., and B.S.M. provided important

normal tissue lines for potential off-tumor reactivity. S.M, D.T.T., D.M.B., and S.A.G.

contributed key patient-derived xenografts. R.J.O. provided important reagents and

intellectual contributions to the construction of the chimeric antigen receptors. T.J.F.

conceived of, designed, and supervised the research, analyzed data, and wrote the

manuscript.

Conflicts of Interest: None.

18

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References

1.

Pui CH, Evans WE. A 50-year journey to cure childhood acute lymphoblastic

leukemia. 2.

Semin Hematol. 2013;50(3):185-196.

Hunger SP, Loh ML, Whitlock JA, et al. Children's Oncology Group's 2013

blueprint for research: acute lymphoblastic leukemia.

Pediatr Blood Cancer.

2013;60(6):957-963. 3.

Inaba H, Greaves M, Mullighan CG. Acute lymphoblastic leukaemia.

Lancet.

2013;381(9881):1943-1955. 4.

Bhojwani D, Pui CH. Relapsed childhood acute lymphoblastic leukaemia.

5.

Raetz EA, Bhatla T. Where do we stand in the treatment of relapsed acute

Lancet Oncol. 2013;14(6):e205-217. lymphoblastic leukemia?

Hematology Am Soc Hematol Educ Program.

2012;2012:129-136. 6.

Oeffinger KC, Mertens AC, Sklar CA, et al. Chronic health conditions in adult

survivors of childhood cancer. 7.

N Engl J Med. 2006;355(15):1572-1582.

Mathisen MS, Kantarjian H, Thomas D, O'Brien S, Jabbour E. Acute

lymphoblastic leukemia in adults: encouraging developments on the way to higher cure rates. 8.

Leuk Lymphoma. 2013;54(12):2592-2600.

Schafer ES, Hunger SP. Optimal therapy for acute lymphoblastic leukemia in

adolescents and young adults. 9.

Nat Rev Clin Oncol. 2011;8(7):417-424.

Davila ML, Riviere I, Wang X, et al. Efficacy and toxicity management of 19-

28z CAR T cell therapy in B cell acute lymphoblastic leukemia.

Sci Transl Med.

2014;6(224):224ra225. 10.

Fry TJ, Mackall CL. T-cell adoptive immunotherapy for acute lymphoblastic

leukemia. 11.

Hematology Am Soc Hematol Educ Program. 2013;2013:348-353.

Grupp SA, Kalos M, Barrett D, et al. Chimeric antigen receptor-modified T

cells for acute lymphoid leukemia. 12.

N Engl J Med. 2013;368(16):1509-1518.

Kochenderfer JN, Dudley ME, Feldman SA, et al. B-cell depletion and

remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells.

Blood. 2012;119(12):2709-

2720. 13.

Lee DW, Kochenderfer JN, Stetler-Stevenson M, et al. T cells expressing CD19

chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. 14.

Maude SL, Frey N, Shaw PA, et al. Chimeric antigen receptor T cells for

sustained remissions in leukemia. 15.

Lancet. 2015;385(9967):517-528.

N Engl J Med. 2014;371(16):1507-1517.

Topp MS, Gokbuget N, Zugmaier G, et al. Phase II trial of the anti-CD19

bispecific T cell-engager blinatumomab shows hematologic and molecular remissions in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia. 16.

J Clin Oncol. 2014;32(36):4134-4140.

Shochat C, Tal N, Bandapalli OR, et al. Gain-of-function mutations in

interleukin-7 receptor-alpha (IL7R) in childhood acute lymphoblastic leukemias.

Exp Med. 2011;208(5):901-908. 17.

J

Roberts KG, Li Y, Payne-Turner D, et al. Targetable kinase-activating lesions

in Ph-like acute lymphoblastic leukemia.

N Engl J Med. 2014;371(11):1005-1015.

19

From www.bloodjournal.org by guest on June 7, 2015. For personal use only.

18.

Loh ML, Zhang J, Harvey RC, et al. Tyrosine kinome sequencing of pediatric

acute lymphoblastic leukemia: a report from the Children's Oncology Group TARGET Project. 19.

Blood. 2013;121(3):485-488.

Hertzberg L, Vendramini E, Ganmore I, et al. Down syndrome acute

lymphoblastic leukemia, a highly heterogeneous disease in which aberrant expression of CRLF2 is associated with mutated JAK2: a report from the International BFM Study Group. 20.

Blood. 2010;115(5):1006-1017.

Mullighan CG, Collins-Underwood JR, Phillips LA, et al. Rearrangement of

CRLF2 in B-progenitor- and Down syndrome-associated acute lymphoblastic leukemia. 21.

Nat Genet. 2009;41(11):1243-1246.

Russell LJ, Capasso M, Vater I, et al. Deregulated expression of cytokine

receptor gene, CRLF2, is involved in lymphoid transformation in B-cell precursor acute lymphoblastic leukemia. 22.

Blood. 2009;114(13):2688-2698.

Yoda A, Yoda Y, Chiaretti S, et al. Functional screening identifies CRLF2 in

precursor B-cell acute lymphoblastic leukemia.

Proc Natl Acad Sci U S A.

2010;107(1):252-257. 23.

Chen IM, Harvey RC, Mullighan CG, et al. Outcome modeling with CRLF2,

IKZF1, JAK, and minimal residual disease in pediatric acute lymphoblastic leukemia: a Children's Oncology Group study. 24.

Blood. 2012;119(15):3512-3522.

Harvey RC, Mullighan CG, Chen IM, et al. Rearrangement of CRLF2 is

associated with mutation of JAK kinases, alteration of IKZF1, Hispanic/Latino ethnicity, and a poor outcome in pediatric B-progenitor acute lymphoblastic leukemia. 25.

Blood. 2010;115(26):5312-5321.

Cario G, Zimmermann M, Romey R, et al. Presence of the P2RY8-CRLF2

rearrangement is associated with a poor prognosis in non-high-risk precursor B-cell acute lymphoblastic leukemia in children treated according to the ALL-BFM 2000 protocol. 26.

Blood. 2010;115(26):5393-5397.

Moorman AV, Schwab C, Ensor HM, et al. IGH@ translocations, CRLF2

deregulation, and microdeletions in adolescents and adults with acute lymphoblastic leukemia. 27.

J Clin Oncol. 2012;30(25):3100-3108.

Yamashita Y, Shimada A, Yamada T, et al. IKZF1 and CRLF2 gene alterations

correlate with poor prognosis in Japanese BCR-ABL1-negative high-risk B-cell precursor acute lymphoblastic leukemia.

Pediatr Blood Cancer. 2013;60(10):1587-

1592. 28.

Attarbaschi A, Morak M, Cario G, et al. Treatment outcome of CRLF2-

rearranged childhood acute lymphoblastic leukaemia: a comparative analysis of the AIEOP-BFM and UK NCRI-CCLG study groups. 29.

Br J Haematol. 2012;158(6):772-777.

Palmi C, Vendramini E, Silvestri D, et al. Poor prognosis for P2RY8-CRLF2

fusion but not for CRLF2 over-expression in children with intermediate risk B-cell precursor acute lymphoblastic leukemia. 30.

Leukemia. 2012;26(10):2245-2253.

van Bodegom D, Zhong J, Kopp N, et al. Differences in signaling through the B-

cell leukemia oncoprotein CRLF2 in response to TSLP and through mutant JAK2.

Blood. 2012;120(14):2853-2863. 31.

Tasian SK, Doral MY, Borowitz MJ, et al. Aberrant STAT5 and PI3K/mTOR

pathway signaling occurs in human CRLF2-rearranged B-precursor acute lymphoblastic leukemia.

Blood. 2012;120(4):833-842. 20

From www.bloodjournal.org by guest on June 7, 2015. For personal use only.

32.

Tasian SK, Loh ML. Understanding the biology of CRLF2-overexpressing

acute lymphoblastic leukemia. 33.

Crit Rev Oncog. 2011;16(1-2):13-24.

Maude SL, Tasian SK, Vincent T, et al. Targeting JAK1/2 and mTOR in murine

xenograft models of Ph-like acute lymphoblastic leukemia.

Blood.

2012;120(17):3510-3518. 34.

Teachey DT, Obzut DA, Cooperman J, et al. The mTOR inhibitor CCI-779

induces apoptosis and inhibits growth in preclinical models of primary adult human ALL.

Blood. 2006;107(3):1149-1155.

35.

Milone MC, Fish JD, Carpenito C, et al. Chimeric receptors containing CD137

signal transduction domains mediate enhanced survival of T cells and increased antileukemic efficacy in vivo. 36.

Mol Ther. 2009;17(8):1453-1464.

Imai C, Mihara K, Andreansky M, et al. Chimeric receptors with 4-1BB

signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia.

Leukemia. 2004;18(4):676-684. 37.

Ziegler SF, Liu YJ. Thymic stromal lymphopoietin in normal and pathogenic T

cell development and function. 38.

Nat Immunol. 2006;7(7):709-714.

Lo Kuan E, Ziegler SF. Thymic stromal lymphopoietin and cancer.

J Immunol.

2014;193(9):4283-4288. 39.

Haso W, Lee DW, Shah NN, et al. Anti-CD22-chimeric antigen receptors

targeting B-cell precursor acute lymphoblastic leukemia.

Blood. 2013;121(7):1165-

1174. 40.

Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer:

harnessing the T cell response. 41.

Nat Rev Immunol. 2012;12(4):269-281.

Roberts KG, Pei D, Campana D, et al. Outcomes of Children With BCR-ABL1-

Like Acute Lymphoblastic Leukemia Treated With Risk-Directed Therapy Based on the Levels of Minimal Residual Disease. 42.

J Clin Oncol. 2014.

Ensor HM, Schwab C, Russell LJ, et al. Demographic, clinical, and outcome

features of children with acute lymphoblastic leukemia and CRLF2 deregulation: results from the MRC ALL97 clinical trial. 43.

lymphoblastic leukemia. 44.

Blood. 2011;117(7):2129-2136.

Mullighan CG, Su X, Zhang J, et al. Deletion of IKZF1 and prognosis in acute

N Engl J Med. 2009;360(5):470-480.

Tal N, Shochat C, Geron I, Bercovich D, Izraeli S. Interleukin 7 and thymic

stromal lymphopoietin: from immunity to leukemia.

Cell Mol Life Sci.

2014;71(3):365-378. 45.

Gauvreau GM, O'Byrne PM, Boulet LP, et al. Effects of an anti-TSLP antibody

on allergen-induced asthmatic responses. 46.

Mullighan CG, Zhang J, Harvey RC, et al. JAK mutations in high-risk childhood

acute lymphoblastic leukemia. 47.

N Engl J Med. 2014;370(22):2102-2110.

Proc Natl Acad Sci U S A. 2009;106(23):9414-9418.

Levine RL, Wadleigh M, Cools J, et al. Activating mutation in the tyrosine

kinase JAK2 in polycythemia vera, essential thrombocythemia, and myeloid metaplasia with myelofibrosis. 48.

Cancer Cell. 2005;7(4):387-397.

Guest RD, Hawkins RE, Kirillova N, et al. The role of extracellular spacer

regions in the optimal design of chimeric immune receptors: evaluation of four different scFvs and antigens.

J Immunother. 2005;28(3):203-211.

21

From www.bloodjournal.org by guest on June 7, 2015. For personal use only.

49.

Hudecek M, Sommermeyer D, Kosasih PL, et al. The non-signaling

extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity.

Cancer Immunol Res. 2014.

Table 1. Genomic alterations in

CRLF2-rearranged Ph-like ALL specimens used

for patient-derived xenograft models.

Specimen

USI

Source

CRLF2

JAK mutation

rearrangement JH331

PAMDKS de novo

IGH@-CRLF2

Other mutations

JAK2 R683G

IKZF1, PAX5, CDKN2A

JH352

PALJCF

de novo

P2RY8-CRLF2

JAK1 L624_R629>W

CDKN2A/B

NH362

PALTWS

de novo

IGH@-CRLF2

Negative

IKZF1, PAX5, CDKN2A

ALL4364

n/a

relapse

P2RY8-CRLF2

JAK2 R683G

USI = unique specimen identifier (Children’s Oncology Group), n/a = not available

22

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Figure Legends

Figure 1. Construction of short and long anti-TSLPR CAR constructs and

lentiviral transduction of T cells. A. Anti-TSLPR hybridoma (3G11) supernatant

binds to the surface of TSLPR-overexpressing ALL. Binding was detected using

phycoerytherin-conjugated goat-anti-mouse antibody. Binding of a commercially

available, directly conjugated anti-TSLPR antibody (Ab) is shown for comparison.

B.

Schematic representation of anti-TSLPR chimeric antigen receptor constructs. Both

constructs contain an anti-TSPLR single chain fragment variable sequence from the

3G11 hybridoma, a CD8 transmembrane domain, a CD137 (41BB) costimulatory

domain, and a CD3 zeta signaling domain. The long version of the CAR also contains

a spacer region derived from an immunoglobulin CH2CH3 domain.

C. Transduction

efficiency of activated, CD3/CD28 bead-expanded human T cells with lentiviral-

based vectors expressing short and long anti-TSLPR constructs. Left panels show

detection of CAR using Protein L. Right panels show detection using a TSLPR protein

Fc construct. D. TSLPR expression on normal pediatric tissues, representative of 48

healthy donors. Colon: Weak cytoplasmic granular staining in crypt base columnar

cells and weak stromal tissue positivity can be seen. Liver: Mild-moderate diffuse

cytoplasmic granular staining in liver sinusoid. Kupffer cells around the lining of

liver sinusoid shows stronger granular staining compared to the surrounding

parenchyma. Heart: Absent or negative staining on myocardial tissue. Thymus:

Weak and diffuse granular staining over thymus tissue. Tonsil: Some, but not all,

lymphoid tissue in the tonsil shows weak granular cytoplasmic staining. Spleen:

Weak cytoplasmic staining over splenic cords. There is also hemosiderin-laden

macrophages and lipofusin (yellow droplets). Kidney: Proximal tubules, distal

tubules, and collecting ducts show mild-to-moderate cytoplasmic reaction, but the

glomeruli are negative. Skin: Lower level of stratum spinosum and stratum basale

(basal layer) show granular cytoplasmic staining. There is no staining of keratinized

squamous cell layer and collagen tissue.

E. Expression level of TSLPR on B-ALL

relative to expression of CD19 and CD22. JH331 is a patient-derived xenograft

model of

CRLF2-rearranged ALL with endogenous TSLPR overexpression.

histograms represent isotype control.

23

Shaded

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Figure 2. Both short and long demonstrate activity

CAR is active

in vivo.

in vitro but only the short

γ and TNFα by short and long anti-TSLPR

A. Production of IFN

CAR T cells as measured by ELISA of supernatant following co-incubation with

TSLPRhi ALL (REH TSLPR or MUTZ5).

B. Lysis of REH-TSLPR and MUTZ5 TSLPRhi

ALL by both short and long CAR T cells measured by

51Cr

release following 4 hour

co-culture. Results show specific lysis calculated as described in Methods.

C. Short

6

TSLPR CAR T cells (15x10 ) administered IV on day 4 following injection of

luciferase-expressing REH-TSLPR demonstrate potent activity as measured by

bioluminescent imaging, whereas long TSLPR CAR given at the same dose fail to

alter leukemia progression. Control mice received the same dose of expanded GFP

transduced T cells. A single animal in the short TSLPR CAR group was sacrificed due

to a wasting syndrome consistent with xenogeneic GVHD. ACT = adoptive cell

transfer.

D. In a separate experiment, mice were treated as in Figure 2C, and

peripheral blood was analyzed on days 16 and 27 following injection of 15x10

6

short or long TSLPR CAR T cells or GFP-transduced control T cells. Representative

dot plots showing increased persistence of short CD8+ TSLPR CAR T cells compared

to long CAR T cells on days 16 and 27 following injection.

E. Significantly increased

absolute number of short TSLPR CAR T cells at day 27 following injection compared

to long CAR T cells as measured using a bead calibration as described in Methods.

Figure 3. Potent activity of short TSLPR CAR T cells is associated with a

relative expansion of CD8+ CAR T cells

in vivo.

A. Mice were injected with high-

6

dose REH-TSLPR (5x10 ) followed by late injection of short TSLPR CAR T cells

6

(10x10 ) on day 16. ACT = adoptive cell transfer.

B. Slightly increased relative

number of CD4+ CAR T cells following CD3/CD28 bead-mediated expansion. This

converts to a predominance CD8+/TSLPR CAR+ (measured by TSLPR Fc) at day 50

following injection.

C. Representative dot plots showing phenotype of short TSLPR

CAR T cells prior to injection at day 20 following injection.

24

D. Relative percentages

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of naïve, central memory and effector memory CAR T cells at day 20 following

injection.

Figure 4. Short TSLPR CAR T cells demonstrate activity over a broad dose

range and mediate durable eradication of TSLPRhi ALL at high leukemic

burden. A. Dose titration of short TSLPR CAR T cells given on day 3 following high-

dose REH TSLPR ALL injection (5x10

6

cells/mouse). ACT = adoptive cell transfer.

Survival plot of mice treated in figure 4A.

B.

C. Flow cytometry of peripheral blood for

the presence of CD45+ leukemia cells at day 12 following injection.

D. Quantitation

of bioluminescence in mice treated in figure 4A.

Figure 5. Leukemia eradication in human

CRLF2-rearranged Ph-like ALL

patient-derived xenograft models by short TSLPR CAR T cells. A . Flow

cytometric surface expression of CD19 and TSLPR in human ALL cells from TSLPRhi

PDX models JH352, NH362, and JH331. Shaded histograms denoted by dotted lines

represent unstained controls. B. Complete eradication of leukemia in a TSLPRhi

luciferase-expressing PDX model (JH331) by short TSLPR CAR T cells. ACT =

adoptive cell transfer.

C and D. Analysis of human CD45+ CD19+ ALL cells in

peripheral blood (C) and bone marrow (D) of TSLPRhi PDX models JH352 and

NH362 injected with 1x10

6

ALL cells/mouse, then treated 22 days later with 15x10

6

short TSLPR CAR T cells. (E-G.) Tissue analyses of an aggressive relapsed TSLPRhi

ALL PDX model (ALL4364) treated with TSLPR CAR T cells. One million ALL

cells/mouse were injected IV on day 1, then animals were treated with 1.2 million of

TSLPR CAR+ T cells IV on day 14. E. Spleens from short TSLPR CAR-treated and

untreated mice on day 35 and day 42 after leukemia injection (2 and 3 weeks after

CAR injection). F. Representative dot plot of bone marrow, spleen, and peripheral

blood on day 35 following leukemia injection.

G. Scatter plots of organ and blood

leukemia infiltration at day 35 following TSLPRhi ALL injection comparing short

TSLPR CAR treated and untreated mice.

H. Separate experiment showing survival

analysis demonstrating prolonged survival of TSLPR CAR T cell-treated ALL4364

PDX animals (n=5/group). ACT=Adoptive cell therapy.

25

I. Expression of TSLPR on

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human CD45+/CD19+ leukemia cells in the bone marrow of a mouse with relapse

following short TSLPR CAR treatment.

Figure 6: TSLPR CAR demonstrates comparable activity to CD19 CAR. A.

Schematic diagram of second-generation CD19 and the CD22 CAR constructs. Both

CARs have identical transmembrane and signaling domains to the TSLPR CAR.

B.

Flow cytometric surface expression of protein L on TSLPR, CD19 and CD22 CAR T

cells at 5 days post-viral transduction demonstrating efficacy of CAR transduction.

C.

TSLPR, CD19 and CD22 CAR-redirected T cells were injected IV into NSG mice

6

(3x10 /mouse) previously engrafted with the patient xenograft cell line JH331-LUC

29 days earlier and followed by bioluminescent imaging. T cells transduced with

GFP were used as a negative control. ACT = adoptive cell transfer. D = day.

D.

JH331-LUC bearing NSG mice were treated as in Figure 6C but including a group

receiving a log lower dose of CAR T cell. Control mice received 3x10

CAR T cells.

26

6

GD2-targeted

A.

REH-TSLPR

B.

MUTZ5 3G11 TSLPR Ab Unstained

Short TSLPR CAR

Anti-TSLPR scFv

CD137 CD3ζ

Long TSLPR CAR

Anti-TSLPR scFv

C.

CD8

CH2CH3

CD8

MFI Untransduced

Short CAR

Long CAR

Short CAR

Protein-L -PE

D.

Colon

Skin

TSLPR-Fc-PE Liver

E.

Kidney

CD19 Myocardium

Long CAR

CD22

Thymus

REH Spleen

Figure 1

Tonsil

JH331 MUTZ5 REH-TSLPR

TSLPR

CD137 CD3ζ

A.

B.

C. ACT

Long

GFP

Short

Long

GFP

D.

Short

Long

E. Day 16

TSLPR Fc

Day 27

Figure 2

CD8

p=0.008 CAR T cells/ ml of blood

Short

Day post Infusion

16

27 Short CAR

16

27 Long CAR

B. CD4/CD8 Ratio

A. ACT

1.4 1.2 1 0.8 0.6 0.4 0.2 0 Before injection

D.

CCR7

C.

CM

NAIVE

EM

CD45RA Bone Marrow

Pre-infusion

Figure 3

Blood

Spleen

Day 20 post-infusion

50 days post injection

A. B. ACT

C.

D. 1 0 11

R E H - T S L P R + L e n ti-G F P

R E H - T S L P R + 1 5 E 6 S h o rt C A R

P h o t o n s /s /c m /s r

1 0 10

2

R E H - T S L P R + 1 0 E 6 S h o rt C A R

10 9

R E H - T S L P R + 5 E 6 S h o rt C A R R E H - T S L P R + 1 E 6 S h o rt C A R

10 8

R E H -T S L P R + 1 5 E 6 L o n g C A R

10 7 10 6 0

10

20

30

D a y P o s t T u m o r C h a lle n g e

Figure 4

40

A.

JH352

NH362

JH331

B. ACT

CD19 JH352

NH362

JH331

TSLPR

C.

Figure 5

D.

Blood

JH352

NH362

Bone Marrow

JH352

NH362

E.

Treated

Untreated

F.

Day 35

Day 42

Bone Marrow Treated

Untreated

H.

Spleen Treated

100

P e r c e n t s u r v iv a l

G.

Untreated

U n tre a te d

C A R T re a te d

80 60 40 20 ACT

0

0

50

100

150

200

250

D a y P o s t T u m o r C h a lle n g e

I.

TSLPR Isotype

Blood

TSLPR

Treated

Untreated

TSLPR

CD19

Figure 5 cont.

A.

B.

FMC63 scFv

CD8

CD137 CD3ζ

CD19 CAR

m971 scFv

CD8

CD137 CD3ζ

CD22 CAR

TSLPR CAR

CD19 CAR

CD22 CAR

Protein-L-PE

C. ACT

TSLPR D29 ADT D32 D38 D46

Figure 6

CD19

CD22

GFP

D.

From www.bloodjournal.org by guest on June 7, 2015. For personal use only.

Prepublished online June 3, 2015; doi:10.1182/blood-2014-11-612903 originally published online June 3, 2015

Eradication of B-ALL using chimeric antigen receptor-expressing T cells targeting the TSLPR oncoprotein Haiying Qin, Monica Cho, Waleed Haso, Ling Zhang, Sarah K. Tasian, Htoo Zarni Oo, Gian Luca Negri, Yongshun Lin, Jizhong Zou, Barbara S. Mallon, Shannon Maude, David T. Teachey, David M. Barrett, Rimas J. Orentas, Mads Daugaard, Poul H.B. Sorensen, Stephan A. Grupp and Terry J. Fry

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