Plenary Paper

CLINICAL TRIALS AND OBSERVATIONS

Clearance of acute myeloid leukemia by haploidentical natural killercells is improved using IL-2 diphtheria toxin fusion proteinVeronika Bachanova,1 Sarah Cooley,1 Todd E. Defor,2 Michael R. Verneris,3 Bin Zhang,1 David H. McKenna,4

Julie Curtsinger,4 Angela Panoskaltsis-Mortari,3 Dixie Lewis,2 Keli Hippen,3 Philip McGlave,1 Daniel J. Weisdorf,1

Bruce R. Blazar,3 and Jeffrey S. Miller1

1Department of Medicine, 2Department of Pediatrics, 3Laboratory Medicine and Pathology, 4Masonic Cancer Center, and Blood and Marrow Transplant

Program, University of Minnesota, Minneapolis, MN

Key Points

• Depletion of host regulatoryT cells with IL2DT improvesefficacy of haploidentical NKcell therapy for refractoryacute myeloid leukemia.

• Depletion of Treg andpersistence of NK cells for$7 days after NK celladoptive transfer predictsbeneficial clinical responses.

Haploidentical natural killer (NK) cell infusions can induce remissions in some patients

with acute myeloid leukemia (AML) but regulatory T-cell (Treg) suppression may reduce

efficacy.We treated 57 refractoryAMLpatientswith lymphodepleting cyclophosphamide

and fludarabine followed by NK cell infusion and interleukin (IL)-2 administration. In 42

patients, donor NK cell expansion was detected in 10%, whereas in 15 patients receiving

host Treg depletionwith the IL-2-diphtheria fusion protein (IL2DT), the rate was 27%,with

amedian absolute count of 1000 NK cells/mL blood. IL2DTwas associated with improved

complete remission rates at day 28 (53% vs 21%; P 5 .02) and disease-free survival at

6 months (33% vs 5%; P < .01). In the IL2DT cohort, NK cell expansion correlated with

higher postchemotherapy serum IL-15 levels (P 5 .002), effective peripheral blood Treg

depletion (<5%) at day 7 (P < .01), and decreased IL-35 levels at day 14 (P 5 .02). In vitro

assays demonstrated that Tregs cocultured with NK cells inhibit their proliferation

by competition for IL-2 but not for IL-15. Together with our clinical observations, this

supports the need to optimize the in vivo cytokine milieu where adoptively transferred

NK cells compete with other lymphocytes to improve clinical efficacy in patients with refractory AML. This study is registered at

clinicaltrials.gov, identifiers: NCT00274846 and NCT01106950. (Blood. 2014;123(25):3855-3863)

Introduction

Tumor lysis by natural killer (NK) cells is limited by inhibitory killerimmunoglobulin receptors (KIRs) that mediate self-tolerance byengaging major histocompatibility complex class I antigens.1 Incontrast, NK cells reconstituting after transplantation can overcomethis major histocompatibility complex barrier by KIR ligand mis-matching to mediate a potent anti-leukemia reaction by decreasedtriggering through inhibitory KIR.2We have previously described thesafety and preliminary efficacy of adoptive transfer of haploidenticalNK cells.3 Patients were treated with lymphodepleting chemotherapyand received haploidentical NK cell infusions from siblings, parents,or children, followed by subcutaneous interleukin (IL)-2 to stimulateNK proliferation and activation. In that study, we found that 26%of poor prognosis acute myeloid leukemia (AML) patients achievedcomplete hematologic remission (CR) after NK cell adoptive transfer.

In subsequent applications of donor NK cell infusions to treatnon-Hodgkin lymphoma, breast cancer, and ovarian cancer, we andothers have found that host regulatory T cells (Tregs) are resistant tocytotoxic therapy and expand rapidlywhen IL-2 is administered afterNK cell infusion.4,5 Tregs are phenotypically distinct CD41CD251

Foxp31 immunosuppressive lymphocytes residing in lymphoid

organs and peripheral blood (PB). They prevent autoimmunity andmediate tolerance by restricting immune responses, including inhibitionof NK-mediated cytotoxicity.6 In the setting of NK cell adoptivetransfer, however,wehypothesize that hostTregs interferewithNK-cellproliferation and expansion. Because Tregs are uniquely dependent onthe high affinity IL-2 receptor a chain (CD25) for their function andsurvival, IL-2 mediates the strongest proliferative signal for Tregs. Wereport here the results of in vitro tests to determine the effect ofcompetition between Tregs and NK cells, which support the in-corporation of Treg depletion into our adoptive transfer platform.

IL-2 diphtheria toxin (IL2DT, Denileukin diftitox; Ontak), isa recombinant cytotoxic fusion protein composed of the amino acidsequences for diphtheria toxin followed by truncated amino acidsequences for IL-2. Therefore, IL2DT should selectively depleteIL-2 receptor (CD251)-expressing cells, including Tregs. IL2DT is100 times more effective in killing cells bearing the IL-2 receptor achain isoform (CD25) compared with cells expressing the lower-affinity IL-2 receptors (ie, CD122 and CD132).7 In murine AMLmodels, depletion of Tregs by anti-IL-2 receptoramonoclonal anti-body or IL-2 diphtheria toxin fusion protein dramatically improved

Submitted October 13, 2013; accepted March 27, 2014. Prepublished online

as Blood First Edition paper, April 9, 2014; DOI 10.1182/blood-2013-10-

532531.

V.B. and S.C. contributed equally to this work.

The online version of this article contains a data supplement.

There is an Inside Blood Commentary on this article in this issue.

The publication costs of this article were defrayed in part by page charge

payment. Therefore, and solely to indicate this fact, this article is hereby

marked “advertisement” in accordance with 18 USC section 1734.

© 2014 by The American Society of Hematology

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the efficacy of adoptive NK or cytotoxic T-cell immunotherapy.8,9

IL2DT is a particularly attractive agent to test for the selectivedepletion of Tregs due to the short half-life, rapid internalizationtime, and induction of apoptosis, thus allowing for dosing regimensthat will not affect adoptive immune therapy (ie, NKcells) infused justhours after IL2DT.10 Thus, we tested host Treg depletion with IL2DTin our platform of lymphodepleting chemotherapy to enhance in vivoNKcell expansion and induction of remissions in refractoryAMLafteradoptive NK cell transfer.

Methods

Patient eligibility and clinical protocol

Patients with relapsed or primary refractory AML with adequate organ func-tion who had failed$2 therapies were eligible for enrollment. The protocol andconsent procedures were approved by the University of Minnesota institutionalreview board (clinicaltrials.gov NCT00274846 and NCT01106950), and in-formed consent was given by all patients and donors for treatment andprospective data collection in accordance with Declaration of Helsinki.Nonmobilized donor PB mononuclear cells (MNCs) were collectedwith the COBE Spectra Apheresis System (TerumoBCT, Lakewood,CO) for 3 (n 5 31) or 5 hours (n 5 26). The trial schema, chemotherapy,and trial end points are detailed in Figure 1, and standard definitions ofresponse were used.11 The primary prospective end point of the study wassuccessful in vivo donor NK cell expansion defined as measurement of.100 donor NK cells/mL of PB at day 114 after NK cell infusion[(absolute lymphocyte count/mL)3 (% of lymphocyte gate that are CD561

/CD32 NK cells) 3 (% donor chimerism using standard short tandemrepeat [STR] testing)].

Preparation of the NK-enriched cell products

The apheresis productswere T-cell (CD3)6B-cell (CD19) depleted6CD56selected using the Miltenyi Biotec CliniMACS Cell Selection System andreagents (Miltenyi Biotec, Bergisch Gladbach, Germany) (Table 1) and cul-tured overnight with 1000 IU/mL IL-2 as previously published.12 An aliquot

of this prepared cell product was analyzed by flow cytometry to determinethe number of T, B, and NK cells by using fluorescein isothiocyanate,Ag-presenting cells, phycoerythrin, and Peridinin-Chlorophyll-Protein-Complex–conjugated antibodies against CD3, CD14, CD56, CD19 or CD20,KIR, and NKG2A (BD PharMingen, San Diego, CA) and tested in a 4-hourCr-release cytotoxicity assay against the K562 cell line.13

Immunophenotyping and enzyme-linked immunosorbent assay

Patient PB was analyzed by flow cytometry before chemotherapy, days0, 7, 14, and 28 after NK cell infusion. Lyphocytes were characterized bymulticolor fluorescent antibodies directed against CD45, CD56, CD3, CD4,and CD20, Ki67 (BP PharMingen, San Diego, CA) and in selected patientsCD25, CD127, Foxp3, Helios, 41BB, and CD40 ligands. Plasma IL-15, IL-7,IL-35, and proliferation assay supernatant for IL-15 and IL-2 concentrationswere determined by commercial enzyme-linked immunosorbent assay (R&DSystems, Minneapolis, MN).

5-Carboxyfluorescein diacetate succinimide ester assay to

assess NK-cell suppression by Tregs

Umbilical cord blood (UCB)-derived Tregs were purified and expanded aspreviously reported.14 The effect of Tregs on NK cells (NK cell isolationkit, MACS; Miltenyi Biotec) or PBMNCs was tested after labeling with5-carboxyfluorescein diacetate succinimide ester (CFSE) to assess proliferationinduced by anti-CD3 mAb-coated beads (Dynal) or cytokines as indicated.We used the same suppression assay to evaluate patient PBMNCs collectedat day 14 with healthy donor NK cells. Acquired data were analyzed usingthe proliferation platform in FlowJo (Treestar, Ashland, OR).

Statistical analysis

Disease-free survival (DFS) was estimated by Kaplan-Meier curves through6months after therapy.15Cumulative incidencewas used to estimate nonrelapsemortality (NRM), treating relapse and disease progression as competing risks.16

Simple proportions were used to describe in vivo donor NK-cell expansion andCR.Associations betweenNK-cell expansion and remission and Treg depletionwere tested by the Fisher’s exact test andWilcoxon rank-sum test, respectively.In vitro results were compared by paired 2-tailed t test.

Figure 1. Clinical trial schema. Patients received fludarabine 25 mg/m2/day intravenously (IV) daily (days26 through22) and cyclophosphamide 60 mg/kg/day IV (days25

and24) to lymphodeplete the recipient and facilitate homeostatic expansion of allogeneic NK cells. One (n5 11) or 2 doses (n 5 4) of IL2DT, 12 (n5 11) or 18 mg/kg (n5 4)

IV, were added at day 21 6 22 to deplete Treg. NK cell products were administered by IV infusion on day 0 followed by subcutaneous IL-2 (9 3 106 units) starting 4 hours

after NK cell infusion and given every other day for 6 doses to facilitate NK cell survival and expansion in vivo. Unseparated PB donor chimerism by STR and lymphocyte

subsets were analyzed at days 7, 14, and 28. Bone marrow (BM) was analyzed for leukemia clearance at days 14 and 28 to assess disease status according to World Health

Organization criteria. Toxicity and adverse events were classified according to National Cancer Institute Common Terminology Criteria for Adverse Events V 3.0. The primary

prospective end point of the study was successful in vivo donor NK cell expansion defined as measurement of.100 donor NK cells/mL of PB at day114 after NK cell infusion

[(absolute lymphocyte count/mL)3 (% of lymphocyte gate that are CD561/CD32 NK cells) 3 (% donor chimerism using standard short tandem repeat testing)]. We evaluated

BM at day 28 and used standard definitions of CR, CRp (,100 000 platelet count/mL), and CRi (,1000 absolute neutrophils/mL).

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Results

Patients and disease characteristics

A total of 57 patients with relapsed and refractory primary or se-condary AMLwere treated from 2003 to 2011 (Table 1). All patientsreceived lymphodepleting chemotherapy with fludarabine andcyclophosphamide, and the 15 patients in cohort 3 also receivedIL2DT (Figure 1). The outcomes of the first 19 patients in cohort 1were published previously3 and are included here to compare theefficacy of different NK-cell products. Patient characteristics in-cluding age, percentage ofmarrow blasts at time of treatment, and thenumber of prior therapies were similar between cohorts. Six patientsin cohort 1 had undergone prior hematopoietic stem cell transplan-tation (HCT; 2 autologous and 4 allogeneic). AllNKcell donorswereHLA-haploidentical relatives and about half were KIR ligandmismatched in the graft-versus-host disease (GVHD) direction.IL2DT-treated patients (Table 2) often had an antecedent hemato-logic malignancy with progression to AML (55%), and 40% hadreceived prior hypomethylating therapy.

NK-cell products

Three different processing methods were used to prepare NK-cellproducts for infusion. These includedCD3 depletion alone (cohort 1;n 5 32), a CD3 depletion followed by CD56 selection (cohort 2;n 5 10), and single step CD3/CD19 depletion (cohort 3; n 5 15).Analysis of the cellular content of each product, summarized inTable 1, showed that the addition of CD56 selection resulted inthreefold fewer NK cells per product comparedwith CD3 depletionalone. Lymphapheresis runs of 3 hours (n5 31) werewell tolerated

andwere extended to 5 hours for donors in cohorts 2 and 3 to obtainhigher NK cell doses. All clinical products were highly cytotoxicagainst K562 targets (data not shown). The highest NK cell doses(mean 2.6 6 1.5 3 107 NK cells/kg) were obtained with theCD3/CD19 depletion method, due to the 5-hour collection timeand reduced cell loss with the single GMP manipulation.

Infusion and long-term toxicities

All 57 patients tolerated NK-cell infusions well. Grade 1 to 2 non-hematologic toxicities (fever, chills, hypertension/hypotension,dyspnea, hypoxemia, headaches)were common, but therewas only 1grade 4 infusion-related hypersensitivity reaction, which promptlyresolved with antihistamines and supportive care. We observed noinfusional toxicity with IL2DT. Later grade 3 to 5 toxicities, whichincluded infection or fevers (n5 5), cytomegalovirus viremia (n5 1),alveolar hemorrhage (n 5 2), pulmonary events (n 5 2), pleuraleffusion (n 5 1), candidemia (n 5 1), fungal pneumonia (n 5 1),atrial fibrillation (n 5 1), left ventricular dysfunction (n 5 1),typhlitis (n5 1), meningitis (n5 1), and Epstein-Barr virus (EBV)-associated lymphoma (n5 1), were most likely related to prolongedcytopenias and immune suppression. One patient (cohort 1) died inremission from complications of EBV-associated lymphoprolifer-ative disorder at day 116. We did not observe acute cytokine releasesyndrome or complications associated with tumor lysis, and nopatients developed acute GVHD or autoimmune disorders.

Addition of host Treg depletion with IL2DT significantly

increases CR rate

Among the 42 patients in cohorts 1 and 2 who did not receive hostTreg depletion with IL2DT, 9 (21%) achieved remissions (5 CR and4 incomplete remission without neutrophil [,1000 cells/mL] andplatelet recovery [CRi]) by day 35. Themedian duration of remissionwas 2.3 months (range, 1.8-15 months). This platform served asa bridging therapy for 4 patients who proceeded to allogeneic HCTbetween days 50 and 65 after NK-cell infusion. Among the other 5patientswho achieved remissions, 1 died ofNRM (EBV event above),and 4 were ineligible for transplant due to comorbidities. Theyrelapsed at 61, 67, 190, and 450 days after NK-cell infusion, dem-onstrating that this is not curative therapy. There was no improvementin the remission rate for the patients who received purified CD56-selected NK cell products (cohort 2) compared with those whoreceived CD3-depleted products.

Augmented lymphocyte and Treg depletionwith IL2DT (cohort 3)resulted in remissions at day 28 for 8 of 15 patients (53%), includingCR(n53),CRwithout platelet recovery (CRp;n52), andCRi (n53),which was significantly better compared with strategies withoutIL2DT (CR rate, 21%; P 5 .02). Six patients in remission sub-sequently received allogeneic donor HCT 45 to 120 days after NK-cell therapy. One transplant-ineligible patient remained in CR untilrelapse at 8 months, and 1 patient who successfully expanded donorNK cells and cleared leukemia died of neutropenic sepsis at day 30.The median duration of remission in cohort 3 was 11.2 months(range, 1-32 months). Patients in CR and CRp attained neutrophilrecovery (ANC. 1000 cells/mL) at a median of 20 days after NK-cellinfusion (range, 15-22 days; n5 5).

There was no correlation between CR and AML blast burden,cytogenetics, number of prior therapies, KIR ligandmismatch, use ofhypomethylating agent, or disease status at the time of enrollment incohorts 1 and 2 (data not shown) or cohort 3 (Table 2). Consideringrelapse/progression as a competing risk, the overall NRMwas similarbetween the groups: 12% (95% confidence interval [CI], 5-18%) in

Table 1. Patients, treatment, and product characteristics

Variable Cohort 1 Cohort 2 Cohort 3

No. of patients enrolled 32 10 15

Time period 2003-2007 2005 2010-2011

Patient age in years (range) 46 (7-68 y) 37 (5-65 y) 51 (3-71 y)

Patient gender (male) 19 (59%) 5 (50%) 8 (55%)

Marrow blasts

(mean %)

45%

(range 2-98)

36%

(range 7-92)

34%

(range 8-69)

Number of prior

therapies (mean)

4 3 3

Prior HCT 2 auto/4 allo 0 0

Recipient CMV1 status 14 (44%) 6 (60%) 7 (45%)

Conditioning Cy/Flu Cy/Flu Cy/Flu

No. of IL-2 doses (mean) 4 (range 3-6) 4 (range 3-6) 4 (range 1-6)

IL2DT received No No Yes*

KIR mismatch in GVHD

direction

6 (17%) 5 (50%) 8 (53%)

Product processing method CD3- CD3-CD561 CD3-CD19-

Final product characteristics

Dose of NC/kg 2.5 6 0.8 x107 0.44 6 0.09 3

1074.7 6 1.8 3

107

Dose of NK cells/kg

infused

0.96 6 0.3 3

1070.34 6 0.05 x107 2.6 6 1.5 3

107

Percentage NK cells 39 6 9% 75 6 6% 54 6 16%

Dose of T-cells/kg 14 3 104 6.2 3 104 9.7 3 104

Percentage T cells 0.7% 1.3% 0.3%

CD32, CD3 depletion; CD32CD561, CD3 depletion followed by CD56-positive

selection; CD192, CD19 negative selection; CMV, cytomegalovirus; NC, nucleated

cell dose.

*Doses of 12 to 18 mg/kg 3 1 (n 5 11) or 2 doses (n 5 4) were used.

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Table

2.Treatm

entdetails,correlativeanalysis,andpatientoutcome

No.

Age

(years)

Prior

disease

WBC*

Cytogenetics/

FISH

Molecular

Marrow

blasts*

No.ofprior

therapies

Disease

status

KIR

ligand

mismatch

PB

donor

chim

erism

atday

7

DonorPB

NK

cellsatday14

(cells/mL)

PB

percentageof

Tregsatday14

Disease

responseatday

28

Tim

eto

death/tim

eofsurvival(m

o)

Survival

status

166

MDS

82

t(1;3),del5q-

EGFR

96%

2†

PIF

No

95%

12390

0.02%

CRi

1.1

Dead

259

MDS

0.2

Complex,del7p-

EGFR

34%

3PIF

No

95%

484

0.1%

PD

0.9

Dead

349

RAEB-2

55

Norm

al

Flt3ITD

49%

3†

PIF

No

13%

071%

CR‡

10

Dead

451

ET/M

DS

10

del5q-,del20q-

EGR1

40%

3PIF

No

QNS

049%

PD

0.8

Dead

55

Primary

AML

110

t(9;11)

MLL

95%

5PIF

Yes

0%

027%

PD

1.1

Dead

651

Primary

AML

32

Monosomy7

EVI1

81%

4†

PIF

Yes

34%

24

1.1%

PD

2.5

Dead

771

MDS

0.2

Complex,del20q-

MYBL2

6%

3†

Relapse

Yes

68%

017%

CR‡

9.4

Alive

864

MPD

53

Monosomy7

EGFR

9%

5†

PIF

No

92%

532

0.06%

CRi‡

2.6

Dead

957

MPD

0.7

Norm

al

Jak21,

Flt3ITD

24%

2†

PIF

Yes

QNS

No

11%

CRi‡

12

Alive

10

37

Primary

AML

2.2

Norm

al

Flt3ITD

10%

3PIF

No

33%

No

28%

CRp‡

12

Alive

11

13

MDS

225

Trisomy6

Flt3ITD

7%

3PIF

No

42%

No

10%

CR

10.2

Dead

12

54

Primary

AML

5.6

t(12;14)

ETV6

36%

4Relapse

Yes

0%

No

NA

PD

0.4

Dead

13

5Primary

AML

NA

t(9;11)

MLL

33%

4Relapse

Yes

0%

No

16%

PD

1.1

Dead

14

16

Primary

AML

50

t(3;22)del9q

n/a

85%

6PIF

Yes

99%

1470

0%

PD

1.2

Dead

15

73

Primary

AML

2.5

Trisomy1

PBX1

24%

2Relapse

Yes

70%

No

20%

CRp‡

12

Alive

ET,essentia

lthrombocythemia;MPD,mye

loproliferativedisorder;NA,notava

ilable;PD,progressivedisease

;PIF,primary

inductionfailure;QNS,quantitynotsu

fficient;RAEB,refractory

anemia

with

excess

blasts;

WBC,white

bloodce

ll.

*Attimeoftherapy(in106cells/mL).

†Includespriordemethylationagent.

‡Subsequentallogeneic

donorstem

celltransplant.

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cohorts1 and2 and13%(95%CI,1-26%) incohort 3.DFSat 6monthswas 5% (95% CI, 1-14%) for cohorts 1 and 2 compared with 33% forcohort 3 (95% CI, 12-56%; P, .01). Thus, the addition of host Tregdepletion with IL2DT into our previously published adoptive NK-celltherapy platform resulted in a remission rate.50% and superior DFSfor patients with refractory/relapsed AML with no increased com-plications or toxicity.

Addition of Treg depletion with IL2DT enhances successful

donor NK-cell expansion

In cohorts 1 and 2, 4 of 42 (10%) patients had detectable donor NKcells in vivo. In contrast, host Treg depletion with IL2TD wasassociatedwith a higher rate (4/15 [27%]) of successful donor NK-cellexpansion. The magnitude of the NK-cell expansion was also higherafter Treg depletion, with median absolute circulating donor-derivedNK-cell counts at day114 of 190 cells/mL (range, 110-240 cells/mL)and 1000 NK cells/mL (range, 480-12 390 cells/mL; P 5 .12),respectively. Failure to expand NK cells did not correlate withdisease burden (WBC counts and percentage blasts) or prior therapy.Immunological parameters such as baseline PB Treg frequencies(measured in cohort 3) or number of IL-2 injections received did notinfluence NK-cell expansion (Table 2). In addition, there was nocorrelation between the incidence of NK-cell expansion and themethod of NK-cell product manufacture (CD3-depleted andCD56-selected [cohort 2] vs CD3-depleted [cohort 1]) or the infusedNK-cell dose (data not shown).

Magnitude of Treg depletion correlates with successful donor

NK-cell expansion

We evaluated the magnitude of Treg depletion achieved with IL2DTto determinewhether it correlatedwith successfulNK-cell expansionand attainment of complete remission. Prior to lymphodepletingchemotherapy, the median absolute lymphocyte count in patientswas 800 cells/mL (range, 0-1300 cells/mL), and Tregs (CD41FoxP31

CD25hi) comprised 4% (range, 0.3-7.5%, n 5 15) of PB lymphocytes(median absolute Treg count, 34/mL; range, 0-60 cells/mL). Followingthe cyclophosphamide and fludarabine (day 22), patients becamelymphopenic (median WBC, 350 cells/mL; range, 0-760 cells/mL).At that time, most residual lymphocytes were T cells (median, 79%;range, 23-95%), predominantly CD31CD41 (median, 89% ofT cells; range, 74-97%), with very fewCD31CD81 effectors surviving(median, 7%; range, 1-9%). Although Tregs comprised 9% ofall lymphocytes (range, 0.03-27%; absolute count, 31 cells/mL; range,10-94 cells/mL), suggesting relative Treg chemoresistance, theywerenot proliferating based on undetectable Ki67 expression (data notshown).Treg levelsweremeasured onday7 following the infusionofIL2DT and the first 3 doses of IL-2. Tregs were depleted in somepatients (n5 6; median PB Tregs, 1%; range, 0.05-4.7%]), but moreoften, host Tregs accumulated (n 5 9; median Tregs, 28%; range,9-52%]). We observed a strong inverse correlation between day 7absolute PB Treg count and successful in vivo donor NK-cellexpansion. Of the 6 patients with ,5% Tregs at day 7 (medianabsolute Treg of 3 cells/mL; range, 0-6 cells/mL), 5 successfullyexpanded donor NK cells. In contrast, none of the 9 patients with

Figure 2. Treg depletion leads to NK-cell persis-

tence and expansion that correlates with remission.

(A) Successful in vivo donor NK cell expansion was

observed in 80% of patients with Treg depletion (shown

as percentage of PB lymphocytes; n 5 6) compared

with 0% in patients with high levels of Tregs (n 5 9). (B)

Rates of complete remission in patients with or without

detectable donor NK cells in PB at day 7. (C) PB flow

cytometry plot of selected subjects who demonstrated

in vivo NK expansion at days 7 and 14.

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higher Treg counts expanded NK cells (median Treg, 32; range,10-69 cells/mL; P , .01; Figure 2A). Treg proliferation measuredby the proportion of cells expressing Ki67 was significantlyhigher in patients without successful in vivo donor NK expan-sion compared with those who did expand (906 2% vs 656 5%;P 5 .004), suggesting that host Tregs were responding to theIL-2 administration given to the patient after NK-cell infusion.The ability of IL2DT to deplete Tregs is further supported byreductions in serum IL-35 concentrations 14 days after adoptivetransfer in patients who received IL2DT compared with thosewho did not (88.2 [range, 5-186] pg/mL, n 5 12 vs 30 [range,10-83] pg/mL, n 5 15; P 5 .02). Tregs collected 14 days fromadoptive transfer from 4 patients who did not expand werefurther characterized by immunophenotyping. They were allCD41CD251CD127lowFoxp31Helioshigh, and of those, 29 6 5.5%were 41BB1CD40L2, consistent with an activated Treg phenotype.17

NK cells from 2 patients who had Treg .25% 41BB1CD40L2

after thawing exhibited .90% suppression of healthy donor NKcells (supplemental Figure 1, available on the Blood Web site).

Early donor NK persistence correlates with AML clearance

To determine whether the presence of low levels of donor NK cellsenhances clinical efficacy, we compared rates of AML clearance inpatients with and without detectable persistence of donor NK cells atday 7 after infusion. Although all patients were leukopenic (,100WBC cells/mL) at day 7, donor chimerism was measurable by STR.Although only 4 patients met the end point of successful in vivodonor NK-cell expansion (at day 14 after adoptive transfer), 10 of 15(66%) had donor NK cells detectable 7 days after infusion (4 patientsshown in Figure 2C). As NK cells were the predominant populationcirculating in blood, they accounted for the majority of donorchimerism in these samples. For the 10 patientswith detectable donorNK cells (mean donor STR chimerism, 49.5% [range, 13-99%]),7 (70%) attained CR by day 28 (Table 2; Figure 2B-C). In contrast,only 1 of 5 patients (20%) who lacked detectable donor NK cells (nodonor chimerism) at day 7 achieved CR (P5 .05; Figure 2B-C).

In vivo expanded donor NK cells are cytotoxic and up-regulate

inhibitory receptors

The cytotoxicity of in vivo expanded donor NK cells collected fromthe PBof patients on day 14 against K562 targetswas higher than that

of NK cells from the IL-2-activated products (Figure 3A). Althoughthis may be explained in part by the composition of NK cells in thePBMNC product (mean 53% NK cells in the product vs 99% in thePB of the patients), it is evident that the cells had potent activity. Acomparison of the phenotype profiles of the 2 demonstrated that invivo expanded NK cells expressed higher levels of the inhibitoryreceptor NKG2A (P 5 .001), with no change in KIR expression(Figure 3B).

Endogenous serum IL15 levels correlate with NK-cell

proliferation and expansion

Because lymphodepleting chemotherapy affects levels of homeo-static cytokines, wemeasured serum concentrations of IL-15 and IL-7at baseline, after chemotherapy, and at day 14.3 In cohort 3, serumIL-15 and IL-7 levelswere low at baseline (mean, 11.8 pg/mL [range,1.5-44] and 3 pg/mL [range, 0-52], respectively). They increasedeightfold after chemotherapy (mean, 93 pg/mL [range, 30-221];P5 .002) and 91 pg/dL [range, 11-151]; P, .001, respectively). Inthe patients receiving host Treg depletion with IL2DT, serum IL-15levels at the time of NK-cell infusion (day 0) correlated withsuccessful donor NK-cell expansion (148 pg/mL [range, 74-221]in expanders vs 33 pg/mL [range, 21-46] in nonexpanders, P5 .002;supplemental Figure 2). In contrast, cohorts 1 and 2 without Tregdepletion both had low IL-15 levels on day 0 (mean, 35 vs 32 pg/dl),and there was no correlation with expansion. By day 7, the IL-15levels in cohort 3 had declined to levels similar to cohorts 1 and 2(32 pg/mL [range, 19-61] in all patients) and had returned to close tobaseline at day 14, suggesting a transient window for IL-15-drivenhomeostatic NK-cell expansion after lymphodepleting chemother-apy. This is supported by high NK-cell proliferation rate at day 7(mean Ki67 expression, 98.2 6 2%), which fell by day 14 (mean,466 24%; P5 .05).

Tregs suppress in vitro NK-cell proliferation in the presence of

IL-2 but not IL-15

To better understand the influence of IL-15 and IL-2 on Treg andNK-cell proliferation, we analyzed cell and cytokine interactions invitro. CFSE-labeled NK cells or T-effector cells were purified fromhealthy donors and incubated with allogeneic UCB-derived Tregs.Tregs were potent inhibitors of CD3 bead-stimulated PB T-effectorproliferation, as we have previously demonstrated.14 Resting NK

Figure 3. In vivo expanded NK cells are potent killers, express high levels of NKG2A, and correlate with endogenous IL-15 prior to NK cell infusion. (A) Cytotoxicity

of in vitro IL-2 activated NK cell products compared with PB NK cells isolated at day 14 after in vivo expansion. Cytotoxicity assay against K526 targets at various effector to

target ratios. Cytotoxicity of the NK cell product (in gray; 15 infusion products; mean 6 standard error of the mean [SEM]) prior to infusion compared with NK cells isolated

from the PB at day 14 of those that expanded (black line; 4 subjects; showed mean6 SEM). (B) Expression of inhibitory receptors on NK cells in product and in vivo expanded

NK cells at day 14. (C) Serum IL-15 levels at various time points after NK cell infusion. Comparison of IL2DT cohort patients with donor NK expansion (n 5 4) vs no NK cell

expansion (n 5 11) and all patients not treated with IL2DT (n 5 42, mean and standard deviation shown).

3860 BACHANOVA et al BLOOD, 19 JUNE 2014 x VOLUME 123, NUMBER 25

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cells in media without cytokines did not proliferate (data not shown),and the addition of IL-2 (0.5 ng/mL) or IL-15 (0.5 ng/mL) or thecombination (both, 0.25 ng/mL) was required to stimulate NK-cellproliferation after 5 days of culture (Figure 4A). Without addedTregs, both cytokines induced equivalent NK-cell proliferation at 5days in a concentration-dependent manner. Proliferation of IL-2- butnot IL-15-activated NK cells was potently suppressed by Tregs in aconcentration-dependentmanner (Figure 4A-B). The proliferation ofIL-2-stimulated NK cells (0.5 ng/mL) was inhibited 50% to 85% byTregs at ratios from 1:8 to 1:1. In contrast, proliferation of IL-15-stimulatedNKcells was not inhibited byTregs (,10%), even at a 1:1

ratio. The addition of Tregs at a 1:1 ratio induced potent suppressionof IL-2-stimulated NK proliferation, which was not observed withIL-15 or the combination of IL-2 and IL-15, supporting a modelwhere competition with Tregs that consume and deplete IL-2 is 1potential mechanism for the observed limited NK-cell proliferation.High concentrations of IL-2 (10 ng/mL) could partially overcomeTreg inhibition (data not shown).

To determine whether Tregs inhibit NK-cell proliferation bycompetition for cytokines, we measured Treg utilization of IL-2 orIL-15 by incubating NK cells (1 3 105 NK cells) with or withoutTreg (1 3 105 Tregs) and 0.25, 0.5, or 10 ng/mL of cytokine for

Figure 4. Cytokine-induced NK-cell proliferation is suppressed by allogeneic Treg cells. (A) Healthy donor purified NK cells were CSFE labeled and cultured alone

or with UCB-derived Tregs at a ratio of 1:1. Suppression of proliferation of NK cells incubated with IL-2 (0.5 ng/mL), IL-15 (0.5 ng/mL), or a combination of IL-2 1 IL-15

(0.25 ng/mL) was measured. Shown is 1 representative donor of 6 experiments. (B) Healthy donor NK cells and UCB-derived Tregs were coincubated with 0.5 ng/mL of

IL-2 or IL-15 and cocultured at various Treg:NK cell ratios. Percent suppression of NK proliferation by Tregs was evaluated by CFSE dilution. Treg suppression of CD3

bead-stimulated PBMNC effector T-cell proliferation was measured as a control. Data are an aggregate of 5 separate experiments. NK cells and Tregs were coincubated

with IL-2 or IL-15 for 4 days, and (C) IL-2 or (D) IL-15 in the supernatant was measured by enzyme-linked immunosorbent assay. Various cytokine concentrations

(0.2, 0.5, and 10 ng/mL) and Treg:NK cell ratios (1:1, 2:1, 4:1, 8:1) were compared. Results from 5 NK cells donors are shown (mean and SEM). *P 5 .05 compared with

NK cells alone.

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4 days (Figure 4C). When starting with 0.5 ng/mL, after 4 days ofculture, the mean remaining concentrations of IL-2 were 78 pg/mLwith no cells, 24 pg/mL with NK cells alone, 55 pg/dL with Tregalone, and 4.5 pg/mL with a 1:1 ratio of NK and Treg cells (P5 .05compared with NK cells alone) . Similar IL-2 consumption by Tregswas observed using low (0.25 ng/mL) or high (10 ng/mL) dose IL-2in the starting media. In contrast, NK consumed IL-15 (concen-trations, 0.25, 0.5, and 10 ng/mL), but the addition of Tregs did notaffect residual IL-15 levels (Figure 4D). This suggests that Tregscompete for or consume IL-2, a finding that may be directly relevantto in vivo platforms where the tolerability of pharmacologic IL-2dosing limits the achievable serum blood concentrations.

Discussion

Adoptive therapy using haploidentical NK cells can induce re-missions in patients with relapsed and refractoryAML. Successful invivo expansion of donor NK cells is correlated with attainment ofremission, and thus methods to enhance NK-cell expansion arecritical tooverall clinical efficacy.The strategy tested here, augmentingthe lymphodepleting platform with an immunotoxin (IL2DT) to dep-lete Tregs, led to improvements in rates of in vivo NK-cell expansionand AML remission in 27% and 53% of patients, respectively, com-pared with cohorts without IL2DT.3,18 Expanded donor NK cellsmaintained proliferative and cytotoxic effector function in vivo withthe setting of sustained Treg depletion and elevated serum levels ofIL-15 and IL-7. In humans, IL-15 is secreted by monocytes, macro-phages, and dendritic cells in response to PB lymphopenia and signalsthrough common IL-2/IL-15 receptorb (CD122) andg chains (CD132)expressed on lymphocytes.19 IL2DT may potentiate IL-15 release bymore profound blood or tissue lymphodepletion by elimination ofactivated (postchemotherapy) lymphocytes expressing CD25, althoughfuture study is needed to understand these interactions.

Our clinical observations are compatible with preclinical studiesfrom Zhou et al showing that a brief course of IL2DT restored theproliferation of transferred cytotoxic T lymphocytes and reduced theleukemia burden in the liver and spleen ofmice, markedly increasingsurvival.8 Hallet et al analyzed the efficacy of NK cells in a murineadoptive transfer model in which Tregs were depleted with an anti-CD25 antibody.9 NK cell-mediated killing and the survival ofleukemia-bearing mice cotreated with IL-2 and anti-CD25 antibodywere markedly improved compared with either treatment alone.Although CD81 T-cell depletion did not change these outcomes,NK-cell depletion completely abrogated all antitumor effects,indicating the essential role of NK cells in mediating these anti-tumor responses.

Our studies show that lack of in vivo NK-cell expansion cor-relates with high numbers of host-derived Tregs with an activatedphenotype and suppressive function. This suggests that the Treg-mediated suppressive environment may, in part, blunt the efficacyof adoptive NK-cell therapy. Our in vitro data demonstrate that thisis mediated in part through Treg competition for IL-2, as has beenreported in animal models.20,21 However, we acknowledge that theabsence of donor NK cells in patients is likely the consequence ofa variety of rejection mechanisms.8,22-25 Direct NK-cell inhibitionvia Treg membrane-bound transforming growth factor-b has beenreported and may also contribute suppression of in vivo NK-cellexpansion.26 Targeting some proposed inhibitory mechanismssuch as blockade of cytotoxic T-lymphocyte antigen 4, the pro-grammed cell death protein 1 pathway, or inhibitors of indoleamine

2,3-dioxygenase may further unleash suppressed NK-mediatedimmunity.22-25 Ultimately, the relative influence of these mecha-nisms may be depend on the specific tumor type and needs furtherstudy.

Although a single dose of IL2DT seems insufficient to entirelydeplete host Tregs, the decreased IL-35 levels (made by Tregs)14 days after adoptive transfer suggest a partial effect. Given theprofound absolute lymphopenia at the time of IL2DT infusion(day21) and the fact that sampling is limited to PB, which maymisseffects related to Tregs trafficking to and from lymphoid tissues, theimmediate depleting effect of IL2TD in tissues is not directlymeasurable. In some patients, we observed rebound in the CD41

Foxp31CD1272 cell compartment and Treg proliferation (measuredbyKi67 expression), whichwas likely induced by the pharmacologicIL-2 given with the intent of promoting in vivo expansion of donorNK cells. This is consistent with the observed proliferative effect ofin vivo low dose IL-2 on Tregs.27

The IL2DT cohort demonstrated that lack of host Tregs is asso-ciatedwith improved in vivo donorNK-cell expansion and remissioninduction. However, in addition to IL2DT, cohort 3 also receivedproducts with higher NK cells doses, which may have contributed tothe better clinical results. Higher numbers of infused cells may beneeded to overwhelm any residual T cell-mediated rejection of thepartially HLA-mismatched NK cells. Although the lymphodepletingchemotherapy was efficient in inducing significant circulating lym-phopenia, it is more difficult to assess secondary lymphoid tissues,such as the lymph nodes and spleen, where such rejection mightoccur. Antitumor effects of lymphodepleting chemotherapy alsoshould be considered, although the Flu/Cy regimen was identical in3 cohorts with widely different response rates. IL2DT could providean additional anti-leukemia effect by targeting CD25 expressed onAML blasts and leukemia stem cells.28-30

Several groups are actively investigating methods to expand NKcells ex vivo using genetically engineered antigen-expressing cellswith membrane-bound IL-15, IL-21, and 41BB ligand expression toovercome the limitations of in vivo NK-cell expansion.31-33 TheseNK cell products should be tested clinically against the fresh acti-vated NK cell products described here.

In summary, we demonstrated that adoptively transferred hap-loidentical adult NK cells that expand in vivo are associated withpromising clinical efficacy in patients with refractory or relapsedAML. High NK-cell doses are obtained after depletion of CD31

and CD191 cells from a 5-hour donor apheresis collection, andthere does not seem to be a negative effect of co-infusedmonocytes.It is also possible that IL-15 receptor a onmonocytes may facilitatetrans-presentation of endogenous IL-15 seen early after lympho-depleting chemotherapy enhanced by IL2DT. Our data suggest thatthe detection of donor NK cells as early as 7 days after infusion mayserve as a surrogate biomarker for clinical response, a method thatshould be further tested and validated in future cellular trials.34

Interrupting pathways that maintain the immunosuppressiveenvironment and supplementing IL-15 should be incorporated tofurther improve NK-cell expansion rates and to increase clinicalbenefit.

Acknowledgments

The authors thank the technical and quality assurance staff at Mole-cular & Cellular Therapeutics, Cytokine Reference Laboratory,University of Minnesota cGMP facility, and our dedicated researchstaff in Center of Experimental Therapeutics at University of

3862 BACHANOVA et al BLOOD, 19 JUNE 2014 x VOLUME 123, NUMBER 25

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Minnesota, in particular Dixie Lewis, TimKrepski, JudyWitte, andJill Aughey for outstanding support and invaluable contributions inconducting the trial. We want to acknowledge the University ofMinnesota Masonic Cancer Center Translational Therapy CoreLaboratory for excellent assistance.

This work is supported by National Institutes of Health, NationalCancer Institute grants P01CA65493 (J.S.M., S.C.,M.R.V., P.B.M.,and B.R.B.), P01 CA111412 (J.S.M., S.C., T.E.D., and D.J.W.),and R01 CA72669 (B.R.B.). Research reported in this publicationwas supported by the National Center for Advancing TranslationalSciences of theNational Institutes ofHealthAward (UL1TR000114).The content is solely the responsibility of the authors and does notnecessarily represent the official views of the National Institutes ofHealth. Partial funding of correlative assays and agent DenileukinDiftitox were provided by Eisai Inc.

Authorship

Contribution: V.B., S.C., M.R.V., P.M., D.J.W., B.R.B., and J.S.M.designed and performed the research, analyzed and interpreted thedata, andwrote themanuscript; T.E.D. performed statistical analysis;B.Z., J.C., A.P.-M. and K.H. performed correlative studies; D.H.M.was responsible for GMP cell processing and manuscript prepara-tion; andD.L. performed the patient research andwas responsible fortoxicity assessments.

Conflict-of-interest disclosure: The authors declare no competingfinancial interests.

Correspondence: Jeffrey S. Miller, Blood and Marrow TransplantProgram, University of Minnesota, Mayo Mail Code 480, 420DelawareStSE,Minneapolis,MN55455; e-mail:mille001@umn.edu.

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online April 9, 2014 originally publisheddoi:10.1182/blood-2013-10-532531

2014 123: 3855-3863  

Daniel J. Weisdorf, Bruce R. Blazar and Jeffrey S. MillerMcKenna, Julie Curtsinger, Angela Panoskaltsis-Mortari, Dixie Lewis, Keli Hippen, Philip McGlave, Veronika Bachanova, Sarah Cooley, Todd E. Defor, Michael R. Verneris, Bin Zhang, David H. cells is improved using IL-2 diphtheria toxin fusion proteinClearance of acute myeloid leukemia by haploidentical natural killer 

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