Growth and Activation of Natural Killer Cells Ex Vivo from ... › content › clincanres › 19 › 8 › 2132.full.pdffactor, IFN-g, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC

Cancer Therapy: Preclinical

Growth and Activation of Natural Killer Cells Ex Vivo fromChildren with Neuroblastoma for Adoptive Cell Therapy

Yin Liu1,3, Hong-Wei Wu1, Michael A. Sheard1, Richard Sposto1,2, Srinivas S. Somanchi4,Laurence J.N. Cooper4, Dean A. Lee4, and Robert C. Seeger1

AbstractPurpose: Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal

antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated

NK(aNK) cells canbe grown ex vivo fromperipheral bloodmononuclear cells (PBMC)of childrenwithhigh-

risk neuroblastoma using artificial antigen-presenting cells (aAPC).

Experimental Design: Irradiated K562-derived Clone 9.mbIL21 aAPCwere cocultured with PBMC, and

propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex multicytokine

assays, and a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of

disseminated neuroblastoma.

Results: Coculturing patient PBMC with aAPC for 14 days induced 2,363- � 443-fold expansion ofCD56þCD3�CD14� NK cells with 83% � 3% purity (n ¼ 10). Results were similar to PBMC from normaldonors (n¼ 5). Expression ofDNAM-1,NKG2D, FcgRIII/CD16, andCD56 increased 6-� 3-, 10-� 2-, 21-�20-, and 18-� 3-fold, respectively, on day 14 compared with day 0, showing activation of NK cells. In vitro,aNK cellswere highly cytotoxic against neuroblastoma cell lines and killingwas enhancedwithGD2-specific

mAb ch14.18. When mediating cytotoxicity with ch14.18, release of TNF-a, granulocyte macrophagecolony-stimulating factor, IFN-g , sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cellsincreased 4-, 5-, 6-, 15-, 265-, 917-, and 363-fold (151–9,121 pg/mL), respectively, comparedwith aNK cells

alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with

thawed and immediately intravenously infused cryopreserved aNK cells compared with untreatedmice and

was further improved when ch14.18 was added.

Conclusion: Propagation of large numbers of aNK cells that maintain potent antineuroblastoma

activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18. Clin Cancer

Res; 19(8); 2132–43. �2013 AACR.

IntroductionAlthoughoutcomehas steadily improvedover the past 20

years for patients with high-risk, metastatic neuroblastoma,long-term event-free survival (EFS) is still only 45% (1–3).

Addition of immunotherapy with antitumor cell disialo-ganglioside (GD2) monoclonal antibody (mAb) ch14.18along with interleukin (IL)-2 and granulocyte macrophagecolony-stimulating factor (GM-CSF) to 13-cis retinoic acidimproves EFS and overall survival for children who have aclinical response after induction chemotherapy and mye-loablative consolidation therapy (3). However, 40% ofpatients still develop disease progression or relapse duringor after immunotherapy (3).

mAb immunotherapy for residual disease may be furtherenhanced by improving the ability of natural killer (NK)cells to function in antibody-dependent cell-mediatedcytotoxicity (ADCC) and to secrete antitumor cytokinesand chemokines. Strategies include modifying mAbs tohave high affinity interaction with NK cell FcgRIII/CD16 orto deliver cytokines via mAb–cytokine fusion proteins tothe tumor microenvironment (4), enhancing activation ofNK cells in vivo using immune-modulating drugs, such aslenalidomide (5, 6), or growing and activating NK cells exvivo for adoptive cell therapy. Some of these approachesalso may be combined with cytotoxic chemotherapy or

Authors' Affiliations: 1Division of Hematology/Oncology and SabanResearch Institute, Children's Hospital Los Angeles; 2Department of Pre-ventive Medicine, Keck School of Medicine, University of Southern Cali-fornia, Los Angeles, California; 3Division of Hematology/Oncology, Shang-hai Children's Medical Center, Shanghai Jiaotong University School ofMedicine, Shanghai, China; and 4Division of Pediatrics, MD AndersonCancer Center, University of Texas, Houston, Texas

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

D.A. Lee and R.C. Seeger are co-senior authors of this article.

Corresponding Author:Robert C. Seeger, Division of Hematology/Oncol-ogy and Saban Research Institute, Children's Hospital Los Angeles, KeckSchool of Medicine, University of Southern California, Los Angeles, CA90027. Phone: 323-361-5618; Fax: 323-361-4902; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-12-1243

�2013 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 19(8) April 15, 20132132

on June 14, 2021. © 2013 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst February 1, 2013; DOI: 10.1158/1078-0432.CCR-12-1243

http://clincancerres.aacrjournals.org/

targeted therapy for more effective treatment of measur-able disease.Adoptive cell therapy with NK cells alone or combined

with mAbs has therapeutic potential for a wide variety ofhuman malignancies, including neuroblastoma (7). Oneapproach for obtaining NK cells has been to harvest largenumbers of peripheral blood lymphocytes by leukapher-esis, deplete allogeneic T cells, and activate the remainingNK cells with IL-2 before reinfusion. In this manner, hap-loidentical NK cell therapy for acutemyelogenous leukemiaattained remission in poor-prognosis adults (8) and main-tained remission in children (9). A second method is togrowNKcells ex vivo (10–14), but clinical testing of suchNKcells has been limited because of the inability to obtain largenumbers of pure NK cells that do not senesce after replica-tion (15, 16).We recently genetically engineered K562 cells that coex-

press CD64/FcgRI, CD86/B7-2, CD137L/4-1BBL, truncatedCD19, and membrane-bound IL-21 (K562 Clone 9.mbIL21) to serve as artificial antigen-presenting cells(aAPC) promoting sustained ex vivo proliferation of humanNK cells (17, 18). The respondingNK cells have a significantincrease in telomere length compared with freshly isolatedNK cells, which may explain their sustained proliferation(18). With this method, large numbers of activated NK cells(aNK) can be generated from normal adult donors withhigh purity and functionality.In this study, we show that K562 Clone 9.mbIL21 cells

allow the generation of large numbers ofNK cells exhibitingactivation characteristics from peripheral blood mononu-

clear cells (PBMC) of children with high-risk neuroblasto-ma. These aNK cells are highly cytotoxic alone or with mAbch14.18 against multidrug sensitive and resistant neuro-blastoma cell lines in vitro and secrete an array of cytokinesand chemokines with antitumor potential while mediatingADCC. These aNK cells maintain their functional activitiesafter viable cryopreservation, and most importantly, retainpotent antitumor activity with ch14.18 when intravenouslyinfused immediately after thawing into nonobese diabetic/severe combined immunodeficient (NOD/SCID)micewithdisseminated human neuroblastoma.

Materials and MethodsCell lines

Neuroblastoma cell lines CHLA-255 andCHLA-136weremaintained in Iscove’s modified Dulbecco’s medium(IMDM)with 20% FBS (Invitrogen), and LA-N-1wasmain-tained in RPMI-1640 (Mediatech) with 10% FBS. CHLA-255-Fluc cells were transduced with the firefly luciferase(Fluc) gene (CHLA-255-Fluc) using a lenti-virus vector (19).CHLA-255-Fluc is sensitive to etoposide and melphalan invitro, whereas CHLA-136 and LA-N-1 are resistant to etopo-side and melphalan [resistance: IC90 > 1,000 ng/mL and>10,000 ng/mL for etoposide and melphalan, respectively;Dr. Nino Keshelava (Division of Hematology/Oncology,Keck School ofMedicine,University of SouthernCalifornia,Los Angeles, CA), personal communication and refs. 20–22]. The K562Clone 9.mbIL21 cell linewas grown inRPMI-1640 with 10% FBS (17, 18).

Preparation of peripheral blood mononuclear cellsPeripheral blood was obtained from 10 patients with

high-risk neuroblastoma and 5 healthy adults, and PBMCwere isolated by density separation using Histopaque-1077(Sigma-Aldrich; ref. 23). Written informed consent wasobtained from healthy donors in accordance with a proto-col approved by the Committee on Clinical Investigation atChildren’s Hospital Los Angeles (Los Angeles, CA) for theuse of cells for cancer and/or blood research. Anonymousspecimens from patients with high-risk, stage IV (metastat-ic) neuroblastoma were obtained from patients enrolledand consented in therapeutic and biology protocols of theChildren’s Oncology Group (COG).

NK cell propagation and activationK562 Clone 9.mbIL21 cells (clinical-grade master cell

bank designated CJLCKT64.86.41BBL.CD19. mbIL21)were derived from Clone 9 cells (generated with Dr. CarlJune, University of Pennsylvania, Philadelphia, PA) at MDAnderson Cancer Center (Houston, TX) using the SleepingBeauty transposon/transposase system to express a mem-brane-bound variant of IL-21 (18). Before initiating cocul-tures of K562 Clone 9.mbIL-21 aAPC and PBMC on day 0,the aAPC were irradiated with 100 Gy using a gammairradiator, washed with PBS, and resuspended in NK cellexpansion medium (NKEM) containing RPMI-1640 and10% FBS with 50 IU/mL recombinant human IL-2

Translational RelevanceAdoptive cell therapy with natural killer (NK) cells

has therapeutic potential for malignancies. We reporthighly efficient ex vivo numeric growth and activation ofNK cells (aNK) from blood of patients with neuroblas-toma. K562-derived artificial antigen-presenting cellsdesignated as Clone 9.mbIL21 act as feeder cells tostimulate NK cells to proliferate more than 2,000-foldin 14 days and to become highly cytotoxic againstmultidrug sensitive and resistant neuroblastoma celllines when alone or when combined with anti-GD2antibody ch14.18. Incubation of aNK cells and ch14.18with neuroblastoma cells markedly increased secretionof TNF-a, granulocyte macrophage colony-stimulatingfactor, IFN-g , sCD40L, CCL2/MCP-1, CXCL9/MIG, andCXCL11/I-TAC. Cryopreserved aNK cells that wereinfused intravenously immediately after thawing intononobese diabetic/severe combined immunodeficientmice bearing disseminated neuroblastoma significantlydecreased tumor growth and increased mouse survival,especially when combined with ch14.18. These resultssupport clinical testing of ex vivo grown and activatedautologous NK cells combined with ch14.18 as treat-ment of neuroblastoma.

Natural Killer Cell Immunotherapy for Neuroblastoma

www.aacrjournals.org Clin Cancer Res; 19(8) April 15, 2013 2133




(PeproTech; addition of at least 20 IU/mL IL-2 to themedium was necessary to induce robust NK cell growth).PBMC (5 � 106) from normal donors were incubated withaAPC (2.5� 106) in T25 flasks (Corning, 25 cm2), whereasPBMC (106) from patients with neuroblastoma were incu-bated with aAPC (0.5 � 106) in 6-well tissue culture plates(Corning, 9.5 cm2), both in NKEM at a total cell concen-tration of 0.5 � 106/mL. An equal-volume of fresh NKEMwas added on day 3. At day 7 of coculture, cells werecounted, new irradiated aAPC were added (total cell:aAPCratio ¼ 2:1), and cells were seeded into T75 or T150 flaskswith additional NKEM (total cell concentration�0.5� 106/mL). An equal-volume of fresh NKEM was added on day11. Cells were grown for 14 days, at which time they werephenotyped by flow cytometry and tested for cytotoxicity.The remainder were aliquoted and viably frozen in a mix-ture of 50% Cryoprotective Medium (Lonza), 25% RPMI-1640, and 25% FBS.

Flow cytometrySurface marker staining was conducted as previously

described (24). Briefly, cells were washed twice in fluores-cence-activated cell sorting (FACS) buffer (PBS with 0.1%NaN3 and 0.1%bovine serumalbumin) and centrifuged for10 minutes at 400 � g. Antibodies listed in SupplementaryTable S1were added in the dark at 4�Cusing concentrationspreviously determined by titration. Isotype-matched irrel-evant mAbs were used to define nonspecific staining. Cellswere incubated at 4�C for 90 minutes and washed twice inFACSbuffer. Flow cytometry analysiswas conducted using aBD LSR II flow cytometer, DIVA software (BD Biosciences)and FlowJo analysis software (Tree Star). The mean fluo-rescence intensity (MFI) ratiowas calculated as follows:MFIof viable cells stained with specific antibody/MFI of viablecells stained with an isotype-matched irrelevant antibody.

In vitro cytotoxicity assaysNKcells that hadbeen grown for 14dayswere seeded into

96-well Costar black tissue culture plates (Corning) in 0.1mL RPMI-1640 with 50 IU/mL IL-2 and 2% FBS. Threeneuroblastoma cell lines (LA-N-1, CHLA-136, and CHLA-255-Fluc) were labeled with calcein-acetomethoxy (calcein-AM; 5 mg/106 cells) for 30 minutes (25). Neuroblastomacells were added to aNK cells at various effector-to-target (E:T) ratios in IMDM for CHLA-255-Fluc and CHLA-136 orRPMI-1640 for LA-N-1 with 2% FBS and without or withanti-GD2 mAb ch14.18 (0.1 mg/mL; provided by theNational Cancer Institute, Frederick, MD). Cells were thencoincubated for 6 hours at 37�C, and live cells, whichcontain calcein-AM, were quantified with digital imagingmicroscopy system (DIMSCAN) as previously described(25).

Cytometric bead array and Luminex assaysNK cells were grown for 14 days, cryopreserved, thawed,

and cultured in NKEM for 72 hours before generatingconditioned media by coculturing aNK cells with CHLA-255-Fluc or CHLA-136 tumor cell lines (aNK:tumor cells¼

1:1) without or with ch14.18 (0.1 mg/mL) for 24 hours.Conditioned media were examined for granzymes A and Busing the cytometric bead array (CBA) assay from BDBiosciences and for cytokines and chemokines using the39-, 23-, and 9-plex Human Cytokine/Chemokine Panelsfrom Millipore according to the manufacturer’s protocols.For granzyme analyses, data were collected on an LSRII flowcytometer using DIVA software (BD Biosciences). FCAPsoftware (BD Biosciences) was used to fit standard curvesto the data obtained from the analyte standards and tocalculate absolute concentration values for each analytefrom its respective standard curve. In the Luminex assay,data were acquired with a Luminex-200 instrument (Lumi-nex Corporation). Cytokine concentrations were deter-mined by referring to a standard curve and expressed aspg/mL using xPonent software (Luminex Corporation).

Murine model of disseminated neuroblastomaNOD/SCID mice were purchased from the Jackson Lab-

oratory. Rat anti-mouse CD122 (200 mg/mouse) wasinjected intraperitoneal 1 day before tumor cell injectionand then every other week to eliminate residual murine NKcells. CHLA-255-Fluc cells were injected intravenously onday 0. Multiple intravenous injections of expanded aNKcells were given together with intravenous IL-2 and withoutor with ch14.18 as described in Results and figure legends.Tumor growth was assessed weekly by bioluminescenceimaging 15 minutes after intraperitoneal injection of a D-luciferin potassium salt solution (1.5 mg/mouse) using aXenogen IVIS-200 system (Caliper Life Sciences). Photonsemitted were quantified with the Living Image 3.0 software(Caliper Life Sciences). Animal experiments were carriedout in accordance with a protocol approved by the Institu-tional Animal Care and Usage Committee of Children’sHospital Los Angeles.

Statistical analysisData were analyzed using the statistical software Stata

(version 11) and are represented as mean � SD unlessotherwise stated. ANOVA was conducted to determine thesignificance of observed differences. Mouse survival timewas defined as the length of time (in days) from the tumorinjection date until the end of the study or time of sacrificedue to disease progression. Censorednormal regressionwasused to examine whether any difference in survival timeexisted because of varying treatments. The censored Wil-coxon test was used to examine the difference in the survivalcurves among the different treatment groups. A P value ofless than 0.05 was considered statistically significant.

ResultsPropagation of NK cells

PBMC from 10 children with high-risk neuroblastomaand from 5 normal adults were cultured with K562-derivedaAPC and IL-2 (Fig. 1). Total cell number in cultures ofPBMC from 10 patients with neuroblastoma increased by amean of 116-fold (range, 41- to -200-fold), and CD56þ

CD3� NK cells increased by a mean of 2,363-fold (range,

Liu et al.

Clin Cancer Res; 19(8) April 15, 2013 Clinical Cancer Research2134




600- to 6,362-fold) by day 14 of coculture. This growth wassimilar in cultures from5healthy adult donors, with ameanof 126-fold (range, 77–175) increase in total cells and2,593-fold (range, 1,051–5,606) increase in NK cells (Fig.1A). The doubling time for NK cells from patients was 1.24days and fromnormal donorswas1.25days. Propagationofthe effector cells could be prolonged for at least 28 dayswithan additional 40-fold increase in total cell number com-pared with day 14 (data not shown). Final cultures frompatients had an average of 83.2%� 2.8% CD56þCD3� NKcells and 9.1%� 2.2% CD3þ T cells of which 6.3%� 2.1%were TCR-gdþ T cells. For normal donors, the final producthad an average of 76.6%� 3.4%CD56þCD3�NK cells and19.4% � 2.5% CD3þ T cells (TCR-gdþ T cells were notevaluated; Fig. 1B–D).The ability of K562-derived aAPC to selectively propagate

NK cells was shown by analyses of the hematopoietic cellsubpopulations on day 0 and then on day 14 of coculture.NK-cell frequency in PBMC from patients and normaldonors on day 0 was similar at 4.7% � 0.5% and 4.7%� 0.9%, respectively. Differences between groups wereobserved for CD3þ T cells (36.9% � 6.9% for patients and63.4% � 5.2% for normal donors) and CD14þ monocytes(27.6%� 7.2% for patients and 14.5%� 3. 7% for normaldonors; P < 0.0001; Fig. 1D). Specimens from patients wereanonymous, and so it is not possible to correlate clinicalvariables such as disease status and treatment with PBMC

subsets. At day 14, large decreases in CD3þ andCD14þ cellswere observed in both groups (Fig. 1D). Additional analysesconducted only on cells from patients showed a decrease inCD4þCD3þ T cells, CD8þCD3þ T cells, CD4þCD25þCD3þ

T cells, 6B11þCD3þ invariant NKT cells, CD14þ mono-cytes, and CD19þ B lymphocytes at day 14 (0.2% � 0.2%,2.4% � 0.4%, 0.05% � 0.02%, 0.07% � 0.01%, 0.2% �0.1%, and 0.6%� 0.4%, respectively) compared with day 0(24.0% � 5.3%, 12.1% � 3.1%, 0.2% � 0.1%, 0.2% �0.1%, 27.6%� 7.2%, and 19.9%� 3.2% respectively). Thedifference in the cell frequency of all cell types over timewassignificant (P < 0.01).

Expressionof cell surface immune-functionmarkers byaNK cells

Expression of natural cytotoxicity receptors DNAM-1,NKG2D, and NKp46, the degranulation marker CD107a/LAMP1, the adhesion molecule CD56, the chemokinereceptor CXCR4, and Fc receptors CD16, CD32, and CD64was quantified by flow cytometry for NK cells from 10patients before (day 0) and after K562-aAPC–stimulatedexpansion (day 14; Fig. 2). The MFI ratio for DNAM-1,NKG2D, CD16, and CD56 increased by 6.2-� 3.2-, 10.3-�2.4-, 20.9- �19.7-, and 17.8- � 2.9-fold, respectively. Onaverage, therewas little or no difference inNKp46, CD107a,CXCR4, CD32, and CD64 expression between NK cells atdays 0 and 14.

Figure 1. Propagation of NK cellsfrom PBMC of patient and normaldonors by coculture with gamma-irradiated K562 Clone 9.mbIL21aAPC (total cells: aAPC ratio ¼ 2:1)and 50 IU/mL IL-2. A, total cellgrowth curves for 5 normal donorsand 10 neuroblastoma patients (seeMaterials and Methods for cellculture details; arrows indicate thedays when aAPC were added tocultures). Recovery of viable cellsafter 7 and 14 days of coculture withaAPC was determined by Trypanblue exclusion. The slopes of thetotal viable cell growth curves forpatients and healthy donors wereidentical (P ¼ 0.33). B and C,representative immunophenotypingresults for a patient's PBMC before(B) and after 14 days (C) coculturewith aAPC supplemented with50 IU/mL IL-2. Aliquots of day0 and 14 cells were viably frozen andthen thawed for analysis on the sameday. D, mean and SD ofimmunophenotyping data fromnormal donors (n ¼ 5) and patients(n ¼ 10). CD19 was not included inthe analysis of specimens fromnormal donors on days 0 and14 (n.d., not done).

A

DPatient donors (n = 10)Healthy donors (n = 5)

100

80

60

40

20

0

Per

cent

age

of to

tal c

ells

n.d. n.d.

Day 0 Day 14 Day 0 Day 14

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l num

ber

(106

)

0 5 10 15

500

100

10

1

Healthy donors (n = 5)

Patient donors (n = 10)

Cell culture duration (d)

B

C

CD

14

After 14 days of coculture with K562 Clone9.mbIL21

Before coculture with K562 Clone9.mbIL21

CD16

CD

56

CD3101102 104103 105

101102103104105

102103104105

101

15.3% 18.1%

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101102103104105

101

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3.7%

101102 104103 105 101102 104103 105

CD14+

CD19+CD3+

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Direct cytotoxicity and ADCC by expanded aNK cellsThe cytotoxicity of aNK cells from 10 patients with

neuroblastoma and 5 normal donors was tested against theneuroblastoma cell lines CHLA-255-Fluc (drug sensitive),LA-N-1 (multidrug resistant), and CHLA-136 (multidrugresistant; refs. 20–22) after a 6-hour incubation using thecalcein-AM assay (Fig. 3; ref. 25). Both multidrug-sensitiveand -resistant cell lines were sensitive to aNK cell directcytotoxicity and to ch14.18-mediated ADCC with signifi-cant killing occurring at 1:1, 1:2, and 1:5 aNK:neuroblas-toma cell ratios (P

Antitumoractivityof cryopreservedaNKcells in aNOD/SCID mouse model of disseminated neuroblastomaThe antitumor activity of cryopreserved aNK cells grown

with K562-mbIL21 aAPCwas tested in vivo using amodel ofdisseminated neuroblastoma inwhichCHLA-255-Fluc cellsare injected intravenously into NOD/SCID mice. Biolumi-nescent imaging of untreatedmice does not detect disease at7 days but does so in at least 50% at 21 days and 100% at 28days, and so treatments were begun at 7 or 21 days tomodeldifferent levels of tumor burden.Initial experiments compared aNK cells from single nor-

mal donors that were cryopreserved, thawed and eithercultured for 3 days before injection, or thawed and imme-diately injected intravenously (Fig. 5). Beginning at 7 days,micewere treatedweekly for 4weekswith aNK cells alone orin combinationwith ch14.18 (107 aNKcells/mouse 1�/wk,3 mg IL-2/mouse 2�/wk, and 15 mg ch14.18/mouse 2�/wk). Tumor growth was reduced and mouse survival waslonger among mice receiving any treatment compared withuntreated mice. Mice receiving treatment that includedch14.18 had an increased survival time compared withthose receiving aNK cells alone (P ¼ 0.01). Mice receivingthawed and immediately injected or thawed and culturedaNKcellswith ch14.18had similar tumor growth (P¼0.26)and survival (P ¼ 0.73). A second experiment using aNKcells from another normal donor with the same schedulesand doses of aNK cells, IL-2, and ch14.18 confirmed nodifference in efficacy between thawed and cultured versusthawed and immediately injected aNK cells (Supplemen-

tary Fig. S2). These results show that cryopreserved aNKcellsinfused immediately after thawing retain their antitumorfunctions.

The next experiment compared the impact of frequencyand duration of aNK treatment using cells from a singlepatient donor that were cryopreserved, thawed, and thenimmediately injected intravenously (Supplementary Fig.S3). aNK cells (107) were injected twice weekly � 3 weeks(group 2, aNK alone and group 3, aNK with ch14.18) oronce weekly � 6 weeks (group 4, aNK with ch14.18)beginning at day 7. IL-2 (3 mg/mouse, 4� or 2�/wk) andch14.18 (15 mg/mouse, 4� or 2�/wk) were given in thesameweeks as aNK cell infusions. Tumor growth in untreat-ed mice was significantly greater than that of all 3 treatedgroups (P < 0.001; Supplementary Fig. S3B). Tumor growthof treatment groups with or without ch14.18 also wassignificantly different (P < 0.001). Tumor growth of treat-ment groups receiving 2�/wk or 1�/wk aNK with ch14.18was significantly different up to day 62 after tumor cellinjection (P¼ 0.006) but not afterwards (P¼ 0.49), and so,overall there was no difference (P¼ 0.10). For mice treatedwith aNK and ch14.18, 3 of 10 in the 2�/wk group and 1 of10 in the 1�/wk group had no detectable tumor by imagingat day 83 (55 and 34 days after the last treatment). Withrespect to survival, the untreated group was significantlyworse than all treatment groups (P < 0.001). Survival of the2 groups receiving aNK and ch14.18 was significantly betterthan that of the group receiving aNKalone (P

Patient donors (n = 5)

aNK + CHLA-255-Fluc aNK

aNK + CHLA-136aNK

aNK + CHLA-255-Fluc + Ch14.18 aNK

aNK + CHLA-136 + Ch14.18 aNK

CCL1/I-309CCL2/MCP-1CCL3/MIP-1aCCL4/MIP-1bCCL7/MCP-3CCL8/MCP-2CCL11/EotaxinCLL13/MCP=4CCL20/MCP3aCCL22/MDCCXCL1-3/GROa+b+gCXCL8/IL-8CXCL9/MIGCXCL10/IP-10CXCL11/I-TACCXCL12/SDF-1a+bXCL1/LymphotactinCX3CL1/FractalkineIL-1aIL-1bIL-2IL-3IL-4IL-5IL-6IL-7IL-9IL-10IL-11IL-12p40IL-12p70IL-13IL-15IL-16IL-29/IFN-I1GrAGrBIL-1RAsIL-2RAEGFFGF-2VEGFM-CSFG-CSFGM-CSFIFNa2IFNgTNFaTNFbTRAILFlt-3LsCD40L

pg/mL347200912111430114214677816

1466919

80455

40849225

8971961154

109644102414

1515

160131

3668022348

44491652

130169585

47451

503342803330

183

Fold2 3554337321325

16031

449124

111129

337742618

211631111781

115256473522

19

Fold2

172221111223

3631

751222426623303146621112351

1022232323127

pg/mL39796

552963757674511

387119

11958

4082742

804142272742249124853

18999

2898821622

1828154554

129040

19425

27222243252072

pg/mL41664

10491612

8510676718

1162194960

5173253

10492138163913479179

1384

22990

2897126504

2358205668

158456

47731

394539813629

115

CCL1/I-309CCL2/MCP-1CCL3/MIP-1aCCL4/MIP-1bCCL7/MCP-3CCL8/MCP-2CCL11/EotaxinCLL13/MCP=4CCL20/MCP3aCCL22/MDCCXCL1-3/GROa+b+gCXCL8/IL-8CXCL9/MIGCXCL10/IP-10CXCL11/I-TACCXCL12/SDF-1a+bXCL1/LymphotactinCX3CL1/FractalkineIL-1aIL-1bIL-2IL-3IL-4IL-5IL-6IL-7IL-9IL-10IL-11IL-12p40IL-12p70IL-13IL-15IL-16IL-29/IFN-I1GrAGrBIL-1RAsIL-2RAEGFFGF-2VEGFM-CSFG-CSFGM-CSFIFNa2IFNgTNFaTNFbTRAILFlt-3LsCD40L

Fold2

265333

11321324

9171

363123

11927

146531516

1282211268194235364422

15

pg/mL38614937221409104911777817

912119

59164

35142204

63315301439844810199724

238116

4067026672

39481745

121129762

42037

438373674527

151

Fold3

134421211225

1001

35133272

10196531415

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101

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11

Fold change0.1 1 10 100 1,000 10,000 0.1 1 10 100 1,000 10,000

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Fold change

Fold change Fold change

Figure 4. Cytokine and chemokine release from K562 Clone 9.mbIL21 aAPC-expanded aNK cells after a 24-hour incubation with neuroblastoma cell linesCHLA-255-Fluc and CHLA-136 alone or with anti-GD2 antibody ch14.18. Day 14 expanded aNK cells from 5 patient donors with neuroblastoma werethawed and cultured for 72 hours with 50 IU/mL IL-2 before coculture with CHLA255-Fluc or CHLA-136 cells (1:1 E:T ratio, 24 hours) without or with ch14.18,and then supernatants were collected for both Luminex and the CBA assays. Means and SD of fold changes are shown in dots and lines, respectively; redsquare dots indicate significant P values (P < 0.05) for each comparison versus aNK cells alone. Fold changes and concentration (pg/mL) of cytokines orchemokines secreted by aNK cells exposed to different conditions (numerator in each function) are shown on the right-hand side of each data point.

Liu et al.





receiving aNK cells and ch14.18 2�/wk was better than thatof mice receiving aNK and ch14.18 1�/wk (P ¼ 0.038).These results confirm in vivo the antineuroblastoma activityof aNK cells that were cryopreserved, thawed, and thenimmediately infused with ch14.18 and suggest a modesteffect of treatment schedule.The last experiment compared different treatments (aNK

alone, ch14.18 alone, and aNK combined with ch14.18)beginning at day 7 or 21 when disease was not or wasdetectible by imaging (Fig. 6 and Supplementary Fig. S4).

Groups ofmice received cryopreserved aNK cells (107) froma single normal donor twice weekly � 4 weeks with IL-2 (3mg/mouse, 2�/wk) and with or without ch14.18 (15 mg/mouse, 2�/wk). Two other groups received ch14.18 alonein the same schedule anddose. Beginning treatment at day 7with NK cells alone was associated with decreased tumorgrowth (P ¼ 0.007) and increased survival (P ¼ 0.032)when compared with untreated control mice (Fig. 6). Treat-ment with ch14.18 alone caused a further decrease in tumorgrowth (P < 0.001) and increase in survival (P < 0.001).

A Day 34 Day 48

B C

aNK cells thawed and immediately injected+ch14.18

aNK cells thawed and immediately

injected

Control

aNK cells thawed,cultured 3

days, and injected+ch14.18

x x x x x x xx

x x x x x x

x x

x

P values for survival

Days after tumor injectionDays after tumor injection

021 28 34 41 48 10 20 30

100

80

60

40

20

0

Flu

x

5001,0003,000

100

10

0

40 50

Pro

babi

lity

of s

urvi

val (

%)

Group 2 3 41 0.004

Tumor growth was most reduced and survival mostincreased when treatment included both ch14.18 and NKcells, and this combination was more effective than eitherNK cells (P < 0.001) or ch14.18 (P < 0.001) alone. Evenwhen treatmentwasbegun21days after tumor cell injection(Supplementary Fig. S4), when disseminated disease wasvisualized in allmice, tumor growthwas less after 2weeks oftreatment with NK and ch14.18 together than after notreatment (P < 0.001), NK cells alone (P ¼ 0.003), orch14.18 alone (P ¼ 0.039). Survival was greater after treat-ment with NK and ch14.18 than no treatment (P¼ 0.004),marginally better than NK cells alone (P ¼ 0.086), and

equivalent to ch14.18 alone (P ¼ 0.297). All treatmentswere more effective when begun on day 7 compared withday 21 (two-way ANOVA; P ¼ 0.009), and tumor growthwas inhibited by the combination of aNK with ch14.18regardless of when treatment was initiated.

DiscussionRepeated infusionsof aNKcells andantitumor antibodies

may provide an effective strategy for treating minimalresidual disease and possibly measurable disease whencombinedwith cytotoxic therapy. Somanchi and colleagues

A Day 20 Day 55

B C

Control x x x x x xx

x x x

1 2 3 4 5 6 7

15 16 17 18 19 20 21

22 23 24 25 26 27 28

ch14.18

aNK

aNK + ch14.18

1 2 3 4 5 6 7

8 9 10 11 12 13 14

15 16 17 18 19 20 21

22 23 24 25 26 27 28

8 9 10 11 12 13 14

x x x x xxx

P values for survival

Group 2 3 41

and Denman and colleagues reported a new method toefficiently grow large numbers of aNK cells ex vivo usingK562Clone 9.mbIL21 cells as aAPC (17, 18). In their study,the number of NK cells from PBMC of normal donorsincreasedby ameanof 47,967-fold in21days, had amarkedincrease in telomere length after stimulation and did notsenesce, even after 6 weeks of culture (17, 18). Using thesamemethod, we show for the first time that large numbersof aNK cells can be grown fromPBMCof patients with high-risk neuroblastoma. These aNK cells alone and with anti-GD2mAb ch14.18 are highly cytotoxic and secretemultiplecytokines with antitumor potential when cultured withmultidrug sensitive and resistant neuroblastoma cell lines.Importantly, these aNK cells retain antitumor function(s) invivo after cryopreservation. These results provide amodel forclinical testing of adoptive cell therapy with activated autol-ogous NK cells and anti-GD2 mAb ch14.18.While a number of strategies are possible for generating

human NK cells for adoptive cell therapy, the low cellnumber available for adoptive transfer has limited clinicaltesting of this immunotherapeutic strategy (7). Leukapher-esis, T-cell depletion, and short-term culture in IL-2 canprovide haploidentical or autologous NK cells but rarely insufficient quantity for more than a single infusion (8, 9, 26,27). Culture of T-cell–depleted products from patients withmelanoma for approximately 21 days in IL-2 resulted in278- to 1,097-fold expansion of autologous NK cells, andreinfusedNK cellswere shown to circulate for at least 1weekwithout causing tumor regression (14). In another clinicalstudy, allogeneic NK cells from related donors were expand-ed ex vivo 3- to 131-fold with hydrocortisone and IL-15 andinfused to treat patients with advanced non–small cell lungcancer (28). Othermethods for ex vivo expansion have beenreported from preclinical studies. NK cells from normaldonors cultured in defined medium with IL-2 expanded amedian of 193-fold (range, 21- to 277-fold; ref. 29) andfrom patients with multiple myeloma amean of 1,625-fold(range, 502- to 2,658-fold; ref. 30). A mixture of IL-7,recombinant human stem cell factor, IL-2, and IL-15 indefined medium stimulated a 3-log increase in NK cellsfrom cord blood (11). aAPC have been genetically engi-neered and used with or without additional cytokines tostimulate expansion. Addition of IL-2 to K562 cells thatweremodified to expressmembrane-bound IL-15 and41BBligand stimulated a mean of 277-fold increase (range, 201-to 1,459-fold) inNK cells fromnormal donors and a similarlevel from patients with acute leukemia (12). KT64.41BBL.A2 cells, which naturally express IL15Ra and MICA/B,stimulated a 1,000-fold increase of NK cells from blood ofnormal donors (13). K562-MICA-41BBL-IL-15 cells stimu-lated the expansion of NK cells by a mean of 550-fold(range, 201–880; ref. 31).Using K562 Clone 9.mbIL21 cells and added IL-2 (17,

18), we show a mean of 2,363-fold (range, 600- to 6,362-fold) expansion of NK cells from patients with high-riskneuroblastoma at day 14, with 83% of the total cells beingCD56þCD3� NK cells. In contrast to other methods, telo-mere length in NK cells significantly increases as compared

with fresh NK cells with this method (18), which allowsculturing for at least 28 days to obtain an even greaternumber of aNK cells. This positive effect on telomere lengthis likely due to IL-21 activation of STAT3 (32) that in turnregulates human telomerase reverse transcriptase (hTERT)expression (33). Importantly, growth of NK cells frompatients and normal donors was equivalent, which stronglysupports the feasibility of preparing autologous aNK cellsfor therapy. Without T-cell depletion, less than 20% of thetotal population at day 14 was T cells, and most of thesewere TCR-gdþ T cells. Because K562 Clone 9.mbIL21 cellsare lethally irradiated before culture and are lysed by theexpandingNKcells, the riskof infusing viableK562Clone9.mbIL21 cells to patients is negligible. Thus, we anticipatethat it will be possible to generate billions of aNK cells fromperipheral blood of children with neuroblastoma for mul-tiple infusion treatments, without the need for apheresis.

Generation of activated CD56þCD3� NK cells fromPBMCofbothpatients andnormal donorswithK562Clone9.mbIL21 cells and IL-2 is highly reproducible. More than80% of cells are CD56þCD3� NK cells, and they display anactivated phenotype as shown by more than a 6-foldincrease in the MFI ratio for NKG2D, DNAM-1, CD16, andCD56 at day 14 compared with day 0. NK cells generatedfrom PBMC of patient and normal donors are highlycytotoxic against chemotherapy sensitive and resistant neu-roblastoma cell lines when alone and even more so withmAb ch14.18. Remarkably, only approximately 20% oftumor cells survived ADCC at a 1:1 aNK:tumor cell ratioafter 6 hours. Our previous study showed increased expres-sion of inhibitory killer cell immunoglobin-like receptor(KIR) KIR2DL2/3 but not of inhibitory KIR2DL1, NKG2A,or NKG2C on expanded compared with freshNK cells (18).NKG2Awas expressed by 85%of expanded NK cells but theother receptors were expressed by only 15% to 30%.ExpandedNK cells killed 721.221 cell line targets expressingHLA group C1 or Bw4, which are ligands for inhibitoryKIR2DL2/3 or KIR3DL1, respectively, equally as well as theparent cell line without these ligands (18). Altogether, ourresults are similar to earlier reports with IL-2 alone or incombinationwithother cytokines, except that amuch largernumber of NK cells are obtained with the K562 Clone 9.mbIL21 model (11, 12, 34).

We showed that a complex array of cytokines and che-mokines are released in vitro upon aNK cell interaction withdrug-sensitive and -resistant tumor cell lines, especiallyduring ADCC mediated by ch14.18. Although it is notpossible to relate these data to what might occur in vivo,on balance, the pattern suggests an antitumor effect. Levelsof TNF-a, GM-CSF, IFN-g , sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC increased 4-, 5-, 6-, 15-, 265-,917-, and363-foldwith concentrations ranging from151 to9,121 pg/mL.However, cytokines that are important forNKcell proliferation and activation, IL-12p40, IL-12p70, andIL-15 were present at low levels (

responses, and activation of the CD40 pathway by sCD40Lmay contribute to antitumor immune responses. sCD40Lalso may have direct effects upon tumor cell proliferationand survival (36, 37). Because the increased release of thesecytokines was only achievedwhen aNK cells interacted withneuroblastoma cells, especially via mAb ch14.18, quantifi-cation of cytokines in blood may provide useful indicatorsof aNK-tumor cell interactions in vivo.

The use of autologous aNK cells for expansion will likelyprevent host reactions against the adoptively transferredcells that may occur with allogeneic aNK cells, potentiallyresulting in impaired survival, migration, and function.Although there is a possibility that autologous aNK antitu-mor function could be suppressed by KIR inhibitory recep-tor/HLA class I molecule interactions as has been suggestedfrom reviews of patients undergoing myeloablative therapyand autologous hematopoietic stem cell transplantation(38) and of patients treated with an anti-GD2/IL-2 fusionprotein (39), available in vitro data suggest that highly aNKcells are not impacted by these interactions (13, 18). Fur-thermore, 60% of individuals have NK cells that expressKIRs but do not express the cognate HLA class I ligands forthe KIRs (missing KIR ligand; refs. 40, 41), and these NKcells can kill neuroblastoma cells with anti-GD2mAb (42).Finally, neuroblastoma cells often do not express surfaceHLA class I molecules, the ligands for inhibitory KIR recep-tors (43–47). To date, the impact of KIR/HLA class I inter-actions on outcome in patients with neuroblastoma treatedwith the ch14.18 mAb has not been evaluated (3).

aNK cells that are grown and activated with K562 clone 9.mbIL21 cells are active against disseminated human neu-roblastoma cells growing in NOD/SCID mice. Viably cryo-preserved aNKcells thatwere thawedand cultured for 3daysor thawed and immediately infused intravenously wereequally effectivewhen combinedwithmAb ch14.18 againstneuroblastoma. Treatment with aNK cells alone, ch14.18alone, or the combination of both was more effective whenbegun at 7 days after tumor cell injection before diseasecould be imaged than when begun at 21 days when dis-seminated diseasewas readily imaged. In either setting, aNKcombined with ch14.18 was more effective than aNK cellsor ch14.18 alone.

The K562 Clone 9.mbIL21 NK cell growth and activationmethod provides an important advance for generating aNKcells in high numbers, purity, and functionality fromPBMCof patients with neuroblastoma for use in NK cell-based

immunotherapy. Because viably cryopreserved aNK can bethawed and immediately infused into patients, it will befeasible to grow and cryopreserve aNK cells in a centrallaboratory for later shipment to institutions participating inmulticenter treatment protocols to evaluate dose and tox-icity as well as aNK cell survival, expansion, migration, and,within the context of such studies, antitumor activity.Adoptive cell therapy with aNK combined with ch14.18may be effective against a relatively small amount of diseasebut likely will need to be combined with cytotoxic therapyto be effective against a large amount of disease.

Disclosure of Potential Conflicts of InterestD.A. Lee is the co-owner of InCellerate, receives research support from

Celgene, and is a consultant/advisory board member of Celgene CellularTherapeutics. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: Y. Liu, H.-W. Wu, L.J.N. Cooper, D.A. Lee, R.C.SeegerDevelopment of methodology: Y. Liu, H.-W. Wu, D.A. Lee, R.C. SeegerAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): Y. Liu, H.-W. Wu, M.A. Sheard, R.C. SeegerAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): Y. Liu, H.-W. Wu, R. Sposto, L.J.N.Cooper, R.C. SeegerWriting, review, and/or revision of the manuscript: Y. Liu, M.A. Sheard,L.J.N. Cooper, D.A. Lee, R.C. SeegerAdministrative, technical, or material support (i.e., reporting or orga-nizingdata, constructingdatabases):Y. Liu,H.-W.Wu, S.S. Somanchi, L.J.N. Cooper, R.C. SeegerStudy supervision: R.C. Seeger

AcknowledgmentsThe authors thank Ms. Jemily Malvar for assistance with statistical anal-

yses and Dr. Martine Torres for editorial assistance.

Grant SupportThis work was supported by grants to R.C. Seeger from the National

Cancer Institute (P01 CA81403-12), the Bogart Pediatric Cancer ResearchProgram, the ThinkCure Foundation, the Al Sherman Foundation, and theAnna Bing Arnold endowment; to M.A. Sheard from BD Biosciences/Immu-nology; to D.A. Lee from the St. Baldrick’s Foundation, the SunbeamFoundation, and the Farrah Fawcett Foundation; and to L.J.N. Cooper fromthe National Cancer Institute (CA141303), Hyundai Hope on Wheels, andthe Pediatric Cancer Research Foundation.

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

Received April 15, 2012; revised January 16, 2013; accepted January 18,2013; published OnlineFirst February 1, 2013.

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2013;19:2132-2143. Published OnlineFirst February 1, 2013.Clin Cancer Res Yin Liu, Hong-Wei Wu, Michael A. Sheard, et al. with Neuroblastoma for Adoptive Cell Therapy

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