gene-modiﬁed human a b-t cells expressing a chimeric z … · research article gene-modiﬁed...

Research Article

Gene-Modified Human a/b-T Cells Expressing a ChimericCD16-CD3z Receptor as Adoptively Transferable EffectorCells for Anticancer Monoclonal Antibody Therapy

FumihiroOchi1,2, Hiroshi Fujiwara1, Kazushi Tanimoto1,6, Hiroaki Asai1, YukihiroMiyazaki1, SachikoOkamoto3,Junichi Mineno3, Kiyotaka Kuzushima4, Hiroshi Shiku5, John Barrett6, Eiichi Ishii2, and Masaki Yasukawa1

AbstractThe central tumoricidal activity of anticancer monoclonal antibodies (mAb) is exerted by FcgR IIIa (CD16)–

expressing effector cells in vivo via antibody-dependent cell-mediated cytotoxicity (ADCC), as observed fornatural killer (NK) cells. In practice, chemotherapy-induced leukopenia and exhaustion of NK cells resulting fromADCC often hamper the clinical efficacy of cancer treatment. To circumvent this drawback, we examined in vivothe feasibility of T cells, gene-modified to express a newly generated affinity-matured (158V/V) chimeric CD16-CD3z receptor (cCD16z-T cells), as a transferable alternative effector for cancer mAb therapy. cCD16z-T cellswere readily expandable in ex vivo culture using anti-CD2/CD3/CD28 beads and recombinant human interleukin-2 (rhIL-2), and they successfully displayed ADCC-mediated tumoricidal activity in vitro. During ADCC, ligation ofopsonized cancer cells to the introduced cCD16z-T cells stimulated the effector cells to produce proinflammatorycytokines and release toxic granules through the activation of the Nuclear factor of activated T cells (NFAT)pathway after phosphorylation of the CD3z chain. In parallel, these stimulated cCD16z-T cells transientlyproliferated and differentiated into effector memory T cells. In contrast, NK cells activated by rhIL-2 displayedsimilar ADCC activity, but failed to proliferate. Human cCD16z-T cells infused concomitantly with anti-CD20mAb synergistically inhibited the growth of disseminated Raji cells, a CD20þ lymphoma cell line, in immuno-deficient mice, whereas similarly infused rhIL-2–treated NK cells survived for a shorter time and displayed lesseffective tumor suppression. Our findings strongly suggest the clinical feasibility of cCD16z-T cells as adoptivelytransferable ADCC effector cells that could potentially enhance the clinical responses mediated by currentlyavailable anticancer mAbs. Cancer Immunol Res; 2(3); 249–62. �2014 AACR.

IntroductionMonoclonal antibodies (mAb) specific for tumor antigens

are efficacious in the treatment of cancers (1); these includeanti-CD20mAb (rituximab) for CD20þ lymphoid malignancies(2) and anti-Her2/Neu mAb (trastuzumab) for Her2/Neuþ

solid tumors (3). A major mechanism for the tumoricidalactivity is antibody-dependent cell-mediated cytotoxicity(ADCC), which is executed by the Fc receptor–bearing innate

immune cells including natural killer (NK) cells, gd-T cells,macrophages, and neutrophils against the opsonized cancercells. NK cells comprise the crucial population of these effectorcells (4).

In practice, as anticancer treatment proceeds, a decline inthe clinical efficacy of the mAb therapy is often observed.This decline in efficacy could be attributed to the decrease inthe number of active effector cells during the ADCC processfrom a combination of chemotherapy-induced leukopeniaand NK cell exhaustion (5). As CD16-expressing effector cellsplay a critical role in ADCC, we hypothesized that thecombined therapeutic regimen of replenishing these cellstogether with the specific anticancer mAb may improvecancer treatment.

Ex vivo–expanded NK cells (6, 7) and gd-T cells (8) have beentested in adoptive cell transfer cancer therapy alone or incombination with tumor antigen-specific mAbs. However, noreliable clinical procedure has yet been established for ex vivoexpansion of autologous effector cells to the numbers that aretherapeutically sufficient (9). Instead, culture-induced killer(CIK) cells (10) and lymphokine-activated killer (LAK) cells(NCT 01329354; ClinicalTrials.gov), both mostly comprise NKcells, are being investigated for adoptive cell therapy in com-bination with rituximab in patients with refractory CD20þ

Authors' Affiliations: Departments of 1Hematology, Clinical Immunologyand Infectious Disease, and 2Pediatrics, EhimeUniversity Graduate Schoolof Medicine, Ehime; 3Center for Cell and Gene Therapy, Takara Bio Inc.,Shiga; 4Division of Immunology, Aichi CancerCenter, Aichi; 5Department ofCancer Vaccine and Immuno-Gene Therapy, Mie University GraduateSchool of Medicine, Mie, Japan; and 6Hematology Branch, National Heart,Lung and Blood Institute, NIH, Bethesda, Maryland

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

Corresponding Authors: Hiroshi Fujiwara, Department of Hematology,Clinical Immunology and Infectious Disease, Ehime University GraduateSchool of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan. Phone:81-89-960-5296; Fax: 81-89-960-5299; E-mail: [email protected];and Masaki Yasukawa, [email protected]

doi: 10.1158/2326-6066.CIR-13-0099-T

�2014 American Association for Cancer Research.

CancerImmunology

Research

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on August 5, 2020. © 2014 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from

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lymphomas, although no conclusive results have yet beenreported.

Recent laboratory studies have demonstrated that bothcirculating CD16þ a/b-T cells and CD16þ g/d-T cells areinvolved in ADCC-mediated antiviral activity in vivo, inde-pendently of human leukocyte antigen (HLA) restriction(11–13). The binding affinity of the FcgRIIIa-158(Valine/Valine) homodimer (158V/V) to immunoglobulin G (IgG)1and IgG3 is much higher than that of the FcgRIIIa-158(Phenylalanine/Phenylalanine) homodimer (158F/F; ref. 14),and that the FcgRIIIa-158V/F biallelic polymorphism influ-ences the efficacy of NK cell activation (15, 16). Unlike that ofNK and g/d-T cells, the ex vivo expansion and gene-modi-fication of CD3þ T cells are clinically established for theapplication to adoptive cell therapy (17). Therefore, wehypothesized that therapeutically infused CD3þ T cells,gene-modified to express CD16 with the 158V/V amino acidsubstitution, might be able to mediate ADCC against opso-nized cancer cells and virus-infected cells in vivo. In addi-tion, these effector cells could be used in combination with

almost all currently available ADCC-mediating anticancermAbs. A similar concept was proposed by Cl�emenceau andcolleagues (18), who validated in vitro the applicability ofCD3þ T cells gene-modified to express a FcgRIIIa-158(V/V)-FceRIg chimeric receptor for ADCC against CD20þ lympho-ma cells in combination with rituximab, although the in vivovalidation has not been described yet. Regarding the risk ofleukemogenesis of infused gene-modified T cells, resultsfrom clinical trials using retroviral or lentiviral vectorgene-modified T cells for adoptive cell therapy have provedthe long-term safety of this approach (19, 20). Therefore, webelieve that adoptive transfer of gene-modified T cellsexpressing the CD16 complex, designed to mediate ADCC,might be clinically acceptable and possibly enhance theclinical efficacy of mAb therapy against cancers.

In the present study, we used lentivirus vectors to establishgene-modified CD3þ T cells expressing the FcgRIIIa-158(V/V)-CD3z chimeric receptor (cCD16z-T cells) as the adoptivetransfer effector cells for ADCC, and examined their functionin detail both in vitro and in vivo. The cCD16z-T cells were

Figure 1. Functional validation of the chimeric CD16 (158V/V)-CD3z receptor. A, lentiviral vector construct of chimeric CD16(158V/V)-CD3z. Asc1 and Sal1 arerestriction enzymes. SP, ECD, TM, and ICP denote the signal peptide, extracellular domain, transmembrane portion, and intracellular portion, respectively.aa158-V indicates amino acid substitution at position 158 to Valine. Number of amino acids at each site is indicated in parentheses. B, parent Jurkat/MA/CD8a/luc cells (left), andcCD16zgene-transducedJurkat/MA/CD8a/luccells (right) are shown.C, assessmentof target-responsivephosphorylation statusofthe CD3z chain (CD247) in cCD16z-Jurkat/MA/CD8a/luc cells by flow cytometry. Solid line denotes the response to Raji cells in the presence of1mg/mLof rituximab.Dotted line denotes the response toRaji cells alone. Dashed line indicates the response toK562 cells similarly treatedwith rituximabas anegative control. Shaded area indicates the response to 1 mg/mL of rituximab alone. D, target-responsive luciferase productionmediated by cCD16z-Jurkat/MA/CD8a/luc cells. The introducedcCD16z receptor successfully discriminated rituximab-opsonizedRaji cells andsuccessively activated theNFATpathwayto produce luciferase, which was obviously inhibited by the anti-CD16 F(ab0)2 mAb, 3G8, in a dose-dependent manner. Clear bar denotes luciferaseproduction in the response to each target cell alone. Black bar indicates the response to rituximab alone, rituximab-opsonized Raji cells, or similarly treatedK562 cells. Gray bar denotes the response to rituximab-opsonized Raji cells in the presence of various concentrations of 3G8.

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Cancer Immunol Res; 2(3) March 2014 Cancer Immunology Research250




Figure 2. Validation of cCD16z-T cells. A, surfaceCD16 expression of cCD16z-T cells assessed byflow cytometry (right). NGM-T cells, non-gene-modified T cells similarly treated using anti-CD2/CD3/CD28 beads and rhIL-2 (left). B,expandability of cCD16z-T cells in ex vivo culturewith 50 U/mL of rhIL-2. Proliferation of expandedcCD16z-Tcells from13differentdonors relative tothe number on day 2 is shown. Mean number isshown as a solid circle (*). Error bars indicateSEs. Proliferation on day X was calculated asnumber of cCD16z-T cells on day X/that on day 2.C, time course of the CD8þ/CD4þ T-cell ratioamong cultured cCD16z-T cells (n¼ 3). Error barsindicateSDs.D, levels ofCD20expression onRajiand Daudi cells (top). Solid line indicates Raji,dotted line indicates Daudi, and the shaded areaindicates K562 cells as a negative control. The51Cr-release assay (bottom) indicated thatcCD16z-T cells successfully and equally exertedADCC activity against rituximab-opsonized Raji(black circles) and Daudi (gray circles) cells, butnot againstK562cells (clearcircles) inaccordancewith the dose of rituximab. Effector cellsestablished from 3 different donors were used atanE:T ratio of 5:1.Experimentswereconducted intriplicate. Error bars represent SDs. E, levels ofHer2/neu expression onSKOV3 andMCF-7 cells.Solid line indicates SKOV3, dotted line indicatesMCF-7 and shaded area indicates K562 cells(top).Similarly toD,cCD16z-Tcells exertedADCCactivity against trastuzumab-opsonized SKOV3(black circles) and MCF-7 cells (gray circles), butnot againstK562cells (clearcircles) inaccordancewith the doseof trastuzumab. In addition, the levelofHer2/neu expressionon tumor cells also limitedthe ADCC activity. Error bars represent SDs. F,levels of CCR4 expression on MT-4 and ATN-1cells. Solid line indicates MT-4, dotted lineindicates ATN-1, and shaded area indicates K562cells (top). Similarly to D and E, cCD16z-T cellsexerted ADCC activity against mogamulizumab-opsonized MT-4 (black circles) and ATN-1 (graycircles), but not against K562 cells (clear circles) inaccordance with the dose of mogamulizumab.The level ofCCR4expressionon tumor cells againlimited the ADCC activity. Error bars representSDs. G, when the rituximab dose was fixed at 0.1mg/mL, which was lower than the pharmacologicrange, the number of effector cCD16z-T cells alsoregulated the ADCC activity against rituximab-opsonized Raji cells. n ¼ 3, E:T ratio ¼ 5:1. Errorbars represent SDs. H, similarly to G (n ¼ 3, E:Tratio ¼ 5:1), at a fixed dose of 0.1 mg/mL oftrastuzumab, also lower than the pharmacologicrange, thenumber of effector cells again regulatedthe ADCC activity against trastuzumab-opsonizedSKOV3cells. Error bars representSDs.I, similarly to G and H (n ¼ 3, E:T ratio ¼ 5:1), at afixed dose of 0.1 mg/mL of mogamulizmab, alsolower than the pharmacologic range, the numberof effector cells again regulated the ADCCactivityagainst mogamulizumab-opsonized ATN-1 cells.Error bars represent SDs. J, as was shown in Gand H (n ¼ 3, E:T ratio ¼ 5:1), ADCC activitymediated by cCD16z-T cells against rituximab-opsonized Raji cells was significantly inhibited by3G8, an anti-CD16 F(ab0)2 mAb (P < 0.01). K562was used as a negative control. Error barsrepresent SDs.

Chimeric CD16-CD3z Receptor-Expressing T Cells for ADCC

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readily expandable in ex vivo short-term culture usingrecombinant human interleukin-2 (rhIL-2), and successfullydisplayed tumoricidal activity via ADCC both in vitro andin vivo. Our experimental observations indicate that thisapproach could be a potential option for enhancing theclinical efficacy of currently available mAb therapy againstcancers.

Materials and MethodsCellsApproval for this study was obtained from the Institutional

Review Board of Ehime University Hospital (Ehime, Japan).Written informed consent was provided by all the healthyvolunteers and patients with B-cell lymphomas in accordancewith the Declaration of Helsinki. The HEK 293T cell line(RIKEN BioResource Center) was maintained in Dulbecco'sModified Eagle Medium (DMEM; Sigma-Aldrich) supplemen-tedwith 10% FBS (Biowest) and 2mmol/L L-glutamine (Sigma-Aldrich), and was used for the production of infectious lenti-virus particles. The CD20þ lymphoma cell lines Raji (21) and>Daudi (21), the human ovarian carcinoma cell lines SKOV3(22) and K562 [American Type Culture Collection (ATCC)], thehuman T-lymphotropic virus type-I (HTLV-1)–immortalized Tcell lineMT-4 (23), and the adult T-cell leukemia (ATL) cell lineATN-1 (23) were cultured in RPMI-1640 (Sigma-Aldrich) sup-plemented with 10% FBS, 2 mmol/L L-glutamine, and penicil-lin/streptomycin (Biowest). The human breast cancer cell lineMCF-7 (ATCC) was maintained in DMEM with 10% FBS, 2mmol/L L-glutamine, and penicillin/streptomycin. The Jurkat/MA cell line is a Jurkat subclone previously established byCalogero and colleagues that lacks the endogenous T-cellreceptor (TCR) expression and stably expresses both thehuman CD8a gene (hCD8a) and an NFAT-luciferase geneconstruct for the detection of signaling via the newlyintroduced TCRs (24). Jurkat/MA cells were cultured inIscove's modified Dulbecco's medium (IMDM) with 8% fetalcalf serum (FCS) and 0.05 mg/mL hygromycin B (Invitro-gen). Peripheral blood mononuclear cells (PBMC) fromhealthy volunteers and patients with B-cell lymphomas,and autologous lymphoma cells from biopsied pathologiclymph nodes were stored in liquid nitrogen until use.Except for confirming the expression and function of thetransgenes described below, no additional validation testswere performed on the cell lines used.

Construction of a plasmid carrying the chimeric FcgRIII-CD3z chain

A lentiviral vector expressing a novel chimeric CD16 with a158V/V-CD3z (cCD16z) construct was synthesized (Fig. 1A).cDNA encoding the signal peptide and extracellular domain ofCD16 (accession no.: NM_000569) with a gene alteration atF158V was directly connected to the CD3z gene (accession no.:NM_198053) at the second amino acid (P) of the extracellulardomain. After codon-optimization (GeneArt), the cCD16z con-struct was inserted into the lentiviral vector, pRRLSIN.cPPT.MSCV/GFP.WPRE (25).

ChemicalsRituximab (Rituxan) and trasutsuzumab (Herceptin) were

purchased from Chugai Pharmaceutical Co., Ltd. Mogamuli-zumab (Poteligeo), a new defucosylated anti-C–C chemokinereceptor 4 (CCR4) mAb exploiting ADCC activity againstCCR4þ ATL tumor cells (26), was purchased from Kyowa-Hakko-Kirin, Ltd.

MiceAll in vivo mouse experiments were approved by the Ehime

University Animal Care Committee. Six-week-old NOD/scid/gcnull (NOG) female mice were purchased from the CentralInstitute for Experimental Animals (Kanagawa, Japan; ref. 27)and maintained in the institutional animal facility at EhimeUniversity (Ehime, Japan).

Flow cytometryTransfectants obtained using cCD16z gene transfer,

non-gene-modified T cells, NK cells isolated from PBMCsusing MACS beads (Miltenyi Biotec), and cell lines werelabeled with anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD45, anti-CD56, anti-CD62L, anti-CD194 (also known asCCR4; BD Biosciences), anti-CD45RA (eBioscience), anti-CD16, anti-CD69, and anti-CD340 (also known as erbB2/HER-2; BioLegend) mAbs. Flow cytometry was conductedusing a Gallios flow cytometer (Coulter), and data analysiswas performed using FlowJo Version 7.2.2 software(TreeStar).

Establishment of cCD16z gene-transduced T cellsCD3þ T cells isolated from PBMCs of healthy volunteers

and patients with B-cell lymphomas using MACS beads(Miltenyi Biotec) were cultured in GT-T503 medium

Figure 3. Cellular outputs mediated by cCD16z-T cells during ADCC. A, cCD16z-T cells from 3 different donors produced proinflammatory cytokines inexclusive response to rituximab-opsonized Raji cells, as determined by ELISA assay. Clear bar depicts IL-2 and filled bar depicts IFN-g . Error barsrepresent SDs. �, A less than detectable level. NGM-T cells, non-gene-modified T cells. B, to demonstrate the reproducibility, an experiment similar tothat in A was conducted against trastuzumab-opsonized SKOV3 cells. cCD16z-T cells (n ¼ 3) exclusively produced IL-2 (clear bar) and IFN-g(filled bar) in response to trastuzumab-opsonizedSKOV3 cells. Error bars represent SDs. NGM-T cells, non-gene-modified T cells. C, cCD16z-T cells releasedcytotoxic granules exclusively in response to rituximab-opsonized Raji cells, but not K562 cells. D, cCD16z-T cells released cytotoxic granules againexclusively in response to trastuzumab-opsonized SKOV3 cells, but not K562 cells. E, CFSE dilution assay demonstrated the proliferative responsemediatedby cCD16z-T cells in response to rituximab-opsonized Raji cells (middle), but not to similarly treated K562 cells as a negative control (bottom).OKT3as apositive control led to obvious proliferationof cCD16z-T cells, but rituximabalone did not (top). A representative result from three experiments usingdifferent donor-derived effector cells is shown. F, after ligation with rituximab-opsonized Raji cells (*) at the indicated dose of rituximab and E:T ratio,cCD16z-T cells transiently proliferated for 7 days, then declined thereafter. On the other hand, similarly treated K562 cells (*) did not. G, in this series ofexperiments, during the proliferative response to rituximab-opsonized Raji cells in similar experiments, cCD16z-T cells tended to differentiate towardCD45RA�/CD62L� effector memory T cells.






(Takara Bio Inc.) supplemented with 5% human serum, 0.2%human albumin, and 50 U/mL of rhIL-2 (R&D Systems), andstimulated with anti-CD2/CD3/CD28 MACSiBeads (MiltenyiBiotec) in a 24-well culture plate (Coster). On day 4, afterstimulation with the beads, the cCD16z gene was lentivirallytransduced into the cultured T cells using 10 mg/mL ofprotamine sulfate (Mochida Pharmaceutical Co., Ltd.). Thecells were transferred to another 24-well plate, and centri-fuged at 1,000 � g for 90 minutes. One week later, CD16þ

transfectants were isolated with MACS beads (MiltenyiBiotec). Phenotypes of the CD16þ transfectants were exam-ined by flow cytometry, and then subjected to furtherexpansion and functional assays.

Phosphorylation status of the CD3z chain in Jurkat/MAcells gene-modified to express the cCD16z receptor afterligation with opsonized cancer cells

The phosphorylation status of CD3z (Tyr142) in Jurkat/MA cells lentivirally gene-modified to express the cCD16zreceptor (cCD16z-Jurkat/MA/CD8a/luc) after ligation withopsonized target cells was assessed by flow cytometry usingthe Alexa Fluor 674–conjugated mouse anti-CD247(pY142)mAb in accordance with the manufacturer's instructions(BD Phosflow, BD Bioscience).

Luciferase production by Jurkat/MA cells engineered toexpress the cCD16z receptor upon ligation withopsonized cancer cells

To demonstrate the functionality of the introduced cCD16zreceptor in gene-modified T cells, CD16þ Jurkat/MA/CD8a/luc cells were isolated, expanded, and subjected to luciferaseassay as described previously (28). One million CD16þ Jurkat/MA/CD8a/luc cells were coincubated with 1� 106 Raji cells ornegative control K562 cells, with or without rituximab (1 mg/L),for 12 hours in advance at an effector:target ratio of 1:1. Forblocking experiments, the F(ab0)2 fragment of the anti-humanCD16-specific mAb 3G8 (Ancell) was added at several con-centrations (10, 25, and 50 mg/mL).

CFSE dilution assayTo assess the proliferative response to opsonized target cells

mediated by effector cells during the ADCC process, cCD16z-Tcells or NK cells labeled with CFSE (Molecular Probes Inc.)were cocultured with Raji, primary lymphoma cells, or K562cells with or without 1 mg/mL of rituximab. After 4 days, CFSEdilution among cCD16z-T cells orNKcellswas assessed byflowcytometry as described previously (28).

In vitro ADCC assayTo determine the ADCC activity mediated by the cCD16z-T

cells or NK cells, a standard 51Cr-release assay was performed(29). Briefly, 104 target cells were labeled with 5 � 103 51Cr(Na2CrO4: MP Bio Japan) and incubated at various concentra-tions of each mAb or effector:target (E:T) ratios with effectorcells in 200 mL of culture medium in 96-well round-bottomedplates. After 4 hours of incubation with effector cells at 37�C,100 mL of the supernatant was collected from each well. Thepercentage of specific lysis was calculated as: (experimental

release cpm � spontaneous release cpm)/(maximal releasecpm � spontaneous release cpm) � 100 (%). In some experi-ments, we conducted ADCC assays with a fixed E:T ratio of 5:1or a 0.1 mg/mL concentration of mAb. For blocking experi-ments, 10 mg/mL 3G8 mAb was used.

In vitro complement-dependent cytotoxicity assayTo exclude the complement-dependent cytotoxicity

(CDC) activity of rituximab in a xenografted mouse model,we examined in vitro the rituximab-mediated CDC activityagainst 1 � 104/well Raji cells in 10% mouse serum using the51Cr-release assay during 1.5 hours of culture with variousconcentrations of rituximab. Experiments were conductedin triplicate.

IFN-g and IL-2 secretion assayFifty thousand cCD16z-T cells were coincubated with Raji,

Daudi, SKOV3, MCF-7, or K562 cells for 24 hours with orwithout 2 mg/mL of rituximab or trastuzumab. cCD16z-Tcells treated with 1 mg/mL of OKT-3 (BioLegend) and 1mmol/L of ionomycin (Cell Signaling Technology) were usedas a positive control. IFN-g and IL-2 in culture supernatantswere measured using an ELISA kit (Thermo Scientific).

CD107a assayCD107a expression mediated by cCD16z-T cells in

response to opsonized cancer cells was examined asdescribed previously (30). Briefly, 1 � 105 target cells pre-incubated with or without 4 mg/mL of rituximab for 20minutes were incubated with 2 � 105 cCD16z-T cells for3 hours in a 96-well round-bottomed plate. After beinglabeled with fluorescein isothiocyanate (FITC)–conjugatedCD107a mAb (BioLegend), anti-CD3, anti-CD8 (BD Bios-ciences), and anti-CD16 (BioLegend) mAbs, the cells wereanalyzed by flow cytometry.

In vivo antitumor activity mediated by intravenouslyinfused cCD16z-T cells in a xenografted mouse model

To examine the in vivo antitumor effect of cCD16z-T cells,we prepared luciferase gene-transduced Raji cells (Raji/luc)whose CD20 expression was equal to that of the parentalRaji cells. All NOG mice of ages 6 weeks (n ¼ 11) wereexposed to 1 Gy of irradiation, and then intravenouslyinoculated with 5 � 105 Raji/luc cells via tail vein on day0. These mice were divided into three cohorts: (i) nontreatedmice (n ¼ 3), (ii) mice subjected to intravenous adminis-tration of 5 � 106 cCD16z-T cells alone (n ¼ 3), and (iii)mice subjected to intravenous administration of both 5 �106 cCD16z-T cells and 40 mg/body of rituximab (n ¼ 5).Intravenous administration of cCD16z-T cells with or with-out rituximab was done twice on days 4 and 24. In this seriesof experiments, no exogenous rhIL-2 was administered.Serial acquisition of the luciferase photon counts of inoc-ulated Raji/luc cells was carried out as described previously(31). The photon count relative to that on day 4 before celltherapy, which indicated the residual tumor mass burden,was calculated for each mouse. In some experiments,mice were similarly treated with activated NK cells from

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healthy donors (n ¼ 3) using 3-day culture with 50 U/mL ofrhIL-2. The same experiments were carried out twiceindependently.

Statistical analysisData were analyzed using the statistical computing package

SPSS 15.0J for Windows. Data were expressed as mean� SE or

Table 1. Patient demographics

PatientNo.

Age,y Sex Pathologic diagnosis

CD20þ

tumorcellsa

PreviousTx. (number)

WBC(/mL)

CD3þ

T cellNKcell

#1 60 M NHL (DLBCL);CD20(þ)CD79a(þ)CD3(�)CD56(�)CD10(�)Bcl-2(þ)

96.9% None 6,700 69.1% 1.43%

#2 60 M NHL (FL);CD20(þ)CD79a(þ)CD3(�)CD56(�)CD10(þ)Bcl-2(þ)

46.2% R-CHOP (1) 5,300 72.7% 11.6%

#3 55 M NHL (FL);CD20(þ)CD79a(þ)CD3(�)CD10(þ)Bcl-2(þ)

67.0% R-CHOP (5) 3,700 69.4% 2.92%

Abbreviations: DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; M, male; NHL, non-Hodgkin lymphoma; R-CHOP,rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, prednisolone; Tx, treatment.aDetermined by flow cytometer.

Figure 4. cCD16z-T cells generated from patients with B-cell lymphomas successfully display ADCC activity against rituximab-opsonized autologouslymphoma cells. A, cCD16z-T cells generated from all 3 patients with B-cell lymphomas successfully killed rituximab-opsonized autologouslymphoma cells and Raji cells (as a positive control), but not un-opsonized autologous lymphoma cells or K562 (as a negative control). In addition, the ADCCactivity mediated by patient-derived cCD16z-T cells did not seem to be affected by the number of chemotherapy cycles. Auto. lymphoma cells,autologous lymphoma cells; Pt. lymphoma, patient lymphoma cells. B, cCD16z-T cells generated from 2 patients (nos. 1 and 2) also displayed a proliferativeresponse to rituximab-opsonized autologous lymphoma cells.






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SD. Differences were assessed using paired t test or one-wayANOVA followed by Tukey post hoc analysis. Overall survivalrate was estimated by the Kaplan–Meier method and analyzedusing the log-rank test. Differences at P < 0.05 were consideredsignificant.

ResultsFunctional validation of chimeric CD16 with the 158V/V-CD3z receptorThe newly devised cCD16z gene construct (Fig. 1A) was

lentivirally introduced into Jurkat/MA/CD8a/luc cells(cCD16z-Jurkat/MA/CD8a/luc; Fig. 1B). CD3z phosphorylatedat 142Y tended, without statistical significance, to increase incCD16z-Jurkat/MA/CD8a/luc cells ligated with opsonizedCD20þ Raji cells in the presence of 1 mg/mL of rituximab (MFI¼ 8.34� 3.4, mean� SE), but not in cCD16z-Jurkat/MA/CD8a/luc cells ligated with Raji cells alone (MFI¼ 3.23� 1.4, mean�SE), 1 mg/mL of rituximab alone (MFI¼ 3.15� 1.6, mean� SE),or similarly treated K562 cells as a control with 1 mg/mL ofrituximab (MFI ¼ 3.58 � 1.4, mean � SE; Fig. 1C). Thus, theintroduced cCD16z construct successfully recognized the ritux-imab-opsonized Raji cells, evoking phosphorylation of the CD3zchain. Subsequently, activation of the NFAT pathway in thecCD16z-Jurkat/MA/CD8a/luc was triggered in response to therituximab-opsonized Raji cells, as demonstrated by themaximalproduction of luciferase, which was inhibited by the anti-CD16(Fab0)2 mAb (3G8) in a dose-dependent manner (Fig. 1D). Insummary, the introduced cCD16z receptor inT cells successfullyrecognized the opsonized target cells and transmitted thestimulatory signal to the intracellular NFAT pathway to activateT cells via phosphorylation of the CD3z chain. Thereafter, weused this cCD16z construct in all subsequent experiments.

ADCCactivity of cCD16z-T cells against opsonized cancercellsLentivirally gene-modified cCD16z-T cells successfully

expressed CD16 on their surface (96.2% of cCD16z-T cells vs.0.6% of NGM-T cells; Fig. 2A). cCD16z-T cells were readilyexpandable in ex vivo culture, showing a 3.4 � 0.8 � 102-foldincrease on day 14, and a 1.01� 0.25� 103-fold increase on day28 (mean� SE; Fig. 2B). The absolute cCD16z-T cell count was7.82 � 1.83 � 105 on day 2 of culture (mean � SE), eventuallyincreasing to 11.9 � 5.1 � 108 on day 28. The CD8þ T-cellpopulation became more predominant in ex vivo–expandedcCD16z-T cells as culture progressed, 32.3% � 3.2% on day 2,69.7% � 3.1% on day 14, and 87.2% � 2.0% on day 28 (mean �SE; Fig. 2C). cCD16z-T cells derived from 3 healthy individuals

displayed ADCC activity in vitro against opsonized CD20þ

lymphoma cell lines (Raji and Daudi) with rituximab, but notsimilarly treated K562 cells (Fig. 2D). cCD16z-T cells similarlydisplayed ADCC activity against the opsonized Her2/neuþ

breast cancer cell line MCF-7 and the ovarian cancer cell lineSKOV3 with trastuzumab (Fig. 2E), and against the opsonizedCCR4þ HTLV-1 infection-immortalized CD4þ T cell line MT-4and theATL cell lineATN-1withmogamulizumab (Fig. 2F), butnot against similarly treatedK562 cells, respectively. TheADCCactivities observed in the presence of rituximab, trastuzumab,andmogamulizumabwere uniformly dependent on the dose ofthe antibody. In addition, the cell-surface expression level oftarget antigen also limited the ADCC activity mediated bycCD16z-T cells. The ADCC-mediated cytocidal activity dis-played by cCD16z-T cells became almost maximal in thepresence of 0.1 mg/mL therapeutic antibodies. Therefore, weexamined the effect of effector cell number on this ADCCactivity in the presence of 0.1 mg/mL of each mAb (n ¼ 3). Asshown in Fig. 2G–I, the cytocidal activity mediated by cCD16z-T cells against opsonized target cells in the presence of 0.1 mg/mLof eachmAbwas dependent on the number of effector cells.Again, in a blocking test using the Raji–rituximab combinationas a representative, the ADCC activity mediated by cCD16z-Tcells against similarly opsonized Raji cells was inhibited sig-nificantly by the 3G8 mAb at 10 mg/mL (Fig. 2J).

Responsive functionalities displayed by cCD16z-T cellsfollowing ligation with opsonized cancer cells

As a result of recognizing opsonized Raji and Daudi, but notK562 cells in the presence of rituximab, cCD16z-T cells, but notNGM-T cells, produced high amounts of both IFN-g and IL-2(Fig. 3A). This observation was reproducible using the com-bination of SKOV3, MCF-7, and trastuzumab (Fig. 3B). Inaddition, cCD16z-T cells exhibited cytotoxic degranulation,reflected as CD107a expression, exclusively in response torituximab-opsonized Raji and trastuzumab-opsonized SKOV3,but not similarly opsonized K562, cells in both CD4þ and CD8þ

T-cell subsets of cCD16z-T cells (Fig. 3C and D). These cellularoutputs mediated by cCD16z-T cells were directly involved inthe cytocidal activity. In addition, the CFSE dilution assaydemonstrated a proliferative response mediated by cCD16z-Tcells in response to rituximab-opsonized Raji cells, but notrituximab alone or similarly treated K562 cells in the presenceof rituximab (Fig. 3E). The proliferationmediated by cCD16z-Tcells lasted for 7 days following single ligation with rituximab-opsonized Raji cells, but not with similarly treated K562 cells(Fig. 3F). This observation was replicated using the SKOV3–

Figure 5. Target-responsive functionality mediated by NK cells against opsonized tumor cells. A, freshly isolated NK cells from healthy individuals (n¼ 3) werebarely positive for CD69 and showed slight cytocidal activity only against K562 cells (so-called NK cell activity), but not the ADCC activity at all againstrituximab-opsonized Raji cells. B, the sameNK cells as those used in Awere additionally incubatedwith 50 U/mL of rhIL-2 for 3 days. This treatment renderedthe NK cells (n ¼ 3) positive for CD69, a cell-surface activation marker, and became capable of ADCC activity against rituximab-opsonized Raji cells.These activated NK cells exerted enhanced cytocidal activity against K562 cells at the same time. C, cCD16z-T cells generated from the same individuals asthose in A and B displayed ADCC activity against rituximab-opsonized Raji cells comparable with that of the activated NK cells in B, althoughtheir CD69 expression varied. Unlike the activated NK cells, cCD16z-T cells did not exert any cytocidal activity against K562 cells. D, CFSE dilution assaydemonstrated that rhIL-2–treated NK cells did not proliferate at all after ligation without target cells (top). These activated NK cells did not proliferate even inresponse to rituximab-opsonized Raji cells (middle), or K562 cells (bottom), both types of target cells being recognized and sufficiently killed by theseactivated NK cells, as shown in B. A representative result from three experiments is shown. E, after ligation with rituximab-opsonized Raji cells (*) or K562cells (*) at the indicated dose of rituximab and E:T ratio, rhIL-2–treated NK cells did not proliferate at all.






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trastuzumab combination (data not shown). Furthermore,after single ligation of rituximab-opsonized Raji cells, theeffector memory (CD45RA-CD62L-) subset of cCD16z-T cellspredominated (66.4% � 5.0%), in comparison with thoseligated with rituximab alone (25.7% � 10.6%), or similarlytreated K562 cells (39.6% � 3.3%; mean � SD; Fig. 3G). Thus,both the proliferative response and differentiation into aneffector memory T-cell subset might contribute to the persis-tence of therapeutically infused cCD16z-T cells following therecognition of opsonized cancer cells in vivo.

cCD16z-T cells generated from PBMCs of patients withCD20þ B-cell lymphomas successfully display ADCCactivity against rituximab-opsonized autologouslymphoma cellsPBMCs and autologous tumor cells were obtained from 3

patients with CD20þ B-cell lymphomas. The demographics ofthese patients are shown in Table 1. As shown in Fig. 4A,cCD16z-T cells generated from all 3 patients successfully killedrituximab-opsonized, but not un-opsonized autologous lym-phoma cells. Raji andK562were used as a positive and negativecontrol, respectively. Next, limited by the number of lymphomacells available, cCD16z-T cells from2of 3 patients (nos. 1 and 2)were subjected to CFSE dilution assay. The cCD16z-T cellsfrom these 2 patients similarly proliferated in response torituximab-opsonized autologous lymphoma cells, as did thepositive control, rituximab-opsonized Raji cells (Fig. 4B). Col-lectively, cCD16z-T cells generated from patients functionedsimilarly as those from healthy individuals.

Activated NK cells exert ADCC activity comparable withthat of cCD16z-T cells, but do not proliferate in responseto opsonized tumor cellsWe found that CD3�CD56þCD16þNK cells accounted for

13.6%� 2.0% (mean� SE) of the peripheral blood lymphocytes(PBL) from healthy individuals (n ¼ 10), but only 3.7% � 1.6%(mean � SE) from patients with lymphoma (n ¼ 5). Thus, dueto the difficulty in preparing a sufficient number of NK cellsfrom patients with lymphoma, we only compared the func-tionality of NK cells with that of cCD16z-T cells generated fromthe same healthy donors (n¼ 3). After activation using a 3-dayculture with 50 U/mL of rhIL-2, NK cells became positive forCD69 expression and capable of exerting the ADCC activityagainst rituximab-opsonized Raji cells to a degree comparablewith that of cCD16z-T cells (Fig. 5A–C). We then examined the

proliferative activity in response to rituximab-opsonized Rajicells mediated by these activated NK cells. After ligation withopsonized tumor cells and in the absence of additional rhIL-2,unlike the cCD16z-T cells (Fig. 3E and F), these activated NKcells did not proliferate (Fig. 5D and E).

Successful inhibition of the growth of disseminatedCD20þ lymphoma cells by adoptively transferredcCD16z-T cells in combinationwith rituximab in amousexenograft tumor model

NOGmice intravenously inoculated with Raji/luc cells wereused to assess the in vivo therapeutic effect of cCD16z-T cells.First, we confirmed that rituximab did not exert CDC activityagainst Raji cells in the presence of serum from un-inoculatedNOG mouse (Fig. 6A), as reported previously (32). As shownin Fig. 6B, in the Raji/luc xenograft tumor model, NOG micewere treated with cCD16z-T cells and rituximab intravenouslytwice (on days 4 and 24 after tumor cell inoculation; n ¼ 9),tumor growth was significantly suppressed during the 5-weekobservation period (P < 0.01) in comparison with that ofnontreated mice (n ¼ 3), mice treated with rituximab alone(n ¼ 3), mice treated with NGM-T cells (n ¼ 3), mice treatedwith cCD16z-T cells alone (n ¼ 3), or rhIL-2–treated NK cells(n ¼ 3). Serial examination of mice in all cohorts using in vivobioluminescence assay up to day 14 also demonstrated thesuppression of tumor growth mediated by the combinedregimen of cCD16z-T cells and rituximab (Fig. 6C). This resultwas reflected in the survival period of the mice; all mice in thecontrol cohort died within 14 days and all mice treated withactivated NK cells and rituximab died within 18 days, whereasmice treated with cCD16z-T cells and rituximab survived formore than 35 days (data not shown). In some experiments,mice were euthanized on day 14 when almost all mice in thecontrol cohorts had died, and residual Raji/luc cells in autop-sied organswere assessed using luciferase assay (Fig. 6D andE).In mice treated with cCD16z-T cells alone (solid bar), massivetumor burdens were detected in the liver, spleen, lung/heart,and uterus/ovary/fallopian tube. In contrast, in mice treatedwith cCD16z-T cells and rituximab (clear bar), tumor growthwas suppressed in all the organs.

We examined the persistence of human NK cells and Rajicells in mouse spleen and bone marrow using flow cytometry(Supplementary Fig. S1). On day 14, a number of CD20þ Rajicells in the bone marrow were observed in a mouse treatedwith NK cells (Supplementary Fig. S1C, left), but not in amouse

Figure6. In vivoantitumor effectmediatedbycCD16z-Tcells infusedconcomitantlywith rituximab.A, 51Cr-releaseassay revealed that rituximabexerteddose-dependent CDC activity against Raji cells exclusively in human serum, but not NOG mouse serum. K562 was used as a negative control. B, therapeuticadoptive transfer model. NOGmice were intravenously inoculated with 5� 105 Raji/luc cells on day 0. Thereafter, intravenous administration of either 5� 106

cCD16z-T cellswith 40mg/bodyof rituximab (n¼5), rhIL-2–treatedNKcells (n¼3) orNGM-T cellswith thesamedoseof rituximab (n¼3)wasconducted twiceon days 4 and 24. NOG mice inoculated with Raji/luc cells but not subjected to any cell therapy, NOG mice that had similarly received cCD16z-Tcells alone, and NOG mice that had similarly received rituximab alone (n ¼ 3 per group) were used as controls. The same experiments were independentlyconducted twice. Unlike the situation in all control mice and mice treated with NGM-T cells and activated NK cells, simultaneous infusion of cCD16z-T cellswith rituximab obviously and significantly suppressed tumor cell growth for longer than 32 days (P < 0.01). C, serial images of the bioluminescenceassay until day 14 demonstrate tumor growth in each group. D,mice treatedwith cCD16z-T cells alone (n¼ 3) and those treatedwith cCD16z-T and rituximab(n ¼ 2) were euthanized on day 14 and subsequently autopsied to assess residual tumor cells in organs using luciferase assay. A representative casefrom each group is shown. Each organ in the picture corresponds to an identical number. E, residual tumor cells determined on the basis of luciferase photoncounts are shown as a bar graph. Clear bar (&) represents mice treated with cCD16z-T cells and rituximab, and black bar (&) indicates those treatedwith cCD16z-T cells alone. Error bars indicate SDs.






treated with cCD16z-T cells (Supplementary Fig. S1D). Fur-thermore, as shown in Supplementary Fig. S1C (right), inanother mouse treated with NK cells on day 18, Raji cellsincreased in number in both the spleen and the bone marrow,but the number of NK cells remained constant.

These observations suggested that cCD16z-T cells infusedalong with rituximab successfully inhibited the growth ofCD20þ Raji cells in vivo, and that their tumor-suppressiveactivity was superior to that of NK cells.

DiscussionCancer antigen-specificmAbs have demonstrated efficacy in

the treatment of cancer (1). The central tumoricidalmachineryof this strategy in vivo is ADCC executed by FcgRIII (CD16)–expressing effector cells, such as NK cells (4). In practice,however, a decline in the clinical efficacy of this treatment isoften observed, and this involves both chemotherapy-inducedleukopenia and NK cell exhaustion due to ADCC evoked by theapplied mAb (5). Given that effector cells play important rolesin ADCC, instead of escalating the dose of therapeutic mAb,replenishment of effector cells might circumvent this draw-back. In the present study, we focused on three factors: (i) theexpression level of target antigen on the surface of cancer cells,(ii) the dose of mAb used, and (iii) the number of effector cellsrestricting the ADCC activity (Fig. 2D–I). When the number ofeffector cells was fixed, the maximal ADCC mediated bycCD16z-T cells was achieved (Fig. 2D and E) at a rituximab,trastuzumab, or mogamulizumab concentration of 0.1 to 1.0mg/mL, which is lower than the clinically effective concentra-tions in serum (26, 33, 34). When each mAb concentration wasfixed at 0.1 mg/mL, ADCC activities against opsonized tumorcells were dependent on the number of cCD16z-T cells (Fig.2G–I). At the clinically relevant concentrations of mAb, thenumber of effector cells seems to be a crucial determinant ofthe efficacy of this form of therapy.

However, the anticancer effect mediated by ex vivo–expand-ed NK cells in clinical trials seems controversial (9), and theclinical efficacy of such cells administered along with antican-cer mAbs, that is, as infused effector cells for ADCC, has not yetbeen evaluated clinically. There is a need to establish a stan-dard procedure to expand clinically acceptable NK cells (6, 7).The other potential candidate CD16þ gd-T cells (8) still awaitclinical evaluation. A clinical trial of adoptive cell therapy usingculture-induced killer cells (CIK) in conjunction with ritux-imab against refractory lymphomas is ongoing (NCT01329354;ClinicalTrials.gov). In this setting, the central effector cellsamong infused CIKs have been defined as NK cells (10),although no conclusive results have been reported yet.

With regard to clinical feasibility, T cells have a provenrecord in terms of ex vivo expansion and genetic modificationfor adoptive cell therapy (17). Therefore, in the present study,we generated CD3þ T cells gene-modified to express thechimeric CD16 (158V/V)-CD3z receptor as an adoptive trans-fer effector cell mediating ADCC in the setting of anticancermAb therapy. It is known that CD16 naturally associates withimmunoreceptor tyrosine-based activatingmotif (ITAM)–con-taining homodimers of FcRIg in humanNK cells (35). Although

in this study we did not make a direct comparison with thechimeric CD16/FcRIg construct, in view of the lower prolifer-ation mediated by activated NK cells against opsonized Rajicells (Fig. 5A–E), we expected this chimeric CD16 (158V/V)-CD3z to show some degree of enhancement of proliferativeactivity after ligation with opsonized tumor cells. The chimericCD16 (158V/V)-CD3z construct introduced into Jurkat/MAcells successfully recognized opsonized Raji cells in the pres-ence of rituximab. This opsonized cell ligation led to thephosphorylation of the CD3z chain (Fig. 1C), followed byactivation of the NFAT pathway, as reflected by luciferaseproduction, which was blocked by the anti-CD16 (Fab0)2 mAb(3G8; Fig. 1D). As expected, through this mechanism, duringADCC, the cCD16z-T cells produced IFN-g and IL-2 (Fig. 3Aand B), released cytotoxic granules (Fig. 3C andD), proliferated(Fig. 3E and F), and differentiated into an effector memory T-cell phenotype (Fig. 3G). The former two cellular outputsdirectly accounted for tumoricidal activity, and the latter twocellular activities were able to contribute to the durableantitumor effect observed in vivo. To our knowledge, noprevious reports describing a similar concept have includedin vivo experimental observations (18, 34). Therefore, afterconfirming that rituximab-induced CDC was not present inNOG mouse serum (Fig. 6A), we used an in vivo biolumines-cence assay to demonstrate that cCD16z-T cells concomitantlyinfused with rituximab significantly inhibited the growth ofdisseminated Raji/luc cells in NOG mice, compared with theother cohorts (P < 0.01; Fig. 6B and C). The luciferase assay ofautopsied organs from mice treated simultaneously withcCD16z-T cells and rituximab demonstrated inhibition oftumor growth in the liver, spleen, lung/heart, and uterus/ovary/fallopian tube (Fig. 6D and E). Although cCD16z-T cellsand activated NK cells displayed comparable ADCC activity invitro (Fig. 5B and C), cCD16z-T cells demonstrated a superiortumor-suppressive effect in vivo (Fig. 6B and C and Supple-mentary Fig. S1). In Fig. 6B, slower tumor progression wasobserved in mice treated with cCD16z-T cells plus rituximab.The progression of the Raji/luc tumor in NOG mice was moreobvious in head lesions, but remained localized and sup-pressed at truncal sites (Supplementary Fig. S2A). Further-more, in a cCD16z-T–treated mouse on day 32, we observeddurably persistent cCD16z-T cells, defined as hCD45þ/CD3þ/CD16þ, accounting for 9.0% of total spleen cells and 0.14% oftotal bone marrow cells, but no CD20þ Raji cells (Supplemen-tary Fig. S2B), and these cCD16z-T cells retained their respon-siveness against rituximab-opsonized Raji cells (Supplemen-tary Fig. S2C). In addition, Raji cells in mice showing tumorprogression were still positive for CD20 (data not shown).Taken together, these data indicate that NOG mice inoculatedintravenously with Raji cells may be more likely to develophead metastasis, and that head lesions seem to be less acces-sible to cCD16z-T cells (and/or rituximab) than those in thetruncal sites. The localization and persistence of effector cellsor the loss of CD20 by Raji cells may be specific to thisparticular model.

In this series of in vivo experiments, exogenous rhIL-2, whichhad been defined as an important determinant in this typeof murine experiment (36, 37), was not administered. Our

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findings strongly suggested that the proliferation and differ-entiation mediated by the infused cCD16z-T cells in responseto rituximab-opsonized Raji cells (Fig. 3E–G) contributed tothe persistent efficacy observed in vivo. Overall, the datasuggest that our newly generated cCD16z-T cells would bepromising for clinical application.At present, chimeric antigen receptor (CAR)–transduced T

cells, whose target recognition is mediated by an introducedsingle-chain variable fragment (scFv) of cancer-associatedantigen-specific mAb, especially in CD19-CAR T-cell therapyfor CD19þ leukemia, have shown clinical efficacy and safety(38, 39). However, the range of applications for the CARstrategy remains narrow, and the preparation of a huge seriesof CAR-T cells covering various kinds of cancer does not seemrealistic. From this viewpoint, cCD16z-T cells might be advan-tageous, because almost all anticancer mAbs currently avail-able for ADCC would be theoretically applicable.Because thefirst-generationCAR constructs containing only

CD3-z were unable to exert fully the anticipated antitumoreffect (40), the incorporation of a costimulatorymolecule, suchas 4.1BB, into the cytoplasmic portion of our chimeric CD16-CD3z construct, may improve the persistence and antitumortrafficking in vivo, and possibly providing a better clinicaloutcome (41). Therefore, we plan to develop a "second-gener-ation" chimeric CD16-CD3z construct. In the present study, wehave successfully demonstrated that our therapeutic approachusing novel cCD16z-T cells as adoptive transfer anticancereffector cells is feasible, and can mediate ADCC both in vitroand in vivo.Although further studies are warranted, our experimental

observations strongly suggest that cCD16z-T cells might pro-vide another option to improve the currently available anti-cancer immunotherapy.

Disclosure of Potential Conflicts of InterestS. Okamoto and J. Mineno are employed as Senior Scientist and General

Manager, respectively, for Takara Bio Inc. H. Shiku has received a commercialresearch grant. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: F. Ochi, H. Fujiwara, K. Tanimoto, H. ShikuDevelopment of methodology: H. Fujiwara, K. Tanimoto, H. ShikuAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Ochi, H. Fujiwara, H. Asai, Y. Miyazaki, J. BarrettAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Ochi, H. Fujiwara, H. Asai, Y. MiyazakiWriting, review, and/or revision of the manuscript: F. Ochi, H. Fujiwara, K.Tanimoto, J. Barrett, E. Ishii, M. YasukawaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): H. Fujiwara, S. Okamoto, J. Mineno, K.KuzushimaStudy supervision: H. Fujiwara, H. Shiku, E. Ishii, M. Yasukawa

AcknowledgmentsThe authors thank Dr. Kenji Kameda (Ehime University) for skilled technical

assistance. The authors also thank Drs. Erik Hooijberg (Vrije UniversiteitMedisch Centrum, Amsterdam, the Netherlands) for supplying the Jurkat/MAcell line and Akihiro Nawa (Department of Obstetrics and Gynecology, EhimeUniversity Graduate School of Medicine, Ehime, Japan) for supplying the SKOV3cell line.

Grant SupportThis work was supported in part by grants from the Ministry of Education,

Culture, Sports, Science and Technology of Japan to H. Fujiwara and M.Yasukawa, and a Grant-in-Aid for Cancer Research from the Ministry of Health,Labor and Welfare to M. Yasukawa.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 18, 2013; revised November 13, 2013; accepted November 27,2013; published OnlineFirst January 3, 2014.

References1. Weiner LM, Surana R, Wang S. Monoclonal antibodies: versatile

platforms for cancer immunotherapy. Nat Rev Immunol 2010;10:317–27.

2. Quintas-Cardama A, Wierda W, O'Brien S. Investigational immu-notherapeutics for B-cell malignancies. J Clin Oncol 2010;28:884–92.

3. Spector NL, Blackwell KL. Understanding the mechanisms behindtrastuzumab therapy for human epidermal growth factor receptor 2-positive breast cancer. J Clin Oncol 2009;27:5838–47.

4. Houot R, Kohrt HE, Marabelle A, Levy R. Targeting immune effectorcells to promote antibody-induced cytotoxicity in cancer immunother-apy. Trends Immunol 2011;32:510–6.

5. Kohrt HE, Houot R, Marabelle A, Cho HJ, Osman K, Goldstein M, et al.Combination strategies to enhance antitumor ADCC. Immunotherapy2012;4:511–27

6. Besser MJ, Shoham T, Harari-Steinberg O, Zabari N, Ortenberg R,Yakirevitch A, et al. Development of allogeneic NK cell adoptivetransfer therapy in metastatic melanoma patients: in vitro preclinicaloptimization studies. PLoS ONE 2013;8:e57922.

7. Liu Y, Wu H-W, Sheard MA, Sposto R, Somanchi SS, Cooper LJN,et al. Growth and activation of natural killer cells ex vivo from childrenwith neuroblastoma for adoptive therapy. Clin Cancer Res 2013;19:2142–3.

8. Capietto AH, Martinet L, Fournie JJ. Stimulated gd T cells increase thein vivo efficacy of trastuzumab in HER-2þ breast cancer. J Immunol2011;187:1031–8.

9. Parkhurst MR, Riley JP, Dudley ME, Rosenberg SA. Adoptive transferof autologous natural killer cells leads to high levels of circulatingnatural killer cells but does not mediate tumor regression. Clin CancerRes 2011;17:6287–97.

10. Pievani A, Belussi C, Klein C, Rambaldi A, Golay J, Introna M.Enhanced killing of human B-cell lymphoma targets by combined useof cytokine-induced killer cell (CIK) cultures and anti-CD20 antibodies.Blood 2011;117:510–8.

11. ClemenceauB, Vivien R, BerthomeM, Robillard N, Garand R, Gallot G,et al. Effector memory ab T lymphocytes can express FcgRIIIa andmediate antibody-dependent cellular cytotoxicity. J Immunol 2008;180:5327–34.

12. Bjorkstrom NK, Gonzalez VD, Malmberg K-J, Falconer K, Alaeus A,Nowak G, et al. Elevated number of FcgRIIIAþ(CD16þ) effector CD8 Tcells with NK cell-like function in chronic hepatitis C virus infection. JImmunol 2008;181:4219–28.

13. Couzi L, Pitard V, Sicard X, Garrigue I, Hawchar O, Merville P, et al.Antibody-dependent anti-cytomegalovirus activity of human gd T cellsexpressing CD16 (FcgRIIIa). Blood 2012;119:1418–27.

14. Koene HR, Kleijer M, Algra J, Roos D, von dem Borne AE, de Haas M.FcgRIIIa-158V/F polymorphism influences the binding of IgG by nat-ural killer cell FcgRIIIa, independently of the FcgRIIIa-48L/R/H pheno-type. Blood 1997;90:1109–14.

15. Hatjiharissi E, Xu L, Santos DD, Hunter ZR, Ciccarelli BT, Verselis S,et al. Increased natural killer cell expression of CD16, augmented






binding and ADCC activity to rituximab among individuals expressingthe FcgRIIIa-158V/V and V/F polymorphism. Blood 2007;110:2561–4.

16. Veeramani S, Wang S-Y, Dahle C, Blackwell S, Jacobus L, KnutsonT, et al. Rituximab infusion induces NK activation in lymphomapatients with the high-affinity CD16 polymorphism. Blood 2011;118:3347–9.

17. Rosenberg SA. Raising the bar: the curative potential of human cancerimmunotherapy. Sci Transl Med 2012;4:127ps8

18. Cl�emenceau B, Congy-Jolivet N, Gallot G, Vivien R, Gaschet J,Thibault G, et al. Antibody-dependent cellular cytotoxicity (ADCC) ismediated by genetically modified antigen-specific human T lympho-cytes. Blood 2006;107:4669–77.

19. Scholler J, Brady TL, Binder-Scholl G, HwangWT, Plesa G, Hege KM,et al. Decade-long safety and function of retroviral modified chimericantigen receptor T cells. Sci Transl Med 2012;4:132ra53.

20. Tebas P, Stein D, Binder-Scholl G, Mukherjee R, Brady T, Rebello T,et al. Antiviral effects of autologous CD4 T cells genetically modifiedwith a conditionally replicating lentiviral vector expressing long anti-sense to HIV. Blood 2013;121:1524–33.

21. Ohminami H, Yasukawa M, Fujita S. HLA class I-restricted lysis ofleukemia cells by a CD8(þ) cytotoxic T-lymphocyte clone specific forWT1 peptide. Blood 2000;95:286–93.

22. Maeda O, Shibata K, Hosono S, Fujiwara S, Kajiyama H, Ino K, et al.Spectrin aII and bII tetramers contribute to platinum anticancerdrug resistance in ovarian serous adenocarcinoma. Int J Cancer2012;130:113–21.

23. Miyazaki Y, Fujiwara H, Asai H, Ochi F, Ochi T, Azuma T, et al.Development of a novel redirected T-cell-based adoptive immuno-therapy targeting human telomerase reverse transcriptase for adult T-cell leukemia. Blood 2013;121:4894–901.

24. Calogero A, Hospers GA, Kr€use KM, Schrier PI, Mulder NH, HooijbergE, et al. Retargeting of a T cell line by antiMAGE-3/HLA-A2 alpha betaTCR gene transfer. Anticancer Res 2000;20:1793–9.

25. Yang S, Rosenberg SA, Morgan RA. Clinical-scale lentiviral vectortransduction of PBL for TCR gene therapy and potential for expressionin less-differentiated cells. J Immunother 2008;31:830–9.

26. Ishida T, Joh T, Uike N, Yamamoto K, Utsunomiya A, Yoshida S, et al.Defucosylated anti-CCR4monoclonal antibody (KW-0761) for relapseadult T-cell leukemia: a multicenter phase II study. J Clin Oncol 2012;30:837–42.

27. Ito M, Hiramatsu H, Kobayashi K, Suzue K, Kawahata M, Hioki K, et al.NOD/SCID/gamma(c)(null)mouse: an excellent recipientmousemodelfor engraftment of human cells. Blood 2002;100:3175–82.

28. Nagai K, Ochi T, Fujiwara H, An J, Shirakata T, Mineno J, et al. AurorakinaseA-specificT-cell receptor gene transfer redirects T lymphocytesto display effective antileukemia reactivity. Blood 2012;119:368–76.

29. Ochi T, Fujiwara H, Suemori K, Azuma T, Yakushijin Y, Hato T, et al.Aurora-A kinase: a novel target of cellular immunotherapy for leukemia.Blood 2009;113:66–74.

30. Ochi T, Fujiwara H, Okamoto S, An J, Nagai K, Shirakata T, et al. Noveladoptive T-cell immunotherapy using a WT1-specific TCR vectorencoding silencers for endogenous TCRs showsmarked antileukemiareactivity and safety. Blood 2011;118:1495–503.

31. Asai H, Fujiwara H, An J, Ochi T, Miyazaki Y, Nagai K, et al. Co-introduced functional CCR2 potentiates in vivo anti-lung cancer func-tionality mediated by T cells double gene-modified to express WT1-specific T-cell receptor. PLoS ONE 2013;8:256820.

32. Baxter AG, Cooke A. Complement lytic activity has no role in thepathogenesis of autoimmune diabetes in NOD mice. Diabetes 1993;42:1574–8.

33. Tobinai K, Kobayashi Y, Narabayashi M, Ogura M, Kagami Y, Mor-ishimaY, et al. Feasibility andpharmacokinetic studyof a chimeric anti-CD20 monoclonal antibody (IDEC-C2B8, rituximab) in relapsed B-celllymphoma. Ann Oncol 1998;9:527–34.

34. Tokuba Y, Watanabe T, Omuro Y, Ando M, Katsumata N, Okumura A,et al. Dose escalation and pharmacokinetic study of a humanized anti-HER2monoclonal antibody in patientswithHER2/neu-overexpressingmetastatic breast cancer. Br J Cancer 1999;81:1419–25.

35. Vivier E, Rochet N, Ackerly M, Petrini J, Levine H, Daley J, et al.Signaling function of reconstituted CD16:z:g receptor complex iso-forms. Int Immunol 1992;4:1313–23.

36. Iwashiro M, Jinyan W, Toda M, Linan W, Kato T, Kuribayashi K.Effective anti-tumor adoptive immunotherapy: utilization of exogenousIL-2-independent cytotoxic T lymphocyte clones. Int Immunol 2002;14:1459–68.

37. Klebanoff CA, Gattinoni L, Palmer DC, Muranski P, Ji Y, Hinrichs CS,et al. Determinants of successful CD8þ T-cell adoptive immunotherapyfor large established tumors inmice. Clin Cancer Res 2011;17:5343–52.

38. Kalos M, Levine BL, Porter DL, Katz S, Grupp SA, Bagg A, et al. T cellswith chimeric antigen receptors have potent antitumor effects and canestablish memory in patients with advanced leukemia. Sci Transl Med2011;3:95ra73.

39. GruppSA, KalosM, Barrett D, AplencR, Porter DL, Rheingold SR, et al.Chimeric antigen receptor-modified T cells for acute lymphoid leuke-mia. N Engl J Med 2013;368:1509–18.

40. Gong MC, Latouche JB, Krause A, Heston WD, Bander NH, SadelainM. Cancer patient T cells genetically targeted to prostate-specificmembrane antigen specifically lyse prostate cancer cells and releasecytokines in response to prostate-specific membrane antigen. Neo-plasia 1999;1:123–7.

41. Sadelain M, Brentjens R, Riviere I. The basic principles of chimericantigen receptor design. Cancer Discov 2013;3:388–98.

Ochi et al.





2014;2:249-262. Published OnlineFirst January 3, 2014.Cancer Immunol Res Fumihiro Ochi, Hiroshi Fujiwara, Kazushi Tanimoto, et al. Anticancer Monoclonal Antibody Therapy

Receptor as Adoptively Transferable Effector Cells forζCD16-CD3-T Cells Expressing a Chimericβ/αGene-Modified Human

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