t lymphocytes expressing a cd16 signaling receptor exert ...signaling mediated by 4-1bb-cd3z induced...

Microenvironment and Immunology

T Lymphocytes Expressing a CD16 Signaling Receptor ExertAntibody-Dependent Cancer Cell Killing

Ko Kudo1, Chihaya Imai4, Paolo Lorenzini1, Takahiro Kamiya1, Koji Kono2, Andrew M. Davidoff5,Wee Joo Chng3, and Dario Campana1

AbstractTo expand applications for T-cell–based immunotherapy in cancer, we designed a receptor that binds the Fc

portion of human immunoglobulins and delivers activation signals. The construct included the high-affinity CD16(FCGR3A) V158 variant, CD8a hinge, and transmembrane domains, alongwith signaling domains fromCD3z and4-1BB (TNFRSF9), forming a chimeric receptor termed CD16V-BB-z. After retrovirus-mediated expression inhuman T cells, CD16V-BB-z bound humanized antibodies with higher affinity than a control receptor containingthe more common F158 variant. Engagement of CD16V-BB-z provoked T-cell activation, exocytosis of lyticgranules, and sustained proliferation, with amean cell recovery after 4-week coculturewithDaudi lymphoma cellsand rituximab of nearly 70-fold relative to input cells. In contrast, unbound antibody alone produced no effect.CD16V-BB-z T cells specifically killed lymphoma cells and primary chronic lymphocytic leukemia cells incombinationwith rituximab at a low effector:target ratio, evenwhen assayed onmesenchymal cells. Trastuzumabtriggered CD16V-BB-z–mediated killing of HER2 (ERBB2)þ breast and gastric cancer cells; similar results wereobtainedwith an anti-GD2 antibody in neuroblastoma and osteosarcoma cells. Furthermore, coadministration ofCD16V-BB-z T cells with immunotherapeutic antibodies exerted considerable antitumor activity in vivo.Signaling mediated by 4-1BB-CD3z induced higher T-cell activation, proliferation, and cytotoxicity than CD3zor FceRIg , and the receptor was expressed effectively after mRNA electroporation without viral vectors,facilitating clinical translation. Our results offer preclinical proof of concept for CD16V-BB-z as a universal,next-generation chimeric receptor with the potential to augment the efficacy of antibody therapies for cancer.Cancer Res; 74(1); 93–103. �2013 AACR.

IntroductionImmunotherapy is a promising option for cancer treatment

because of its potential to evade genetic and cellular mechan-isms of drug resistance, and to target tumor cells while sparingnormal tissues. T lymphocytes can exert major antitumoreffects, as demonstrated by results of allogeneic hematopoieticstem cell transplantation for hematologic malignancies, whereT-cell–mediated graft-versus-host disease is inversely associ-ated with disease recurrence, and immunosuppression with-drawal or infusion of donor lymphocytes can contain relapse(1–5). The expression of chimeric signaling receptors with

antibody recognition properties can skew the reactivity of Tlymphocytes towards cancer cells: ligation of the cognatetarget results in T-cell activation and triggers cytotoxicity(6–10). Recent results of clinical trials with infusions of chi-meric receptor-expressing autologous T lymphocytes providedcompelling evidence of their clinical potential (11–16).

Another approach to cancer immunotherapy is the admin-istrationofmonoclonal antibodies, which can exert cytotoxicitythrough a variety of mechanisms, including induction of proa-poptotic signals, complement fixation, and antibody-depen-dent cell cytotoxicity (ADCC; refs. 17–22). Amajor role is playedby the lattermechanism, which results from the engagement ofFc receptors (FcgR) expressed on the surface of natural killer(NK cells) and other cells, such as neutrophils and macro-phages, by the Fc portion of the antibody (23). Polymorphismsof FcgR genes can have marked functional consequences,which, in turn, influence response to antibody treatment. Tothis end, allotypes of the gene coding FcgRIIIa (FCRG3A orCD16), expressed by NK cells, can result in receptors havingeither a phenylalanine (F) or a valine (V) residue at position 158;CD16 V158, which occurs in a minority of individuals, hashigher Fc binding and is associated with increased tumor cellkilling and superior responses in patients (18, 24–32).

We sought to combine the anticancer potential of T-cell andantibody therapy. T lymphocytes expressing ab T-cell recep-tors (the vast majority of T cells) lack activating FcgR and do

Authors' Affiliations: Departments of 1Pediatrics and 2Surgery, and3Cancer Science Institute, National University of Singapore, Singapore;4Department of Pediatrics, Niigata University, Niigata, Japan; and 5Depart-ment of Surgery, St. Jude Children's Research Hospital, Memphis,Tennessee

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Dario Campana, Department of Pediatrics, YongLoo Lin School of Medicine, National University of Singapore, Centre forTranslational Medicine, 14 Medical Drive, Level 9 South, Singapore117599. Phone: 65-6601-2666; Fax: 65-6779-7486; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-13-1365

�2013 American Association for Cancer Research.

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Published OnlineFirst November 6, 2013; DOI: 10.1158/0008-5472.CAN-13-1365

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not mediate ADCC (23). We reasoned that expression of achimeric receptor composed of FcgR and T-cell–signalingmolecules should confer ADCC capability to these cells and,if so, they should significantly augment the antitumor potentialof monoclonal antibodies, regardless of the targeted tumorantigen. We here describe the antibody-guided antitumoractivity of T lymphocytes expressing such receptor.

Materials and MethodsCells

The human cell lines Daudi and Ramos (B-cell lymphoma),Jurkat (T-cell acute lymphoblastic leukemia), and CHLA-255,NB1691 and SK-N-SH (neuroblastoma) were available at St.Jude Children's Research Hospital (Memphis, TN). MCF-7 andSK-BR-3 (breast carcinoma), and U2-OS (osteosarcoma) wereobtained from the American Type Culture Collection; MKN7(gastric carcinoma) was from the National Institute of Bio-medical Innovation (Osaka, Japan). Daudi, CHLA-255,NB1691, SK-N-SH, SK-BR-3,MCF-7, U2-OS, and MKN7 werealso transduced with a murine stem cell virus (MSCV)-inter-nal ribosome entry site (IRES)-green fluorescent protein(GFP) retroviral vector containing the firefly luciferase gene(33) and selected for GFP expression with a FACSAria (BDBiosciences) or a MoFlo cell sorter (Beckman Coulter).Cell lines were characterized by the providers for molecularand/or gene expression features; the cell marker profile ofleukemia and lymphoma cell lines was periodically tested byflow cytometry to ensure that no changes had occurred. Celllines were expanded after receipt, cryopreserved and cells forexperiments were obtained from recently thawed vials.Peripheral blood or bone marrow samples from newly diag-nosed and untreated patients with B-chronic lymphocyticleukemia (CLL) were obtained following informed consentand approval from the Domain Specific Ethics Board govern-ing Singapore's National University Hospital.

Peripheral blood samples were obtained from deidentifiedby-products of platelet donations from adult donors. Mono-nuclear cells were enriched by centrifugation on Accu-PrepHuman Lymphocytes Cell Separation Media (Accurate Chem-ical & Scientific Corp.), and cultured with anti-CD3/CD28beads (Invitrogen) in RPMI-1640 with 10% FBS, antibiotics,100 IU/mL interleukin (IL)-2 (Roche) for 3 days. Onday 4, T cellswere purified by negative selection with a mixture of CD14,CD16, CD19, CD36, CD56, CD123, and CD235a antibodies andmagnetic beads (Pan T Cell Isolation Kit II; Miltenyi Biotec;purity, >98%). Purified T cells were maintained in the abovemedium, with the addition of 100 IU/mL IL-2 every other day.

Plasmids, virus production, and gene transductionThe pMSCV-IRES-GFP, pEQ-PAM3(-E), and pRDF were

obtained from the St. Jude Children's Research Hospital VectorDevelopment and Production Shared Resource (10). TheFCRG3A (CD16) and FCER1G cDNA were obtained from Ori-gene; the FCRG3A V158 variant was generated using site-directedmutagenesis by PCR. The CD8a hinge and transmem-brane domain, and the intracellular domains of 4-1BB andCD3z were subcloned from an anti-CD19-41BB-CD3z cDNApreviously made in our laboratory (10). These molecules were

assembled in various combinations (Supplementary Fig. S1),using splicing by overlapping extension by PCR (SOE-PCR).The constructs and expression cassette were subcloned intoEcoRI and MluI sites of the MSCV-IRES-GFP vector.

Preparation of retroviral supernatant and transductionwereperformed as previously described (10, 34). Briefly, T cells wereincubated with RD114-pseudotyped retroviral supernatant inthe presence of RetroNectin (Takara) at 37�C for 24 hours andthen maintained in RPMI-1640 with FBS, antibiotics, and 100IU/mL IL-2 until the time of the experiments, 7 to 21 days aftertransduction.

Surface expression of CD16 was analyzed by flow cytometryusing R-phycoerythrin (PE)-conjugated anti-human CD16(clones B73.1 or 3G8, BD Biosciences). Western blotting wasperformed as previously described (34), using a mouse anti-human CD3z (clone 8D3; BD Biosciences) and then with a goatanti-mouse IgG horseradish peroxidase-conjugated secondaryantibody (Cell Signaling Technology). Antibody binding wasrevealed by using the Amershan ECL Prime detection reagent(GE Healthcare).

mRNA electroporationThe pVAX1 vector (Invitrogen) was used as a template for in

vitro mRNA transcription. The CD16V-BB-z cDNA was sub-cloned into EcoRI and XbaI sites of pVAX1. The correspondingmRNA was transcribed in vitro with T7 mScript mRNA pro-duction system (CellScript; ref. 35).

For electroporation, we used the Amaxa Nucleofector(Lonza); 1 � 107 of purified T cells activated with 200 IU/mLIL-2 overnight were mixed with 200 mg/mL mRNA in Cell LineNucleofector Kit V, transferred into the processing chamber,and transfected using the program X-001. Immediately afterelectroporation, cells were transferred from the processingchamber into a 24-well plate and then cultured in RPMI-1640with 10% FBS, antibiotics, and 100 IU/mL IL-2.

Antibody binding, cell activation, conjugation, and cellproliferation assays

Tomeasure the chimeric receptors' antibody-binding capac-ity, T lymphocytes (5 � 105) transduced with chimeric recep-tors or a vector containing GFP only were incubated withrituximab (Rituxan, Roche; 0.1–1 mg/mL), trastuzumab (Her-ceptin; Roche; 0.1–1 mg/mL) or purified human IgG (R&DSystems; 0.1–1 mg/mL) for 30 minutes at 4�C. After washingtwice with PBS, cells were incubated with goat anti-human IgGconjugated to PE (Southern Biotechnology Associates, Bir-mingham, AL) for 10 minutes at room temperature and cellstaining was measured using an Accuri C6 flow cytometer (BDBiosciences).

To prepare immobilized antibody, we added 100 mL ofantibody (1 mg/mL) to the wells of a 96-well plate (Costar)that was then kept at 4�C for 16 hours; the plate was washedonce with PBS before use. Expression of IL-2 receptors wasmeasured by staining with anti-CD25 PE (BD Biosciences). Todetermine whether antibody binding to the receptor promotedcell aggregation, CD20 (MS4A1)-positive Daudi cells werelabeled with CellTrace calcein red-orange AM (Invitrogen) andthen incubated with rituximab (0.1 mg/mL) for 30 minutes at

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4�C. After washing twice in PBS, Daudi cells were mixed withJurkat cells transduced with the chimeric receptor or mock-transduced at 1:1 E:T ratio in 96 round bottom plates (Costar)for 60 minutes at 37�C. The proportion of cells formingheterologous cell aggregates (calcein AM-GFP double positive)was determined by flow cytometry.To measure cell proliferation, 1 � 106 of T cells transduced

with the chimeric receptor or mock-transduced were placed inthe wells of a 6-well plate (Costar) in RPMI-1640 with FBS,antibiotics and 50 IU/mL IL-2. Daudi, SK-BR-3, or NB1691 cellswere treatedwith Streck cell preservative (Streck Laboratories)to stop proliferation and labeled with rituximab, trastuzumab,or hu14.18K322A (gift of Dr. James Allay, Children's GMP,Memphis, TN; ref. 36), respectively (all at 0.1 mg/mL), for 30minutes at 4�C. Theywere added to thewells, at 1:1 ratio with Tcells, on days 0, 7, 14, and 21. Then, the number of viable T cellsafter culture was measured by flow cytometry.

CD107a degranulation and cytotoxicity assaysTodeterminewhether CD16 cross-linking caused exocytosis

of lytic granules, we placed chimeric receptor- and mock-transduced T cells (1 � 105) into each well of a rituximab-coated 96-well flat bottom plate and cultured cells for 4 hoursat 37�C. In some experiments, T cells were cocultured withDaudi cells preincubated with rituximab. Anti-human CD107aPE antibody (BD Biosciences) was added at the beginning ofthe cultures; one hour later GolgiStop (0.15mL; BDBiosciences)was added. CD107a-positive T cells were analyzed by flowcytometry.To test cytotoxicity, target cells were suspended in RPMI-

1640 with 10% FBS, labeled with calcein AM green fluores-cent (Invitrogen), and plated into 96-well round bottomplates (Costar). T cells were added at various E:T ratios asindicated in Results, and cocultured with target cells for 4hours, with or without the antibodies rituximab, trastuzu-mab, or hu14.18K322A at 0.1 mg/mL. In some experiments,we also used cells transduced with a chimeric receptor inwhich the hinge and transmembrane domains of CD8a, andthe signaling domains of 4-1BB and CD3z were combinedwith the single-chain variable region of anti-CD19 (10).Cells were then collected, resuspended in an identical vol-

ume of PBS, and propidium iodide was added. The number ofviable target cells (calcein AM-positive, propidium-iodide neg-ative) was counted using the Accuri C6 flow cytometer (33).Cytotoxicity tests with primary CLL cells were also performedafter 24 hours of coculture with immortalized bone marrow-derived mesenchymal stromal cells (37), as previouslydescribed (10); numbers of viable CLL cells after culture wereenumerated by flow cytometry after anti-CD19 PE (BD Bios-ciences) staining. Tomeasure cytotoxicity against the adherentcell lines NB1691, CHLA-255, SK-BR-3, MCF-7, U2-OS, andMKN7, we used their luciferase-labeled derivatives. After plat-ing for at least 4 hours, T cells were added as described above.After 4 hours of coculture, we added the Promega Bright-Gloluciferase reagent (Promega) to each well; 5 minutes later,luminescence was measured using a plate reader BiotekFLx800 (BioTek) and analyzed with the Gen5 2.0 Data Analysissoftware.

Xenograft experimentsDaudi cells expressing luciferase were injected intraperito-

neally (i.p.; 0.3 � 106 cells/mouse) in NOD.Cg-Prkdcscid

IL2rgtm1Wjl/SzJ (NOD/scid IL2RGnull) mice (Jackson Labora-tory). Some mice received rituximab (150 mg) i.p. Four daysafter Daudi inoculation, with or without intraperitoneal injec-tion of T cells on days 5 and 6. T cells had been activated withanti-CD3/CD28 beads for 3 days, transduced with the CD16V-BB-z receptor, resuspended in RPMI-1640 plus 10% FBS andthen injected at 1 � 107 cells per mouse. Rituximab injectionwas repeated weekly for 4 weeks, with no further T lymphocyteinjection. A group of mice received tissue culture mediuminstead of rituximab or T cells. All mice received intraperito-neal injections of 1,000–2,000 IU of IL-2 twice a week for 4weeks. Similar experiments were also performed with miceengrafted with NB1691 cells (i.p.; 0.3 � 106 cells/mouse) andtreated with hu14.18K322A (25 mg).

Tumor engraftment and growth was measured using aXenogen IVIS-200 system (Caliper Life Sciences). Imagingcommenced 5 minutes after intraperitoneal injection of anaqueous solution of D-luciferin potassium salt (3 mg/mouse)and photons emitted from luciferase-expressing cells werequantified using the Living Image 3.0 software.

ResultsExpression of the CD16V-BB-z receptor

The V158 polymorphism of FCRG3A (CD16) encodes ahigh-affinity immunoglobulin Fc receptor and is associatedwith favorable responses to antibody therapy (25, 26, 29–31).We generated a V158 variant of the FCGR3A gene andcombined it with the hinge and transmembrane domain ofCD8a, the T-cell stimulatory molecule CD3z, and the costi-mulatory molecule 4-1BB (Fig. 1A), which we previouslyfound to significantly enhance chimeric receptor–mediatedT-cell activation (10). We used an MCSV retroviral vectorcontaining the CD16V-BB-z construct and GFP to transduceperipheral blood T lymphocytes from 12 donors: medianGFP expression in CD3þ cells was 89.9% (range, 75.3%–97.1%); in the same cells, median chimeric receptor surfaceexpression as assessed by anti-CD16 staining was 83.0%(67.5%–91.8%; Fig. 1B). T lymphocytes from the same donorstransduced with a vector containing only GFP had a medianGFP expression of 90.3% (67.8%–98.7%) but only 1.0% (0.1%–2.7%) expressed CD16 (Fig. 1B). Expression of the receptordid not differ significantly between CD4þ and CD8þ T cells:69.8% � 10.8% CD4þ cells were CD16þ after transductionwith CD16V-BB-z, as compared with 77.6% � 9.2% CD8þ

cells (Supplementary Fig. S2).CD16V-BB-z –transduced T lymphocytes expressed CD3z at

levels much higher than those expressed by mock-transducedcells: mean (�SD) of the mean fluorescence intensity was45,985 � 16,365 in the former versus 12,547 � 4,296 in thelatter (P¼ 0.027 by t test; n¼ 3; Fig. 1B).We also determined thepresence of the chimeric protein by Western blotting probedwith an anti-CD3z antibody. CD16V-BB-z–transduced T lym-phocytes expressed a chimeric protein of approximately 25 kDaunder reducing conditions, in addition to the endogenousCD3zof 16 kDa; under nonreducing conditions, the CD16V-BB-z

A Universal Chimeric Receptor for T-Cell Therapy

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protein was shown to be expressed as either a monomer or adimer of 50 kDa (Fig. 1C).

Antibody-binding capacity of V158 versus F158 CD16receptors

To test the capacity of the CD16V-BB-z chimeric receptor tobind immunoglobulin (Ig), we transduced peripheral blood Tlymphocytes from 3 donors: CD16V-BB-z–expressing T lym-phocytes were coated with the antibody after incubation withrituximab (Fig. 2A). Similar results were obtained with tras-tuzumab and human IgG (not shown).

We then compared the Ig-binding capacity of the CD16V-BB-z receptor, which contained the high-affinity V158 poly-morphism of FCRG3A (CD16), to that of an identical receptorcontaining the F158 variant instead ("CD16F-BB-z"). Aftertransducing Jurkat cells with either receptor, we incubatedthem with rituximab and an anti-human Ig PE antibody(binding rituximab) and related the PE fluorescence intensityto that of GFP. As shown in Fig. 2B, at any given level of GFP,cells transduced with the CD16V-BB-z receptor had a higherPE fluorescence intensity than that of cells transducedwith theCD16F-BB-z receptor, indicating that the former had a signif-icantly higher antibody-binding affinity. Trastuzumab andhuman IgG were also bound by CD16V-BB-z receptors witha higher affinity (Supplementary Fig. S3).

To determine whether antibody binding to the CD16V-BB-zreceptor could promote aggregation of effector and target cells,we mixed Jurkat cells expressing CD16V-BB-z (and GFP) at a1:1 ratio with the CD20þ Daudi cell line (labeled with CalceinAM red-orange) for 60minutes, andmeasured the formation ofGFP-Calcein doublets with or without addition of rituximab. In3 experiments, 39.0% � 1.9% of events in the coculture were

doublets if Jurkat cells expressed CD16V-BB-z receptors andrituximab was present (Fig. 2C and D). In contrast, there wereless than 5% doublets with human IgG instead of rituximab, orwithmock-transduced Jurkat cells regardless of whether ritux-imab was present.

Binding of Ig to CD16V-BB-z induces T-cell activation,degranulation, and cell proliferation

We assessed whether CD16V-BB-z receptor cross-linking byan immobilized antibody could induce activation signals in Tlymphocytes. Indeed, T lymphocytes transduced with CD16V-BB-z markedly increased IL-2 receptor expression (CD25)when cultured on plates coated with rituximab, whereas nochanges were detected in the absence of antibody, or in mock-transduced cells regardless of whether the antibody was pres-ent (Fig. 3A and B).

In addition to expression of IL-2 receptors, CD16V-BB-zreceptor cross-linking triggered exocytosis of lytic granules inT lymphocytes, as detected by CD107a staining. Thus, in 6experiments in which T lymphocytes from 4 donors were eitherseeded onto microtiter plates coated with rituximab (n ¼ 3) orcoculturedwithDaudicells in thepresenceofrituximab(n¼3),TlymphocytesexpressingCD16V-BB-zbecameCD107aþ (Fig.3C).

T lymphocytes expressing CD16V-BB-z expanded in thepresence of rituximab and Daudi cells (at a 1:1 ratio with Tlymphocytes): in 3 experiments, mean T-cell recovery after 7days of culture was 632% (�97%) of input cells; after 4 weeks ofculture, it was 6,877% (�1,399%; Fig. 3D). Of note, unboundrituximab, even at a very high concentration (1–10mg/mL), hadno significant effect on cell proliferation in the absence oftarget cells, and no cell growth occurred without rituximab, orin mock-transduced T cells regardless of the presence of the

Figure 1. Expression of CD16V-BB-z receptors in T cells. A, schematicrepresentation of the CD16V-BB-zreceptor construct. B, flowcytometric dot plots illustrateexpression of CD16 (B73.1antibody) in combination with GFPorCD3z in activatedT lymphocytestransduced with a vectorcontaining GFP alone (Mock) orGFPandCD16V-BB-z. Percentageof positive cells in each quadrant isshown. C, Western blotting of celllysates from T lymphocytestransduced with GFP alone orCD16V-BB-z, probed with ananti-CD3z antibody.

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antibody and/or target cells (Fig. 3D). Thus, CD16V-BB-zreceptor cross-linking induces signals that result in sustainedproliferation.

T lymphocytes expressing CD16V-BB-zmediate ADCC invitro and in vivoThe observation that CD16V-BB-z cross-linking provoked

exocytosis of lytic granules implied that CD16V-BB-z T lym-phocytes should be capable of killing target cells in the presenceof specific antibodies. Indeed, in 4-hour in vitro cytotoxicityassays, CD16V-BB-z T lymphocytes were highly cytotoxicagainst the B-cell lymphoma cell lines Daudi and Ramos in thepresence of rituximab: more than 50% target cells were typicallylysed after 4 hours of coculture at a 2:1 E:T ratio (Fig. 4A andSupplementary Fig. S4). In contrast, target cell killing was low inthe absence of the antibody or with mock-transduced T cells(Fig. 4A and Supplementary Fig. S4). Notably, the effector cellsused in these experiments were highly enriched with CD3þ Tlymphocytes (>98%) and contained no detectable CD3� CD56þ

NK cells. Rituximab-mediated cytotoxicity of CD16V-BB-z Tlymphocytes was also clear with CD20þ primary CLL cells (n¼5); cytotoxicity typically exceeded 70% after 4 hours of cocultureat a 2:1 E:T ratio (Fig. 4B). Bone marrow mesenchymal stromalcells have been shown to exert immunosuppressive effects (38,39). To test whether this would affect the cytotoxic capacity ofCD16V-BB-z T lymphocytes, we cocultured themwith CLL cells

in the presence of bone marrow-derived mesenchymal stromalcells for 24 hours at a 1:2 E:T. As shown in Fig. 4C, mesenchymalcells did not diminish the killing capacity of the ADCC-medi-ating lymphocytes.

Next, we determined whether different immunotherapeuticantibodies could trigger similar cytotoxicities against tumorcells expressing the corresponding antigen. Thus, we tested thecytotoxicity of CD16V-BB-zT lymphocytes against solid tumorcells expressing HER2 (the breast cancer cell lines MCF-7 andSK-BR-3 and the gastric cancer cell line MKN7) or GD2 (theneuroblastoma cell lines CHLA-255, NB1691 and SK-N-SH, andthe osteosarcoma cell line U2-OS). We used the antibodiestrastuzumab to target HER2 and hu14.18K322A to target GD2.CD16V-BB-z T lymphocytes were highly cytotoxic againstthese cells in the presence of the corresponding antibody (Fig.4A and Supplementary Fig. S4). In experiments with NB1691,we also tested whether cytotoxicity could be achieved at evenlower E:T ratios by prolonging the culture to 24 hours. Cyto-toxicity exceeded 50% at 1:8 ratio in the presence ofhu14.18K322A (Supplementary Fig. S5A). To further test thespecificity of the CD16V-BB-z –mediated cell killing, we cul-tured the CD20þ Daudi cells with CD16V-BB-z T lymphocytesand antibodies of different specificity: only rituximabmediatedcytotoxicity, while there was no increase in cytotoxicity in thepresence of trastuzumab or hu14.18K322A (SupplementaryFig. S5B). Importantly, CD16V-BB-z-mediated cell killing

Figure 2. Antibody-binding capacity of CD16V-BB-z receptors. A, T lymphocytes transduced with a vector containing GFP (Mock) or GFP and CD16V-BB-zwere incubatedwith rituximab for 30minutes; the amount of antibody boundwas visualizedwith a goat-anti human IgG antibody conjugated to phycoerythrin(GAH IgG) and flow cytometry. B, Jurkat cells transduced with CD16V-BB-z (V158) or CD16F-BB-z (F158) were incubated with rituximab for 30 minutes.The plot compares the relation between mean fluorescence intensity (MFI) of GFP and MFI of GAH IgG obtained with cells expressing the tworeceptors. C, mock- or CD16V-BB-z–transduced Jurkat cells were cocultured with Daudi cells labeled with calcein AM orange-red in the presence ofrituximab. Cell aggregates are quantified in the top right quadrants of each dot plot. D, summary of the aggregation assays illustrated in C (mean � SDof 3 experiments). Aggregation measured with Jurkat cells transduced with CD16V-BB-z in the presence of rituximab (Ab) was significantly higher than thatmeasured in the three other culture conditions (P < 0.001 by t test).






with immunotherapeutic antibodies was not inhibited bythe presence of unbound monomeric IgG, even if this waspresent at a concentration 1,000 times higher than that ofthe cell-bound immunotherapeutic antibody (SupplementaryFig. S5C).

To gauge the antitumor capacity of CD16V-BB-z T lym-phocytes in vivo, we performed experiments with NOD/scidIL2RGnull mice engrafted with luciferase-labeled Daudicells. We measured tumor growth by live imaging in micereceiving CD16V-BB-z T lymphocytes plus rituximab, andcompared their outcome to mice receiving either rituximabor T lymphocytes alone, or no treatment. Tumor cellsexpanded in all mice except those that received rituximabfollowed by CD16V-BB-z T lymphocytes (Fig. 5). All 5 micetreated with this combination were still in remission morethan 120 days after tumor injection, in contrast to 0 of 12mice that were untreated or received antibody or cells alone.A strong antitumor activity was also observed in miceengrafted with the neuroblastoma cell line NB1691 andtreated with hu14.18K322A and CD16V-BB-z T lymphocytes(Supplementary Fig. S6).

Comparison of CD16V-BB-z with other receptorsIn linewith their higher affinity for Ig, CD16V-BB-z receptors

induced significantly higher T cell proliferation andADCC than

that triggered by the lower affinity CD16F-BB-z receptors(Supplementary Fig. S7).

Next, we compared the function of T cells bearing CD16V-BB-z with that of T cells expressing other receptors withdifferent signaling properties. These included a receptor withno signaling capacity (CD16V-truncated), one with CD3z butno 4-1BB (CD16V-z), and a previously described receptor thatcombined CD16V with the transmembrane and cytoplasmicdomains of FceRIg (CD16V-FceRIg ; ref. 40; Supplementary Fig.S1). After retroviral transduction in activated T cells, all recep-tors were highly expressed (Supplementary Fig. S8). CD16V-BB-z induced significantly higher activation, proliferation, andspecific cytotoxicity than all other constructs (Fig. 6).

Finally, we determined how ADCC exerted by T cells expres-sing CD16V-BB-z compared with the cytotoxicity of T cellsexpressing a chimeric antigen receptor. For this purpose, wetransduced T cells with an anti-CD19 chimeric receptor ("anti-CD19-BB-z") composed by an anti-CD19 ScFv and transmem-brane and signaling domains identical to those of CD16V-BB-z(10). T cells expressing this receptor were tested against Daudicells (which express high levels of CD19 in addition to CD20) inparallel to T cells expressing CD16V-BB-z in the presence ofrituximab. Under these conditions, CD16V-BB-z cells exertedcytotoxicity thatwas higher than that of anti-CD19-BB-zT cells(Supplementary Fig. S9).

Figure 3. Immunoglobulin binding to CD16V-BB-z receptors induces T-cell activation, exocytosis of lytic granules, and cell proliferation. A, T lymphocytestransduced with a vector containing GFP (Mock) or GFP and CD16V-BB-z were cultured in microtiter plates coated with rituximab for 48 hours without IL-2;expression of CD25 was measured by flow cytometry. B, summary of the results of the test illustrated in A; bars show CD25 expression in GFPþ cells(mean � SD of experiments with T cells from 3 donors); CD25 expression was significantly higher in T lymphocytes transduced with CD16V-BB-z in thepresence of rituximab (Ab) than in the other experimental conditions (P � 0.003). C, T lymphocytes from 4 donors transduced with a vector containingGFP (mock) or GFP and CD16V-BB-z were cultured as in A (n ¼ 3) or with Daudi cells (n ¼ 3) for 4 hours; CD107a staining was measured by flowcytometry (mean� SD of the 6 experiments); CD107a expression was significantly higher in T lymphocytes transduced with CD16V-BB-z in the presence ofrituximab (Ab) than in the other conditions (P < 0.0001). D, mock- or CD16V-BB-z-transduced T lymphocytes were cultured alone, or with rituximabwith or without Daudi cells for up to 4 weeks. Symbols indicate percentage of cell recovery as compared with the number of input cells (mean � SD ofexperiments with T cells from three donors).

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Expression of CD16V-BB-z receptors by mRNAelectroporationIn all the above experiments, CD16V-BB-z expression was

enforced by retroviral transduction. We tested whether analternative method, electroporation of mRNA, could also con-fer ADCC capacity to T lymphocytes. We electroporated acti-vated T lymphocytes from 2 donors and obtained high expres-sion efficiencies: 55% and 82% of T lymphocytes becameCD16þ 24 hours after electroporation (Fig. 7A). In the seconddonor, receptor expression was also tested on day 3, when itwas 43%, a result similar to those of previous experiments withanother receptor where expression persisted for 72 to 96 hours(35). ADCC was activated in T lymphocytes electroporatedwith CD16V-BB-z mRNA: in the presence of rituximab, 80%Ramos cells were killed after 4 hours at a 2:1 E:T ratio, whereascells electroporated without mRNAs were ineffective (Fig. 7B).

DiscussionWe developed a chimeric receptor that endows T lympho-

cytes with the capacity to exert ADCC. When the CD16V-BB-z

receptor is engaged by an antibody bound to tumor cells, ittriggers T-cell activation, sustained proliferation, and specificcytotoxicity against cancer cells targeted by antibody. CD16V-BB-z T lymphocytes were highly cytotoxic against a wide rangeof tumor cell types, including B-cell lymphoma, breast andgastric cancer, neuroblastoma and osteosarcoma, as well asprimary CLL cells. Cytotoxicity was entirely dependent on thepresence of a specific antibody bound to target cells; unboundantibodies did not provoke nonspecific cytotoxicity nor affectedcytotoxicitywith cell-boundantibodies. CD16V-BB-zTcells alsokilled CLL cells when these were cultured on mesenchymal celllayers, regardless of the known immunosuppressive effects ofthis microenvironment (38, 39). Moreover, CD16V-BB-z T lym-phocytes infused after rituximab eradicated B-cell lymphomacells engrafted in immunodeficient mice, and had considerableantitumor activity inmice engraftedwithneuroblastomacells inthe presence of an anti-GD2 antibody. In sum, T cells expressingCD16V-BB-z effected strong ADCC in vitro and in vivo.

The affinity of CD16 for the Fc portion of Ig is a criticaldeterminant of ADCC and, thus, influences clinical responses

Figure 4. ADCC mediated by CD16V-BB-z T lymphocytes in vitro. A, representative examples of cytotoxicity against cancer cell lines mediated by mock- orCD16V-BB-z-transduced T lymphocytes in the presence of the corresponding antibody. Each symbol indicates the mean of triplicate cultures (P < 0.01 bypaired t test for all 3 comparisons). The full set of data is shown in Supplementary Fig. S4. B, cytotoxicity ofmock- or CD16V-BB-z-transducedT lymphocytes,with or without rituximab (Ab), against primary cells from patients with chronic lymphocytic leukemia (CLL). Each bar (with a different shade for eachpatient) corresponds tomean (�SD) cytotoxicity as determined in triplicate 4-hour assays at 2:1 E:T ratio. Cytotoxicity with CD16V-BB-z T cells and antibodywas significantly higher than that measured in any of the other three conditions (P < 0.0001 by t test); with mock-transduced T cells, the addition ofantibody increased cytotoxicity (P ¼ 0.016); all other comparisons, P > 0.05. C, cytotoxicity against the same CLL samples tested in B after 24 hoursat 1:2 E:T in the presence of mesenchymal stromal cells (MSC). Each bar corresponds to the average of two tests. Cytotoxicity with CD16V-BB-z T cells plusantibodywas significantly higher than thatwith antibody alone (P¼0.0002) or cells alone (P < 0.0001); cytotoxicitywith antibody alonewas significantly higherthan that with cells alone (P ¼ 0.0045).






to antibody immunotherapy. Hence, considerable efforts arebeing made to further enhance the affinity of Fc fragments forFcgR, for example, by glycoengineering (23, 41). To constructour receptor, we selected the FCRG3A (CD16) gene with the158V polymorphism. This variant encodes a receptor withhigher binding affinity for Ig and has been shown to mediatesuperior ADCC (18, 24–32). Indeed, in side-to-side compar-isons with a chimeric receptor containing the more commonF158 variant, the CD16V-BB-z had a significantly higher capac-ity to bind human Ig Fc, and induced more vigorous prolifer-ation and cytotoxicity, evoking results of recent studies addres-sing the role of affinity in chimeric antigen receptor function(42, 43). Clemenceau and colleagues described a receptorcontaining the CD16 158V combined with the transmembraneand cytoplasmic domains of FceRIg , and found that selected T-cell clones expressing this receptor could lyse Epstein–Barrvirus derived B-lymphoblastoid cells in the presence of ritux-imab (40). Current "second generation" chimeric receptorscombine a stimulatory molecule with a costimulatory one toaugment signaling and prevent activation-induced apoptosis.Therefore, we combined CD16 V158 with a stimulatory molec-ular tandem constituted by CD3z and 4-1BB (CD137). Wepreviously found the addition of the costimulatory molecule4-1BB to CD3z significantly improved the expansion andcytotoxicity of T lymphocytes when incorporated into a chi-

meric receptor directed against CD19 (10). This was also thecase with the CD16V-BB-z receptor, which induced amarkedlysuperior T-cell activation, proliferation, and cytotoxicity thandid receptors acting through CD3z alone, or of FceRIg . Of note,in side-by-side cytotoxicity assays targeting cells expressingboth CD19 and CD20, CD16V-BB-z plus rituximab was moreeffective than a receptor with identical signaling properties butbinding directly to CD19.

The clinical potential of genetically modified T cells expres-sing receptors that recognize antigens of the surface of tumorcells and can transduce stimulatory signals is being increas-ingly demonstrated by results of clinical trials (11–16). Mostnotably, significant tumor reductions and/or complete remis-sions have been reported in patients with B-cell malignancieswho received autologous T lymphocytes expressing chimericantigen receptors against CD19 or CD20 by viral transduction(12–16). Expanding this strategy to other tumors involvesconsiderable effort, including the development of anotherchimeric antigen receptor construct, and the optimization oflarge-scale transduction conditions in compliance with regu-latory requirements. In this regard, the receptor described inthis study should facilitate the implementation of T-cell ther-apy by allowing one single receptor to be used for multiplecancer cell types. It should also allow the targeting of multipleantigens simultaneously, a strategy that may ultimately be

Figure 5. T lymphocytes expressing CD16V-BB-z receptors exert antitumor activity in vivo. NOD-SCID-IL2RGnull mice were injected intraperitoneallywith 3� 105 Daudi cells labeled with luciferase. Rituximab (150 mg) was injected intraperitoneally once weekly for 4 weeks starting on day 4. In four mice, noother treatment was given, whereas in five other mice, the first rituximab injection was followed by T lymphocytes expressing CD16V-BB-z receptors(1 � 107 i.p.; n ¼ 5) on days 5 and 6; the other two groups of four mice each received CD16V-BB-z T lymphocytes preceded by intraperitonealinjection of RPMI-1640 instead of rituximab or intraperitoneal injection of RPMI-1640 medium only (Control). A, results of in vivo imaging of tumor growth.Each symbol corresponds to one bioluminescence measurement; lines connect all measurements in one mouse. B, representative mice (2 per group)for each experimental condition. Ventral images on day 3 were processed with enhanced sensitivity to demonstrate the presence of tumors in mice of theCD16V-BB-zþ rituximab group. Mice were euthanized when bioluminescence reached 5� 1010 photons/second. C, overall survival comparisons of mice inthe different treatment groups.

Kudo et al.





advantageous given immunoescape mechanisms exploited bytumors, as illustrated by the recent report of a leukemia relapsedriven by a subclone lacking themarker targeted by a chimericreceptor with single specificity (44). Antibody-directed cyto-

toxicity could be stopped whenever required by simple with-drawal of antibody administration. Because the T cells expres-sing CD16V-BB-z are only activated by antibody bound totarget cells, soluble immunoglobulin should not exert any

Figure 6. CD16V-BB-z induces higher T-cell activation, proliferation, and cytotoxicity than CD16V receptors with different signaling properties. A, CD25meanfluorescence intensity (MFI) by flow cytometry plotted against green fluorescent protein (GFP) MFI in T lymphocytes expressing different chimeric receptorsafter 48-hour coculture with Daudi cells and rituximab (0.1 mg/mL). CD25 expression with CD16V-BB-z was significantly higher than that triggered byCD16V-z, CD16V-FceRIg , or CD16V with no signaling capacity (CD16V-trunc.; P < 0.0001). B, T lymphocytes transducedwith various CD16V receptors werecultured with Daudi, SK-BR-3, or NB1691 cells in the presence of rituximab, trastuzumab, and hu14.18K322A, respectively (all at 0.1 mg/mL). Symbolsindicate percentage of cell recovery as compared with number of input cells (mean � SD of 3 experiments); cell counts for weeks 1–3 of culture weresignificantly higher with CD16V-BB-z receptors than with all other receptors by paired t test for all 3 cultures (P < 0.0001). C, 4-hour ADCC of T lymphocytesexpressing various CD16V receptors or mock-transduced T cells against Daudi, SK-BR-3, and NB1691 in the presence of rituximab, trastuzumab,and hu14.18K322A, respectively. Symbols aremean�SDof triplicate cultures at the E:T shown. Cytotoxicities withCD16V-BB-z receptors were significantlyhigher than thosewith all other receptors (P < 0.0001 by t test in all comparisons), whereas cytotoxicities of lymphocytesmock-transduced or transducedwiththe CD16V-truncated receptor were not significantly different (P > 0.05) from each other; cytotoxicity with CD16V-FceRIg was significantly higher thanthat with CD16-z against Daudi (P ¼ 0.006) and SK-BR-3 (P ¼ 0.019); lymphocytes expressing either receptor had higher cytotoxicities than thosemock-transduced or transduced with CD16V-truncated (P < 0.01 for all comparisons).

Figure 7. Expression ofCD16V-BB-z receptors by mRNAelectroporation. A, activated Tlymphocytes were electroporatedwith CD16V-BB-z mRNA orwithout mRNA (mock); expressionof CD16 was tested 24 hours laterby flow cytometry. B, cytotoxicityof mock or CD16V-BB-zelectroporated T cells was testedagainst the Ramos cell line in thepresence of rituximab. Symbolsshow mean � SD percentagecytotoxicity (n ¼ 3; P < 0.001).






stimulation on the infused T cells. Nevertheless, we suggestthat it would be important to test the clinical safety of thisstrategy by usingmRNA electroporation to express the CD16V-BB-z receptor transiently, thus limiting any potential autoim-mune reactivity. In our study, we found that mRNA electro-poration can express the receptor very effectively.

Antibody therapy has become standard-of-care for manycancer subtypes; its clinical efficacy ismostly determined by itscapacity to trigger ADCC through the engagement of Fcreceptors (18, 45). The main effectors of ADCC are NK cellsbut their function can be impaired in patients with cancer. Forexample, it was reported that trastuzumab-mediated ADCC ofgastric cancer cells overexpressing HER2 was significantlylower with peripheral blood mononuclear cells from patientswith gastric cancer and advanced disease as compared withthat obtained with samples from patients with early diseaseor healthy donors (46). Moreover, responses are likely to beinfluenced by other factors, including the genotype of NK cellinhibitory receptors and their ligands (47). The results of ourstudy suggest that the infusion of autologous T cells geneticallyengineered with the CD16V-BB-z receptor should significantlyboost ADCC. Because the combinedCD3z/4-1BB signaling alsocauses T-cell proliferation, there should be an accumulation ofactivated T cells at the tumor site, which may further poten-tiate their activity. CD16V-BB-z receptors can be expressed bymRNA electroporation not only in activated T lymphocytes butalso in resting peripheral bloodmononuclear cells, a procedure

that would take only a few hours from blood collection toinfusion of CD16V-BB-z–expressing cells and is therefore wellsuited for clinical application.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: K. Kudo, D. CampanaDevelopment of methodology: K. Kudo, C. Imai, P. Lorenzini, K. Kono, A.M.Davidoff, D. CampanaAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Kudo, T. Kamiya, W.J. ChngAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K. Kudo, D. CampanaWriting, review, and/or revision of the manuscript: K. Kudo, C. Imai, P.Lorenzini, T. Kamiya, K. Kono, A.M. Davidoff, W.J. Chng, D. CampanaAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): P. Lorenzini, T. Kamiya, K. Kono, A.M.DavidoffStudy supervision: D. Campana

Grant SupportThis work was supported by the American Lebanese Syrian Associated

Charities (ALSAC) and by a Singapore Translational Research InvestigatorAward from the National Medical Research Council of Singapore.

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 May 8, 2013; revised September 17, 2013; accepted October 3, 2013;published OnlineFirst November 6, 2013.

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2014;74:93-103. Published OnlineFirst November 6, 2013.Cancer Res Ko Kudo, Chihaya Imai, Paolo Lorenzini, et al. Antibody-Dependent Cancer Cell KillingT Lymphocytes Expressing a CD16 Signaling Receptor Exert

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