Memory T Cells Expressing an NKG2D-CAR Ef ciently Target … · Cancer Therapy: Preclinical Memory T Cells Expressing an NKG2D-CAR Efﬁciently Target Osteosarcoma Cells Lucía Fernandez1,2,

Cancer Therapy: Preclinical

Memory T Cells Expressing an NKG2D-CAREfficiently Target Osteosarcoma CellsLucía Fern�andez1,2, Jean-Yves Metais3, Adela Escudero2,4, María Vela2,Jaime Valentín2, Isabel Vallcorba4, Alejandra Leivas1,5, Juan Torres6,7,Antonio Valeri5, Ana Pati~no-García8, Joaquín Martínez1,5,Wing Leung9, andAntonio P�erez-Martínez2,10

Abstract

Purpose: NKG2D ligands (NKG2DL) are expressed on varioustumor types and immunosuppressive cells within tumor micro-environments, providing suitable targets for cancer therapy. Var-ious immune cells express NKG2D receptors, including naturalkiller (NK) cells and CD8þ T cells. Interactions between NKG2DLand NKG2D receptors are essential for NK-cell elimination ofosteosarcoma tumor-initiating cells. In this report, we usedNKG2D–NKG2DL interactions to optimize an immunotherapeu-tic strategy against osteosarcoma. We evaluated in vitro and in vivothe safety and cytotoxic capacity against osteosarcoma cells ofCD45RA� memory T cells expressing an NKG2D-4-1BB-CD3zchimeric antigen receptor (CAR).

Experimental Design: CD45RA� cells from healthy donorswere transduced with NKG2D CARs containing 4-1BB and CD3zsignaling domains. NKG2DCAR expressionwas analyzed by flowcytometry. In vitro cytotoxicity of NKG2D-CARþ CD45RA� T cellsagainst osteosarcoma was evaluated by performing conventional

4-hour europium-TDA release assays. For the in vivo orthotopicmodel, 531MII YFP-luc osteosarcoma cells were used as targets inNOD-scid IL2Rgnull mice.

Results: Lentiviral transduction of NKG2D-4-1BB-CD3zmarkedly increasedNKG2D surface expression in CD45RA� cells.Genetic stability was preserved in transduced cells. In vitro,NKG2D-CARþ memory T cells showed significantly increasedcytolytic activity than untransduced cells against osteosarcomacell lines, while preserving the integrity of healthy cells. NKG2D-CARþ memory T cells had considerable antitumor activity in amouse model of osteosarcoma, whereas untransduced T cellswere ineffective.

Conclusions: Our results demonstrate NKG2D-4-1BB-CD3zCAR–redirected memory T cells target NKG2DL-expressing oste-osarcoma cells in vivo and in vitro and could be a promisingimmunotherapeutic approach for patients with osteosarcoma.Clin Cancer Res; 23(19); 5824–35. �2017 AACR.

IntroductionOsteosarcoma is the most frequently observed malignant pri-

mary bone tumor, typically affecting children andadolescents (1).The long-term survival rate for the nonmetastatic type varies from65% to 70%. However, current combined surgical and neoadju-

vant chemotherapy are inefficient for themetastatic type, inwhichthe 5-year survival rate is less than 20% (2). The rapid develop-ment ofmetastatic lesions and resistance to chemotherapy are themajor causes of treatment failure and poor survival rates. Over thepast few decades, efforts have beenmade to intensify dosing, varytiming, and to use multicombinational chemotherapy; however,no substantial improvement in survival has been achieved. Inaddition, several adverse effects accompany high-dose chemo-therapy (3). Therefore, the development of alternative noveltherapies is essential.

Immune system effector cells have the potential to attack andeliminate cancer cells. Primary and metastatic osteosarcoma cellsheterogeneously express ligands for NKG2D receptor (4, 5). In aprevious study, we demonstrated the efficacy of natural killer(NK) cell–based immunotherapies against osteosarcoma in pre-clinical studies (5). We observed that activated and expanded NKcells (NKAE) target osteosarcoma tumor-initiating cells (TIC)using NKG2D receptor and NKG2D ligand (NKG2DL) interac-tions, both in vitro and in an orthotopic osteosarcoma murinemodel. We reported that exposing osteosarcoma cells to NKAEcells in vitro decreased both their capacity to form sarcospheresand their TIC compartment. In an in vivo model, NKAE cellsdelayed tumor growth and inhibited development of lungmetas-tases (5). The preclinical success of this immunotherapeuticapproach, the importance of NKG2D-NKG2DL interactions andthe clinical limitations of NK-based therapies that we and others

1Haematological Malignancies H12O, Clinical Research Department, SpanishNational Cancer Research Centre (CNIO), Madrid, Spain. 2Translational Researchin Pediatric Oncology, Hematopoietic Transplantation and Cell Therapy, IdiPAZ,Hospital Universitario La Paz, Madrid, Spain. 3Haematology Department, St.Jude Children's Research Hospital, Memphis, Tennessee. 4Paediatric OncologyDepartment, Instituto de Gen�etica M�edica y Molecular (INGEMM), HospitalUniversitario La Paz, Madrid, Spain. 5Haematology Department, Hospital Uni-versitario 12 de Octubre, Madrid, Spain. 6Department of Clinical Immunology,Hospital Universitario La Paz, Madrid, Spain. 7Physiopathology of Lymphocytesin Immunodeficiencies Group, IdiPAZ, Hospital Universitario La Paz, Madrid,Spain. 8Paediatrics Laboratory, Universidad de Navarra, Pamplona, Spain. 9Clin-ical Development, Miltenyi Biotec, Cambridge, Massachusetts. 10PaediatricHemato-Oncology, Hospital Universitario La Paz, Madrid, Spain.

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

Corresponding Author: Antonio P�erez-Martínez, Hospital Universitario La Paz,Paseo de la Castellana, 261, Madrid 28046, Spain. Phone: 349-1727-7223; Fax:349-1727-7042; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-0075

�2017 American Association for Cancer Research.

ClinicalCancerResearch

Clin Cancer Res; 23(19) October 1, 20175824

on May 21, 2021. © 2017 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst June 28, 2017; DOI: 10.1158/1078-0432.CCR-17-0075

http://crossmark.crossref.org/dialog/?doi=10.1158/1078-0432.CCR-17-0075&domain=pdf&date_stamp=2017-9-7

http://clincancerres.aacrjournals.org/

have observed (the need to infuse large numbers of cells, disper-sion of therapy, absence of memory, limited in vivo expansion;refs. 6–8) encouraged us to explore the efficacy of an NKG2Dchimeric antigen receptor (CAR) to target osteosarcoma.

CARs are artificial receptors that combine the specific recogni-tion of tumor-associated antigens (TAA) with T-cell–activatingsignals (9). CAR specificity is typically based on antibody single-chain fragment variable (scFv) regions or T-cell receptor (TCR)–binding domains; however, NK-cell receptors have also been used(10–15). The NKG2D receptor and its ligands have attractedconsiderable interest as a potential strategy to attack tumor cellsas NKG2DL are expressed on most types of tumor cells and theydemonstrate relative selectivity of ligand expression on tumorcells compared with healthy cells (16). Endogenous NKG2Dreceptors recognize NKG2DL expressed on healthy tissues with-out triggering an immune response.

CARs are usually expressed on autologous T cells; however, NKcells (12, 17) or different T-cell subsets (18, 19) can also be used.Most clinical trials use autologous T cells to express CARs; how-ever, T cells from some patients fail to grow, rendering thisapproach useless for patients with rapid disease progression.Allogeneic T cells expressing CARs have also been infused intopatients after a hematopoietic stem cell transplant (HSCT) fromthe same healthy donor (20, 21). However, the universal avail-ability of large numbers of healthy donor T cells to express CARsand their infusion into patientswithout the requirement of a priorHSCTwould bemajor challenges of CAR–T-cell immunotherapy.One potential complication of the use of allogeneic T cells is theundesirable graft versus host (GvH) reactions. To avoid GVHeffects, T cells without a reactive T-cell receptor (TCR) can beused. One method to enrich for non-allogeneic T cells is byusing antigen-experienced memory T cells for CAR transduc-tion. One of the markers that can distinguish memory fromna€�ve T cells is CD45RA. CD45RA is expressed on na€�ve T cells,whereas CD45RO is expressed on memory T cells (22).CD45RAþ na€�ve T cells have high potential for alloreactivity

against recipient-specific antigens upon adoptive transfer, caus-ing clinical graft versus host disease (GvHD; refs. 23, 24). Incontrast, CD45RA�CD45ROþ T cells exert a memory responseto prior pathogens or vaccines and can mediate graft-versus-tumor effects without inducing GvHD (25, 26).

Although CAR-T therapies have shown remarkable clinicalresponses, they can also over activate the immune system andcause harm. One life-threatening complications of CAR T-celltherapies is cytokine release syndrome (CRS) that is related to T-cell activation and expansion. To diminish the risk of CRS, wepropose to use memory T cells to express an NKG2D CAR. This T-cell subpopulation has been demonstrated tomaintain allogeneicT-cell immunity producing less CRS and to lack alloreactiveresponse, mild "GvHD" like (27).

In this report, we provide preclinical data to support ourhypothesis that CD45RA� memory T cells expressing anNKG2D-4-1BB-CD3z CAR could be effective and safe for thetreatment of osteosarcoma. We found that NKG2D CAR-redir-ected CD45RA� cells lysed osteosarcoma cells in vitro, and theyreduced tumor burden and prolonged survival in mouse modelsof metastatic osteosarcoma. Furthermore, they had no GvHDactivity and showed no lytic activity against healthy tissues.

Materials and MethodsOsteosarcoma cells

The 531MII primary cell linewas provided byDr. Pati~no-García(University Clinic of Navarra, Spain) and was cultured in mini-mum essential medium (GIBCO, 22571-020) supplementedwith 10% heat-inactivated FBS (GIBCO, 10270-098) and peni-cillin-streptomycin (P/S; GIBCO, 15140-122). TheMG-63 (ATCCCRL-1427) and U-2 OS (ATCC HTB-96) osteosarcoma cell lineswere purchased from the ATCC. The MG-63 cells were cultured inEagle minimum essential medium (EMEM; ATCC, 30-2003) andU-2 OS cells were cultured in McCoy 5A Medium (ATCC,30-2007), both supplemented with 10% FBS and P/S. For thein vivo experiments, 531MII cells were transduced with gammar-etroviral vector containing MSCV-luciferase-IRES-YFP (VectorLaboratory). All cell lines were routinely tested for Mycoplasmaby using the LookOut Mycoplasma PCR Detection Kit (MP0035-1KT) and Jump Start Taq Polymerase (D9307), both from Sigma-Aldrich, following the manufacturer's instructions.

Healthy primary cells and cell linesPeripheral bloodmononuclear cells (PBMC) andbonemarrow

samples were collected from healthy donors with approval fromthe St Jude Children's Research Hospital, Hospital La Paz andHospital 12 de Octubre institutional review boards and writteninformed consent from the donors. Hematopoietic progenitors(CD34þ cells) were sorted by labeling of cells with anti-CD34(Supplementary Table S1) and a FACS AriaFusion flow cytometer(BDBiosciences). Hs 1.Int (ATCC, CRL7820) normal fetal humanintestine fibroblasts and CCD-18 Co (ATCC, CRL1459) normalhuman colon fibroblasts were purchased from ATCC. Hs 1.Intcells were cultured in EMEM and 10% FBS. CCD-18 Co cells werecultured in DMEM (GIBCO 61965-026) and 10% FBS. NuLi-1(ATCC, CRL-4011) normal human lung epithelial cells werekindly provided by Dr. Paz-Ares (Hospital 12 de Octubre), andcultured in a serum-free medium: Airway Epithelial Cell BasalMedium (ATCC, PCS-300-030) supplemented with BronchialEpithelial Cell Growth Kit (ATCC, PCS-300-040) additives.

Translational Relevance

Chimeric antigen receptors (CAR) are being increasinglyconsidered as anticancer treatment. We previously demon-strated an essential role of NKG2D receptor and NKG2Dligand interactions for NK-cell elimination of osteosarcomatumor-initiating cells. In an effort to develop new therapies forpatients with osteosarcoma, we tested the cytotoxic ability ofCD45RA�memory T cells expressing anNKG2D-4-1BB-CD3zCAR in vitro and in anorthotopic osteosarcomamurinemodel.We found that NKG2D-4-1BB-CD3z–expressing CD45RA�

memory T cells had enhanced cytotoxicity against osteosar-coma cells in vitro compared with untransduced T cells. In vivo,mice receiving NKG2D CAR-redirected memory T cellsshowed limited tumor growth and prolonged survival. Inaddition, NKG2D CAR–redirected memory T cells showed nolytic activity against healthy cells, and no chromosomal aber-rations due to lentiviral transduction. Hence, we provideevidence to support our hypothesis that NKG2D CAR–redirected memory T cells are effective and safe for osteosar-coma control.

NKG2D CAR Memory T Cells for Osteosarcoma Treatment

www.aacrjournals.org Clin Cancer Res; 23(19) October 1, 2017 5825




Cell isolation and vector productionFor the depletion of CD45RAþ cells, PBMCs were labeled with

CD45RAmicrobeads (Miltenyi Biotec, 130-045-901) and deplet-ed using CliniMACS or AutoMACs (both from Miltenyi Biotec)following themanufacturer's instructions. T cells were isolated byusing the Pan T-cell Isolation Kit (Miltenyi Biotec, 130-095-535)following the manufacturer's instructions and AutoMACs device.The purity of the CD3-positive and CD45RA-negative fractionswas always �99%, as determined by flow cytometry. TheHL20i4r-MNDantiCD19bbz lentiviral vectors were derived fromthe clinical vector CL20i4r-EF1a-hgcOPT27 but expressed anNKG2D CAR. The anti–CD19-41BB-CD3z CAR designed by Imaiand colleagues (28) was used as backbone to build the NKG2DCAR construct. It contained the extracellular domain of NKG2D(designed byWai-Hang Leung andWing Leung), the hinge regionof CD8a and the signaling domains of 4-1BB and CD3Z. Thecassette was driven by a prMND.

The viral supernatant was produced by transient transfection ofHEK293T cells with the vector genome plasmid and lentiviralpackaging helper plasmids pCAGG-HIVgpco, pCAGG-VSVG, andpCAG4-RTR2. The supernatant was concentrated by ultracentri-fugation and titrated onHeLa cells by serial dilution followed by aquantitative PCR to determine vector genome copy number.

CAR transductionCells were primed at 2 � 106 cells/mL overnight in X-VIVO-15

(Lonza, 04-418Q) supplemented with 250 IU/mL IL2 (130-097-746, Miltenyi Biotec), 10 ng/mL anti-CD3 (clone OKT3; 317303)and 10 ng/mL anti-CD28 antibody (clone 28.2; 302913), bothfrom BioLegend. The following day, transduction was performedon RetroNectin (T100B; Takara Bio, Clontech Laboratories) coat-ed plates using MOI¼ 2. The cells were harvested 4 days later forexperiments.

Flow cytometryCell phenotypes were analyzed by flow cytometry (FCM).

Details of the different fluorochrome-labeled mAbs are summa-rized in Supplementary Table S1. Analyses were performed withBD FACSCanto II or LSR Fortessa (BD Biosciences). The meanfluorescence intensity (MFI) ratio was defined as the MFI of thespecific staining relative to the MFI of the appropriate isotypecontrol staining. FlowJo vX.0.7 software (TreeStar) was used fordata analysis.

Cytotoxicity assaysThe cytotoxicity of NKG2D CAR-expressing T cells against OS

cells was evaluated at 20:1, 10:1, 5:1 and 2.5:1 effector-to-target(E:T) ratios by performing conventional 4-hour europium-TDArelease assays (PerkinElmer; AD0116) as described previously(5, 29). In another set of experiments, the cytotoxicity of NKG2DCAR-expressing T cells was tested against PBMCs, bone marrow–derived mesenchymal stem cells (MSC) from healthy donors,Hs1.Int, CCD-18 Co and NuLi-1 healthy cell lines. To rule outNKG2D CAR-T cells could cause hematologic toxicity, colony-forming assayswere performed.CD34þ cellswere coculturedwithCD45RA�NKG2D-CARþ at 20:1, 10:1, 5:1, and 1:1 E:T ratios, orcultured alone (control) for 2 hours at 37�C and 5% CO2. Cellswere then suspended in Methylcellulose (Methocult Express,StemCell Technologies, 04437) and seeded in triplicates, in ahumid chamber to avoid methylcellulose dehydration. Plateswere incubated at 37�C and 5% CO2 for 10 days, and colonies

were counted. Representative images were acquired in a transi-luminator G:BOX Chemi XX6 transiluminator (Syngene, Synop-tics). GeneSys software was used for analysis (GeneSys imageacquisition software, Syngene, Synoptics).

Allogeneic mixed leukocyte reactionTo compare the alloreactivity of CD45RAþ andCD45RA� cells,

mixed leukocyte reactions (MLR) were performed. Respondercells were seeded at 1� 105 cells per well into 96-well flat-bottomplates in triplicate. PBMCs from an unrelated third party wereg-irradiated with 3,000 rad and mixed with responder cells at a10:1 responder/stimulator ratio. Wells containing responders orstimulators alone were used to set the background signals. Cellproliferation was measured by the incorporation of bromodeox-yuridine (BrdUrd) using the DELFIA Proliferation Kit (PerkinEl-mer; AD0200), according to the manufacturer's instructions. A1420 VICTOR3 Multilabel counter plate (PerkinElmer) was usedfor analysis. The production of IFNg in the supernatant of theMLRs was measured by the Human IFN-g Flex set (CBA; BDBiosciences; 560111), following the manufacturer's instructions.The data were collected and analyzed by flow cytometry using aBD FACSCalibur flow cytometer (BD Biosciences).

In vivo studiesTwo different in vivo assays were performed at St. Jude and the

Instituto de Investigaciones Biom�edicas Albert Sols (IIB). All theprocedures were approved by the St. Jude Animal Care and UseCommittee and by the ethics committee of the Consejo Superiorde Investigaciones Científicas (CSIC) and the Comunidad deMadrid (CAM), respectively. In both experiments, 531MII YFP-luc cells (5 � 105) were engrafted by injection through the tibialplateau in the primary spongiosa of both tibias of 10- to 12-week-old NOD/scid IL2rgnull (NSG)mice with or without T cells (5 �106 T cells/tibia). Human IL2 (100 IU/mouse, Miltenyi Biotec)was intraperitoneally injected starting one day after injection ofeffector cells and continued every other day for 3weeks. In the firstassay, we explored the efficacy of an NKG2D CAR–T-cell therapyagainst osteosarcoma. The mice were divided into 3 treatmentgroups: Group A (control), receiving 531MII YFP-luc osteosarco-ma cells alone; Group B, receiving 531MII YFP-luc cells and 5 �106 untransducedCD45RA� cells; andGroupC, receiving 531MIIYFP-luc cells and 5 � 106 NKG2D-CAR transduced CD45RA�

cells. Tumor progression was regularly evaluated using a XenogenIVIS-200 system (124262, PerkinElmer Life and AnalyticalSciences), with imaging beginning 5minutes after intraperitonealinjection of an aqueous solution of D-luciferin potassium salt(3 mg/mouse). Photons emitted from luciferase-expressing cellswere quantified using the Living Image 3.0 software (Xenogen). Atday 60 postinfusion, survivor mice were rechallenged with anintravenous injection of 1� 105 of 531MII YFP-luc cells. Themicewere euthanized when they displayed signs of significant osteo-sarcoma symptoms, including paralysis or tumor volumes largerthan 1,000 mm3. In the second assay, we studied CAR–T-cellpersistency and specific symptoms of NKG2D-CAR–related tox-icity. The mice were divided into 3 treatment groups: group A(control), with no treatment; group B, receiving 5 � 106

CD45RA�NKG2D-CARþ cells injected intratibially (IT), andgroupC, receiving 5 � 106CD45RA�NKG2D-CARþ cells injectedintravenously. Weight loss was monitored every other day untildayþ21. At day 21 mice were sacrificed and samples were

Fern�andez et al.

Clin Cancer Res; 23(19) October 1, 2017 Clinical Cancer Research5826




collected. To explore CAR–T cells persistence on treated mice,blood was analyzed by FCM at dayþ14 and þ21. To evaluatehepatotoxicity in the serum of treated mice, the levels of albumin(ALB), alkaline aminotransferase (ALT), alkaline phosphatase(ALP), bile acids (BA), cholesterol (CHOL), gamma glutamyltransferase (GGT), total bilirubin (TBIL), and urea nitrogen(BUN) weremeasured by using VetScanMammalian Liver profileand VetScan Chemistry Analyzer, both fromABAXIS (500–1,040)and (200–7,013). Serum from healthy and untreated NSG micewere used as controls.

Genomic stability assaysTo test whether lentiviral transduction could cause any chro-

mosomal abnormality, 5 million NKG2D-CAR–transducedmemory T cells were harvested at days 14 and 30 after transduc-tion for cytogenetic analysis in two separate experiments. Fivemillion untransduced CD45RA� cells from the same donors wereused as controls (20 metaphase cells of each type were examinedper experiment). Comparative genomic hybridization (CGH)wasperformed to rule out chromosomal aberrations in the genomicDNAofNKG2DCARanduntransducedCD45RA� cells.DNAwasisolated from cell cultures using the AllPrep DNA/RNA Micro Kitfrom Qiagen (80284) and frozen at �80�C until analysis. CGHmicroarray was performed using Agilent's eArray (Agilent Tech-nologies) probe library in a custom high-resolution format of 8�60K that is enriched in 320 genes related with hematologicneoplasm. The average resolution of the probe coverage was 1oligo every 175 kb to detect losses or gains of more than 400 kb.For target regions, 1 oligo every 9 kb to identify losses or gainsmore than 25 kb. Array experiments were performed as recom-mendedby themanufacturer (5190-3399,G2534-60003,G5188-5222, all from Agilent Technologies). DNAs (500 ng) from thespecimen and a sex-matched DNA control (G1521, Promega)were double-digested with RsaI and AluI for 2 hours at 37�C. Afterheat inactivation of the enzymes, samples were labeled by ran-dom priming using Genomic DNA enzymatic labelling kit (Agi-lent 5190-0449) for 2 hours using Cy5-dUTP for donor's DNAsand Cy3-dUTP for control DNA. Hybridization was performed at65�Cwith rotation for 24hours. After twowashing steps, the arraywas analyzed with the DNA Micro array scanner with Surescanhigh-resolution technology (Agilent Technologies) using the Fea-ture Extraction software (v9.1 Agilent Technologies) The datawere analyzed with Aberration Detection Method 2 (ADM-2)algorithm and the minimum number of consecutive oligos hasbeen established in 3 to consider a copy-number variation.

Statistical analysisResults are shown as means � SEM. Statistical difference

between two groups was calculated using the Student t test. TheStudent paired t test was usedwhen untransduced or transduced Tcells from the same donor were compared. In the mouse model,survival was estimated by using the univariate Kaplan–Meiermethod and compared by using the log-rank test. Statisticalsignificance was defined as P � 0.05.

ResultsOsteosarcoma cells express NKG2DL

Cell surface expression of ligands for the NKG2D receptor onthe 531MII, U-2 OS, and MG-63 osteosarcoma cell lines wasassessed by flow cytometry. We found low to very high levels of

expression of all NKG2DLs (MICA, MICB, or ULBP1, 2 or 3;Fig. 1).Other primary bone andmetastatic osteosarcoma cell lineshad been previously tested for NKG2DL expression in our labo-ratory, and showed similar expression patterns (5).

CD45RA� fraction contains memory T-cell subsets and showsminimal alloreactivity

The phenotype of cells contained in the CD45RA� fraction wasanalyzed by flow cytometry. We found CD4þ subset counted for74.8%, whereas CD8þ subset counted for 14.48% of the totalcells. Most CD4 and CD8 cells showed effector memory andcentralmemoryphenotypes.CD4- andCD8-na€�ve T cellswere lessthan 0.2% of the total cells (Supplementary Table S2).

To test the alloreactivity of CD45RAþ and CD45RA� cells(responders), they were cocultured with irradiated PBMCs fromthe same unrelated donor (stimulator). The CD45RAþ cellsproliferated much more than the CD45RA� counterparts in theMLR assays (Fig. 2A). Furthermore, only CD45RAþ cells exhibitedhigh IFNg production in the culture supernatant (Fig. 2B).

NKG2D CAR transduction and expressionUntransduced CD45RA� cells already express endogenous

NKG2D receptor and were used as a control of baseline expres-sion. After transduction, expression of the NKG2D receptorincreased both in percentage of positive cells and in MFI. Toensure NKG2D CAR expression was stable, and that transductiondid not affect cell survival, we monitored viability and NKG2Dexpression by flow cytometry at different time points after trans-duction.We found the transduced cells had similar viability as theuntransduced cells, and they maintained a higher expression ofNKG2D (both in percentage and in MFI) up to 20 days aftertransduction (Fig. 3).

Cytotoxicity of NKGD CAR-expressing CD45RA� cellsTo test the cytotoxic capacity of transduced and untransduced

cells against osteosarcoma, we used 531MII (n¼ 8), MG-63 (n ¼5), and U-2 OS (n¼ 6) osteosarcoma cells as targets. The NKG2DCAR-expressing CD45RA� cells showed significantly higher cyto-toxic capacity against osteosarcoma cells compared with theuntransduced CD45RA� cells (P ¼ 0.0009, P ¼ 0.001, P ¼0.007 and P ¼ 0.007 for ratios 20:1, 10:1, 5:1 and 2.5:1, respec-tively, in 531MII cells; P ¼ 0.0019 for ratio 20:1 in MG-63 cells,and P¼ 0.0034 and P¼ 0.044 for ratios 20:1 and 10:1 in U-2 OScells, Fig. 4). We did not observe differences on cytolytic ability ofdifferent NKG2D-CAR T-cell subsets (Supplementary Table S3).There was no apparent relation between NKG2DL expression andsensitivity to NKG2D-CAR T cells' cytotoxicity.

Antitumoral effect of NKG2D-CARþ CD45RA� cells in anorthotopic osteosarcoma murine model

Having shown the ability of NKG2D CARþ CD45RA� cells totarget osteosarcoma cells in vitro, we tested their in vivo efficacy in axenograft orthotopic osteosarcoma murine model. Comparedwith the control groups corresponding to mice receiving osteo-sarcoma cells (531MII) alone or in combination with untrans-duced CD45RA� cells, the mice receiving NKG2D CARþ

CD45RA� cells and osteosarcoma cells (E:T ratio, 10:1) showedlower tumor burden, as seen with significantly lower averagedorsal and ventral bioluminescent signals up to 56 days post-infusion (Fig. 5A and B). At day 60 postinfusion, we rechallenged






all mice groups with an intravenous injection of osteosarcomacells, and could observe a significant extended survival time onlyfor those mice receiving NKG2D CARþ cells (120 days). All othermice from the control groups died from tumor burden within 2weeks of the rechallenge (70.4 days for the untreated controlgroup and 83.8 days for the group receiving CD45RA� untrans-duced cells; Fig. 5A and C). 531MII YFP-luc cells were only foundin the lungs of mice from control group or those treated withuntransduced CD45RA� cells (Supplementary Fig. S1). We fol-lowed up thesemice up to 120 days and did not detect treatment-related toxicity as CRS or any GVHD symptoms. Differentparameters indicative of toxicity related to therapy andNKG2D-CAR T cells persistency were explored. Mice treatedwith NKG2D-CAR memory T cells showed similar weightcurves than those in the control group (Fig. 5D). In addition,biochemical analysis of the serum showed no differences in thelevel of the studied liver enzymes between treated and controlgroups, indicating no liver toxicity is produced by the therapyeither orthotopically or intravenously injected (SupplementaryTable S4). Human CD45þNKG2D-CAR T cells were detectablein the peripheral blood of treated mice 14 and 21 days after thetherapy was administered either locally or systemically (Sup-plementary Fig. S2).

Safety of NKG2D CAR–expressing memory T cellsGenome instability was studied by karyotype or by array-based

CGH. Normal karyotype and no copy number alterations were

found in NKG2D-CARþ memory T cells compared with untrans-duced T cells up to 30 days after transduction (Fig. 6A).

To verifyNKG2DCARþmemory T cells showed no lytic activityagainst healthy tissues, PBMCs and MSCs from healthy donors,Hs 1. Int, CCD-18 Co and NuLi1 healthy cell lines were used astargets for in vitro cytotoxicity assays. No specific lytic activity wasdetected against healthy PBMCs, MSCs and CCD-18 Co. How-ever,NKG2DCARþmemory T cells showedhigh cytotoxic activityagainstHs 1. Int. cells, and a low level of cytotoxicity against NuLi-1 cell line (Fig. 6B). To rule outNKG2D-CARmemory T cells couldcause hematologic toxicity, colony-forming assays were per-formed after culture of healthy CD34þ progenitor cells alone(control) or at different E:T ratios. We observed the ability ofCD34þ cells to form colonies was unaffected after coculture withNKG2D-CAR redirected memory T cells, indicating no hemato-logic toxicity was caused by the therapy (Fig. 6C). Expression ofNKG2DL on healthy cells is shown in Supplementary Table S5.

DiscussionDespite multimodal therapy, osteosarcoma outcomes have

not improved over the past 3 decades, with patients withmetastatic disease having an overall survival of only 20%.CAR–T-cell–based immunotherapy is a promising therapeuticapproach that is currently being tested in several hematologicmalignancies as well as in solid tumors, including osteosarco-ma, opening a new possibility for those cancer patients resistantto traditional therapies.

Figure 1.

Expression of NKG2D ligands by MG-63 (top), 531MII (middle), and U-2 OS (bottom) cells. Mean fluorescence intensity of cells stained with the specificantibody is represented in dark gray, isotype controls are represented with a bright dotted gray line. Percentage of positive cells and MFI ratio of specificstaining versus staining of isotype control are detailed in the histograms.

Fern�andez et al.





The NKG2D cell receptor and its ligands are currently gaininginterest as a potential strategy to target tumor cells. NKG2DL isexpressed in the vast majority of tumor types, including osteo-sarcoma (4, 5), whereas its expression on healthy cells is rare (16).In fact, NKG2D receptor-NKG2DL interactions have been shownto be essential for NK-cell targeting of osteosarcoma TICs (5). Inthis study, we took advantage of NKG2D–NKG2DL interactionsto explore the efficacy and safety of NKG2D–CAR–redirectedmemory T cells as immunotherapy for osteosarcoma treatment.

NKG2D CAR–redirected T and NK cells have shown potentanticancer effects against various tumors such as T-cell lymphoma(30), multiple myeloma (31) osteosarcoma, Ewing sarcoma,ovarian, and pancreatic carcinoma (11, 12, 32, 33). In addition,NKG2D CAR–redirected effector cells are able to target NKG2DLexpressed not only on the tumor cells, but also in the immuno-suppressive cells within the tumor microenvironment, inducingan immunologic response in the host (34). In the current report,

we show for the first time that NKG2D-CAR–redirectedmemory Tcells target osteosarcoma cells both in vitro and in vivo. Onepotential complication of the use of NKG2D CAR T-cell therapiescould be the "on target–off tumor" toxicity, given NKG2DLs arenot expressed exclusively on tumor cells. Recently, VanSeggelenand colleagues (35) described lethal toxicity in mice treated withmurine NKG2D CAR T cells. This toxicity was both CAR constructand strain-dependent, and was exacerbated upon lymphodeplet-ing conditioning with cyclophosphamide. In this study, T cellsexpressing a construct containingDAP10 showed the highest CARexpression and toxicity, followed by a second-generation CARcontaining CD28 and CD3Z. A first-generation CARwith NKG2Dand CD3Z showed the lowest toxicity. A construct similar to ours(NKG2D-4-1BB-CD3z) that lacks DAP10 and contains 4-1BBinstead of CD28, was not tested in this report, and may behavedifferently. BALB/c T cells showed highest expression of NKG2DCAR, as well as cytokine production and cytotoxicity againstNKG2DLþ targets, and also produced amore severe toxicity wheninjected into mice. These data indicate NKG2DL expression isvariable among different mice strains, and will expectedly bedifferent between mice and humans as NKG2DL genes differsignificantly. However, the results showed in this study indicateNKG2DL-targeted therapies should be used with caution. In thisstudy, we did not observe a correlation between NKG2DL expres-sion and in vitro cytotoxicity of NKG2D–CAR–redirected T cells.This may indicate that other signals on the tumor cells, besidesNKG2DL expression, are needed for triggering NKG2D-CAR Tcells cytolytic activity. In addition, different NKG2DL bindNKG2D receptor with a wide range of affinities, and may varyin how they trigger or engage NKG2D, in such a way that differentoutcomes occur (36). This is in accordance with the lack ofcytolytic activity of NKG2D CAR–redirected effector cells againstvarious healthy tissues, thatwe andothers have observed (12).Weobserved NKG2D–CAR T cells had cytotoxic activity against Hs 1.Int (normal fetal intestine) cells in vitro. The expression ofNKG2DL on embryonic cells is well documented (16, 37). How-ever, during pregnancy, placenta has an immunosuppressive roleexpressing and secreting NKG2DL that downmodulate NKG2Dreceptor expression (38). For this reason, the in vitro cytotoxicityagainst normal fetal intestine we have observed may not beextrapolated to their behavior in vivo. NKG2D–CAR T cells alsoshowed a low level of cytotoxicity against NuLi-1 (normal epi-thelial lung) cells. NuLi-1 cell line is immortalized with HPV-16E6/E7-LXSN and some of the cells show chromosomal abnor-malities, such as trisomy 5 and 20. Therefore, it cannot beconsidered as an entirely "normal" tissue. This could explain thecytotoxicity observed. The low level of cytotoxicity of NKG2D–

CAR T cells observed against NuLi-1 cells (around 22%) contrastswith the high level of cytotoxicity observed against thefetal intestine cell line Hs 1.Int (around 86%), indicating adulthealthy tissues could be much less susceptible to NKG2D–CAR Tcells cytotoxicity. All these data taken together suggest thatNKG2D CAR T-cell therapy might be safe in humans; however,more studies are needed to define the extent of ligand expressionon normal tissues and to optimize the dose of therapeutic cellsto rule out any toxicity. An ongoing phase I dose-escalationstudy is currently testing the safety of NKG2D CAR-T cells inpatients withmyeloid malignancies (ClinicalTrials.gov Identifier:NCT03018405). The results are encouraging. To date, 9 patientshave been treatedwith a single dose of up to 3�107of autologousNKG2D–CAR T cells without lymphodepleting conditioning. No

Figure 2.

CD45RA� cells show no alloreactivity in vitro. A, Allogeneic MLR withCD45RAþ or CD45RA� cells as responders and gamma-irradiated third-partyPBMCs as stimulators in a 10:1 ratio. B, IFNg production from allogeneicMLRs of CD45RAþ and CD45RA� cells; ��, P < 0.001.






cases of CRS, cell-related neurotoxicity, autoimmunity or CAR-T–related death have been recorded (39).

In a recent publication by Sentman and colleagues (40), theyattribute the acute toxicity observed in mice treated withNKG2D-CAR-T cells by a CRS-like response similar to thoseobserved in patients treated with CAR-T cells. We did notobserve treatment-related toxicity in the treated mice. This canbe explained by the incapability of murine NKG2DL to trigger ahuman NKG2D-CAR. In addition, xenograft models usingimmunocompromised mice are useful to evaluate the directcytotoxic effect of CAR-redirected effector cells on cancer cells;however, they cannot be used to explore the adaptive immuneresponse elicited by CAR-based immunotherapies. Because the

animals are immunosuppressed, CAR therapy side-effects suchas CRS or tumor lysis syndrome cannot be evaluated. Theanticancer effects of an NKG2D–CAR-based cell immunother-apy have been previously reported using these animal models(12, 13). However, these models do not mirror the currenthuman cancer scenario (41).

CRS is the most prevalent adverse effect following infusion ofCAR-T cells and is correlated with expansion and immune acti-vation of adoptively transferred cells, which provokes the eleva-tion of inflammatory cytokines such as IFNg , IL6, and GM-CSF.These inflammatory cytokines are mainly secreted upon activa-tion, differentiation and clonal expansion of na€�ve T cells. Todiminish the severity of CRS, we used differentiated memory T

Figure 3.

Effects of lentiviral transduction on CD45RA� cells.A, Purity of transduced cells. B,NKG2D expression increases both in the percentage as in MFI after transduction,and the expression is kept along the time. C, Transduction of CD45RA� cells does not affect viability.

Fern�andez et al.





cells to express our CAR, as they show an impaired ability toclonally expand and to secrete inflammatory cytokines like IFNg .

Other potential targets have been proposed for CAR–T-celltherapy against osteosarcoma. HER-2–modified T cells have beenshown to target osteosarcoma TICs (42), however, HER-2 isexpressed at low levels in the lung, and a patient died afterinfusion of HER-2–redirected CAR-T cells (43). IL11 Receptora-chain (IL11Ra) has been proposed as target for CAR–T-celltherapy for osteosarcoma treatment. IL11Ra-CAR–redirected Tcells have been shown to effectively kill osteosarcoma cells in vitroand to induce regression of established metastasis in vivo (44). Aphase I clinical trial is currently testing the safety of a third-generation CAR directed against GD2 (ClinicalTrials.gov Identi-fier NCT: NCT02107963).

CARs can be inserted into effector cells by gammaretroviral,lentiviral, mRNA, transposon/transposase and Sleeping Beautysystems. The lentiviral vectors used in this report providedenhanced transduction efficiency and long-term NKG2D CARexpression. In addition, no chromosomal aberrations were foundonNKG2D-CARmemory T cells, indicating the safety of lentiviraltransduction. This observation is in accordance to a decade-longsafety profile of the use of genetically modified T cells (45).However, in case of toxicity, temporal expression of theNKG2D-CAR provided bymRNA transfectionmight be desirable.

AlthoughCARs canbe expressed in autologous andallogeneic Tcells, autologous infusions are not always an option due to lowlymphocyte numbers or poor lymphocyte quality (and ex vivoexpansion).Donor-derivedCART cells are currently being infusedinto patients after allo-HSCT. However, having off-the-shelf cellproducts to potentially administer without a requirement for apriorHSCT from the same donorwould be amajor goal regardingCAR–T-cell therapies. Onepotential risk of the use of allogeneic T-cell–based therapies is the T-cell response against normal tissue:GvHD. To avoid undesirable GVH reactions, T-cell productslacking an alloreactive TCR are needed. Several methods haveattempted to intensify GVT effects while minimizing GVHresponses to lower toxicity and improve the outcome of treatment(19, 46–48). One approach to enrich nonallogeneic T cells is byusing antigen-experienced memory T cells for CAR transduction(18, 19). Predictably, the vast majority of T cells with a memoryphenotype are likely to have encountered antigens other than theallogeneic type. Thus, selection for memory phenotype cellsshould enrich for a nonalloreactive repertoire. Indeed, memoryT cells showed less potential to generate GVHD inmurinemodels(26, 49), in part due to nonalloreactive TCR enrichment alongwith the evidence that memory T cells are less likely to traffic toGVHD organs such as the gastrointestinal tract. One marker todistinguish na€�ve from memory T cells is CD45RA (22). Severalpreclinical models have used CD45RA memory T cells (19) orCD45RA� T (18) cells for CAR transduction. In our study, we usedCD45RA� T cells because they have shown antipathogenmemoryresponse and antipotent leukemic activity without causing xeno-geneic GvHD, circumventing a major survival limitation with theuse of immunodeficient mice treated with human T cells (18).Although allogeneic memory T cells are nonalloreactive, they canbe immunogenic, thus they could be eliminated by the hostimmune system once is recovered from a lymphodepleting con-ditioning. This could limit the efficacy of the therapy to a shortperiod of time, so several infusions may be needed to maintainantitumor effects. However, a limited life span can be advanta-geous, so CAR-T cells would cause minimal or no toxicity. In this

Figure 4.

NKG2D CAR–transduced CD45RA� cells show enhanced cytotoxicityagainst 531MII, MG-63, and U-2 OS osteosarcoma cell lines. Cytotoxicity ofNKG2D CARþ and untransduced CD45RA� cells against osteosarcomacells at different effector to target (E:T) ratios; �� , P < 0.005;� , P < 0.05.






Figure 5.

Mice receiving NKG2D CAR T cells show prolonged survival and less tumor bioluminescent signal along the time. A, Representative bioluminescencepictures of mice for 531MII-alone control, 531MIIþCD45RA� untransduced and 531MIIþCD45RA�NKG2D-CARþ MOI ¼ 2 groups. B, Comparison of ventral anddorsal bioluminescence signals among 531MII-alone control (dotted gray line), 531MII-CD45RA� untransduced (bold gray line), and 531MII-CD45RA�NKG2D-CARþ MOI ¼ 2 (bold black line) at different time points. C, Kaplan–Meier survival curves for 531MII-alone control (dotted gray line), 531MII-CD45RA�

untransduced (bold gray line), and 531MII-CD45RA�NKG2D-CARþ MOI ¼ 2 (bold black line). D, Weight curve of mice injected intratibially with 531MII-alonecontrol (dotted gray line), with 531MII-CD45RA�NKG2D-CARþ (both intratibial; bold gray line), or with 531MII (intratibial) and CD45RA�NKG2D-CARþ

(intravenous; bold black line).

Fern�andez et al.





Figure 6.

Safety of CD45RA�NKG2D CARþ cells. A, No chromosomal alterations are found in T cells after NKG2D CAR transduction. Representative karyotype ofcontrol untransduced T cells (left) and NKG2DCAR transduced T cells (right). B,NKG2D CAR T cells show no cytolytic activity against healthy PBMC, MSC, and CCD-18Co. Hs1. Int and NuLi-1 cell lines are sensitive to NKG2D-CAR T cells lysis in vitro.C,NKG2D-CAR T cells show no cytotoxicity against CD34þ hematologic progenitorcells. Top, representative picture of the colonies grown at 10 days after coculture with NKG2D-CAR T cells at different E:T ratios. Bottom, number of coloniesobtained in each condition.






report, we described preclinical data to support the use of allo-geneic NKG2D CAR–redirected CD45RA� cells for the treatmentof osteosarcoma. NKG2D CAR-CD45RA� cells showed anti-oste-osarcoma activitywhile showing noGVHDeffects. NKG2D-CARþ

memory T cells failed to completely eradicate the tumor (asobserved by the increase of the bioluminescence signal in treatedmice after day 42),most likely because IL2 injections were limitedto the first 3 weeks after T-cell injection, given it is an essentialsurvival factor for T cells. In future experiments, repeated infu-sions of NKG2D-CAR memory T cells along with IL2 adminis-tration can be done to eradicate tumor progression.

In summary, our results showNKG2DCAR can be expressed inCD45RA� memory T cells with high transduction efficiency, theexpression of CAR remains throughout, and it does not affect theviability or genetic stability of transduced cells. Most importantly,NKG2D–CARþ CD45RA� cells had potent anti-osteosarcomaeffects while remaining noncytotoxic against healthy tissues, asshown in in vitro Europium-TDA conventional assays and colony-forming assays. In vivo, mice treated with NKG2DCAR-expressingT cells showed less tumor burden and longer survival than thoseuntreated or treated with control untransduced CD45RA� T cellswithout any toxicity related to CAR T-cell memory treatment.These data suggest that osteosarcoma patients could benefit froma new NKG2D CAR memory T-cell–based therapy.

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

Authors' ContributionsConception and design: L. Fern�andez, A. Pati~no-García, J. Martínez, W. Leung,A. P�erez-Martínez

Development of methodology: L. Fern�andez, A. Escudero, M. Vela, A. Leivas,W. LeungAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Fern�andez, A. Escudero, M. Vela, J. Valentín,I. Vallcorba, A. Pati~no-García, J. Martínez, W. LeungAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Fern�andez, J.-Y. Metais, J. Valentín, J. Torres,A. P�erez-MartínezWriting, review, and/or revision of the manuscript: L. Fern�andez, J.-Y. Metais,A. Escudero, I. Vallcorba, J. Torres, A. Valeri, A. Pati~no-García, J. Martínez,W. Leung, A. P�erez-MartínezAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): L. Fern�andezStudy supervision: J. Martínez, A. P�erez-Martínez

AcknowledgmentsThe authors thank Jim Houston, Barbara Rooney, Martha Holladay, Patricia

Gonz�alez, María Udriste, María Coronel, Manuel P�erez, Enrique Hern�andez,Victor Toledano, and Jes�us S�anchez for technical support.

Grant SupportThis work was supported in part by the National Health Service of Spain,

Instituto de SaludCarlos III (ISCIII), a FONDOSFEDERgrant (FIS) PI15/00973,Asociaci�on Espa~nola Contra el C�ancer to Antonio P�erez Martínez and a CRISFoundation to Beat Cancer grant to Lucía Fern�andez and Jaime Valentín. We areespecially grateful to the family of 531MII, MEZ, whose generosity and capacityto see beyond, has allowed so many scientific achievements

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

Received January 12, 2017; revised April 27, 2017; accepted June 21, 2017;published OnlineFirst June 28, 2017.

References1. Sakamoto A, Iwamoto Y. Current status and perspectives regarding the

treatment of osteosarcoma: chemotherapy. Rev Recent Clin Trials 2008;3:228–31.

2. Athanasou N, Bielack S, De Alava E, Tos APD, Ferrari S, Paulussen M, et al.Bone sarcomas: ESMOClinical PracticeGuidelines for diagnosis, treatmentand follow-up. Ann Oncol 2010;21Suppl 5:204–13.

3. Wachtel M, Sch€afer BW. Targets for cancer therapy in childhood sarcomas.Cancer Treat Rev 2010;36:318–27.

4. Buddingh EP, SchilhamMW,Ruslan SEN, BerghuisD, Szuhai K, SuurmondJ, et al. Chemotherapy-resistant osteosarcoma is highly susceptible to IL-15-activated allogeneic and autologousNK cells. Cancer Immunol Immun-other 2011;60:575–86.

5. Fern�andez L, Valentín J, Zalacain M, Leung W, Pati~no-garcía A, P�erez-Martínez A. Activated and expanded natural killer cells target osteosarcomatumor initiating cells in anNKG2D –NKG2DL dependentmanner. CancerLett 2015;368:54–63.

6. Bodduluru LN, Kasala ER, Mohan R, Madhana R, Sriram CS. Natural killercells: The journey from puzzles in biology to treatment of cancer. CancerLett 2014;357:454–67.

7. Klingemann H. Challenges of cancer therapy with natural killer cells.Cytotherapy 2015;17:245–9.

8. P�erez-Martínez A, Fern�andez L, Valentín J, Martínez-Romera I, Corral MD,Ramírez M, et al. A phase I/II trial of interleukin-15–stimulated naturalkiller cell infusion after haplo-identical stem cell transplantation forpediatric refractory solid tumors. Cytotherapy 2008;17:1594–603.

9. Barrett DM, Singh N, Porter DL, Grupp SA, June CH. Chimeric antigenreceptor therapy for cancer. Annu Rev Med 2014;65:333–47.

10. Zhang T,WuM-R, SentmanCL. AnNKp30-based chimeric antigen receptorprotomotes T-cell effector functions and anti-tumor efficacy in vivo. JImmunol 2013;189:2290–9.

11. Song D-G, Ye Q, Santoro S, Fang C, Best A, Powell DJ. Chimeric NKG2DCAR-expressing T cell-mediated attack of human ovarian cancer isenhanced by histone deacetylase inhibition. Hum Gene Ther 2013;24:295–305.

12. Chang Y, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. Achimeric receptor with NKG2D specificity enhances natural killer cellactivation and killing of tumor cells. Cancer Res 2013;73:1777–87.

13. Kudo K, Imai C, Lorenzini P, Takahura K, Kono K, Davidoff A, et al. Tlymphocytes expressing a CD16 signaling receptor exert antibody-depen-dent cancer cell killing. Cancer Res 2014;74:93–103.

14. Wu MR, Zhang T, Alcon A, Sentman CL. DNAM-1-based chimericantigen receptors enhance T cell effector function and exhibit in vivoefficacy against melanoma. Cancer Immunol Immunother 2015;64:409–18.

15. Wu MR, Zhang T, DeMars LRSentman CL. B7H6-specific chimeric antigenreceptors lead to tumor elimination and host anti-tumor immunity. GeneTher 2015;22:675–84.

16. Spear P,WuMR, SentmanML, Sentman CL. NKG2D ligands as therapeutictargets. Cancer Immunity 2013;13:8.

17. Oberoi P, Wels WS. Arming NK cells with enhanced antitumor activityCARs and beyond. Oncoimmunology 2013;2:10–2.

18. Chan WK, Suwannasaen D, Throm RE, Li Y, Eldridge PW, Houston J, et al.Chimeric antigen receptor-redirected CD45RA-negative T cells have potentantileukemia and pathogen memory response without graft-versus-hostactivity. Leukemia 2014;29:387–95.

19. Terakura S, Yamamoto TN, Gardner RA, Turtle CJ, Jensen MC, Riddell SR,et al. Generation of CD19-chimeric antigen receptor modified CD8 þ Tcells derived from virus-specific central memory T cells Generation ofCD19-chimeric antigen receptor modified CD8þ T cells derived fromvirus-specific central memory T cells. Blood 2012;119:72–82.


Fern�andez et al.




20. Cruz CRY, Micklethwaite KP, Savoldo B, Ramos CA, Lam S, Ku S, et al.Infusion of donor-derived CD19-redirected virus-specific T cells for B-cellmalignancies relapsed after allogeneic stem cell transplant: a phase 1 study.Blood 2013;122:2965–74.

21. Brudno JN, Somerville RPT, Shi V, Rose JJ, Halverson DC, Fowler DH, et al.Allogeneic T cells that express an anti-CD19 chimeric antigen receptorinduce remissions of B-cell malignancies that progress after allogeneichematopoietic stem-cell transplantation without causing graft-versus-hostdisease. J Clin Oncol 2016;34:1112–21.

22. Mahnke YD, Brodie TM, Sallusto F, Roederer M, Lugli E. The who's who ofT-cell differentiation: human memory T-cell subsets. Eur J Immunol2013;43:2797–809.

23. Chen BJ, Deoliveira D, Cui X, Le NT, Son J, Whitesides JF, et al. Inability ofmemory T cells to induce graft-versus-host disease is a result of an abortivealloresponse. Blood 2012;109:3115–23.

24. Ch�erel M, Choufi B, Trauet J, Cracco P, Dessaint JP, Yakoub-Agha I, et al.Na€�ve subset develops the most important alloreactive response amonghumanCD4þ T lymphocytes in human leukocyte antigen-identical relatedsetting. Eur J Haematol 2014;92:491–6.

25. ZhengH,Matte-Martone C, Li H, Anderson BE, Venketesan S, TanHS, et al.Effector memory CD4þ T cells mediate graft-versus-leukemia withoutinducing graft-versus-host disease. Blood 2008;111:2476–84.

26. Anderson B, McNiff J, Yan J. Memory CD4þ T cells do not induce graft-versus-host disease. J Clin Invest 2003;112:101–8.

27. Cai B, GuoM,Wang Y, Zhang Y, Yang J, Guo Y, et al. Co-infusion of haplo-identical CD19- chimeric antigen receptor T cells and stem cells achievedfull donor engraftment in refractory acute lymphoblastic leukemia. JHematol Oncol 2016;9:1–6.

28. Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH, Geiger L, et al.Chimeric receptors with 4-1BB signaling capacity provoke potent cytotox-icity against acute lymphoblastic leukemia. Leukemia 2004;1640:676–84.

29. Blomberg K, Hautala R, L€ovgren J, Mukkala VM, Lindqvist C, Akerman K.Time-resolved fluorometric assay for natural killer activity using target cellslabelled with a fluorescence enhancing ligand. J Immunol Methods 1996;193:199–206.

30. Zhang T, Barber A, Sentman CL. Chimeric NKG2D – modified T cellsinhibit systemic T-cell lymphoma growth in a manner involving multiplecytokines and cytotoxic pathways. Cancer Res 2007;67:11029–36.

31. Barber A, Meehan KR, Sentman CL. Treatment of multiple myeloma withadoptively transferred chimeric NKG2D receptor-expressing T cells. GeneTher 2012;18:509–16.

32. Morisaki T, Hirano T, Koya N, Kiyota A, Tanaka H, Umebayashi M, et al.NKG2D-directed cytokine-activated killer lymphocyte therapy combinedwith gemcitabine for patients with chemoresistantmetastatic solid tumors.Anticancer Res 2014;34:4529–38.

33. LehnerM,GabrielG, Proff J, SchaftN,Do J, Full F, et al. Redirecting T cells toEwing's Sarcoma Family of Tumors by a chimeric NKG2D receptorexpressed by lentiviral transduction or mRNA transfection. PLoS ONE2012;7:e31210.

34. Barber A, Rynda A, Sentman CL. Chimeric NKG2D Expressing T cellseliminate immunosuppression and activate immunity within the ovariantumor microenvironment. J Immunol 2009;183:6939–47.

35. VanSeggelen H, Hammill J, Dvorkin-Gheva A, Tantalo DGM, Kwiecien JM,Denisova GF, et al. T cells engineered with chimeric antigen receptorstargeting NKG2D ligands display lethal toxicity in mice. Mol Ther 2015;23:1600–10.

36. Raulet DH,Gasser S, Gowen BG, DengW,Heiyoun J. Regulation of ligandsfor the NKG2D activating receptor. Annu Rev Immunol 2013;31:413–41.

37. Eagle RA, Jafferji I, Barrow AD. Beyond stressed self: Evidence for NKG2Dligand expression on healthy cells. Curr Immunol Rev 2009;5:22–34.

38. Hedlund M, Stenqvist A, Nagaeva O, Kjellberg L, Wulff M, Baranov V, et al.Human placenta expresses and secretes NKG2D ligands via exosomes thatdown-modulate the cognate receptor expression: evidence for immuno-suppressive function. J Immunol 2009;183:340–51.

39. Nikiforow S,Werner L, Murad J, JacobsM, Johnston L, Patches S, et al. et al.Safety data from a first-in-human phase 1 trial of NKG2D chimeric antigenreceptor-t cells in AML/MDS and multiple myeloma. Blood 2016;128.22.

40. Sentman M, Murad JM, Cook WJ, Wu M, Reder J, Baumeister SH, et al.Mechanisms of acute toxicity in NKG2D chimeric antigen receptor T-cell–treated mice. J Immunol 2016;197:4674–85.

41. Murphy WJ. Being "penny-wise but pound foolish" in cancer immuno-therapy research: the urgent need formouse cancermodels to reflect humanmodifying factors. J Immunother Cancer 2016;4:1–3.

42. Rainusso N, Brawley VS, Ghazi A, Hicks MJ, Gottschalk S, Rosen JM, et al.Immunotherapy targeting HER2 with genetically modified T cells elim-inates tumor-initiating cells in osteosarcoma. Cancer Gene Ther 2012;19:212–7.

43. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA.Case Report of a serious adverse event following the administration of Tcells transduced with a chimeric antigen receptor recognizing ErbB2. MolTher 2009;18:843–51.

44. Huang G, Yu L, Cooper LJN, Hollomon M, Huls H, Kleinerman ES.Genetically modified T cells targeting Interleukin-11 receptor a -chain killhuman osteosarcoma cells and induce the regression of established oste-osarcoma lung metastases. Cancer Res 2011;130:271–81.

45. Scholler J, Brady TL, Binder-scholl G, Hwang W, Hege KM, Vogel AN, et al.Decade-long safety and function of retroviral-modified chimeric antigenreceptor T-cells. Sci Transl Med 2012;4:132ra53.

46. Savoldo B, Rooney CM, Di Stasi A, Abken H, Hombach A, Foster AE, et al.Epstein Barr virus specific cytotoxic T lymphocytes expressing the anti-CD30zeta artificial chimeric T-cell receptor for immunotherapy of Hodg-kin disease. Blood 2007;110:2620–30.

47. Pule MA, Savoldo B, Myers GD, Rossig C, Russell HV, Dotti G, et al. Virus-specific T cells engineered to coexpress tumor-specific receptors: persistenceand antitumor activity in individuals with neuroblastoma. J Exp Med2009;14:1264–70.

48. Sun J, Huye LE, Lapteva N, Mamonkin M, Hiregange M, Ballard B, et al.Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a ProductionAssistant for Cell Therapy (PACT) translational application. J ImmunotherCancer 2015;3:5.

49. Foster AE, Marangolo M, Sartor MM, Alexander SI, Hu M, Bradstock KF,et al. Human CD62L—memory T cells are less responsive to alloantigenstimulation than CD62L þ naive T cells: potential for adoptive immuno-therapy and allodepletion. Cell 2008;104:2403–9.






2017;23:5824-5835. Published OnlineFirst June 28, 2017.Clin Cancer Res Lucía Fernández, Jean-Yves Metais, Adela Escudero, et al. Osteosarcoma CellsMemory T Cells Expressing an NKG2D-CAR Efficiently Target

Updated version

10.1158/1078-0432.CCR-17-0075doi:

Access the most recent version of this article at:

Material

Supplementary

http://clincancerres.aacrjournals.org/content/suppl/2017/06/27/1078-0432.CCR-17-0075.DC1

Access the most recent supplemental material at:

Cited articles

http://clincancerres.aacrjournals.org/content/23/19/5824.full#ref-list-1

This article cites 49 articles, 13 of which you can access for free at:

Citing articles

http://clincancerres.aacrjournals.org/content/23/19/5824.full#related-urls

This article has been cited by 3 HighWire-hosted articles. Access the articles at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected]

To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/23/19/5824To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/lookup/doi/10.1158/1078-0432.CCR-17-0075

http://clincancerres.aacrjournals.org/content/suppl/2017/06/27/1078-0432.CCR-17-0075.DC1

http://clincancerres.aacrjournals.org/content/23/19/5824.full#ref-list-1

http://clincancerres.aacrjournals.org/content/23/19/5824.full#related-urls

http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]

http://clincancerres.aacrjournals.org/content/23/19/5824


Documents

Memory T Cells Expressing an NKG2D-CAR Ef ciently Target … · Cancer Therapy: Preclinical Memory T Cells Expressing an NKG2D-CAR Efﬁciently Target Osteosarcoma Cells Lucía Fernandez1,2,