Targeting CD20+ Aggressive B-cell Non-Hodgkin Lymphoma by Anti-CD20 CAR mRNA-Modified Expanded Natural Killer Cells In Vitro and in NSG Mice.

Published OnlineFirst December 9, 2014; DOI: 10.1158/2326-6066.CIR-14-0114

Research Article

Targeting CD20þ Aggressive B-cell Non–Hodgkin Lymphoma by Anti-CD20 CAR mRNA-Modified Expanded Natural Killer Cells In Vitro and in NSG Mice

Cancer Immunology Research

Yaya Chu1, Jessica Hochberg1, Ashlin Yahr1, Janet Ayello1, Carmella van de Ven1, Matthew Barth2, Myron Czuczman3,4, and Mitchell S. Cairo1,5,6,7,8

Abstract The prognosis is very dismal for patients with relapsed CD20þ B-cell non-Hodgkin lymphoma (B-NHL). Facilitating the development of alternative novel therapeutic strategies is required to improve outcomes in patients with recurrent/refractory CD20þ B-NHL. In this study, we investigated functional activities of anti-CD20 CAR-modified, expanded peripheral blood NK cells (exPBNK) following mRNA nucleofection against CD20þ B-NHL in vitro and in vivo. CARþ exPBNK had significantly enhanced in vitro cytotoxicity, compared with CAR exPBNK against CD20þ Ramos (P < 0.05), Daudi, Raji, and two rituximab-resistant cell lines, Raji-2R and Raji-4RH (P < 0.001). As expected, there was no significant difference against CD20 RS4;11 and Jurkat cells. CD107a degranulation and intracellular IFNg production were

also enhanced in CARþ exPBNK in response to CD20þ B-NHL– specific stimulation. In Raji-Luc and Raji-2R-Luc xenografted NOD/SCID/g-chain/ (NSG) mice, the luciferase signals measured in the CARþ exPBNK-treated group were significantly reduced, compared with the signals measured in the untreated mice and in mice treated with the CAR exPBNK. Furthermore, the CAR exPBNK-treated mice had significantly extended survival time (P < 0.001) and reduced tumor size, compared with those of the untreated and the CAR exPBNK-treated mice (P < 0.05). These preclinical data suggest that ex vivo–exPBNK modified with anti-CD20 CAR may have therapeutic potential for treating patients with poor-risk CD20þ hematologic malignancies. Cancer

Introduction

Study Group (UKCCSG), that children and adolescents with newly diagnosed mature B-NHL [French–American–British (FAB)/Lymphome malins de Burkitt (LMB) 96], have a 90% 5year overall survival (OS) following treatment with a short, intensive course of chemotherapy (4). Unfortunately, for children and adolescents who relapse or progress with de novo mature BNHL, the prognosis is dismal due to chemoradiotherapy resistance (4, 5). Similarly, the prognosis in adults with recurrent/ refractory Burkitt lymphoma is dismal (6). Therefore, development of alternative cellular targeted therapeutic strategies is required to improve outcomes in children, adolescents, and adults with recurrent/refractory de novo mature B-NHL. CD20 is a glycosylated phosphoprotein expressed on the surface of B cells on all developmental stages, except pro-B cells or plasma cells (7). It is also expressed in >98% of childhood, adolescent, and adult mature B-cell NHLs and therefore is an attractive cancer therapeutic target (6, 8, 9). Rituximab, a chimeric anti-CD20 antibody, has been used successfully in the treatment of childhood and adolescent mature B-NHL as well as in adults with diffuse large B-cell lymphoma and Burkitt lymphoma (9–11). However, CD20 could also be used as a target for genetically engineered immune cell–based therapies. Natural killer (NK) cells are bone marrow–derived cytotoxic lymphocytes that play a major role in the rejection of tumors and cells infected by viruses, even without specific immunization (12, 13). Various activating and inhibitory receptors on the NK-cell surface are engaged to regulate NK-cell activities and to discriminate target cells from healthy "self" cells (14, 15). However, factors limiting NK therapy include small numbers of active NK

B-cell non-Hodgkin lymphoma (B-NHL), including Burkitt lymphoma, makes up approximately 60% of all malignant NHL that occurs in children and adolescents (1). The outcome for children and adolescents with de novo mature B-NHL has improved significantly, as we previously demonstrated that short but intensive chemotherapy is associated with a 90% 5-year eventfree survival (EFS; refs. 2–4). We demonstrated in an international multi-cooperative group study, which comprised the Children's Oncology Group (COG), The Societe Fran¸caise d'Oncologie Pediatrique (SFOP), and the United Kingdom Children's Cancer

1 Department of Pediatrics, Maria Fareri Children's Hospital, New York Medical College, Valhalla, New York. 2Department of Pediatrics, State University of New York at Buffalo, Buffalo, New York. 3Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York. 4Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York. 5 Department of Medicine, New York Medical College, Valhalla, New York. 6Department of Pathology, New York Medical College, Valhalla, New York. 7Department of Microbiology and Immunology, New York Medical College, Valhalla, New York. 8Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York.

Note: Supplementary data for this article are available at Cancer Immunology Research Online (http://cancerimmunolres.aacrjournals.org/). Corresponding Author: Mitchell S. Cairo, New York Medical College, 40 Sunshine Cottage Road, Skyline 1N-D12, Valhalla, NY 10595. Phone: 914-594-2150; Fax: 914-594-2151; E-mail: [email protected] doi: 10.1158/2326-6066.CIR-14-0114 2014 American Association for Cancer Research.

Immunol Res; 3(4); 333–44. 2014 AACR.

www.aacrjournals.org

Downloaded from cancerimmunolres.aacrjournals.org on August 12, 2015. © 2015 American Association for Cancer Research.

333


Chu et al.

cells in unmodified peripheral blood and a lack of specificity in tumor targeting (16). Our group and others have successfully expanded active NK cells in vitro by short-term culture with cytokines alone, using cytokines and coculture with irradiated Epstein–Barr virus (EBV)–transformed lymphoblastoid cell lines as feeder cells, or using cytokines and coculture with K562 cells expressing transfected cell-membrane bound IL15 and 4-1BBL (17–20). Chimeric antigen receptors (CAR) usually include a singlechain variable fragment (Fv) from a monoclonal antibody, a transmembrane hinge region, and a signaling domain such as CD28, CD3z, 4-1BB (CD137), or 2B4 (CD244) endodimers (21–24). The advantage of the CAR strategy is that no human leukocyte antigen (HLA) expression on the target cell is required for the epitope to be accessible to CARþ T or NK cells. Thus, it is not limited to only a subset of patients with a specific HLA type (22). Several clinical trials testing CARþ T cells in patients have shown promising clinical outcomes. Porter, Grupp, Brentjens, and their colleagues (23–25) have engineered patients' T cells with a lentiviral vector expressing anti-CD19 CAR and reinfused the anti-CD19 CAR autologous T cells into patients with refractory chronic lymphocytic leukemia and acute lymphoblastic leukemia. However, significant toxicities, including hypotension, fevers, renal insufficiency, cytokine-release syndrome, and B-cell aplasia, have occurred after infusions of CAR T cells (23–26). The hallmark toxicity of CAR T-cell therapies is cytokine-release syndrome, a potentially life-threatening complication of inflammatory symptoms resulting from elevated plasma cytokine levels associated with T-cell activation and proliferation (23–26). In contrast, NK cell–based immunotherapy has not been associated with a cytokine-release syndrome in patients; it has been associated with a significant NK versus leukemic effect in the absence of graft-versus-host disease (GvHD) and a significant decrease in leukemia relapse following haploidentical allogeneic stem cell transplantation in which the donor/recipient NK killer immunoglobulin-like receptor (KIR)/malignant major histocompatibility complex (MHC) class I mismatch may occur (27). Our approach to NK cell–based immunotherapy is to expand and activate NK cells and redirect them specifically against resistant lymphoma/ leukemia cells. This approach might circumvent resistance to NKcell tumor cytotoxicity independent of HLA class I ligand expression on leukemia cells, target NK cells to specific lymphoma/ leukemic tumor antigens, enhance NK-cell activation, and provide an alternative to adoptive allogeneic T-cell immunotherapy by circumventing the risk of acute GvHD that commonly occurs. Here, we investigated in vitro and in vivo activities of anti-CD20 CAR-modified expanded peripheral blood NK cells (exPBNK) following mRNA nucleofection, against CD20þ B-NHL.

Materials and Methods Cell lines and tumor targets The human cell lines Ramos, Daudi, RS4;11, U698M, K562, and the T-cell ALL lines, Jurkat, were purchased from the American Type Culture Collection (ATCC). Raji, rituximab-resistant Raji2R, and Raji-4RH cells were generously provided by Matthew Barth and Myron Czuczman from Roswell Park Cancer Institute (Buffalo, NY; ref. 28). All tumor cells were maintained in RPMI1640 (Gibco) supplemented with 10% FBS (Gibco) and antibiotics. The K562-mb15-41BBL cell line was generously provided by Dr. Dario Campana (St Jude Children's Research Hospital,

334 Cancer Immunol Res; 3(4) April 2015

University of Tennessee College of Medicine, Memphis, TN; ref. 17). NK-cell expansion and isolation Leukocytes were obtained after informed consent from healthy adult donors at the New York Blood Center. Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll gradient (Amersham Biosciences) separation. NK-cell expansion and isolation were performed as previously described (17) and are detailed in the Supplementary Methods. Production of anti-CD20-BB-z mRNA Anti-CD20 CAR mRNA was generated as previously described with modifications (29). Briefly, the anti-CD20-4-1BB-CD3z was cut from an MSCV-anti-CD20-BB-z-IRES-GFP vector and subcloned into the pcDNA3 vector. Anti-CD20-4-1BB-CD3z mRNA was transcribed in vitro using the mMESSAGE mMACHINE T7 Ultra Kit (Life Technologies) following the manufacturer's instructions. The resulting product was dissolved in nuclease-free H2O. RNA concentrations were measured using a Nano-spectrophotometer (Thermo Fisher) at 260 nm. Nucleofection Six to 8 106 expanded purified NK cells were suspended in 100-mL nucleofection solution (Lonza); anti-CD20-BB-z mRNA was added at 80 to 100 mg/mL. The mixture was placed in the Nucleofector Cuvette and nucleofected using the Amaxa Nucleofector II Device with U-001 program. CAR expression was detected using FITC-conjugated or AF647-conjugated goat anti-mouse IgG, F(ab0 )2 fragment-specific antibody (The Jackson Laboratory), and flow cytometry. Intracellular CD107a and IFNg assays Intracellular CD107a expression and IFNg were measured by flow cytometry, as previously described (20). Intracellular CD107a and IFNg assays are detailed in the Supplementary Methods. In vitro cytotoxicity NK cytotoxic activity was determined by europium release assays with a standard kit (PerkinElmer), as previously described (20). In vitro cytotoxicity assays are detailed in the Supplementary Methods. Animal studies Six- to 8-week-old NOD/SCID/g-chain/ (NSG) mice were purchased (Stock #5557; The Jackson Laboratory), bred, and maintained under pathogen-free conditions in-house at the Department of Comparative Medicine at New York Medical College (Valhalla, NY). All protocols were approved by the Institutional Animal Care and Use Committee. Xenograft models of human Burkitt lymphoma and rituximabresistant Burkitt lymphoma The mammalian expression construct ffLUCZeo-pcDNA3.1 (generously supplied by L. Cooper, University of Texas MD Anderson Cancer Center, Houston, TX) was electroporated into tumor cells (Raji and Raji-2R), and stable clones were selected with Zeocin (Invitrogen). Raji or Raji-2R cells (5 105) expressing luciferase (Raji-Luc or Raji-2R-Luc) were i.p. or s.c. injected into the NSG mice (NOD.Cg-PRkdcscidIl2rgtmWjl/SzJ; 6 weeks; The Jackson Laboratory). Tumor engraftment and progression were




mRNA CAR exPBNK Therapy for CD20þ B-NHL

evaluated using the Xenogen IVIS-200 System (Caliper Life Sciences) after i.p. injection of 150 mg D-luciferin/kg/mouse. Photons emitted from luciferase-expressing cells were quantified using Living Image software. After tumor engraftment was verified, freshly prepared 5 106 CAR exPBNK, mock exPBNK, or medium was i.p. injected into each mouse once a week for 3 weeks. Tumor regression and/or progression of xenografted mice were monitored weekly by tumor volume measurement and by in vivo bioluminescent imaging (BLI; refs. 30, 31). Tumor size was estimated according to the following formula (32): tumor size (cm3) ¼ length (cm) width2 (cm) 0.5. Mice were followed until death or sacrificed if tumor size reached 2 cm3 or larger. Histology (H&E) staining and Immunofluorescence staining Fresh-frozen tumor samples were sectioned for hematoxylin and eosin (H&E) analysis and immunofluorescence staining as described in the Supplementary Methods. TruCOUNT analysis of absolute cell counts TruCOUNT tubes (Becton Dickinson) were used to determine the absolute counts of tumor cells and NK cells in the peripheral blood of xenografted mice. Peripheral blood was obtained via face-vein bleeding and stained for the presence of human CD45, CD20, and CD56 NK cells. After gating on the human CD45þ population, the CD20þ and CD56þ subsets were quantified using TruCOUNT tubes (BD Biosciences) with known numbers of fluorescent beads as described in the manufacturer's instructions. In vitro organ imaging Raji-2R-Luc cells (5 105) were intravenously (i.v.) injected into NSG mice. Mice were sacrificed when hind-leg paralysis was observed. Organs (lung, liver, spleen, and kidney) were collected and soaked in D-PBS with 300 mg/mL D-luciferin for 5 minutes before BLI using the Xenogen IVIS-200 system (Caliper Life Sciences). Statistical analyses Statistical analyses were performed using the INSTAT statistical program (GraphPad). Average values are reported as the mean SEM. Results were compared using the one-tailed unpaired Student t test, with P < 0.05 considered as statistically significant. Probability of survival in animal studies was determined by the Kaplan–Meier method using the Prism program 5.0 (GraphPad Software, Inc.).

Results PBNK (CD3/56þ) expansion and expression of anti-CD20-BBz receptors in exPBNK by mRNA nucleofection PBMNCs from healthy donors were cultured with irradiated geK562 feeder cell lines, as described (17), in the presence of 40 IU/mL IL2. As reported previously, CD56þCD3 PBNK were significantly increased compared with those grown in media alone at day 7 (mean, 66.87% vs. 8.84%; n ¼ 3; Fig. 1A). There was no significant enrichment of CD56þCD3þ PB NKT cells compared with those grown in media alone at day 7 (data not shown). CD56CD3þ PB T cells were significantly reduced compared with those grown in media alone at day 7 (mean, 19.16% vs. 77.00%; n ¼ 3; Fig. 1B). The absolute NK numbers were enhanced with irradiated geK562 as feeders compared with irra-


diated K562 and IL2 alone after normalized to the INPUT NK cell numbers (mean, 44.4-fold 1.47 vs. 12.49-fold 1.50 vs. 0.239fold 0.09; n ¼ 3; P < 0.001; Fig. 1C). We also observed that sustained activation and proliferation by irradiated geK562 induced morphologic changes in NK cell shape and size (Fig. 1D). We examined the expression of CD3 and CD56 in the cells cocultured with media alone, K562, and geK562 by flow cytometry analysis (Fig. 1E). The exPBNK by geK562 were negatively selected with more than 96% purity (Fig. 1E). The anti-CD20-BB-z chimeric receptor (anti-CD20 CAR) was originally constructed in the retroviral plasmid pMSCV-IRES-antiCD20-BB-z (kindly provided by Dr. Dario Campana, St Jude Children's Research Hospital, University of Tennessee College of Medicine; Supplementary Fig. S1A). The expression of anti-CD20 CAR was confirmed in virus-infected Jurkat cells by flow cytometry and by Western blot analysis (Supplementary Fig. S1B and S1C). Anti-CD20 CAR-mediated cytotoxicity was first examined using NK-92 cells and CD20þ NHL. NK92 cells expressing anti-CD20 CAR had enhanced cytotoxicity against CD20þ NK-sensitive Ramos (74.66% 8.01% vs. 49.63% 5.10%; P < 0.05) and CD20þ NK-resistant Daudi (43.15% 4% vs. 16.15% 6.4%; P < 0.01) cells at E:T ¼ 10:1 ratio but not against the CD20 pre-B-ALL cell line RS4;11 (40.46% 1.14% vs. 41.21% 1.02%: P ¼ ns) using europium release assays (Supplementary Fig. S1D). After functional confirmation, the anti-CD20 CAR fragment was excised with restriction enzymes from the retroviral vector pMSCV-IRES-anti-CD20-BB-z and ligated into a T7 promoter– driven plasmid (Fig. 2A). Anti-CD20 CAR mRNA was in vitro transcribed, as described previously (Fig. 2B; ref. 33). Expanded CD56þCD3 PBNK purified from the peripheral blood of 10 healthy donors were nucleofected in the presence of anti-CD20 CAR mRNA to generate anti-CD20 CAR exPBNK or in nucleasefree H2O to generate mock exPBNK. Flow cytometry was used to detect the expression of anti-CD20 CAR in 66.73% 6.817% of viable exPBNK after 16 hours of nucleofection (Fig. 2C). Background from the exPBNK nucleofected with nuclease-free H2O was 2.89% 0.947%. As expected, the anti-CD20 CAR expression was transient and was reduced from 94.1% at 20 hours to 60.1% at day 6 and 2.99% at day 11 after nucleofection (Fig. 2D). CAR mRNA nucleofection, however, did not affect the expression of exPBNK-activating receptors (CD16, CD69, NKG2D, CD244, NKp30, NKp44, and NKp46) or inhibitory receptors (NKG2A, KIR2DS4, CD94, CD158a, CD158b, and CD158e; Supplementary Fig. S2). Cytotoxic activity of anti-CD20 CAR mRNA-modified exPBNK in vitro against B-NHL and rituximab-resistant cells We examined the functionality of anti-CD20 CAR mRNAmodified exPBNK against CD20þ B-NHL target cells. CD20 expression on the target tumor cells was confirmed (Supplementary Table S1). Anti-CD20 CAR exPBNK cytotoxicity was significantly enhanced compared with that of mock exPBNK against CD20þ B-NHL at 10:1 (n 3): Ramos (97.25% 2.61% vs. 82.5% 4.058%; P < 0.05), Daudi (71.5% 3.26% vs. 36.34% 6.31%; P < 0.001), and U-698-M (82.84% 1.17% vs. 26.2% 0.776%; P < 0.001; Fig. 3A). There was no significant difference against CD20 RS4;11 or Jurkat cells (Fig. 3B). Tumor-cell recovery experiments further confirmed the result. Anti-CD20 CAR exPBNK significantly reduced Ramos cell recovery compared with those of medium and mock exPBNK (P < 0.01; Fig. 3C). These data

Cancer Immunol Res; 3(4) April 2015


335


Chu et al.

Figure 1. Irradiated genetically modified K562 cells significantly expanded NK cells from whole PBMC cultures. PBMCs were cultured at a ratio of 1.5:1 without (i.e., IL2 þ þ alone) or with irradiated geK562 feeder cell lines in the presence of 40 U/mL IL2. CD56 CD3 NK cell (A) and CD56 CD3 T cell (B) percentages at day 7 are shown. C, the fold changes of the absolute number of NK cells cultured with IL2 alone, with irradiated K562 or with irradiated geK562 cells, to the input NK-cell number are compared. D, cell phenotypic changes under light microscopy (Axiovert 200M; Carl Zeiss) are shown during ex vivo NK expansion in 24-well plates (original magnification, 200). E, expression of CD3 and CD56 is shown in flow cytometric density plots during NK ex vivo expansion and after NK isolation. Average values are reported as the mean SEM (n ¼ 3). P values using the unpaired Student t test are noted in A, B, and C, respectively.

demonstrate the targeting specificity of anti-CD20 CAR mRNAmodified exPBNK against CD20þ tumor cells. We examined the expression of CD20 in rituximab-sensitive Raji and rituximab-resistant Raji-2R and Raji-4RH cells by flow cytometry analysis (Supplementary Table S2). In addition, the standard europium release assay experiments demonstrated that the cytotoxicity of CAR exPBNK was significantly higher than that of mock exPBNK (P < 0.001) against Raji, Raji-2R, and Raji-4RH cells (Fig. 3D). Daudi and Raji cells have been demonstrated previously to be resistant to NK-mediated cytotoxicity (34). NK resistance of Raji cells is, in large part, mediated through the interaction of KIRs and histocompatibility class I antigens (HLA; ref. 35). Unlike Raji cells, Daudi cells do not synthesize b2-microglobulin and lack the cell-surface HLA antigens (36). Therefore, resistance of Daudi cells to NK-mediated lysis has not been thought to be mediated through HLA antigens. The mechanism of NK resistance in Daudi cells is currently unknown. We found that our exPBNK express KIR2DL1, KIR2DL2/3, KIR3DL1, and NKG2A (Supplementary Fig. S2 and data not shown). Raji, Raji-


2R, and Raji-4RH cells used in our experiments express high levels of HLA class I antigens by flow cytometry analysis (Fig. 3E and Supplementary Table S3). The anti-CD20 CAR exPBNK showed enhanced cytolytic activities against NK-resistant Daudi and Raji cells (Fig. 3), indicating that the expression of anti-CD20 CAR on NK cells can overcome inhibitory signals from KIR–HLA interactions and other unknown mechanismsmediated resistance. We also observed similarly enhanced expression of exPBNKactivating receptors (CD69, NKp44, and NKG2D) after incubation with U-698-M, compared with that with medium (Supplementary Fig. S3), and the inhibitory receptors (CD94 and KIR2DL2/3) were unchanged in mock exPBNK and CAR exPBNK. Intracellular CD107a expression and IFNg production in vitro The lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) is a marker of NK-cell functional activity (37). Intracellular CD107a expression in exPBNK was detected by flow cytometry. CD107a degranulation was enhanced in anti-CD20 CAR exPBNK, compared with that in mock exPBNK, in response





Figure 2. Expression of anti-CD20 CAR in ex vivo–exPBNK by CAR mRNA nucleofection. A, schematic representation of the mRNA constructs encoding the anti-CD20 CAR receptor used in this study. B, a representative ethidium bromide–stained gel showing in vitro–transcribed anti-CD20 CAR mRNA. C, anti-CD20 mRNA was nucleofected into exPBNK and the transgene expression was followed by flow cytometry. Flow cytometric density plots illustrate expression of the anti-CD20 CAR in one of the 10 donors and the percentage of NK cells expressing anti-CD20 CAR as determined by flow cytometry 16 hours after nucleofection (P < 0.001). Average values are reported as the mean SEM (n ¼ 10). P value using the unpaired Student t test is noted. D, anti-CD20 CAR expression was monitored by flow cytometry at the indicated times following nucleofection.

to CD20þ Ramos (31.47% 1.74% vs. 15.2% 0.26%; P < 0.001; n ¼ 3) and Daudi (38.9% 2.7% vs. 19.73% 0.58%; P < 0.001; n ¼ 3) stimulation; however, there was no significant difference in response to RS4;11 (5.69% 0.45% vs. 3.9% 0.06%; P ¼ ns; n ¼ 3) or medium (4.86% 0.066% vs. 4.75% 0.59%; P ¼ ns; n ¼ 3; Fig. 4A). Similarly, intracellular IFNg production was also enhanced in CAR exPBNK compared with that in mock exPBNK in response to CD20þ Ramos-specific (3.07% 0.42% vs. 1.39% 0.26%; P < 0.05; n ¼ 3) and Daudi-specific (5.62% 0.52% vs. 1.42% 0.43%; P < 0.05; n ¼ 3) stimulation in a short 4-hour incubation (Fig. 4B). In vivo cytotoxicity in humanized Burkitt lymphoma xenografted NSG mice We assessed the antitumor activity of exPBNK nucleofected with anti-CD20 CAR mRNA using a humanized Burkitt lymphoma xenograft NSG model. First, we used a dissected tumor model. The purity and kinetics of BLI signal in Raji-Luc were examined (Supplementary Fig. S4A and S4B). Raji-Luc cells (5 105) were injected i.p. into NSG mice. After successful engraftment of RajiLuc cells in mice at day 7, freshly prepared 5 106 mock or CAR exPBNK were injected i.p. into each mouse once a week for 3 weeks (days 9, 16, and 23). Mice that received culture medium were used as controls. We demonstrated that after the third NK-


cell injection, the luciferase signals measured in the CAR exPBNKtreated Raji-Luc group (n ¼ 6) were significantly reduced, compared with those in the medium-treated mice (P ¼ 0.0087; n ¼ 5) and in the mock exPBNK-treated mice (P ¼ 0.0128; n ¼ 8; Fig. 5A and B). Consistent with the reduced luciferase signals, the CAR exPBNK-treated mice appeared to be more healthy and active than the medium-treated and the mock exPBNK-treated mice (data not shown). The survival curve of mice was built based on death of mice caused by tumor cells disseminated in the whole body from the injection site or sacrifice if tumor size reached 2 cm3 or larger. We found that the CAR exPBNK-treated Raji-Luc mice had significantly extended survival time with a median of 40 days compared with that of the untreated mice (29 days, P < 0.001) and of the mock exPBNK-treated mice (30 days, P < 0.001; Fig. 5C). There was no significant difference in survival between the untreated Raji-Luc mice and the mock exPBNK-treated Raji-Luc mice with the current scheduled NK doses. The proliferation of transferred CAR T cells is highly correlated with tumor regression (23–25). To examine whether the antitumor efficacy of anti-CD20 CAR NK cells is correlated with increased proliferation and long persistence of anti-CD20 CAR PBNK compared with that of the mock PBNK in NSG mice, peripheral blood was collected from Raji-Luc xenografted mice after 7 days of the third anti-CD20 CAR NK injection. Circulated



337


Chu et al.

Figure 3. þ Anti-CD20 CAR mRNA enhances exPBNK in vitro cytolytic activity against CD20 B-NHL cells and rituximab-resistant cells. exPBNK were electroporated in the absence (mock, gray) or in the presence of anti-CD20 CAR mRNA (CAR, black). A, MOCK and CAR exPBNK were incubated for 4 hours with the þ þ BATDA-labeled CD20 NK-sensitive Ramos (top left), CD20 NK-resistant Daudi (top middle), and U-698-M (top right) cells at the indicated E:T ratios. In vitro cytotoxicity was measured by standard europium release assay. At indicated by E:T ratios, cytotoxicity of exPBNK expressing anti-CD20 chimeric receptors was significantly higher than that of mock exPBNK. B, MOCK and CAR exPBNK were incubated for 4 hours with the BATDA-labeled CD20 NK-sensitive Jurkat (bottom left) and CD20 NK-resistant Rs4;11 (bottom right) cells at the indicated E:T ratios. In vitro cytotoxicity was measured by standard europium release assay. C, anti-CD20 CAR expressing NK cells inhibit Ramos-cell recovery. MOCK or CAR exPBNK were incubated overnight with Ramos cells at E:T ¼ 3:1. Ramos cells þ incubated with medium only were used as controls. The total living cells were gated by 7-AAD negative. The living Ramos cells were gated by CD19 7-AAD by flow þ cytometry analysis. Each data point represents the mean ( SEM; n ¼ 3) percentage of living CD19 cells versus total living cells. P values using the unpaired Student þ t test are noted in A, B, and C, respectively. D, MOCK and CAR exPBNK were incubated for 2 hours with the BATDA-labeled CD20 rituximab-sensitive Raji and rituximab-resistant Raji-2R and Raji-4RH cells at the E:T ratio ¼ 10:1. In vitro cytotoxicity was measured by standard europium release assay. As indicated by E:T ratios, cytotoxicity of exPBNK expressing anti-CD20 chimeric receptors was significantly higher than that of exPBNK without anti-CD20 CAR (P < 0.001) against Raji, Raji-2R, and Raji-4RH cells. Each data point represents the mean ( SEM; n ¼ 4) percentage of specific europium release after culture. P values using the unpaired Student t test are noted. E, expression of HLA class I antigens in Raji, Raji-2R, and Raji-4RH cells was determined by flow cytometry with allophycocyanin-conjugated anti-human HLA-ABC antibody (BD Biosciences). Representative staining results are shown.

tumor cells and anti-CD20 CAR NK cells were quantified with TruCOUNT beads by flow cytometry analysis with anti-CD20-PE and anti-CD56-FITC antibodies (Fig. 5D). CD20 Raji cell counts were significantly reduced in mice receiving CAR exPBNK, compared with medium (P < 0.001) and mock (P < 0.05) treatment groups (Fig. 5D, left). Mock exPBNK also significantly reduced the CD20 Raji cell counts, compared with the medium treatment


group (P < 0.001; Fig. 5D, left). However, there was no significant difference in the number of NK cells between mice receiving CAR exPBNK and mock exPBNK (Fig. 5D, right). We examined whether the delayed tumor growth was due to the colocalization of CAR exPBNK with the solid tumor masses. Solid tumor masses were dissected from the medium-treated group, the mock exPBNK-treated group, and the CAR exPBNK-treated group, and





Figure 4. þ þ þ Intracellular CD107a and INFg expression was enhanced in CAR exPBNK compared with CAR exPBNK after incubation with CD20 Ramos and CD20 Daudi but not CD20 RS4;11. Expanded NK cells were electroporated in the absence (mock, gray solid, and gray gradient colors) or in the presence of anti-CD20 CAR þ þ mRNA (CAR, black solid and black gradient color). A, MOCK exPBNK or CAR exPBNK were cultured with medium only, CD20 Ramos (left), CD20 Daudi (middle), or CD20 RS4;11 (right) cells at a 10:1 ratio for 4 hours. CD107a expression in exPBNK was detected with anti-human CD107a–FITC antibody (BD Biosciences) gated on þ CD56 cells (labeled with an anti-CD56–PE antibody; BD Biosciences) by flow cytometry. The same cells stained with isotype-matched controls were used for gating. þ þ Each data point represents the mean ( SEM; n ¼ 3). B, MOCK exPBNK, or CAR exPBNK were cultured with medium only, CD20 Ramos (left) or CD20 þ Daudi (right) cells at a 10:1 ratio for 4 hours. IFNg expression in exPBNK was detected with anti-human IFNg –PE antibody (BD Biosciences) gated on CD56 cells (labeled with an anti-CD56–PE-Cy5 antibody; BD Biosciences) by flow cytometry. The same cells stained with isotype-matched controls were used for gating. Each data point represents the mean ( SEM; n ¼ 3). , P < 0.05; , P < 0.001; ns, not statistically significant.

confirmed by BLI (Fig. 5E, top). H&E staining was used to examine the morphology of tumor tissues from each treatment group (Fig. 5E, bottom). Tumor burden of the CAR exPBNK-treated group appeared to be less compact with tumor cells compared with the other two groups. A fluorescent immunostaining analysis was conducted to detect the presence of NK cells in the tumor masses. We found a few green fluorescent Alexa Fluor 488–labeled NK cells accumulating around the edge of the tumors (Fig. 5F) in mock exPBNK-treated mice and in CAR exPBNK-treated mice but not inside of the tumor masses. We used the localized tumor model to examine the antitumor effect of CAR exPBNK against rituximab-resistant Burkitt lymphoma Raji-2R cells. The purity and kinetics of BLI signal in Raji-2R-Luc was examined (Supplementary Fig. S4C and S4D). Raji-2R-Luc cells (5 105) were injected s.c. into NSG mice. Mock or CAR exPBNK (5 106) were i.p. injected into each mouse once a week for 3 continuous weeks (days 3, 10, and 17). Mice that received culture medium were used as control. After the third NK injection, luciferase signal was significantly reduced in the CAR exPBNK-treated Raji-2R-Luc group, compared with the mock exPBNK-treated mice (P < 0.01; Fig. 6A and B). Tumor size measured in the CAR exPBNK-treated Raji2R group (n ¼ 7) was also significantly smaller than that in the medium-treated mice (P ¼ 0.0175; n ¼ 6) and the mock exPBNK-treated mice (P ¼ 0.0122; n ¼ 7; Fig. 6C). Thereafter, the survival curve was established based on the endpoint of 2 cm3 tumor size. The CAR exPBNK-treated Raji-2R-Luc (n ¼ 7) mice had significantly extended survival time with a median of 24 days, compared with that of the medium-treated (18 days; n


¼ 6; P < 0.001) and of the mock exPBNK-treated mice (22 days; n ¼ 7; P < 0.05; Fig. 6D). Anti-CD20 CAR exPBNK inhibit Raji-2R cells migration and dissemination to multiple organs in xenografted mice To study the effect of anti-CD20 CAR exPBNK in tumor migration and multiorgan dissemination in xenografted mice, 5 105 Raji-2R-Luc cells were i.v. injected into NSG mice. Seven days later, 5 106 mock or CAR exPBNK in culture medium with 200 IU/mL of IL2 were i.v. injected into NSG mice with similar tumor burdens once a week for 2 weeks. In the three pairs of NSG mice treated with mock exPBNK or CAR exPBNK, CAR exPBNK-treated NSG mice had less bioluminescence signal in the pointed organs than that in the mock exPBNK-treated NSG mice (Fig. 7A), suggesting that CAR exPBNK therapy inhibited Raji-2R-Luc dissemination to other organs. Consistent with this observation, DiD (a lipophilic fluorescent dye, C67H103CIN2O3S)-labeled exPBNK were able to migrate to the lung, liver, and spleen after i.v. injection to NSG mice, as confirmed by fluorescent imaging using the IVIS imaging system (Supplementary Fig. S5A) and by flow cytometry analysis (Supplementary Fig. S5B). To confirm this, organs (lung, liver, spleen, and kidney) were collected in D-PBS with D-luciferin for BLI. CAR exPBNK-treated NSG mice had less bioluminescence in the lung, liver, kidney, and spleen than that of the mock exPBNKtreated NSG mice (Fig. 7B).

Discussion Adoptive immunotherapy with NK cells has been limited in the past by the low numbers of activated NK cells in the peripheral



339


Chu et al.

Figure 5. Anti-CD20 CAR exPBNK significantly inhibit growth of Raji cells in xenografted mice. A, Live-imaging demonstrating the extent of Raji-Luc progression. 5 6 Raji-Luc cells (5 10 ) were i.p. injected into NSG mice on day 0. Three doses of (5 10 cells/dose) of either anti-CD20 CAR exPBNK (with anti-CD20 CAR mRNA electroporation) or mock exPBNK (without anti-CD20 CAR mRNA electroporation) cells were injected i.p. on days 9, 16, and 23, respectively. Mice treated with medium and mice without tumor inoculation served as controls. Whole mouse luciferase activity was measured once weekly at various time points. B, photons emitted from luciferase-expressing cells were measured in regions of interest that encompassed the entire body and quantified using Living Image software. Signal intensities (total Flux) are shown at the time points indicated in untreated (medium), anti-CD20 CAR exPBNK þ (MOCK)-, and anti-CD20 CAR exPBNK (CAR)-treated mice and plotted as mean SEM. C, mice were followed until death or sacrificed when tumor size 3 reached 2 cm . The Kaplan–Meier survival curves for all groups were generated following therapy initiation using animal sacrifice as the terminal event. Comparison of survival for the four groups showed a statistically significant difference. The CAR exPBNK-treated Raji-Luc (n ¼ 6) mice had significantly extended survival time compared with the untreated mice (n ¼ 5; P < 0.001) and the mock exPBNK-treated mice (n ¼ 8; P < 0.001). There was no statistically significant difference in survival between the untreated Raji mice and the mock exPBNK-treated Raji mice. Five NSG mice without tumor inoculation þ were used as controls. D, peripheral blood was collected from Raji-Luc xenografted mice 7 days after the last NK injection. Circulating CD20 tumor cells (left) and NK cells (right) from the medium-only control group (n ¼ 2), mock exPBNK group (n ¼ 6), and CAR exPBNK group (n ¼ 5), were quantified using the TruCOUNT method by flow cytometry analysis with anti-CD20-PE and anti-CD56-FITC antibodies. , P < 0.05; , P < 0.01; , P < 0.001; ns, not statistically significant. E, tumor masses were dissected from 3 mice in each group, washed with D-PBS, and soaked in D-PBS with D-luciferin. Bioluminescent images were acquired and analyzed with Living Image software (Xenogen; top). Then the tumor masses were embedded in optimal cutting temperature (OCT) compound and frozen at 80 C. Of note, 10 mm sections were cut in a cryostat microtome, fixed with cold acetone, and stained with H&E (bottom; original magnification, 200). F, frozen tumor sections from 3 mice of each group were stained with rabbit anti-mouse CD56 and then with goat anti-rat Alexa fluor 488 secondary antibodies. Rabbit serum and tumor sections without NK treatment were used as negative controls. Nuclei were counterstained with DAPI (original magnification, 100). Data shown are representative of three independent assays.






Figure 6. 5 Anti-CD20 CAR exPBNK significantly inhibit growth of Raji-2R cells in xenografted mice. Raji-2R-Luc cells (5 10 ) were s.c. injected into the right flanks of NSG mice. 6 Three days after tumor inoculation, mice were randomized to equalize tumor burden and injected weekly i.p. with 5 10 anti-CD20 CAR exPBNK or mock exPBNK from the same healthy donor for 3 consecutive weeks; mice treated with medium and mice without tumor inoculation served as controls. A, bioluminescence images were taken once weekly. Live imaging demonstrating the extent of Raji-2R progression is shown. B, photons emitted from luciferase-expressing cells were measured in regions of interest that encompassed the entire body and quantified using Living Image software. Signal intensities (total Flux) are shown at the þ time points indicated in medium untreated, mock exPBNK (mock)-, and anti-CD20 CAR exPBNK (CAR)-treated mice and plotted as mean SEM. C, tumor size was 3 measured with a caliper once a week and plotted as the mean SEM for each group. D, mice were sacrificed when tumor size reached 2 cm . The Kaplan– Meier survival curves for all groups were generated following therapy initiation using animal sacrifice as the terminal event. Comparison of survival for the four groups showed a statistically significant difference. The CAR exPBNK-treated Raji-2R (n ¼ 7) mice had significantly extended survival time compared with the mediumtreated mice (n ¼ 8; P < 0.001) and the mock exPBNK-treated mice (n ¼ 7; P < 0.05). The mock exPBNK treatment also significantly extended the survival time of Raji-2R xenografted mice compared with that of the medium-treated mice (P < 0.01). Five NSG mice without tumor inoculation were used as controls. , P < 0.05; , P < 0.01; , P < 0.001; ns, not statistically significant.

blood and concomitantly by the lack of tumor-targeting specificity. In this study, we established conditions that expanded activated PBNK ex vivo and modified the exPBNK efficiently with anti-CD20 CAR mRNA in a nonviral, clinically relevant method. The antiCD20 CAR expression in exPBNK by mRNA nucleofection was associated with a statistically significant increase in CD107a degranulation and INFg production after stimulation with CD20þ B-NHL. Consequently, CAR exPBNK treatment results in significant and specific in vitro cytotoxicity against CD20þ B-NHL, including both rituximab-sensitive and rituximab-resistant B-NHL cells. Multiple injections of anti-CD20 CAR mRNA-electroporated exPBNK mediated significant tumor regression and inhibited tumor growth in both disseminated and localized tumor models in NSG mice. More importantly, multiple injections of anti-CD20 CAR-modified exPBNK also extended survival in B-NHL tumorxenografts and inhibited tumor-cell migration. These results indicate the therapeutic potential of multiple injections of anti-CD20 CAR mRNA-modified exPBNK against CD20þ B-NHL in patients. Gene-engineered T cells with viral methods have been used for cancer therapy with success, but concerns about safety has arisen after X-SCID patients developed cancerous T cells after receiving hematopoietic stem cells transduced with recombinant retrovirus (38), and manufacturing challenges may also hamper clinical progress. The development of safe, efficient, and nonviral methods with a lower tendency to insert near oncogenes will significantly facilitate targeted cellular therapies (33, 39). Synthetic modified


mRNA in vitro has great therapeutic potential for transient, targeted protein expression with limited toxicity. In addition, large quantities of RNA can be easily prepared by in vitro transcription, which makes it possible to expedite manufacturing and scaling to current Good Manufacturing Practices (cGMP) products. Successful electroporation of CAR mRNA into primary T cells and NK cells has now been documented for a variety of cancers, such as anti-CD19 CAR against leukemia (29, 39) and mesothelin CAR against pancreatic cancer (clinical trial: NCT01897415). However, these synthesized mRNAs result in only short-lived CAR expression in immune cells. As shown in Fig. 2D, anti-CD20 CAR expression lasted less than 2 weeks after nucleofection in exPBNK. Considering the lifespan of adoptively transferred NK cells is only approximately 10 to 12 days even with cytokine support (Supplementary Fig. S5C; refs. 40–42), transient expression of CAR in exPBNK may not be a significant disadvantage and it does not need a suicide gene to limit the risk of the long-term side effect using retroviral or lentiviral vectors. In our xenograft models, three injections of anti-CD20 CAR mRNA-electroporated exPBNK significantly mediated CD20þ tumor regression, extended survival (Fig. 5B and C), reduced tumor size of CD20þ tumor xenografted mice (Fig. 6C), and inhibited tumor-cell migration to other organs in NSG mice (Fig. 7). However, under current NK doses and injection frequencies, we did not obtain tumor-free animals. The ratio of CAR NK cells to target cells may be critical to the success, as shown in our in vitro cytotoxicity assays (Fig. 3). The remaining



341


Chu et al.

Figure 7. 5 Anti-CD20 CAR exPBNK inhibit Raji-2R cell migration and dissemination to multiple organs in xenografted mice. Raji-2R-LUC cells (5 10 ) were i.v. injected into NSG 6 mice. Seven days later, mice with similar tumor burden (measured by BLI) were chosen for NK therapy. Mock exPBNK or CAR exPBNK (5 10 ) in culture medium with 200 IU/mL IL2 were i.v. injected into NSG mice once a week for 2 consecutive weeks. Mice were monitored daily for hind-leg paralysis. A, tumor engraftment and progression in the whole body of a mouse were evaluated using the Xenogen IVIS-200 system (Caliper Life Sciences) after i.p. injection of 150 mg D-luciferin/kg per mouse weekly. The arrows point to the organs that have more bioluminescence intensity in the mock exPBNK-treated NSG mice compared with the CAR exPBNK-treated NSG mice. B, for organ imaging, mice were sacrificed once hind-leg paralysis was observed and the organs (lung, liver, spleen, and kidney) were collected and washed with D-PBS and soaked in D-PBS with D-luciferin. Organ bioluminescent images were acquired and analyzed with Living Image software (Xenogen).

tumor cells in the xenografted mice after three NK injections are still CD20þ (data not shown), and the relapse is not likely a consequence of CD20 deregulation to escape anti-CD20 CAR NK cytotoxicity. In the future, NK cells expanded with a new feeder K562-mb21- 41BBL and increased injection frequency of higher doses of CAR NK will be considered to enhance NK in vivo expansion and antitumor effect. To explore the possible mechanisms of antitumor efficacy of mRNA CAR exPBNK, we first examined whether CAR exPBNK proliferate better and persist longer than mock exPBNK. Within our expectations, CAR exPBNK significantly reduced the number of circulating tumor cells in the peripheral blood of mice, compared with that in the medium (P < 0.001) and mock (P < 0.05) treatment groups (Fig. 5D, left), but there was no significant difference in NK cell numbers among mice receiving CAR exPBNK and mock exPBNK (Fig. 5D, right), which may be due to the lack of proliferative capacities of NK cells. Second, we examined whether mRNA CAR exPBNK colocalize better than mock exPBNK in solid tumor masses to inhibit tumor growth. The tumor burden of the CAR exPBNK-treated group appeared to be less compact with tumor cells than that of the other two groups (Fig. 5E), and the tumor size is smaller in the CAR exPBNK-treated group than that in the other two groups (Fig. 6C). We found a few green fluorescent Alexa Fluor


488–labeled NK cells around the edge of the tumors (Fig. 5F) in both mock exPBNK-treated mice and CAR exPBNK-treated mice. We did not find mock or mRNA CAR exPBNK inside of the tumor masses (data not shown). This is consistent with the recent study in which Singh and colleagues (43) have substantiated the difference between permanent CAR T cells and mRNA-based CAR T cells, and the transiently modified T cells were unable to significantly penetrate inside of the tumor masses. Therefore, anti-CD20 CAR mRNA-modified exPBNK can mediate effective antitumor responses in vivo; however, to achieve complete tumor eradication with mRNA-based CAR exPBNK therapies, we will pursue studies combinating CAR exPBNK therapy with other approaches, such as bispecific antibodies. Tumor cells may escape from NK-cell immunosurveillance and develop resistance by several mechanisms. One mechanism is associated with the downregulation of natural cytotoxicity receptor expression on NK cells (44, 45). Our ex vivo–expanded human PBNK with geK562 upregulated activating receptors, including CD69, NKp30, NKp44, and NKG2D (Supplementary Fig. S6). These exPBNK can mediate cytotoxicity of allogeneic and autologous cancer cell lines by specifically activating receptor–ligand interactions. Another mechanism of tumor-cell resistance is by upregulating the expression of KIR-inhibitory ligands (46). In our





study, we found that the expanded NK cells express KIRs, such as KIR2DL1, KIR2DL2/3, and KIR3DL1, and Raji, Raji-2R, and Raji4RH cells express high-level HLA-ABC (Supplementary Fig. S2 and Fig. 3E). The KIR receptor–HLA ligand interactions mediate resistance to NK-mediated lysis. Previous studies reported that resistance to NK-mediated lysis can be overcome by preincubation of NK cells with IL2 and the generation of "lymphokine-activated killer cells" (34). Our study demonstrates that anti-CD20 CARmodified exPBNK have significantly enhanced cytotoxicity against NK-resistant CD20þ Raji, Raji-2R, and Raji-4RH cells but not against NK-resistant CD20-RS;11 cells (Fig. 3), demonstrating that anti-CD20 CAR-mediated CD20-dependent signaling can overcome the inhibitory signaling from the interaction between inhibitory receptors and HLA ligands. The third mechanism of tumor cells escaping from NK-cell immunosurveillance is by downregulating the NKG2D activation signals such as shedding of NKG2D ligands from tumor cells (47). We are currently investigating the combination of enhancing NKG2D ligand expression on tumor cells and treating with anti-CD20 CARmodified exPBNK (48). Promising clinical results have been obtained using rituximab to treat B-NHL because rituximab was approved by the FDA in 1997 (49). However, approximately 60% of patients who responded to their first rituximab therapy, relapsed following rituximab retreatment (50). One of the relapsed mechanisms is the reduced expression of CD20 on the surface of cancer cells after they are repeatedly exposed to rituximab. Despite significantly reduced CD20 antigen expression in rituximab-resistant cells, compared with that in sensitive cells, CD20 is still relatively high in rituximab-resistant cells (Supplementary Table S2; ref. 28). We attempted to assess whether anti-CD20 CAR-mediated exPBNK cytotoxicity would overcome rituximab resistance. Interestingly, anti-CD20 CAR exPBNK had significantly enhanced cytotoxicity against rituximab-resistant Burkitt lymphoma cells in vitro (Fig. 3D), and anti-CD20 CAR exPBNK significantly inhibited the growth of rituximab-resistant Burkitt lymphoma cells in xenografted NSG mice (Fig. 6), compared with that in mice treated with mock exPBNK. Our results indicate that anti-CD20 CAR NK cells can bind more efficiently to the reduced level of CD20 on resistant tumor cells than rituximab, and the anti-CD20 CAR NK cells can be activated to lyse the resistant tumor cells efficiently. Moreover, we observed that the anti-CD20 CAR exPBNK limited the dissemination/migration of rituximab-resistant Raji-2R tumor cells to multiple organs, compared with the mock exPBNK (Fig. 7). In conclusion, our data confirm that activated expanded allogeneic PBNK become highly cytolytic, killing resistant CD20þ B-

leukemia/lymphoma after nucleofection with anti-CD20 CAR mRNA. We propose that the interaction between anti-CD20 CAR on exPBNK and CD20 on the surface of B-leukemia/lymphoma transmits an activating signal back to the exPBNK and significantly stimulates exPBNK degranulation and release of cytokines such as IFNg to lyse resistant tumor targets (Supplementary Fig. S7). Results from our preclinical studies should assist in designing clinical trials with CAR exPBNK to treat patients with CD20þ Bleukemia/lymphoma without the associated safety concerns of integrating viral vectors. In future studies, we will examine the persistence of expanded NK cells in vivo and assess the combined therapeutic effect of anti-CD20 CAR-modified NK cell–based therapies with the new generation of antibody-based therapies.

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Authors' Contributions Conception and design: Y. Chu, J. Hochberg, M.S. Cairo Development of methodology: Y. Chu, J. Hochberg, A. Yahr, J. Ayello Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Y. Chu, A. Yahr, M. Barth Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Y. Chu, J. Hochberg, A. Yahr Writing, review, and/or revision of the manuscript: Y. Chu, J. Hochberg, J. Ayello, C. van de Ven, M. Barth, M. Czuczman, M.S. Cairo Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Chu, J. Ayello, C. van de Ven, M. Czuczman, M.S. Cairo Study supervision: M.S. Cairo

Acknowledgments The authors thank Erin Morris, RN, for her excellent assistance with the preparation of this article; Dr. Dario Campana (St. Jude Children's Research Hospital) and Dr. Terrence Geiger (St. Jude Children's Research Hospital) for kindly providing anti-CD20 scFv; Dr. Carl Hamby (New York Medical College) for sharing equipment and reagents; and Anne Sollas (Core Histology Lab, Pathology Department, New York Medical College) for technical support.

Grant Support This research was supported by grants from the Pediatric Cancer Research Foundation (PCRF), Children's Cancer Fund (CCF), and a New York Medical College Intramural Research Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received June 12, 2014; revised November 22, 2014; accepted November 25, 2014; published OnlineFirst December 9, 2014.

References 1. Pinkerton R CM. Childhood non-Hodgkin lymphoma. In: Mitchell S, Cairo SLP, editors. Hematological malignancies in children, adolescents and young adults. Singapore: World Scientific; 2012. p. 299–328. 2. Cairo MS, Gerrard M, Sposto R, Auperin A, Pinkerton CR, Michon J, et al. Results of a randomized international study of high-risk central nervous system B non-Hodgkin lymphoma and B acute lymphoblastic leukemia in children and adolescents. Blood 2007;109:2736–43. 3. Gerrard M, Cairo MS, Weston C, Auperin A, Pinkerton R, Lambilliote A, et al. Excellent survival following two courses of COPAD chemotherapy in children and adolescents with resected localized B-cell non-Hodgkin's lymphoma: results of the FAB/LMB 96 international study. Br J Haematol 2008;141:840–7. 4. Cairo MS, Sposto R, Gerrard M, Auperin A, Goldman SC, Harrison L, et al. Advanced stage, increased lactate dehydrogenase, and primary site, but not


5. 6. 7. 8.

adolescent age (>/ ¼ 15 years), are associated with an increased risk of treatment failure in children and adolescents with mature B-cell nonHodgkin's lymphoma: results of the FAB LMB 96 study. J Clin Oncol 2012;30:387–93. Miles RR, Arnold S, Cairo MS. Risk factors and treatment of childhood and adolescent Burkitt lymphoma/leukaemia. Br J Haematol 2012;156:730–43. Blum KA, Lozanski G, Byrd JC. Adult Burkitt leukemia and lymphoma. Blood 2004;104:3009–20. LeBien TW, Tedder TF. B lymphocytes: how they develop and function. Blood 2008;112:1570–80. Perkins SL, Lones MA, Davenport V, Cairo MS. B-Cell non-Hodgkin's lymphoma in children and adolescents: surface antigen expression and clinical implications for future targeted bioimmune therapy: a children's cancer group report. Clin Adv Hematol Oncol 2003;1:314–7.



343


Chu et al.

9. Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2002;346:235–42. 10. Goldman S, Smith L, Anderson JR, Perkins S, Harrison L, Geyer MB, et al. Rituximab and FAB/LMB 96 chemotherapy in children with Stage III/IV Bcell non-Hodgkin lymphoma: a Children's Oncology Group report. Leukemia 2013;27:1174–7. 11. Dunleavy K, Pittaluga S, Shovlin M, Steinberg SM, Cole D, Grant C, et al. Low-intensity therapy in adults with Burkitt's lymphoma. N Engl J Med 2013;369:1915–25. 12. Paust S, von Andrian UH. Natural killer cell memory. Nat Immunol 2011;12:500–8. 13. Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, et al. Innate or adaptive immunity? The example of natural killer cells. Science 2011;331:44–9. 14. Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural killer cells. Nat Immunol 2008;9:503–10. 15. Raulet DH, Vance RE. Self-tolerance of natural killer cells. Nat Rev Immunol 2006;6:520–31. 16. Shereck E, Satwani P, Morris E, Cairo MS. Human natural killer cells in health and disease. Pediatr Blood Cancer 2007;49:615–23. 17. Imai C, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 2005;106:376–83. 18. Berg M, Lundqvist A, McCoy P Jr, Samsel L, Fan Y, Tawab A, et al. Clinicalgrade ex vivo-expanded human natural killer cells up-regulate activating receptors and death receptor ligands and have enhanced cytolytic activity against tumor cells. Cytotherapy 2009;11:341–55. 19. Robinson KL, Ayello J, Hughes R, van de Ven C, Issitt L, Kurtzberg J, et al. Ex vivo expansion, maturation, and activation of umbilical cord blood-derived T lymphocytes with IL-2, IL-12, anti-CD3, and IL-7. Potential for adoptive cellular immunotherapy post-umbilical cord blood transplantation. Exp Hematol 2002;30:245–51. 20. Ayello J, van de Ven C, Cairo E, Hochberg J, Baxi L, Satwani P, et al. Characterization of natural killer and natural killer-like T cells derived from ex vivo expanded and activated cord blood mononuclear cells: implications for adoptive cellular immunotherapy. Exp Hematol 2009;37:1216–29. 21. Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH, Geiger TL, et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia 2004;18:676–84. 22. Kohn DB, Dotti G, Brentjens R, Savoldo B, Jensen M, Cooper LJ, et al. CARs on track in the clinic. Mol Ther 2011;19:432–8. 23. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptormodified T cells in chronic lymphoid leukemia. N Engl J Med 2011;365: 725–33. 24. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013;368:1509–18. 25. Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, et al. CD19targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia. Sci Transl Med 2013;5:177ra38. 26. Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, et al. B-cell depletion and remissions of malignancy along with cytokineassociated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells. Blood 2012;119:2709–20. 27. Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, et al. Effectiveness of donor natural killer cell alloreactivity in mismatched hematopoietic transplants. Science 2002;295:2097–100. 28. Czuczman MS, Olejniczak S, Gowda A, Kotowski A, Binder A, Kaur H, et al. Acquirement of rituximab resistance in lymphoma cell lines is associated with both global CD20 gene and protein down-regulation regulated at the pretranscriptional and posttranscriptional levels. Clin Cancer Res 2008;14: 1561–70. 29. Shimasaki N, Fujisaki H, Cho D, Masselli M, Lockey T, Eldridge P, et al. A clinically adaptable method to enhance the cytotoxicity of natural killer cells against B-cell malignancies. Cytotherapy 2012;14:830–40. 30. Nishimura R, Baker J, Beilhack A, Zeiser R, Olson JA, Sega EI, et al. In vivo trafficking and survival of cytokine-induced killer cells resulting in minimal GVHD with retention of antitumor activity. Blood 2008;112:2563–74.


31. Zheng J, Xu L, Zhou H, Zhang W, Chen Z. Quantitative analysis of cell tracing by in vivo imaging system. J Huazhong Univ Sci Technolog Med Sci 2010;30:541–5. 32. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24: 148–54. 33. Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL, et al. Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor. Cancer Res 2010;70:9053–61. 34. North J, Bakhsh I, Marden C, Pittman H, Addison E, Navarrete C, et al. Tumor-primed human natural killer cells lyse NK-resistant tumor targets: evidence of a two-stage process in resting NK cell activation. J Immunol 2007;178:85–94. 35. Hasenkamp J, Borgerding A, Wulf G, Uhrberg M, Jung W, Dingeldein S, et al. Resistance against natural killer cell cytotoxicity: analysis of mechanisms. Scand J Immunol 2006;64:444–9. 36. de Preval C, Mach B. The absence of beta 2-microglobulin in Daudi cells: active gene but inactive messenger RNA. Immunogenetics 1983;17: 133–40. 37. Alter G, Malenfant JM, Altfeld M. CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods 2004;294:15–22. 38. Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348: 255–6. 39. Barrett DM, Zhao Y, Liu X, Jiang S, Carpenito C, Kalos M, et al. Treatment of advanced leukemia in mice with mRNA engineered T cells. Hum Gene Ther 2011;22:1575–86. 40. Somanchi SS, Somanchi A, Cooper LJ, Lee DA. Engineering lymph node homing of ex vivo–expanded human natural killer cells via trogocytosis of the chemokine receptor CCR7. Blood 2012;119:5164–72. 41. Guimaraes F, Guven H, Donati D, Christensson B, Ljunggren HG, Bejarano MT, et al. Evaluation of ex vivo expanded human NK cells on antileukemia activity in SCID-beige mice. Leukemia 2006;20:833–9. 42. Parkhurst MR, Riley JP, Dudley ME, Rosenberg SA. Adoptive transfer of autologous natural killer cells leads to high levels of circulating natural killer cells but does not mediate tumor regression. Clin Cancer Res 2011;17:6287–97. 43. Singh N, Liu X, Hulitt J, Jiang S, June CH, Grupp SA, et al. Nature of tumor control by permanently and transiently modified GD2 chimeric antigen receptor T cells in xenograft models of neuroblastoma. Cancer Immunol Res 2014;2:1059–70. 44. Pietra G, Manzini C, Rivara S, Vitale M, Cantoni C, Petretto A, et al. Melanoma cells inhibit natural killer cell function by modulating the expression of activating receptors and cytolytic activity. Cancer Res 2012; 72:1407–15. 45. Garcia-Iglesias T, Del Toro-Arreola A, Albarran-Somoza B, Del Toro-Arreola S, Sanchez-Hernandez PE, Ramirez-Due~ nas MG, et al. Low NKp30, NKp46 and NKG2D expression and reduced cytotoxic activity on NK cells in cervical cancer and precursor lesions. BMC Cancer 2009;9:186. 46. Ciccone E, Pende D, Viale O, Than A, Di Donato C, Orengo AM, et al. Involvement of HLA class I alleles in natural killer (NK) cell-specific functions: expression of HLA-Cw3 confers selective protection from lysis by alloreactive NK clones displaying a defined specificity (specificity 2). J Exp Med 1992;176:963–71. 47. Waldhauer I, Goehlsdorf D, Gieseke F, Weinschenk T, Wittenbrink M, Ludwig A, et al. Tumor-associated MICA is shed by ADAM proteases. Cancer Res 2008;68:6368–76. 48. Chu Y, Yahr A, Ayello J, Lo L, Katz J, Cairo M. Effectively targeting sensitive and resistant Burkitt lymphoma by anti-CD20 chimeric antigen receptor (CAR) modified expanded natural killer (NK) cells combined with a histone deacetylase inhibitor, romidepsin. Bone Marrow Transpl 2013; 48:S15-S. 49. Scott SD. Rituximab: a new therapeutic monoclonal antibody for nonHodgkin's lymphoma. Cancer Pract 1998;6:195–7. 50. Davis TA, Grillo-Lopez AJ, White CA, McLaughlin P, Czuczman MS, Link BK, et al. Rituximab anti-CD20 monoclonal antibody therapy in nonHodgkin's lymphoma: safety and efficacy of re-treatment. J Clin Oncol 2000;18:3135–43.




Targeting CD20+ Aggressive B-cell Non−Hodgkin Lymphoma by Anti-CD20 CAR mRNA-Modified Expanded Natural Killer Cells In Vitro and in NSG Mice Yaya Chu, Jessica Hochberg, Ashlin Yahr, et al. Cancer Immunol Res 2015;3:333-344. Published OnlineFirst December 9, 2014.

Updated version Supplementary Material

Cited articles

E-mail alerts Reprints and Subscriptions Permissions

Access the most recent version of this article at: doi:10.1158/2326-6066.CIR-14-0114 Access the most recent supplemental material at: http://cancerimmunolres.aacrjournals.org/content/suppl/2014/12/06/2326-6066.CIR-14-0114.DC1.html

This article cites 49 articles, 20 of which you can access for free at: http://cancerimmunolres.aacrjournals.org/content/3/4/333.full.html#ref-list-1

Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected].


Are natural killer cells superior CAR drivers?

Adoptive transfer of osteoclast-expanded natural killer cells for immunotherapy targeting cancer stem-like cells in humanized mice.

PD1 blockade enhances cytotoxicity of in vitro expanded natural killer cells towards myeloma cells.

Advantages and applications of CAR-expressing natural killer cells.

-) (NSG) Mice.

Combating non-Hodgkin lymphoma by targeting both CD20 and HLA-DR through CD20-243 CrossMab.

T-cell lymphoma with cutaneous involvement.

Natural killer cells. In vitro and in vivo.

Natural killer cells attenuate cytomegalovirus-induced hearing loss in mice.

T cell lymphoma? a case with nasal involvement.

Enhancement by interferon of natural killer cell activity in mice.

T-cell lymphoma, nasal type, with aggressive and indolent course.

Natural killer cells and natural killer T cells in Lyme arthritis.

Upregulation of NKG2D ligands in acute lymphoblastic leukemia and non-Hodgkin lymphoma cells by romidepsin and enhanced in vitro and in vivo natural killer cell cytotoxicity.

t cell lymphoma.

Activation of Natural Killer Cells by Probiotics.

Inhibition of type I natural killer T cells by retinoids or following sulfatide-mediated activation of type II natural killer T cells attenuates alcoholic liver disease in mice.

A bispecific antibody directly induces lymphoma cell death by simultaneously targeting CD20 and HLA-DR.

Human natural killer cells prevent infectious mononucleosis features by targeting lytic Epstein-Barr virus infection.

Hemophagocytic syndrome associated with aggressive natural killer cell leukemia.

Targeting VEGF-A in myeloid cells enhances natural killer cell responses to chemotherapy and ameliorates cachexia.

T cell lymphoma of true natural killer cell origin.

The optimized anti-CD20 monoclonal antibody ublituximab bypasses natural killer phenotypic features in Waldenström macroglobulinemia.

Immunotherapeutic strategies targeting natural killer T cell responses in cancer.