Targeting CD20 Aggressive B-Cell Non–Hodgkin Lymphoma by Anti

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

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

þ þ þ The prognosis is very dismal for patients with relapsed CD20 also enhanced in CAR exPBNK in response to CD20 B-NHL– B-cell non-Hodgkin lymphoma (B-NHL). Facilitating the devel- specific stimulation. In Raji-Luc and Raji-2R-Luc xenografted opment of alternative novel therapeutic strategies is required NOD/SCID/g-chain / (NSG) mice, the luciferase signals mea- þ to improve outcomes in patients with recurrent/refractory sured in the CAR exPBNK-treated group were significantly þ CD20 B-NHL. In this study, we investigated functional activities reduced, compared with the signals measured in the untreated of anti-CD20 CAR-modified, expanded peripheral blood NK cells mice and in mice treated with the CAR exPBNK. Furthermore, the þ (exPBNK) following mRNA nucleofection against CD20 B-NHL CAR exPBNK-treated mice had significantly extended survival þ in vitro and in vivo. CAR exPBNK had significantly enhanced in time (P < 0.001) and reduced tumor size, compared with those þ vitro cytotoxicity, compared with CAR exPBNK against CD20 of the untreated and the CAR exPBNK-treated mice (P < 0.05). Ramos (P < 0.05), Daudi, Raji, and two rituximab-resistant cell These preclinical data suggest that ex vivo–exPBNK modified with lines, Raji-2R and Raji-4RH (P < 0.001). As expected, there was no anti-CD20 CAR may have therapeutic potential for treating þ significant difference against CD20 RS4;11 and Jurkat cells. patients with poor-risk CD20 hematologic malignancies. Cancer CD107a degranulation and intracellular IFNg production were Immunol Res; 3(4); 1–12. 2014 AACR.

Introduction Study Group (UKCCSG), that children and adolescents with newly diagnosed mature B-NHL [French–American–British B-cell non-Hodgkin lymphoma (B-NHL), including Burkitt (FAB)/Lymphome malins de Burkitt (LMB) 96], have a 90% 5- lymphoma, makes up approximately 60% of all malignant NHL year overall survival (OS) following treatment with a short, that occurs in children and adolescents (1). The outcome for intensive course of chemotherapy (4). Unfortunately, for children children and adolescents with de novo mature B-NHL has and adolescents who relapse or progress with de novo mature B- improved signiﬁcantly, as we previously demonstrated that short NHL, the prognosis is dismal due to chemoradiotherapy resis- but intensive chemotherapy is associated with a 90% 5-year event- tance (4, 5). Similarly, the prognosis in adults with recurrent/ free survival (EFS; refs. 2–4). We demonstrated in an international refractory Burkitt lymphoma is dismal (6). Therefore, develop- multi-cooperative group study, which comprised the Children's ment of alternative cellular targeted therapeutic strategies is Oncology Group (COG), The Societe Francaise¸ d'Oncologie required to improve outcomes in children, adolescents, and Pediatrique (SFOP), and the United Kingdom Children's Cancer 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 1 Department of Pediatrics, Maria Fareri Children's Hospital, New York or plasma cells (7). It is also expressed in >98% of childhood, Medical College, Valhalla, New York. 2Department of Pediatrics, State University of New York at Buffalo, Buffalo, New York. 3Department of adolescent, and adult mature B-cell NHLs and therefore is an Medicine, Roswell Park Cancer Institute, Buffalo, New York. 4Depart- attractive cancer therapeutic target (6, 8, 9). Rituximab, a chimeric ment of Immunology, Roswell Park Cancer Institute, Buffalo, New York. anti-CD20 antibody, has been used successfully in the treatment 5Department of Medicine, New York Medical College, Valhalla, New York. 6Department of Pathology, New York Medical College, Valhalla, of childhood and adolescent mature B-NHL as well as in New York. 7Department of Microbiology and Immunology, New York adults with diffuse large B-cell lymphoma and Burkitt lymphoma 8 Medical College, Valhalla, New York. Department of Cell Biology and (9–11). However, CD20 could also be used as a target for Anatomy, New York Medical College, Valhalla, New York. genetically engineered immune cell–based therapies. Note: Supplementary data for this article are available at Cancer Immunology Natural killer (NK) cells are bone marrow–derived cytotoxic Research Online (http://cancerimmunolres.aacrjournals.org/). lymphocytes that play a major role in the rejection of tumors and Corresponding Author: Mitchell S. Cairo, New York Medical College, 40 Sun- cells infected by viruses, even without speciﬁc immunization (12, shine Cottage Road, Skyline 1N-D12, Valhalla, NY 10595. Phone: 914-594-2150; 13). Various activating and inhibitory receptors on the NK-cell Fax: 914-594-2151; E-mail: [email protected] surface are engaged to regulate NK-cell activities and to discrim- doi: 10.1158/2326-6066.CIR-14-0114 inate target cells from healthy "self" cells (14, 15). However, 2014 American Association for Cancer Research. factors limiting NK therapy include small numbers of active NK

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cells in unmodified peripheral blood and a lack of specificity in University of Tennessee College of Medicine, Memphis, TN; tumor targeting (16). Our group and others have successfully ref. 17). expanded active NK cells in vitro by short-term culture with cytokines alone, using cytokines and coculture with irradiated NK-cell expansion and isolation Epstein–Barr virus (EBV)–transformed lymphoblastoid cell lines Leukocytes were obtained after informed consent from healthy as feeder cells, or using cytokines and coculture with K562 cells adult donors at the New York Blood Center. Peripheral blood expressing transfected cell-membrane bound IL15 and 4-1BBL mononuclear cells (PBMC) were obtained by Ficoll gradient (17–20). (Amersham Biosciences) separation. NK-cell expansion and iso- Chimeric antigen receptors (CAR) usually include a single- lation were performed as previously described (17) and are chain variable fragment (Fv) from a monoclonal antibody, a detailed in the Supplementary Methods. transmembrane hinge region, and a signaling domain such as CD28, CD3z, 4-1BB (CD137), or 2B4 (CD244) endodimers Production of anti-CD20-BB-z mRNA (21–24). The advantage of the CAR strategy is that no human Anti-CD20 CAR mRNA was generated as previously described leukocyte antigen (HLA) expression on the target cell is required with modifications (29). Briefly, the anti-CD20-4-1BB-CD3z was þ for the epitope to be accessible to CAR T or NK cells. Thus, it is cut from an MSCV-anti-CD20-BB-z-IRES-GFP vector and sub- not limited to only a subset of patients with a specific HLA type cloned into the pcDNA3 vector. Anti-CD20-4-1BB-CD3z mRNA þ (22). Several clinical trials testing CAR T cells in patients was transcribed in vitro using the mMESSAGE mMACHINE T7 have shown promising clinical outcomes. Porter, Grupp, Brent- Ultra Kit (Life Technologies) following the manufacturer's jens, and their colleagues (23–25) have engineered patients' instructions. The resulting product was dissolved in nuclease-free T cells with a lentiviral vector expressing anti-CD19 CAR and H2O. RNA concentrations were measured using a Nano-spectro- reinfused the anti-CD19 CAR autologous T cells into patients with photometer (Thermo Fisher) at 260 nm. refractory chronic lymphocytic leukemia and acute lymphoblastic leukemia. However, significant toxicities, including hypotension, Nucleofection fevers, renal insufficiency, cytokine-release syndrome, and B-cell Six to 8 106 expanded purified NK cells were suspended in aplasia, have occurred after infusions of CAR T cells (23–26). The 100-mL nucleofection solution (Lonza); anti-CD20-BB-z mRNA hallmark toxicity of CAR T-cell therapies is cytokine-release syn- was added at 80 to 100 mg/mL. The mixture was placed in the drome, a potentially life-threatening complication of inflamma- Nucleofector Cuvette and nucleofected using the Amaxa Nucleo- tory symptoms resulting from elevated plasma cytokine levels fector II Device with U-001 program. CAR expression was detected associated with T-cell activation and proliferation (23–26). In using FITC-conjugated or AF647-conjugated goat anti-mouse IgG, 0 contrast, NK cell–based immunotherapy has not been associated F(ab )2 fragment-specific antibody (The Jackson Laboratory), and with a cytokine-release syndrome in patients; it has been associ- flow cytometry. ated with a significant NK versus leukemic effect in the absence of graft-versus-host disease (GvHD) and a significant decrease in Intracellular CD107a and IFNg assays leukemia relapse following haploidentical allogeneic stem cell Intracellular CD107a expression and IFNg were measured by transplantation in which the donor/recipient NK killer immuno- flow cytometry, as previously described (20). Intracellular CD107a globulin-like receptor (KIR)/malignant major histocompatibility and IFNg assays are detailed in the Supplementary Methods. complex (MHC) class I mismatch may occur (27). Our approach In vitro to NK cell–based immunotherapy is to expand and activate NK cytotoxicity cells and redirect them specifically against resistant lymphoma/ NK cytotoxic activity was determined by europium release leukemia cells. This approach might circumvent resistance to NK- assays with a standard kit (PerkinElmer), as previously described In vitro cell tumor cytotoxicity independent of HLA class I ligand expres- (20). cytotoxicity assays are detailed in the Supplementary sion on leukemia cells, target NK cells to specific lymphoma/ Methods. leukemic tumor antigens, enhance NK-cell activation, and pro- Animal studies vide an alternative to adoptive allogeneic T-cell immunotherapy Six- to 8-week-old NOD/SCID/g-chain / (NSG) mice were by circumventing the risk of acute GvHD that commonly occurs. purchased (Stock #5557; The Jackson Laboratory), bred, and Here, we investigated in vitro and in vivo activities of anti-CD20 maintained under pathogen-free conditions in-house at the CAR-modified expanded peripheral blood NK cells (exPBNK) þ Department of Comparative Medicine at New York Medical following mRNA nucleofection, against CD20 B-NHL. College (Valhalla, NY). All protocols were approved by the Institutional Animal Care and Use Committee. Materials and Methods Cell lines and tumor targets Xenograft models of human Burkitt lymphoma and rituximab- The human cell lines Ramos, Daudi, RS4;11, U698M, K562, resistant Burkitt lymphoma and the T-cell ALL lines, Jurkat, were purchased from the American The mammalian expression construct ffLUCZeo-pcDNA3.1 Type Culture Collection (ATCC). Raji, rituximab-resistant Raji- (generously supplied by L. Cooper, University of Texas MD 2R, and Raji-4RH cells were generously provided by Matthew Anderson Cancer Center, Houston, TX) was electroporated Barth and Myron Czuczman from Roswell Park Cancer Institute into tumor cells (Raji and Raji-2R), and stable clones were selected (Buffalo, NY; ref. 28). All tumor cells were maintained in RPMI- with Zeocin (Invitrogen). Raji or Raji-2R cells (5 105) expressing 1640 (Gibco) supplemented with 10% FBS (Gibco) and anti- luciferase (Raji-Luc or Raji-2R-Luc) were i.p. or s.c. injected into biotics. The K562-mb15-41BBL cell line was generously provided the NSG mice (NOD.Cg-PRkdcscidIl2rgtmWjl/SzJ; 6 weeks; The by Dr. Dario Campana (St Jude Children's Research Hospital, Jackson Laboratory). Tumor engraftment and progression were

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evaluated using the Xenogen IVIS-200 System (Caliper Life diated K562 and IL2 alone after normalized to the INPUT NK cell Sciences) after i.p. injection of 150 mg D-luciferin/kg/mouse. numbers (mean, 44.4-fold 1.47 vs. 12.49-fold 1.50 vs. 0.239- Photons emitted from luciferase-expressing cells were quantified fold 0.09; n ¼ 3; P < 0.001; Fig. 1C). We also observed that using Living Image software. After tumor engraftment was verified, sustained activation and proliferation by irradiated geK562 freshly prepared 5 106 CAR exPBNK, mock exPBNK, or medium induced morphologic changes in NK cell shape and size (Fig. was i.p. injected into each mouse once a week for 3 weeks. 1D). We examined the expression of CD3 and CD56 in the cells Tumor regression and/or progression of xenografted mice cocultured with media alone, K562, and geK562 by flow cyto- were monitored weekly by tumor volume measurement and by metry analysis (Fig. 1E). The exPBNK by geK562 were negatively in vivo bioluminescent imaging (BLI; refs. 30, 31). Tumor size selected with more than 96% purity (Fig. 1E). was estimated according to the following formula (32): tumor The anti-CD20-BB-z chimeric receptor (anti-CD20 CAR) was size (cm3) ¼ length (cm) width2 (cm) 0.5. Mice were originally constructed in the retroviral plasmid pMSCV-IRES-anti- followed until death or sacrificediftumorsizereached2cm3 or CD20-BB-z (kindly provided by Dr. Dario Campana, St Jude larger. Children's Research Hospital, University of Tennessee College of Medicine; Supplementary Fig. S1A). The expression of anti-CD20 Histology (H&E) staining and Immunofluorescence staining CAR was confirmed in virus-infected Jurkat cells by flow cytometry Fresh-frozen tumor samples were sectioned for hematoxylin and by Western blot analysis (Supplementary Fig. S1B and S1C). and eosin (H&E) analysis and immunofluorescence staining as Anti-CD20 CAR-mediated cytotoxicity was first examined using þ described in the Supplementary Methods. NK-92 cells and CD20 NHL. NK92 cells expressing anti-CD20 þ CAR had enhanced cytotoxicity against CD20 NK-sensitive TruCOUNT analysis of absolute cell counts Ramos (74.66% 8.01% vs. 49.63% 5.10%; P < 0.05) and þ TruCOUNT tubes (Becton Dickinson) were used to determine CD20 NK-resistant Daudi (43.15% 4% vs. 16.15% 6.4%; P < the absolute counts of tumor cells and NK cells in the peripheral 0.01) cells at E:T ¼ 10:1 ratio but not against the CD20 pre-B-ALL blood of xenografted mice. cell line RS4;11 (40.46% 1.14% vs. 41.21% 1.02%: P ¼ ns) Peripheral blood was obtained via face-vein bleeding and using europium release assays (Supplementary Fig. S1D). stained for the presence of human CD45, CD20, and CD56 NK After functional confirmation, the anti-CD20 CAR fragment þ þ cells. After gating on the human CD45 population, the CD20 was excised with restriction enzymes from the retroviral vector þ and CD56 subsets were quantified using TruCOUNT tubes (BD pMSCV-IRES-anti-CD20-BB-z and ligated into a T7 promoter– Biosciences) with known numbers of fluorescent beads as driven plasmid (Fig. 2A). Anti-CD20 CAR mRNA was in vitro described in the manufacturer's instructions. transcribed, as described previously (Fig. 2B; ref. 33). Expanded þ CD56 CD3 PBNK purified from the peripheral blood of 10 In vitro organ imaging healthy donors were nucleofected in the presence of anti-CD20 5 Raji-2R-Luc cells (5 10 ) were intravenously (i.v.) injected into CAR mRNA to generate anti-CD20 CAR exPBNK or in nuclease- fi NSG mice. Mice were sacri ced when hind-leg paralysis was free H2O to generate mock exPBNK. Flow cytometry was used observed. Organs (lung, liver, spleen, and kidney) were collected to detect the expression of anti-CD20 CAR in 66.73% 6.817% and soaked in D-PBS with 300 mg/mL D-luciferin for 5 minutes of viable exPBNK after 16 hours of nucleofection (Fig. 2C). before BLI using the Xenogen IVIS-200 system (Caliper Life Background from the exPBNK nucleofected with nuclease-free Sciences). H2O was 2.89% 0.947%. As expected, the anti-CD20 CAR expression was transient and was reduced from 94.1% at 20 Statistical analyses hours to 60.1% at day 6 and 2.99% at day 11 after nucleofec- Statistical analyses were performed using the INSTAT statistical tion (Fig. 2D). program (GraphPad). Average values are reported as the mean CAR mRNA nucleofection, however, did not affect the expres- SEM. Results were compared using the one-tailed unpaired Stu- sion of exPBNK-activating receptors (CD16, CD69, NKG2D, t P fi dent test, with < 0.05 considered as statistically signi cant. CD244, NKp30, NKp44, and NKp46) or inhibitory receptors Probability of survival in animal studies was determined by the (NKG2A, KIR2DS4, CD94, CD158a, CD158b, and CD158e; – Kaplan Meier method using the Prism program 5.0 (GraphPad Supplementary Fig. S2). Software, Inc.). Cytotoxic activity of anti-CD20 CAR mRNA-modified exPBNK Results in vitro against B-NHL and rituximab-resistant cells þ PBNK (CD3 /56 ) expansion and expression of anti-CD20-BB- We examined the functionality of anti-CD20 CAR mRNA- þ z receptors in exPBNK by mRNA nucleofection modified exPBNK against CD20 B-NHL target cells. CD20 PBMNCs from healthy donors were cultured with irradiated expression on the target tumor cells was confirmed (Supplemen- geK562 feeder cell lines, as described (17), in the presence of 40 tary Table S1). Anti-CD20 CAR exPBNK cytotoxicity was signif- þ IU/mL IL2. As reported previously, CD56 CD3 PBNK were icantly enhanced compared with that of mock exPBNK against þ significantly increased compared with those grown in media CD20 B-NHL at 10:1 (n 3): Ramos (97.25% 2.61% vs. alone at day 7 (mean, 66.87% vs. 8.84%; n ¼ 3; Fig. 1A). There 82.5% 4.058%; P < 0.05), Daudi (71.5% 3.26% vs. 36.34% þ þ was no significant enrichment of CD56 CD3 PB NKT cells 6.31%; P < 0.001), and U-698-M (82.84% 1.17% vs. 26.2% compared with those grown in media alone at day 7 (data not 0.776%; P < 0.001; Fig. 3A). There was no significant difference þ shown). CD56 CD3 PB T cells were significantly reduced com- against CD20 RS4;11 or Jurkat cells (Fig. 3B). Tumor-cell recov- pared with those grown in media alone at day 7 (mean, 19.16% ery experiments further confirmed the result. Anti-CD20 CAR vs. 77.00%; n ¼ 3; Fig. 1B). The absolute NK numbers were exPBNK significantly reduced Ramos cell recovery compared with enhanced with irradiated geK562 as feeders compared with irra- those of medium and mock exPBNK (P < 0.01; Fig. 3C). These data

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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 NKexpansionin24-wellplates (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 mRNA- 2R, and Raji-4RH cells used in our experiments express high þ modified exPBNK against CD20 tumor cells. levels of HLA class I antigens by flow cytometry analysis (Fig. 3E We examined the expression of CD20 in rituximab-sensitive and Supplementary Table S3). The anti-CD20 CAR exPBNK Raji and rituximab-resistant Raji-2R and Raji-4RH cells by flow showed enhanced cytolytic activities against NK-resistant cytometry analysis (Supplementary Table S2). In addition, the Daudi and Raji cells (Fig. 3), indicating that the expression of standard europium release assay experiments demonstrated that anti-CD20 CAR on NK cells can overcome inhibitory signals the cytotoxicity of CAR exPBNK was significantly higher than that from KIR–HLA interactions and other unknown mechanisms- of mock exPBNK (P < 0.001) against Raji, Raji-2R, and Raji-4RH mediated resistance. cells (Fig. 3D). We also observed similarly enhanced expression of exPBNK- Daudi and Raji cells have been demonstrated previously to activating receptors (CD69, NKp44, and NKG2D) after incuba- be resistant to NK-mediated cytotoxicity (34). NK resistance of tion with U-698-M, compared with that with medium (Supple- Raji cells is, in large part, mediated through the interaction of mentary Fig. S3), and the inhibitory receptors (CD94 and KIRs and histocompatibility class I antigens (HLA; ref. 35). KIR2DL2/3) were unchanged in mock exPBNK and CAR exPBNK. Unlike Raji cells, Daudi cells do not synthesize b2-microglobulin and lack the cell-surface HLA antigens (36). Therefore, Intracellular CD107a expression and IFNg production in vitro resistance of Daudi cells to NK-mediated lysis has not been The lysosomal-associated membrane protein-1 (LAMP-1 or thought to be mediated through HLA antigens. The mechanism CD107a) is a marker of NK-cell functional activity (37). Intra- of NK resistance in Daudi cells is currently unknown. We found cellular CD107a expression in exPBNK was detected by flow that our exPBNK express KIR2DL1, KIR2DL2/3, KIR3DL1, and cytometry. CD107a degranulation was enhanced in anti-CD20 NKG2A (Supplementary Fig. S2 and data not shown). Raji, Raji- CAR exPBNK, compared with that in mock exPBNK, in response

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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.

þ to CD20 Ramos (31.47% 1.74% vs. 15.2% 0.26%; P < cell injection, the luciferase signals measured in the CAR exPBNK- 0.001; n ¼ 3) and Daudi (38.9% 2.7% vs. 19.73% 0.58%; P < treated Raji-Luc group (n ¼ 6) were significantly reduced, com- 0.001; n ¼ 3) stimulation; however, there was no significant pared with those in the medium-treated mice (P ¼ 0.0087; n ¼ 5) difference in response to RS4;11 (5.69% 0.45% vs. 3.9% and in the mock exPBNK-treated mice (P ¼ 0.0128; n ¼ 8; Fig. 5A 0.06%; P ¼ ns; n ¼ 3) or medium (4.86% 0.066% vs. 4.75% and B). Consistent with the reduced luciferase signals, the CAR 0.59%; P ¼ ns; n ¼ 3; Fig. 4A). Similarly, intracellular IFNg exPBNK-treated mice appeared to be more healthy and active than production was also enhanced in CAR exPBNK compared with the medium-treated and the mock exPBNK-treated mice (data not þ that in mock exPBNK in response to CD20 Ramos-specific shown). The survival curve of mice was built based on death of (3.07% 0.42% vs. 1.39% 0.26%; P < 0.05; n ¼ 3) and mice caused by tumor cells disseminated in the whole body from Daudi-specific (5.62% 0.52% vs. 1.42% 0.43%; P < 0.05; the injection site or sacrifice if tumor size reached 2 cm3 or larger. n ¼ 3) stimulation in a short 4-hour incubation (Fig. 4B). We found that the CAR exPBNK-treated Raji-Luc mice had significantly extended survival time with a median of 40 days In vivo cytotoxicity in humanized Burkitt lymphoma compared with that of the untreated mice (29 days, P < 0.001) xenografted NSG mice and of the mock exPBNK-treated mice (30 days, P < 0.001; Fig. We assessed the antitumor activity of exPBNK nucleofected 5C). There was no significant difference in survival between the with anti-CD20 CAR mRNA using a humanized Burkitt lympho- untreated Raji-Luc mice and the mock exPBNK-treated Raji-Luc ma xenograft NSG model. First, we used a dissected tumor model. mice with the current scheduled NK doses. The purity and kinetics of BLI signal in Raji-Luc were examined The proliferation of transferred CAR T cells is highly correlated (Supplementary Fig. S4A and S4B). Raji-Luc cells (5 105) were with tumor regression (23–25). To examine whether the antitu- injected i.p. into NSG mice. After successful engraftment of Raji- mor efficacy of anti-CD20 CAR NK cells is correlated with Luc cells in mice at day 7, freshly prepared 5 106 mock or CAR increased proliferation and long persistence of anti-CD20 CAR exPBNK were injected i.p. into each mouse once a week for 3 PBNK compared with that of the mock PBNK in NSG mice, weeks (days 9, 16, and 23). Mice that received culture medium peripheral blood was collected from Raji-Luc xenografted mice were used as controls. We demonstrated that after the third NK- after 7 days of the third anti-CD20 CAR NK injection. Circulated

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Figure 3. Anti-CD20 CAR mRNA enhances exPBNK in vitro cytolytic activity against CD20þ 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 BATDA-labeled CD20þ NK-sensitive Ramos (top left), CD20þ 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 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 CD19þ 7-AAD by flow cytometry analysis. Each data point represents the mean ( SEM; n ¼ 3) percentage of living CD19þ 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. Invitro 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 group (P < 0.001; Fig. 5D, left). However, there was no significant TruCOUNT beads by flow cytometry analysis with anti-CD20-PE difference in the number of NK cells between mice receiving and anti-CD56-FITC antibodies (Fig. 5D). CD20 Raji cell counts CAR exPBNK and mock exPBNK (Fig. 5D, right). We examined were significantly reduced in mice receiving CAR exPBNK, com- whether the delayed tumor growth was due to the colocalization pared with medium (P < 0.001) and mock (P < 0.05) treatment of CAR exPBNK with the solid tumor masses. Solid tumor masses groups (Fig. 5D, left). Mock exPBNK also significantly reduced the were dissected from the medium-treated group, the mock CD20 Raji cell counts, compared with the medium treatment exPBNK-treated group, and the CAR exPBNK-treated group, and

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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 mRNA (CAR, black solid and black gradient color). A, MOCK exPBNK or CAR exPBNK were cultured with medium only, CD20þ Ramos (left), CD20þ 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 ¼ 6; P < 0.001) and of the mock exPBNK-treated mice (22 days; the morphology of tumor tissues from each treatment group (Fig. n ¼ 7; P < 0.05; Fig. 6D). 5E, bottom). Tumor burden of the CAR exPBNK-treated group appeared to be less compact with tumor cells compared with the Anti-CD20 CAR exPBNK inhibit Raji-2R cells migration and other two groups. A fluorescent immunostaining analysis was dissemination to multiple organs in xenografted mice conducted to detect the presence of NK cells in the tumor masses. To study the effect of anti-CD20 CAR exPBNK in tumor migra- 5 We found a few green fluorescent Alexa Fluor 488–labeled NK tion and multiorgan dissemination in xenografted mice, 5 10 cells accumulating around the edge of the tumors (Fig. 5F) in Raji-2R-Luc cells were i.v. injected into NSG mice. Seven days later, 6 mock exPBNK-treated mice and in CAR exPBNK-treated mice but 5 10 mock or CAR exPBNK in culture medium with 200 IU/mL not inside of the tumor masses. of IL2 were i.v. injected into NSG mice with similar tumor burdens We used the localized tumor model to examine the antitu- once a week for 2 weeks. In the three pairs of NSG mice treated mor effect of CAR exPBNK against rituximab-resistant Burkitt with mock exPBNK or CAR exPBNK, CAR exPBNK-treated NSG lymphoma Raji-2R cells. The purity and kinetics of BLI signal in mice had less bioluminescence signal in the pointed organs than Raji-2R-Luc was examined (Supplementary Fig. S4C and S4D). that in the mock exPBNK-treated NSG mice (Fig. 7A), suggesting Raji-2R-Luc cells (5 105) were injected s.c. into NSG mice. that CAR exPBNK therapy inhibited Raji-2R-Luc dissemination to Mock or CAR exPBNK (5 106) were i.p. injected into each other organs. Consistent with this observation, DiD (a lipophilic fl mouse once a week for 3 continuous weeks (days 3, 10, and uorescent dye, C67H103CIN2O3S)-labeled exPBNK were able 17). Mice that received culture medium were used as control. to migrate to the lung, liver, and spleen after i.v. injection to NSG fi fl After the third NK injection, luciferase signal was significantly mice, as con rmed by uorescent imaging using the IVIS imaging fl reduced in the CAR exPBNK-treated Raji-2R-Luc group, com- system (Supplementary Fig. S5A) and by ow cytometry analysis fi pared with the mock exPBNK-treated mice (P < 0.01;Fig.6A (Supplementary Fig. S5B). To con rm this, organs (lung, liver, and B). Tumor size measured in the CAR exPBNK-treated Raji- spleen, and kidney) were collected in D-PBS with D-luciferin for 2R group (n ¼ 7) was also significantly smaller than that in the BLI. CAR exPBNK-treated NSG mice had less bioluminescence in medium-treated mice (P ¼ 0.0175; n ¼ 6) and the mock the lung, liver, kidney, and spleen than that of the mock exPBNK- exPBNK-treated mice (P ¼ 0.0122; n ¼ 7; Fig. 6C). Thereafter, treated NSG mice (Fig. 7B). the survival curve was established based on the endpoint of 2cm3 tumor size. The CAR exPBNK-treated Raji-2R-Luc (n ¼ 7) Discussion mice had significantly extended survival time with a median of Adoptive immunotherapy with NK cells has been limited in the 24 days, compared with that of the medium-treated (18 days; n past by the low numbers of activated NK cells in the peripheral

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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. Raji-Luc cells (5 105) were i.p. injected into NSG mice on day 0. Three doses of (5 106 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 reached 2 cm3.TheKaplan–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 80C. 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.

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Figure 6. Anti-CD20 CAR exPBNK significantly inhibit growth of Raji-2R cells in xenografted mice. Raji-2R-Luc cells (5 105) were s.c. injected into the right flanks of NSG mice. Three days after tumor inoculation, mice were randomized to equalize tumor burden and injected weekly i.p. with 5 106 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 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 cm3. 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 medium- treated 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. mRNA in vitro has great therapeutic potential for transient, targeted In this study, we established conditions that expanded activated protein expression with limited toxicity. In addition, large quan- PBNK ex vivo and modified the exPBNK efficiently with anti-CD20 tities of RNA can be easily prepared by in vitro transcription, which CAR mRNA in a nonviral, clinically relevant method. The anti- makes it possible to expedite manufacturing and scaling to current CD20 CAR expression in exPBNK by mRNA nucleofection was Good Manufacturing Practices (cGMP) products. Successful elec- associated with a statistically significant increase in CD107a troporation of CAR mRNA into primary T cells and NK cells has degranulation and INFg production after stimulation with now been documented for a variety of cancers, such as anti-CD19 þ CD20 B-NHL. Consequently, CAR exPBNK treatment results in CAR against leukemia (29, 39) and mesothelin CAR against þ significant and specific in vitro cytotoxicity against CD20 B-NHL, pancreatic cancer (clinical trial: NCT01897415). However, these including both rituximab-sensitive and rituximab-resistant B-NHL synthesized mRNAs result in only short-lived CAR expression in cells. Multiple injections of anti-CD20 CAR mRNA-electroporated immune cells. As shown in Fig. 2D, anti-CD20 CAR expression exPBNK mediated significant tumor regression and inhibited lasted less than 2 weeks after nucleofection in exPBNK. Consid- tumor growth in both disseminated and localized tumor models ering the lifespan of adoptively transferred NK cells is only in NSG mice. More importantly, multiple injections of anti-CD20 approximately 10 to 12 days even with cytokine support (Sup- CAR-modified exPBNK also extended survival in B-NHL tumor- plementary Fig. S5C; refs. 40–42), transient expression of CAR in xenografts and inhibited tumor-cell migration. These results indi- exPBNK may not be a significant disadvantage and it does not need cate the therapeutic potential of multiple injections of anti-CD20 a suicide gene to limit the risk of the long-term side effect using þ CAR mRNA-modified exPBNK against CD20 B-NHL in patients. retroviral or lentiviral vectors. In our xenograft models, three Gene-engineered T cells with viral methods have been used for injections of anti-CD20 CAR mRNA-electroporated exPBNK sig- þ cancer therapy with success, but concerns about safety has arisen nificantly mediated CD20 tumor regression, extended survival þ after X-SCID patients developed cancerous T cells after receiving (Fig. 5B and C), reduced tumor size of CD20 tumor xenografted hematopoietic stem cells transduced with recombinant retrovirus mice (Fig. 6C), and inhibited tumor-cell migration to other organs (38), and manufacturing challenges may also hamper clinical in NSG mice (Fig. 7). However, under current NK doses and progress. The development of safe, efficient, and nonviral methods injection frequencies, we did not obtain tumor-free animals. The with a lower tendency to insert near oncogenes will significantly ratio of CAR NK cells to target cells may be critical to the success, as facilitate targeted cellular therapies (33, 39). Synthetic modified shown in our in vitro cytotoxicity assays (Fig. 3). The remaining

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Figure 7. Anti-CD20 CAR exPBNK inhibit Raji-2R cell migration and dissemination to multiple organs in xenografted mice. Raji-2R-LUC cells (5 105) were i.v. injected into NSG mice. Seven days later, mice with similar tumor burden (measured by BLI) were chosen for NK therapy. Mock exPBNK or CAR exPBNK (5 106) 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, micewere sacriﬁced oncehind-leg paralysiswas observedand theorgans(lung, liver, spleen, and kidney)were collected andwashedwithD-PBSandsoakedinD-PBSwithD-luciferin. Organ bioluminescent images were acquired and analyzed with Living Image software (Xenogen).

tumor cells in the xenografted mice after three NK injections are 488–labeled NK cells around the edge of the tumors (Fig. 5F) in þ still CD20 (data not shown), and the relapse is not likely a both mock exPBNK-treated mice and CAR exPBNK-treated mice. consequence of CD20 deregulation to escape anti-CD20 CAR NK We did not find mock or mRNA CAR exPBNK inside of the tumor cytotoxicity. In the future, NK cells expanded with a new feeder masses (data not shown). This is consistent with the recent study in K562-mb21- 41BBL and increased injection frequency of higher which Singh and colleagues (43) have substantiated the difference doses of CAR NK will be considered to enhance NK in vivo between permanent CAR T cells and mRNA-based CAR T cells, and expansion and antitumor effect. the transiently modified T cells were unable to significantly pen- To explore the possible mechanisms of antitumor efficacy of etrate inside of the tumor masses. Therefore, anti-CD20 CAR mRNA CAR exPBNK, we first examined whether CAR exPBNK mRNA-modified exPBNK can mediate effective antitumor proliferate better and persist longer than mock exPBNK. Within our responses in vivo; however, to achieve complete tumor eradication expectations, CAR exPBNK significantly reduced the number of with mRNA-based CAR exPBNK therapies, we will pursue studies circulating tumor cells in the peripheral blood of mice, compared combinating CAR exPBNK therapy with other approaches, such as with that in the medium (P < 0.001) and mock (P < 0.05) treatment bispecificantibodies. groups (Fig. 5D, left), but there was no significant difference in NK Tumor cells may escape from NK-cell immunosurveillance and cell numbers among mice receiving CAR exPBNK and mock develop resistance by several mechanisms. One mechanism is exPBNK (Fig. 5D, right), which may be due to the lack of prolif- associated with the downregulation of natural cytotoxicity recep- erative capacities of NK cells. Second, we examined whether mRNA tor expression on NK cells (44, 45). Our ex vivo–expanded human CAR exPBNK colocalize better than mock exPBNK in solid tumor PBNK with geK562 upregulated activating receptors, including masses to inhibit tumor growth. The tumor burden of the CAR CD69, NKp30, NKp44, and NKG2D (Supplementary Fig. S6). exPBNK-treated group appeared to be less compact with tumor These exPBNK can mediate cytotoxicity of allogeneic and autol- cells than that of the other two groups (Fig. 5E), and the tumor size ogous cancer cell lines by specifically activating receptor–ligand is smaller in the CAR exPBNK-treated group than that in the other interactions. Another mechanism of tumor-cell resistance is by two groups (Fig. 6C). We found a few green fluorescent Alexa Fluor upregulating the expression of KIR-inhibitory ligands (46). In our

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study, we found that the expanded NK cells express KIRs, such as leukemia/lymphoma after nucleofection with anti-CD20 CAR KIR2DL1, KIR2DL2/3, and KIR3DL1, and Raji, Raji-2R, and Raji- mRNA. We propose that the interaction between anti-CD20 CAR 4RH cells express high-level HLA-ABC (Supplementary Fig. S2 on exPBNK and CD20 on the surface of B-leukemia/lymphoma and Fig. 3E). The KIR receptor–HLA ligand interactions mediate transmits an activating signal back to the exPBNK and significantly resistance to NK-mediated lysis. Previous studies reported that stimulates exPBNK degranulation and release of cytokines such as resistance to NK-mediated lysis can be overcome by preincubation IFNg to lyse resistant tumor targets (Supplementary Fig. S7). of NK cells with IL2 and the generation of "lymphokine-activated Results from our preclinical studies should assist in designing þ killer cells" (34). Our study demonstrates that anti-CD20 CAR- clinical trials with CAR exPBNK to treat patients with CD20 B- modified exPBNK have significantly enhanced cytotoxicity against leukemia/lymphoma without the associated safety concerns of þ NK-resistant CD20 Raji, Raji-2R, and Raji-4RH cells but not integrating viral vectors. In future studies, we will examine the against NK-resistant CD20-RS;11 cells (Fig. 3), demonstrating persistence of expanded NK cells in vivo and assess the combined that anti-CD20 CAR-mediated CD20-dependent signaling can therapeutic effect of anti-CD20 CAR-modified NK cell–based overcome the inhibitory signaling from the interaction between therapies with the new generation of antibody-based therapies. inhibitory receptors and HLA ligands. The third mechanism of tumor cells escaping from NK-cell immunosurveillance is by Disclosure of Potential Conflicts of Interest downregulating the NKG2D activation signals such as shedding No potential conflicts of interest were disclosed. of NKG2D ligands from tumor cells (47). We are currently investigating the combination of enhancing NKG2D ligand Authors' Contributions expression on tumor cells and treating with anti-CD20 CAR- Conception and design: Y. Chu, J. Hochberg, M.S. Cairo modified exPBNK (48). Development of methodology: Y. Chu, J. Hochberg, A. Yahr, J. Ayello Promising clinical results have been obtained using rituximab to Acquisition of data (provided animals, acquired and managed patients, treat B-NHL because rituximab was approved by the FDA in 1997 provided facilities, etc.): Y. Chu, A. Yahr, M. Barth Analysis and interpretation of data (e.g., statistical analysis, biostatistics, (49). However, approximately 60% of patients who responded to computational analysis): Y. Chu, J. Hochberg, A. Yahr their first rituximab therapy, relapsed following rituximab retreat- Writing, review, and/or revision of the manuscript: Y. Chu, J. Hochberg, ment (50). One of the relapsed mechanisms is the reduced expres- J. Ayello, C. van de Ven, M. Barth, M. Czuczman, M.S. Cairo sion of CD20 on the surface of cancer cells after they are repeatedly Administrative, technical, or material support (i.e., reporting or organizing exposed to rituximab. Despite significantly reduced CD20 antigen data, constructing databases): Y. Chu, J. Ayello, C. van de Ven, M. Czuczman, expression in rituximab-resistant cells, compared with that in sen- M.S. Cairo Study supervision: M.S. Cairo sitive cells, CD20 is still relatively high in rituximab-resistant cells (Supplementary Table S2; ref. 28). We attempted to assess whether Acknowledgments anti-CD20 CAR-mediated exPBNK cytotoxicity would overcome The authors thank Erin Morris, RN, for her excellent assistance with the rituximab resistance. Interestingly, anti-CD20 CAR exPBNK had preparation of this article; Dr. Dario Campana (St. Jude Children's Research fi signi cantly enhanced cytotoxicity against rituximab-resistant Bur- Hospital) and Dr. Terrence Geiger (St. Jude Children's Research Hospital) for kitt lymphoma cells in vitro (Fig. 3D), and anti-CD20 CAR exPBNK kindly providing anti-CD20 scFv; Dr. Carl Hamby (New York Medical College) significantly inhibited the growth of rituximab-resistant Burkitt for sharing equipment and reagents; and Anne Sollas (Core Histology Lab, lymphoma cells in xenografted NSG mice (Fig. 6), compared with Pathology Department, New York Medical College) for technical support. that in mice treated with mock exPBNK. Our results indicate that anti-CD20 CAR NK cells can bind more efficiently to the Grant Support reduced level of CD20 on resistant tumor cells than rituximab, This research was supported by grants from the Pediatric Cancer Research and the anti-CD20 CAR NK cells can be activated to lyse the Foundation (PCRF), Children's Cancer Fund (CCF), and a New York Medical resistant tumor cells efficiently. Moreover, we observed that the College Intramural Research Award. The costs of publication of this article were defrayed in part by the payment of anti-CD20 CAR exPBNK limited the dissemination/migration page charges. This article must therefore be hereby marked advertisement in of rituximab-resistant Raji-2R tumor cells to multiple organs, accordance with 18 U.S.C. Section 1734 solely to indicate this fact. compared with the mock exPBNK (Fig. 7). In conclusion, our data confirm that activated expanded allo- Received June 12, 2014; revised November 22, 2014; accepted November 25, þ geneic PBNK become highly cytolytic, killing resistant CD20 B- 2014; published OnlineFirst December 9, 2014.

References 1. Pinkerton R CM. Childhood non-Hodgkin lymphoma. In: Mitchell S, adolescent age (>/ ¼ 15 years), are associated with an increased risk of Cairo SLP, editors. Hematological malignancies in children, adolescents treatment failure in children and adolescents with mature B-cell non- and young adults. Singapore: World Scientiﬁc; 2012. p. 299–328. Hodgkin's lymphoma: results of the FAB LMB 96 study. J Clin Oncol 2. Cairo MS, Gerrard M, Sposto R, Auperin A, Pinkerton CR, Michon J, et al. 2012;30:387–93. Results of a randomized international study of high-risk central nervous 5. Miles RR, Arnold S, Cairo MS. Risk factors and treatment of childhood and system B non-Hodgkin lymphoma and B acute lymphoblastic leukemia in adolescentBurkittlymphoma/leukaemia.BrJHaematol2012;156:730–43. children and adolescents. Blood 2007;109:2736–43. 6. Blum KA, Lozanski G, Byrd JC. Adult Burkitt leukemia and lymphoma. 3. Gerrard M, Cairo MS, Weston C, Auperin A, Pinkerton R, Lambilliote A, Blood 2004;104:3009–20. et al. Excellent survival following two courses of COPAD chemotherapy in 7. LeBien TW, Tedder TF. B lymphocytes: how they develop and function. children and adolescents with resected localized B-cell non-Hodgkin's Blood 2008;112:1570–80. lymphoma: results of the FAB/LMB 96 international study. Br J Haematol 8. Perkins SL, Lones MA, Davenport V, Cairo MS. B-Cell non-Hodgkin's 2008;141:840–7. lymphoma in children and adolescents: surface antigen expression and 4. Cairo MS, Sposto R, Gerrard M, Auperin A, Goldman SC, Harrison L, et al. clinical implications for future targeted bioimmune therapy: a children's Advanced stage, increased lactate dehydrogenase, and primary site, but not cancer group report. Clin Adv Hematol Oncol 2003;1:314–7.

www.aacrjournals.org Cancer Immunol Res; 3(4) April 2015 OF11

Chu et al.

9. Coiffier B, Lepage E, Briere J, Herbrecht R, Tilly H, Bouabdallah R, et al. 31. Zheng J, Xu L, Zhou H, Zhang W, Chen Z. Quantitative analysis of cell CHOP chemotherapy plus rituximab compared with CHOP alone in tracing by in vivo imaging system. J Huazhong Univ Sci Technolog Med Sci elderly patients with diffuse large-B-cell lymphoma. N Engl J Med 2010;30:541–5. 2002;346:235–42. 32. Tomayko MM, Reynolds CP. Determination of subcutaneous tumor 10. Goldman S, Smith L, Anderson JR, Perkins S, Harrison L, Geyer MB, et al. size in athymic (nude) mice. Cancer Chemother Pharmacol 1989;24: Rituximab and FAB/LMB 96 chemotherapy in children with Stage III/IV B- 148–54. cell non-Hodgkin lymphoma: a Children's Oncology Group report. Leu- 33. Zhao Y, Moon E, Carpenito C, Paulos CM, Liu X, Brennan AL, et al. Multiple kemia 2013;27:1174–7. injections of electroporated autologous T cells expressing a chimeric 11. Dunleavy K, Pittaluga S, Shovlin M, Steinberg SM, Cole D, Grant C, et al. antigen receptor mediate regression of human disseminated tumor. Cancer Low-intensity therapy in adults with Burkitt's lymphoma. N Engl J Med Res 2010;70:9053–61. 2013;369:1915–25. 34. North J, Bakhsh I, Marden C, Pittman H, Addison E, Navarrete C, et al. 12. Paust S, von Andrian UH. Natural killer cell memory. Nat Immunol Tumor-primed human natural killer cells lyse NK-resistant tumor targets: 2011;12:500–8. evidence of a two-stage process in resting NK cell activation. J Immunol 13. Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, Lanier LL, et al. 2007;178:85–94. Innate or adaptive immunity? The example of natural killer cells. Science 35. Hasenkamp J, Borgerding A, Wulf G, Uhrberg M, Jung W, Dingeldein S, 2011;331:44–9. et al. Resistance against natural killer cell cytotoxicity: analysis of mechan- 14. Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S. Functions of natural isms. Scand J Immunol 2006;64:444–9. killer cells. Nat Immunol 2008;9:503–10. 36. de Preval C, Mach B. The absence of beta 2-microglobulin in Daudi cells: 15. Raulet DH, Vance RE. Self-tolerance of natural killer cells. Nat Rev Immu- active gene but inactive messenger RNA. Immunogenetics 1983;17: nol 2006;6:520–31. 133–40. 16. Shereck E, Satwani P, Morris E, Cairo MS. Human natural killer cells in 37. Alter G, Malenfant JM, Altfeld M. CD107a as a functional marker for the health and disease. Pediatr Blood Cancer 2007;49:615–23. identification of natural killer cell activity. J Immunol Methods 17. Imai C, Iwamoto S, Campana D. Genetic modification of primary natural 2004;294:15–22. killer cells overcomes inhibitory signals and induces specific killing of 38. Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, leukemic cells. Blood 2005;106:376–83. McIntyre E, et al. A serious adverse event after successful gene therapy for 18. Berg M, Lundqvist A, McCoy P Jr, Samsel L, Fan Y, Tawab A, et al. Clinical- X-linked severe combined immunodeficiency. N Engl J Med 2003;348: grade ex vivo-expanded human natural killer cells up-regulate activating 255–6. receptors and death receptor ligands and have enhanced cytolytic activity 39. Barrett DM, Zhao Y, Liu X, Jiang S, Carpenito C, Kalos M, et al. Treatment of against tumor cells. Cytotherapy 2009;11:341–55. advanced leukemia in mice with mRNA engineered T cells. Hum Gene Ther 19. Robinson KL, Ayello J, Hughes R, van de Ven C, Issitt L, Kurtzberg J, et al. Ex 2011;22:1575–86. vivo expansion, maturation, and activation of umbilical cord blood-derived 40. Somanchi SS, Somanchi A, Cooper LJ, Lee DA. Engineering lymph node T lymphocytes with IL-2, IL-12, anti-CD3, and IL-7. Potential for adoptive homing of ex vivo–expanded human natural killer cells via trogocytosis of cellular immunotherapy post-umbilical cord blood transplantation. Exp the chemokine receptor CCR7. Blood 2012;119:5164–72. Hematol 2002;30:245–51. 41. Guimaraes F, Guven H, Donati D, Christensson B, Ljunggren HG, Bejarano 20. Ayello J, van de Ven C, Cairo E, Hochberg J, Baxi L, Satwani P, et al. MT, et al. Evaluation of ex vivo expanded human NK cells on antileukemia Characterization of natural killer and natural killer-like T cells derived from activity in SCID-beige mice. Leukemia 2006;20:833–9. ex vivo expanded and activated cord blood mononuclear cells: implications 42. Parkhurst MR, Riley JP, Dudley ME, Rosenberg SA. Adoptive transfer of for adoptive cellular immunotherapy. Exp Hematol 2009;37:1216–29. autologous natural killer cells leads to high levels of circulating natural 21. Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH, Geiger TL, et al. killer cells but does not mediate tumor regression. Clin Cancer Res Chimeric receptors with 4-1BB signaling capacity provoke potent cytotox- 2011;17:6287–97. icity against acute lymphoblastic leukemia. Leukemia 2004;18:676–84. 43. Singh N, Liu X, Hulitt J, Jiang S, June CH, Grupp SA, et al. Nature of tumor 22. Kohn DB, Dotti G, Brentjens R, Savoldo B, Jensen M, Cooper LJ, et al. CARs control by permanently and transiently modified GD2 chimeric antigen on track in the clinic. Mol Ther 2011;19:432–8. receptor T cells in xenograft models of neuroblastoma. Cancer Immunol 23. Porter DL, Levine BL, Kalos M, Bagg A, June CH. Chimeric antigen receptor- Res 2014;2:1059–70. modified T cells in chronic lymphoid leukemia. N Engl J Med 2011;365: 44. Pietra G, Manzini C, Rivara S, Vitale M, Cantoni C, Petretto A, et al. 725–33. Melanoma cells inhibit natural killer cell function by modulating the 24. Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, et al. expression of activating receptors and cytolytic activity. Cancer Res 2012; Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N 72:1407–15. Engl J Med 2013;368:1509–18. 45. Garcia-Iglesias T, Del Toro-Arreola A, Albarran-Somoza B, Del Toro-Arreola 25. Brentjens RJ, Davila ML, Riviere I, Park J, Wang X, Cowell LG, et al. CD19- S, Sanchez-Hernandez PE, Ramirez-Duenas~ MG, et al. Low NKp30, NKp46 targeted T cells rapidly induce molecular remissions in adults with che- and NKG2D expression and reduced cytotoxic activity on NK cells in motherapy-refractory acute lymphoblastic leukemia. Sci Transl Med cervical cancer and precursor lesions. BMC Cancer 2009;9:186. 2013;5:177ra38. 46. Ciccone E, Pende D, Viale O, Than A, Di Donato C, Orengo AM, et al. 26. Kochenderfer JN, Dudley ME, Feldman SA, Wilson WH, Spaner DE, Maric I, Involvement of HLA class I alleles in natural killer (NK) cell-specific et al. B-cell depletion and remissions of malignancy along with cytokine- functions: expression of HLA-Cw3 confers selective protection from lysis associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-recep- by alloreactive NK clones displaying a defined specificity (specificity 2). tor-transduced T cells. Blood 2012;119:2709–20. J Exp Med 1992;176:963–71. 27. Ruggeri L, Capanni M, Urbani E, Perruccio K, Shlomchik WD, Tosti A, et al. 47. Waldhauer I, Goehlsdorf D, Gieseke F, Weinschenk T, Wittenbrink M, Effectiveness of donor natural killer cell alloreactivity in mismatched Ludwig A, et al. Tumor-associated MICA is shed by ADAM proteases. hematopoietic transplants. Science 2002;295:2097–100. Cancer Res 2008;68:6368–76. 28. Czuczman MS, Olejniczak S, Gowda A, Kotowski A, Binder A, Kaur H, et al. 48. Chu Y, Yahr A, Ayello J, Lo L, Katz J, Cairo M. Effectively targeting sensitive Acquirement of rituximab resistance in lymphoma cell lines is associated and resistant Burkitt lymphoma by anti-CD20 chimeric antigen receptor with both global CD20 gene and protein down-regulation regulated at the (CAR) modified expanded natural killer (NK) cells combined with a pretranscriptional and posttranscriptional levels. Clin Cancer Res 2008;14: histone deacetylase inhibitor, romidepsin. Bone Marrow Transpl 2013; 1561–70. 48:S15-S. 29. Shimasaki N, Fujisaki H, Cho D, Masselli M, Lockey T, Eldridge P, et al. A 49. Scott SD. Rituximab: a new therapeutic monoclonal antibody for non- clinically adaptable method to enhance the cytotoxicity of natural killer Hodgkin's lymphoma. Cancer Pract 1998;6:195–7. cells against B-cell malignancies. Cytotherapy 2012;14:830–40. 50. Davis TA, Grillo-Lopez AJ, White CA, McLaughlin P, Czuczman MS, Link 30. Nishimura R, Baker J, Beilhack A, Zeiser R, Olson JA, Sega EI, et al. In vivo BK, et al. Rituximab anti-CD20 monoclonal antibody therapy in non- trafficking and survival of cytokine-induced killer cells resulting in minimal Hodgkin's lymphoma: safety and efficacy of re-treatment. J Clin Oncol GVHD with retention of antitumor activity. Blood 2008;112:2563–74. 2000;18:3135–43.

OF12 Cancer Immunol Res; 3(4) April 2015 Cancer Immunology Research

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 Published OnlineFirst December 9, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/2326-6066.CIR-14-0114

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