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Research Article

Nature of Tumor Control by Permanently and TransientlyModifiedGD2ChimericAntigenReceptor TCells inXenograftModels of Neuroblastoma

Nathan Singh1, Xiaojun Liu2, Jessica Hulitt3, Shuguang Jiang2, Carl H. June2,4, Stephan A. Grupp3,5,David M. Barrett3, and Yangbing Zhao2,4

AbstractChimeric antigen receptor (CAR) therapy has begun to demonstrate success as a novel treatment modality for

hematologicmalignancies. The success observed thus far has beenwith T cells permanently engineered to expresschimeric receptors. T cells engineered using RNA electroporation represent an alternative with the potential forsimilar efficacy and greater safety when initially targeting novel antigens. Neuroblastoma is a common pediatricsolid tumorwith the potential to be targeted using immunotherapy.We performed xenograft studies in NSGmicein which we assessed the efficacy of both permanently modified and transiently modified CAR T cells directedagainst the neuroblastoma antigen GD2 in both local and disseminated disease models. Disease response wasmonitored by tumor volume measurement and histologic examination, as well as in vivo bioluminescence. RNA-modified GD2 CAR T cells mediated rapid tumor destruction when delivered locally. A single infusion oflentivirally modified GD2 CAR T cells resulted in long-term control of disseminated disease. Multiple infusions ofRNAGD2 CAR T cells slowed the progression of disseminated disease and improved survival, but did not result inlong-term disease control. Histologic examination revealed that the transiently modified cells were unableto significantly penetrate the tumor environment when delivered systemically, despite multiple infusions ofCARTcells. Thus,we demonstrate that RNA-modifiedGD2CARTcells canmediate effective antitumor responsesin vivo, and permanentlymodified cells are able to control disseminated neuroblastoma in xenograftmice. Lack oflong-term disease control by RNA-engineered cells resulted from an inability to penetrate the tumor microen-vironment. Cancer Immunol Res; 2(11); 1059–70. �2014 AACR.

IntroductionNeuroblastoma is the most common extracranial pediatric

solid tumor. Derived from neuroendocrine tissue of the sym-pathetic nervous system, it accounts for 9%of cancer diagnosesand 15% of cancer-related deaths in children (1). The currentstandard of care for high-risk disease consists of chemother-apy, surgery, consolidation chemotherapy followed by stem-cell transplant, tumor-directed radiation, and antibody-based

therapy. This exhaustive regimen still only yields a 3-yearevent-free survival rate of approximately 45% (2).

Historically, poor tumor immunogenicity and immune eva-sion (3, 4) have resulted in largely disappointing T-cell therapytrials. Advances in the understanding of T-cell and tumorinteraction, cell production techniques, and engineering plat-forms, as well as the recognition of the need for host condi-tioning, have recently allowed for significant improvements.Chimeric antigen receptors (CAR) are molecules composed ofan extracellular antigen-binding domain derived from theimmunoglobulin variable chain (scFv) linked to intracellularT-cell activating and costimulatory domains (5, 6). The anti-gen-specific scFv allows for MHC-independent tumor antigenrecognition that initiates robust T-cell stimulation, even inresponse to previously immunoevasive malignancies (7).

In the most successful clinical trial of CARs to date, Kalosand colleagues (8) and Porter and colleagues (9) demonstratedcomplete remissions lasting >3 years in 2 of 3 adults withchronic lymphocytic leukemia (CLL) using CARs directedagainst the B-cell antigen CD19. Translating this success toacute leukemia, our group recently reported the induction ofcomplete remissions in 2 children with acute lymphoblasticleukemia (ALL) using the same CD19 CAR T cells (10).

The dramatic clinical responses observed in these studieshave employed T cells permanently engineered to express

1Department ofMedicine, PerelmanSchool ofMedicine at the University ofPennsylvania, Philadelphia, Pennsylvania. 2Abramson Family CancerResearch Institute, University of PennsylvaniaCancerCenter, Philadelphia,Pennsylvania. 3Division of Oncology, Children's Hospital of Philadelphia,Philadelphia, Pennsylvania. 4Department of Pathology and LaboratoryMedicine, Perelman School of Medicine at the University of Pennsylvania,Philadelphia, Pennsylvania. 5Department of Pathology, Children's Hospitalof Philadelphia, Philadelphia, Pennsylvania.

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

D.M. Barrett and Y. Zhao contributed equally to this article.

Corresponding Author: Nathan Singh, Hospital of the University of Penn-sylvania, Department of Medicine, 100 Centrex Building, 3400 SpruceStreet, Philadelphia, Pennsylvania 19104. Phone: 215-827-9554;Fax: 215-590-3770; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0051

�2014 American Association for Cancer Research.

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CARs using lentiviral vectors. This process involves ex vivoexposure of harvested autologous lymphocytes to self-inacti-vating lentiviral vector encoding a CAR, resulting in genomicintegration of the CAR transgene. While >500 patient-years ofdata suggest that this modification is extremely unlikely toresult in insertional mutagenesis in mature lymphocytes (11),these data are from adults and the increased life-span ofmodified cells in children raises additional theoretical safetyconcerns. More importantly, when targeting solid tumor anti-gens, the risk of on-target off-tumor toxicity becomes a sig-nificant concern. Several adverse events have demonstratedthe potential risks of uncontrolled CAR T cells (12, 13), andhave highlighted the need for safer CARTcellsmoving forward,especially in early clinical testing (14, 15). Given these con-siderations, we and other groups have previously reported thedevelopment of an mRNA electroporation–based approach toinduce transient CAR expression (16–18). This strategy createsan efficient CAR expression system that ensures complete lossof CAR-driven T-cell activity in a predictable time framewithout the need to administer other systemic agents toeliminate modified T cells. We have reported the efficacy oftransiently modified CD19 CAR T cells in a disseminatedxenograft model of systemic ALL (19), and recently demon-strated enhanced efficacy of these transiently modified cellswhen delivered repeatedly in an optimizeddosing strategy (20).This optimized therapeutic regimen approached the antitu-mor responses observed with permanently modified CD19CAR T cells and demonstrated long-term disease control,suggesting that multiple infusions of transiently modified CART cells may present an alternative to genome-modifying T-cellengineering techniques.

RNACART cells have demonstrated in vitro activity (21) andin vivo efficacy in localized models of solid tumors, and havesimilarly shown enhanced efficacy usingmultiple cell infusions(17, 22). On the basis of these findings, as well as our ownexperience with RNA CAR T cells in ALL, we evaluated a CARtargeting GD2, a diasialoganglioside expressed on the surfaceof most neuroblastomas (1) that has already been shown to bean effective target for neuroblastoma immunotherapy (23). Asingle-chain antibody variable region fragment (scFv) target-ing GD2 was linked to the CD3z and 4-1BB intracellularsignaling domains and tested in localized and disseminatedanimal models of neuroblastoma. We demonstrate that mul-tiple infusions of RNA GD2 CAR T cells result in effectiveantitumor responses to disease in vivo, and that a single low-dose infusion of permanentlymodifiedGD2 CART cells resultsin long-term control of disseminated neuroblastoma. Multipleinfusions of RNA GD2 CAR T cells are less effective at con-trolling disseminated disease, and our data highlight thepotential mechanism underlying this lack of efficacy. Together,these data clarify the necessary components for success oftransiently modified CAR T cells in solid tumors.

Materials and MethodsT-cell expansion in vitro

Human T cells were isolated from healthy donors by theUniversity of Pennsylvania Human Immunology Core. Cells

were incubated with microbeads coated with CD3 andCD28 stimulatory antibodies (Life Technologies; catalog#111.32D). T cells were combined at a ratio of 1:3 (cells:beads) and cultured in T-cell culture media. Beads wereremoved on day 7 or 8 of stimulation. Cells were counted,and volumes were measured (Multisizer 3; Beckman Coul-ter) every 48 hours. When cell growth kinetics and volumesuggested that cells had rested down from activation, theywere cryopreserved.

Generation of CAR constructs and RNA electroporationCARs containing scFv domains directed against GD2 or

CD19 linked to CD3z and 4-1BB intracellular signalingdomains were produced as previously described (GD2-z con-struct was generously provided byDr.MalcolmBrenner, BaylorCollege of Medicine, Houston, TX; refs. 24, 25). Development ofconstructs for RNA manufacture was performed as previouslydescribed (17). The mScript RNA System (CellScript; catalog#MSC11625) was used to generate capped in vitro transcribedRNA, which was purified using an RNeasy Mini Kit (Qiagen,Inc.; catalog #74104). T cells were expanded and frozen asdescribed above. Before electroporation, cells were thawed,washed three times with Opti-MEM, and resuspended in Opti-MEMmedium at a final concentration of 1 to 3� 108 cells/mL.T cells were then mixed with transcribed mRNA at a concen-tration of 10mgmRNA/0.1mLT cells and electroporated in a 2-mm cuvette using an ECM830 Electro Square Wave Porator(both from Harvard Apparatus BTX; catalogs #450125 and#450002) 1–2 hours before infusion into animals. Viability aftertransfection ranged from 50% to 80%, and in all cases, T cellsdemonstrated >95% CAR expression.

Production of lentiviral vectors and T-cell transductionHigh-titer, replication-defective lentiviral vectors were pro-

duced using 293T human embryonic kidney (HEK) cells (26).HEK293T cells were seeded at 107 cells per T150 tissue cultureflask 24 hours before transfection. On the day of transfection,cells were treatedwith 7mg of pMDG.1, 18mg of pRSV.rev, 18mgof pMDLg/p.RRE packaging plasmids, and 15 mg of transferplasmid in the presence of either Express-In TransfectionReagent (Open Biosystems) or Lipofectamine 2000 transfec-tion reagent (Life Technologies; catalog #11668019). Transferplasmids containing CAR constructs were modified so thatexpression of the CAR was under the control of the EF-1apromoter as previously described (25). Viral supernatants wereharvested 24 and 48 hours after transfection and concentratedby ultracentrifugation overnight at 10,500 �g. Twenty-fourhours after initial stimulation, T cells were exposed to lentiviralvector at a concentration of 5 to 10 infectious particles perT cell, and then cultured as described above.

CAR detection on modified cellsCells were washed and resuspended in FACS buffer (PBS þ

1% bovine serum albumin), then incubated with biotin-labeledpolyclonal goat anti-mouse F(ab)2 antibody (Jackson Immu-noresearch; catalog #115-066-072) at 4�C for 25 minutes, andthen washed twice. Cells were then incubated with R-phyco-erythrin (PE)–conjugated streptavidin (BD Biosciences;

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Cancer Immunol Res; 2(11) November 2014 Cancer Immunology Research1060




catalog #554061) for 10 minutes at 4�C, then washed twice.Flow cytometry acquisition was performed on an Accuri C6cytometer (BD Biosciences; catalogs #342975 and #653118).Analysis was performed using the FlowJo software (Treestar,Inc.).

Mouse xenograft studiesSix- to 10-week-old NOD-SCID-gc�/� (NSG) mice were

obtained from The Jackson Laboratory or bred in-house underan approved Institutional Animal Care and Use Committee(IACUC) protocol and maintained in pathogen-free conditions.For flank tumor studies, animals were injected subcutaneouslywith 2 � 107 SY5Y human neuroblastoma cells suspended in0.2 mL Matrigel (BD Biosciences; catalog #354234). For dissem-inated tumor studies, animalswere injected via tail veinwith 2�106 SY5Ycells in0.1mLsterilePBS.T cellswere injected in 0.1mLsterile PBS. Cyclophosphamide (Baxter Healthcare; catalog#1001995601) was injected i.p. at 60 mg/kg 24 hours before anycell administration following the initial infusion. Animals weremonitored for signs of disease progression and overt toxicity,such as xenogeneic graft-versus-host disease (GVHD), as evi-denced by >10% loss in body weight, loss of fur, diarrhea,conjunctivitis, and disease-related hind-limb paralysis.

Measurement of flank tumorsFlank tumor measurements were made biweekly using

electronic calipers (Fowler-Sylvac; catalog #54-200-777). Thelongest length and width measurements were recorded andtumor volumewas calculated according to the formula [(widthþ length)/2)3]/2. Mice were sacrificed when tumors reached 3cm3, or when tumor burden inhibited animal activity.

Bioluminescent imagingDisease burdens were monitored over time using a Xenogen

IVIS bioluminescent imaging system, as previously described(19, 27).

ImmunohistochemistryHistology was performed by the Pathology Core Facility at

the Children's Hospital of Philadelphia. Subcutaneous tumorsand tumor-infiltrated livers were excised postmortem, andpreserved in 4% paraformaldehyde. Image analysis was per-formed using the ImageScope software (Aperio). Staining wasperformed on a BondMax automated staining systemwith theBond Refine polymer staining kit (both Leica Microsystems).Standard protocol was followed, with the exception of theprimary antibody incubation, which was extended to 1 hour atroom temperature. Anti-human CD3 antibody (Dako; catalog#M7254) was used at 1:50 dilution and antigen retrieval wasperformed with the E1 retrieval solution for 20 minutes. Slideswere rinsed and dehydrated through a series of ascendingconcentrations of ethanol and xylene. Stained slides were thendigitally scanned at �20 magnification on an Aperio OS slidescanner (Aperio).

Cell line identity testingThe SH-SY5Y (SY5Y) tumor cell line was obtained originally

from the American Type Culture Collection (CRL-2266; ATCC)

and generously donated to our laboratory by Dr. John Maris,Children's Hospital of Philadelphia. The parent cell line wasgenotyped by short tandem repeat (STR) analysis (28). Celllines were tested forMycoplasma and verified every 6 months,or after any genetic modification, such as luciferase transduc-tion, to ensure identity and Mycoplasma-free status.

Statistical considerationsAll statistical analysis was performed with Prism 4 (Graph-

pad Software). For comparison among multiple groups,Kruskal–Wallis analysis was performed with Dunn multiplecomparison tests to compare individual groups. Survivalcurves were compared using the log-rank test with theBonferroni correction for multiple comparisons.

ResultsmRNAGD2CARTcellsmediate rapidneuroblastomacellkilling in vivo

Subcutaneous injection of tumor cells in themouse flank is awell-established model of solid tumor therapy in mice. Toassess the antitumor efficacy of our transiently modified GD2CAR T cells in a solid tumor model of neuroblastoma, NSGmice were injected s.c. with 2 � 107 SY5Y (a GD2þ humanneuroblastoma cell line) cells and given 2 weeks to establishlarge flank tumors. On day 15, 5 � 106 human T cells electro-porated with RNA encoding either a GD2 or CD19 CAR bearingthe 4-1BB and CD3z signaling domains (GD2-BB/z or CD19-BB/z) were injected into the tumor. On day 20, several micewere sacrificed, and tumors were excised and probed for thepresence of human T cells by immunohistochemical analysis.Intratumoral (i.t.) injection of RNAGD2 CAR T cells resulted inrapid necrosis of target neuroblastoma, with T cells diffuselypresent throughout the tumor environment (Fig. 1A). RNACD19 CAR T cells, on the other hand, did not alter the tumorarchitecture after i.t. injection, and quickly diffused out of thetumor and localized to the periphery of the tissue (Fig. 1B).Mice not sacrificed for histology received two additional i.t.injections of 1.5 � 107 cells each, and RNA GD2 CAR T cellsmediated significant antitumor responses, whereas RNA CD19CAR T cells permitted rapid tumor growth (P < 0.001; Fig. 1C).Tumor expansion observed in mice treated i.t. with RNACD19 CAR T cells was similar to that in mice treated withsaline (P ¼ 0.7912).

Intravenous injection of neuroblastoma cells results in areproducible model of disseminated neuroblastoma

Subcutaneous tumors have many anatomic and physio-logic differences when compared with spontaneous intra-abdominal tumors, several of which have direct bearing onthe physiology of cellular therapies. Previous studies of CART cells in solid tumors, such as epithelial ovarian cancer (29),have used a peritoneal tumor model in which tumor cells areinjected directly into the peritoneum to establish diseasealong the internal peritoneal wall, mimicking natural dis-ease. We sought to develop a model that would more closelymimic the clinical setting of disseminated neuroblastoma inwhich CAR T cells are likely to be given. SY5Y neuroblastoma

Permanently and Transiently Modified CAR T Cells for Neuroblastoma

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cells were engineered to express click beetle green luciferase(SY5Y-CBG) before delivery, which allowed for longitudinalobservation of a disseminated tumor burden. Tumor cellswere injected i.v., and animals were observed for the estab-lishment and progression of systemic disease. Within 2hours of cell delivery, bioluminescent signal was observedin the lungs, and then rapidly disseminated (SupplementaryFig. S1). As early as day 7, disease was detected in theabdomen, and was subsequently observed in the bonemarrow and lymph nodes. Necropsy revealed that thisabdominal signal resulted from hepatic infiltration of neu-roblastoma, causing diffuse nodular hepatic disease.

Permanently modified GD2 CAR T cells mediateregression of disseminated neuroblastoma

We first tested the efficacy of permanently modifiedGD2 CAR T cells in our disseminated model of neuroblas-toma. CAR T cells were engineered using a well-established

lentiviral vector transduction technique (9, 25). NSGmice were given 2 � 106 SY5Y-CBG cells via tail vein andgiven 1 to 2 weeks to establish a significant disease burden(>106 photons/s/cm2/sr), after which CAR T cells weredelivered systemically. Animals were imaged twice weeklyto monitor disease burden. A single infusion of as few as106 permanently modified GD2 CAR T cells was sufficientto suppress and control disseminated neuroblastoma overtime (P < 0.001; Fig. 2A), whereas tumor growth progressedcomparably after systemically delivered control CD19CAR T cells and saline (P ¼ 0.8872). Not surprisingly, thedisease control observed had a significant impact on thesurvival of animals treated with GD2 CAR T cells (P ¼0.0257; Fig. 2B). Interestingly, all mortality in animals trea-ted with GD2 CAR T cells resulted from xenogeneicGVHD, with 63% of GVHD mortality occurring in diseaseremission and the remaining 37% with minimal diseaseburdens (<2 � 107 photons/sec/cm2/sr).

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Figure 1. RNA GD2 CAR T cellsmediate rapid destruction ofneuroblastoma cells and controltumor growth. NSG mice wereimplanted with 2 � 107 SY5Yneuroblastoma cellssubcutaneously in the flank andgiven 3weeks to establish disease.Mice were then injectedintratumorally with 5� 106 (A) RNACD19 CAR T cells or (B) RNA GD2CAR T cells, and tumors wereexcised 5 days later andimmunostained for human CD3.RNA CD19 CAR T cells do notaffect the tumor architecture andpassively filter out of the tumor totheperiphery of the tissue,whereasRNA GD2 CAR T cells mediaterapid destruction of SY5Y cells anddisperse throughout the tumor.(Inlay, �2 magnification; mainimage, �6 magnification).C, RNA GD2 CAR T cells mediatelong-term disease suppression ascompared with RNA CD19 CART cells (black arrows represent cellinfusions, 5 � 106 on day 15,1.5 � 107 on days 29 and 36).

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Multiple infusions of RNA GD2 CAR T cells delay diseaseprogression, but do not eradicate disseminatedneuroblastomaWe next used this disseminated model system to assess the

efficacy of RNA GD2 CAR T cells against neuroblastoma.Disseminated disease was established as described, and tran-siently modified T cells were delivered intravenously. On thebasis of our experience with ALL (20), we undertook amultipleinfusion strategy using RNA-electroporated T cells. Mice weretreated initially with 5 � 106 cells on day 8, and 1.5 � 107 ondays 22 and 29 after tumor implantation. We observed atransient decline in disease burden after the initial infusion,and modest disease stabilization with the next two infusions(Fig. 3A). Although this strategy significantly slowed diseaseprogression (P¼ 0.0032), it was unable to control the disease inthe long term. This delayed progression did, however, prolonganimal survival (P ¼ 0.0128; Fig. 3B). Again, animals treatedwith CD19 CAR T cells were statistically similar in both diseaseprogression (P ¼ 0.4175) and overall survival (P ¼ 0.9834) tothose treated with saline.Visual representations of disease burden and response to

therapy highlight the variations in antitumor activity betweentransiently and permanently modified CAR T cells (Fig. 4).Animals treated with lentivirally modified GD2 CAR T cellsshowed rapid loss of bioluminescent signal with maintained

disease suppression in the long term. Interestingly, theseanimals occasionally have focal disease recurrence, which islost on subsequent evaluation. This likely represents the long-term, antitumor T-cell memory of permanently modified CART cells. As demonstrated in the quantitative bioluminescentgraph (Fig. 3B), animals treated with GD2 RNA CAR T cells hadan initial decline in disease burden after the first cell infusion,followed by transient stabilizations with the subsequent twoinfusions.

Antigen-targeted RNA CAR T cells are unable topenetrate the tumor microenvironment

To better understand the dynamics of the T cell–tumorinteraction in our disseminated solid tumor model, weexamined tumor sites using immunohistochemical analysis.Animals were given the standard 1 to 2 weeks to establishdisease after i.v. tumor injection, and then given either asingle infusion of lentivirally modified CAR T cells or threeinfusions of RNA CAR T cells, each separated by 1 week, as instudies described previously. Animals were sacrificed on theday indicated, and the excised livers were sent for histo-pathologic examination and immunohistochemical stainingfor human CD3. Animals receiving permanently modifiedGD2 CAR T cells had rapid infiltration of CAR T cells intotumor sites by day 3, and showed a significant T-cell

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Figure 2. Lentivirally modified GD2CAR T cells control disseminatedneuroblastoma in the long termandprolong animal survival. Mice withdisseminated neuroblastoma weregiven a single dose of 1� 107 CART cells (day 14, black arrow; CD19cells 56% CARþ, GD2 CAR cells10.9% CARþ). A, one dose of GD2CAR T cells mediated sustaineddisease control in the long term.B, overall survival of animalstreated with either CD19 or GD2CAR T cells. All animals treatedwith GD2 CAR T cells died ofxenogeneic GVHD,with no animalssuccumbing to cancer. This isfurther illustrated in C, whichdemonstrates mortality attributedto malignancy in animals treatedwith either CD19 or GD2 CAR Tcells. All animals treated with CD19CAR T cells died of malignancy.Five of 8 animals treated with GD2CAR T cells died in diseaseremission, and 3 of 8 died withcontrolled minimal diseaseburdens.






presence in all tumor sites by day 5 (Fig. 5A). Permanentlymodified control CD19 CAR T cells did not show anysignificant tumor infiltration (Fig. 5B). Alternatively, by day3 after infusion of the first dose of RNA-modified GD2 CART cells, minimal infiltration of these CAR T cells into thetumor site was found, and this T-cell presence had largelydissipated by day 6. Twenty-four hours after the secondinfusion of RNA GD2 CAR T cells (on day 7), a minoraccumulation of T cells was found around the periphery ofthe tumor, which had again largely dissipated by day 10. Thispattern was also observed after the third cell infusion, with aperipheral accumulation around the edge of the tumorwithin 24 hours, which was quickly lost (Fig. 6A). Asexpected, control RNA CD19 CAR T cells did not show anysignificant tumor infiltration (Fig. 6B).

DiscussionHigh-risk neuroblastoma remains a significant cause of

cancer-related death in children. Although we have seendramatic improvement in the treatment of both solid andliquid pediatric tumors, survival rates for both have pla-

teaued since the mid-1990s (30). Patients with relapsedneuroblastoma are largely incurable, although temporarycontrol of disease can be achieved for some patients. Thus,a new therapeutic paradigm is needed to improve survival.Not until recently has engineered T-cell therapy begun tomeet its potential. Integration of the powerful cytotoxicity,significant in vivo proliferation, and surveillance mechan-isms of memory T cells with the antigen specificity endowedby CARs has created a platform for the success of celltherapy. As we have suggested, the recent successes of CARtherapy (8–10) may primarily be attributed to improved cell-manufacturing processes that generate T cells armed withrapid and potent in vivo effector and proliferative capacity(31).

Previous reports have demonstrated the efficacy ofGD2CART cells against disseminated GD2þ melanoma using two infu-sions of 107 CAR T cells each (32). In the only other study to ourknowledge of CAR T cells in a model of systemic solid malig-nancy, 2 � 107 prostate-specific membrane antigen (PSMA)–directed T cells demonstrated efficacy in a disseminatedmodelof prostate cancer (33). In this study, we were able to demon-strate disease eradication in more than half of the animals and

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Figure 3. Multiple infusions of RNAGD2 CAR T cells delay diseaseprogression and enhance animalsurvival. Mice with disseminatedneuroblastomawere given an initialinfusion of 5 � 106 CAR T cells onday 8, followed by two infusions of1.5 � 107 CAR T-cells on days 22and 29 (black arrows). A, an initialdose of GD2 CAR T cellsdemonstrates a modest reductionin disease burden, and the next twoinfusions mediated transientdisease stabilization, resulting in acumulative delay in diseaseprogression over time. B, threeinfusions of RNA GD2 CAR T cellsenhance overall animal survival.

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disease suppression in the rest of the animals treated using asingle infusion of only 106 cells, approximately 5% of the doseused in the previous studies of CAR T cells in solid tumors. Notonly were these cells able to control disease, we also observedmassive cell expansion driven by antigen-specific activation.Indeed the T-cell expansion was high enough to cause death ofanimals from xenogeneic GVHD. T cells that could not rec-ognize tumor (CD19 CAR T cells) did not proliferate to the

same degree, as evidenced by the lack of GVHD in theseanimals. Both of these observations highlight the significantproliferative capacity of our CAR T cells, an attribute endowedby a combination of cell-manufacturing processes and CARsignaling constructs. The ability to use fewer cells to achieveclinical efficacy is of particular importance in pediatricpatients who undergo extensive chemotherapy, which createssignificant lymphodepletion as part of standard treatment for

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Figure 4. Effect of GD2 CAR T cells on disseminated neuroblastoma. A, an initial infusion of RNA GD2 CAR T cells reduces disease burden, while thesubsequent infusions modestly stabilize disease. B, lentiviral GD2 CAR T cells mediate long-term disease control. On day 28, a transient disease recurrenceappears but is eliminated, highlighting the memory response of permanently modified CAR T cells.






many malignancies. Reducing the number of cells needed fortreatment would enhance the number of patients eligible forthis therapy.

GD2 serves as an exciting target in neuroblastoma, givenits narrow expression profile. However, as is the case withmost solid-tumor antigens, on-target off-tumor toxicityremains a persistent issue, as GD2 is also expressed onsensory nerves. Clinical experience has demonstrated thatantibody therapy directed at GD2 can result in significantneuropathic pain (34, 35). This toxicity is managed clinicallyduring infusion of the antibody, and when the infusion ends,the toxicity resolves. Previous clinical studies with the first-generation GD2/z CAR T cells found that, of the 19 patientsfollowed for more than 5 years, only 2 patients had local painat the site of tumor necrosis and 1 had unexplained localpain (36), with no patient experiencing pain greater thangrade 3. While suggestive that the physiology of this toxicitymay be distinct when using an antibody versus a cell-basedtherapy comprising a single-chain Fv without an Fc domain,

this remains a potential on-target off-tumor toxicity ofhighly active GD2 CAR T cells. Our cell production techni-ques, structure of CAR signaling domains, in vivo expansion,and persistence all differ from those described in previousclinical trials (31). For this reason, we have been veryinterested in an RNA-based approach.

Having demonstrated cure of leukemic mice using multipleinfusions of RNA-electroporated CAR T cells in ALL, we soughtto assess the efficacy of this engineering platform in treatingsolid tumors. In the traditional flank tumor xenograft model,our RNA GD2 CAR T cells demonstrated rapid and potentantitumor activity when delivered locally. Although this dem-onstrated cytolytic activity against neuroblastoma in vivo, thismodel is more difficult to translate clinically, as local tumorinjectionsmay not be feasible in some patients. Moreover, late-stage cancer patients may have many sites of disease. To morecloselymimic systemic disease, we developed a sensitive in vivobioluminescent model that enabled the establishment andmonitoring of disseminated disease, primarily manifested in

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Figure 5. Permanently modifiedGD2 CAR T cells demonstraterapid tumor infiltration. Animalswere sacrificed on the daysindicated and livers examinedhistologically after staining forhuman CD3. Tumor sites boxed inthe �1 magnification are enlargedin the �10 magnification. A, within3 days of T-cell infusion, GD2 CART cells have infiltrated into tumorsites. By day 5, gross examinationreveals significant T-cell infiltrationand activity within tumors.B, CD19 CAR T cells do notpenetrate tumor sites.

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the liver and bonemarrow.We found that a single small dose ofpermanentlymodified second-generationGD2/4-1BB/zCARTcells could produce long-term disease control, while multiple

infusions of RNA GD2 CAR T cells were able to delay diseaseprogression and enhance animal survival but could not controldisease in the long term.

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Figure 6. RNAGD2CARTcells localize to but do not infiltrate disseminated tumors. Animalswith establisheddisseminated tumorswere sacrificedon the daysindicated andexaminedhistologically after staining for humanCD3.Blackarrows indicate infusion of 1�107T cells. A,with small tumors, someRNAGD2CART cells are able to penetrate and remain within tumors sites in the first 3 days of injection; however, subsequent injections demonstrate peripheral localizationwithout infiltration. B, initial passive infiltration by CD19 CAR T cells is lost by day 3, and subsequent injections do not result in localization to tumor sites.






On the basis of the fundamental differences in tumorbiology, we have hypothesized that the success observed usingRNA CAR T cells for liquid tumors (20) might not directlytranslate to solid tumors. T cells targeting leukemic cellsimmediately traffic to sites of disease, as disease reservoirsare the natural sites of T-cell residence and surveillance.This all but removes the necessity of antigen-driven T-celltrafficking and retention. Trafficking in solid tumors is furthercomplicated, given the disorganized nature of the tumorvasculature, another issue that is averted when targeting liquidtumors. Thus, while creating a proliferative niche and optimaldosing strategy were the primary hurdles in successful target-ing of liquid tumors using RNA CAR T cells (19), solid tumorspresent the additional barriers of trafficking, penetration, andefficient antigen engagement within a 6-day CAR expressionperiod.

Our histologic data support these hypotheses, elucidatingthe dynamics of T cell and tumor interaction in vivo. Within 72hours of the infusion of lentiviral CAR T cells, there is a largeburden of T cells within the tumor site, and by day 5 all tumorsites are heavily infiltrated with CAR T cells. The dynamics ofRNA CAR T-cell activity are quite different: 72 hours after thefirst cell infusion, a few cells have penetrated into the tumorsite, providing a potentially favorable effector:target ratio onday 3, but these cells are lost by day 6. After a second infusion,the T cells localize to site of these expanding tumors, but areunable to penetrate and are lost quickly. The third infusionresults in similar kinetics of T-cell localization to the peripheryof enlarging tumor masses. These observations correlate wellwith our bioluminescent data, which show a transient declinein tumor burden after thefirst cell infusion, followed bymodestdisease stabilization, likely reflecting a short-lived antitumorresponse at the periphery after the second and third T-cellinfusions (Fig. 3B). The diminishing returns seen with eachadditional T-cell infusion reflect the fact that each infusionfaces the same structural and vascular barriers as the first, inaddition to an expanding tumor burden.

The rapid increase in the number of permanently modifiedGD2 CAR T cells present within the tumor seen over days 1 to 5after T-cell infusion raises the question of whether this occursas a result of continued cell recruitment, in situ expansion ofrecruited cells, or both. If indeed this is continued cell recruit-ment of circulating CART cells to the site of tumor antigen overdays 1 to 5, it would suggest that RNA CAR T cells do not havethe same antigen-driven migratory capabilities, as this iswithin the window of RNA CAR expression. If the increasedT-cell infiltration is a result of intratumoral proliferation inresponse to antigen, this suggests a difference in the prolifer-ative capacity of RNA and permanently modified CAR T cells.Although we cannot formally distinguish between these twopossibilities, the latter seems likely to be the dominant mech-anism, given the importance of antigen-driven proliferation toCAR T-cell efficacy.

The observation that three infusions of RNAGD2CART cellswere not able to control systemic disease led us to assess theefficacy of repeated infusions. In another iteration of themultiple infusion strategy, we injected a total of 108 RNA CART cells over the course of 8 weeks and found that while nine

infusions further slowed disease progression as comparedwiththree infusions, disease continued to progress (data notshown). This observation, along with the poor tumor infiltra-tion of RNA CAR T cells, suggests that increasing cell dose willnot increase the efficacy of this therapy, as it did with ALL (20).

The mechanisms underlying this observed difference in T-cell retention and infiltration are unclear. We have evaluatedCAR molecule expression after RNA electroporation in vitroand demonstrated a progressive decay over the course of 6days, but we have not assessed expression kinetics in vivo. It iscertainly possible that infusion into and circulation within aliving organism alters CAR expression. It is known that uponengagement with the target antigen, the T-cell receptor isinternalized as more TCRs are integrated into the cell mem-brane. This receptor recycling may also apply to CAR mole-cules, and whereas lentivirally modified CAR T cells are able tocontinually present new CARs on the surface, RNA-modifiedCARs are more limited. Another manner in which RNA CARsignal may be diluted is during in vivo expansion. As antigenengagement results in T-cell activation and proliferation,permanently modified CAR T cells produce daughter cells thatalso have permanent CAR expression. RNA-modified CAR T-cell proliferation, however, may produce daughter cells with afraction of the originally delivered CAR RNA. These kineticsmay play an essential role in limiting RNACAR T-cell retentionand infiltration at tumor sites.

Mechanisms independent of CAR expression kinetics mayalso account partially for the differences observed. Althoughthe majority of cell production is identical for both RNA andlentivirus CAR T cells, it is unclear whether the process ofelectroporation has an effect on T-cell function. Data fromintratumoral injection studies suggest that these RNA CAR Tcells are functional and able to mount effective antitumorresponses; however, subtle differences in T-cell activity may bepresent that are unmasked when delivering cells systemically.These differences may occur in the form of variations in cellsignaling or variability in expression of surface molecules thataffect T-cell trafficking, retention, and infiltration, such aschemokine receptors, adhesion molecules, and T-cell inhibi-tory receptors. It is possible that a combination of these factorsaccounts for the differences observed, and further character-ization of these mechanisms will allow identification of theprocesses that can be targeted to enhance efficacy.

To translate this therapy, which shows greater efficacy withlocal delivery, into a clinically viable treatment modality,solving the problem T-cell recruitment and expansion inmetastatic tumors is key. One likely option is the prolongationof RNA CAR expression. One such method under study is thebiochemical stabilization of transferred RNA molecules (37).This strategy will produce CAR T cells with more sustainedreceptor expression that retain the self-inactivating qualities ofRNA electroporation, prolonging the critical time period of T-cell infiltration.

Although the RNA CAR T-cell approach will minimizetoxicities that occur over days, such as seen with permanentlymodified CD19 CARs (10), this may not be the case for adverseevents reported in previous clinical trials (12, 13) that occurwithin hours of cell infusion. We can predict, however, that the

Singh et al.





toxicities observed would have been self-limited and, thus,potentially less severe, as antigen-driven cell division wouldresult in restriction and temporally restricted expression ofCAR molecules. Our recent report of cytokine release syn-drome in children treated with CD19 CAR T cells (10) is anexample of toxicity that may have been self-limited had theseT cells been modified with RNA CARs, as opposed to perma-nently modified with lentiviral CARs. Similarly, long-term B-cell aplasia, which we have observed as a necessary compli-cation of CD19 CAR T-cell persistence, would not occur withthis approach. Another approach to limit CAR T-cell persis-tence is to use suicide gene systems that may eliminate 1 to 3logs of cells. However, T cells have robust proliferative capacityand are capable of repopulating from a single cell, and thus aguaranteed "off" system such as RNA electroporation remainsan attractive approach.In conclusion, we demonstrate the efficacy of permanently

modified GD2 CAR T cells in controlling disseminated neuro-blastoma, and that a lack of longer-term control of metastaticdisease by temporarily modified GD2 CAR T cells results fromreduced tumor infiltration in the setting of large tumorburdens.

Disclosure of Potential Conflicts of InterestC.H. June, S.A. Grubb, and Y. Zhao report receiving commercial research

grants from Novartis. S.A. Grubb is a consultant/advisory board member forNovartis. Y. Zhao has ownership interest in a patent from the University of

Pennsylvania. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: N. Singh, S.A. Grupp, D.M. Barrett, Y. ZhaoDevelopment of methodology: N. Singh, X. Liu, S.A. Grupp, D.M. Barrett,Y. ZhaoAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): N. Singh, X. Liu, J. Hulitt, S.A. Grupp, Y. ZhaoAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): N. Singh, S.A. Grupp, D.M. Barrett, Y. ZhaoWriting, review, and/or revision of the manuscript: N. Singh, C.H. June,S.A. Grupp, D.M. Barrett, Y. ZhaoAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): S. Jiang, S.A. GruppStudy supervision: S.A. Grupp, D.M. Barrett, Y. Zhao

AcknowledgmentsThe authors thank Daniel Martinez and Dr. Bruce Pawel for their assistance

with immunohistochemistry, and Dr. Curt Lind for assistance with HLAphenotyping.

Grant SupportThis study was supported by NIH R01CA120409 (to C.H. June and Y. Zhao),

P01CA066726 (to C.H. June and Y. Zhao), and grants from the PennsylvaniaDepartment of Health, Cookies for Kids Cancer, Solving Kids Cancer, the WWSmith Charitable Trust and the Weinberg Funds (to S.A. Grupp)

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

Received March 21, 2014; revised May 26, 2014; accepted July 29, 2014;published OnlineFirst August 7, 2014.

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Singh et al.




2014;2:1059-1070. Published OnlineFirst August 7, 2014.Cancer Immunol Res Nathan Singh, Xiaojun Liu, Jessica Hulitt, et al. NeuroblastomaGD2 Chimeric Antigen Receptor T Cells in Xenograft Models of Nature of Tumor Control by Permanently and Transiently Modified

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