Therapeutically targeting glypican-2 via single-domainantibody-based chimeric antigen receptors andimmunotoxins in neuroblastomaNan Lia, Haiying Fua,b, Stephen M. Hewittc, Dimiter S. Dimitrovd, and Mitchell Hoa,1

aLaboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; bDepartment ofImmunology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China; cLaboratory of Pathology, Center for Cancer Research,National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and dCancer and Inflammation Program, Center for Cancer Research, NationalCancer Institute, National Institutes of Health, Frederick, MD 21702

Edited by Dennis A. Carson, University of California, San Diego, La Jolla, CA, and approved July 3, 2017 (received for review April 11, 2017)

Neuroblastoma is a childhood cancer that is fatal in almost half ofpatients despite intense multimodality treatment. This cancer isderived from neuroendocrine tissue located in the sympatheticnervous system. Glypican-2 (GPC2) is a cell surface heparan sulfateproteoglycan that is important for neuronal cell adhesion andneurite outgrowth. In this study, we find that GPC2 protein is highlyexpressed in about half of neuroblastoma cases and that highGPC2 expression correlates with poor overall survival comparedwith patients with low GPC2 expression. We demonstrate thatsilencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition ofneuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target geneN-Myc, an oncogenic driver of neuroblastoma tumorigenesis. Wehave isolated human single-domain antibodies specific for GPC2 byphage display technology and found that the single-domain anti-bodies can inhibit active β-catenin signaling by disrupting the inter-action of GPC2 and Wnt3a. To explore GPC2 as a potential target inneuroblastoma, we have developed two forms of antibody thera-peutics, immunotoxins and chimeric antigen receptor (CAR) T cells.Immunotoxin treatment was demonstrated to inhibit neuroblastomagrowth in mice. CAR T cells targeting GPC2 eliminated tumors in adisseminated neuroblastoma mouse model where tumor metastasishad spread to multiple clinically relevant sites, including spine, skull,legs, and pelvis. This study suggests GPC2 as a promising therapeutictarget in neuroblastoma.

glypican | neuroblastoma | single-domain antibody |chimeric antigen receptor T-cell therapy | immunotoxin

Neuroblastoma is the most common extracranial solid tumorof children and the most frequently diagnosed neoplasm

during infancy (1). It accounts for 8–10% of all childhood can-cers and 15% of all pediatric cancer mortality (2). This cancer isderived from neuroendocrine tissue located in the sympatheticnervous system. Neuroblastoma is a complex and heterogeneousdisease, with nearly 50% of patients having a high-risk pheno-type. Despite the use of intensive multimodal treatments, thesehigh-risk phenotype cancers often present with widespread dis-semination and poor long-term survival (3). Approximately 45%of patients receiving standard therapy relapse and ultimatelysuccumb from metastatic disease (4). As such, there is an urgentneed for an effective neuroblastoma treatment.One of the major challenges for the treatment of neuroblas-

toma and other pediatric cancers is the lack of effective thera-peutic targets. Interestingly, glypican-2 (GPC2) was recently foundas one of several mRNA transcripts that were highly expressed inmultiple pediatric cancers, including neuroblastoma (5). Whereasthese candidate markers have shown promise at the mRNA level,they have yet to be validated at the protein level. GPC2 belongs toa six member human glypican family of proteins (6). All of theseproteins are attached to the cell surface by a GPI anchor (6).GPC2 is unique among the glypican family because it is expressed

in the nervous system (7). It is known to participate in cellularadhesion and is thought to regulate the growth and guidance ofaxons (8). The role of GPC2 in the pathogenesis of neuroblastomaor any other cancers has not been reported.The Wnt/β-catenin signaling pathway is highly conserved and

plays an essential role in various processes of embryonic devel-opment (9). Additionally, it has been linked to pathogenesis ofnumerous adult and pediatric tumors (10). Wnt/β-catenin signal-ing has been shown to mediate neural crest cell fate and neuralstem cell expansion. This signaling pathway may be of particularrelevance to neuroblastoma cells because they arise from migra-tory neural crest-derived neuroblasts (11–13). Glypicans play animportant role in developmental morphogenesis and are known tobe modulators for the Wnt signaling pathway (14–17). It has beenshown that GPC3, another member of the glypican family, mayfunction as a coreceptor for Wnt and facilitate the Wnt/Frizzledbinding in hepatocellular carcinoma cells (18, 19).Antibody-based therapeutics are of growing significance in the

field of cancer therapy. Antibodies can be used as vehicles todeliver a variety of effector molecules such as cytotoxic drugs ortoxins to tumor cells. Recombinant immunotoxins are chimericproteins composed of an antibody fragment fused to a toxin, suchas the 38-kDa truncated fragment of Pseudomonas exotoxin(PE38). By combining the specificity of an antibody with theprotein synthesis inhibitory domain from the exotoxin, it is pos-sible to directly target cancer cells (20–24). Another antibody-based therapy that is emerging with clinical applications involves

Significance

Neuroblastoma is a childhood cancer that remains an importantclinical challenge. It is fatal in almost half of the patients despiteadvances in multimodal treatments. In this report, we show thatthe cell surface glycoprotein glypican-2 (GPC2) is overexpressed inneuroblastomas when compared with normal tissues and that ahigh expression level is correlated with poor survival of neuro-blastoma. We also describe that GPC2 has proliferative effects inneuroblastoma via activating Wnt signaling and its downstreamtarget genes including N-Myc, a major driver for neuroblastomatumorigenesis. We have produced the immunotoxins and chi-meric antigen receptor T cells that target GPC2 and exhibitpromising antitumor activities in cell and mouse models. Thisstudy suggests GPC2 as a promising target in neuroblastoma.

Author contributions: M.H. designed research; N.L. and H.F. performed research; D.S.D.contributed new reagents/analytic tools; N.L., S.M.H., and M.H. analyzed data; and N.L.and M.H. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: homi@mail.nih.gov.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1706055114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1706055114 PNAS | Published online July 24, 2017 | E6623–E6631

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chimeric antigen receptor (CAR)-expressing T cells. CARs arecomposed of an antibody fragment fused to a transmembranedomain linked to a T-cell signaling moiety. T-cell expressingCARs (CAR T cells) have the ability to bind antigen directly,whereas normal T cells require antigen presented in MHCmolecules. In recent years, CAR T-cell immunotherapy hasemerged as one of the most promising approaches to treat leu-kemia (25–29). CAR T-cell immunotherapy has not been assuccessful in the treatment of solid tumors, in part due to thelack of tumor-specific targets. To improve engineered T-celltherapies in solid tumors, we will need to identify tumor antigensthat can be safely and effectively targeted to discriminate cancersfrom normal tissues.In the present study, we found that GPC2 protein was specifically

overexpressed in neuroblastoma compared with its expression innormal peripheral nerve tissues and its high expression correlatedwith a poor prognosis for patients with neuroblastoma. We alsofound that down-modulation of GPC2 via siRNA or CRISPR-Cas9 suppressed neuroblastoma cell growth by attenuating Wnt/β-catenin signaling and reduced the expression of N-myc, a Wnttarget gene and an oncogenic driver for neuroblastoma pathogen-esis. Furthermore, we identified a group of human single-domainantibodies (LH1–LH7) to GPC2 by phage display technology. Toevaluate their potential use for the treatment of neuroblastoma, weconstructed immunotoxins using these single-domain antibodiesand demonstrated that the LH7–PE38 immunotoxin inhibitedgrowth of neuroblastoma xenograft tumors in mice. In addition, weproduced CARs targeting GPC2 and expressed them in T cellsisolated from multiple healthy donors. Here we found that CART cells could potently kill neuroblastoma cells. Notably, anti-GPC2CAR T cells were effective in suppressing the dissemination ofneuroblastomas in our mouse xenograft model.

ResultsGeneration of Anti-GPC2 Human Single Domain Antibodies. To identifythe antibodies specific for GPC2, we decided to screen a phage-display engineered human VH single-domain antibody library. En-richment was determined by counting the number of phagesrecaptured after each round of panning. Four rounds of panningresulted in an ≈1,000-fold enrichment of eluted phage (Fig. 1A).Phage pools after two rounds of panning exhibited enhancedbinding to GPC2, whereas no binding to BSA was found withpooled phage from any of the four rounds of panning (Fig. 1B). Atthe end of the fourth round of panning, 27 individual clones wereconfirmed to be GPC2 binders by monoclonal phage ELISA.Subsequent sequencing analysis revealed seven unique binders(LH1–LH7). The GPC2–hFc OD450 values of all seven clones wereat least fivefold higher than that of BSA (Fig. 1C), further indicatingthe specificity of the phages to GPC2. Notably, LH1 and LH7 werethe two most abundant binders and combined for 14 of the27 identified (Fig. 1D). The LH5 clone was excluded from furtherstudy due to its low affinity for GPC2. To determine whether theidentified clones would bind to other members of the human gly-pican family, we performed monoclonal phage ELISA usingrecombinant human GPC1 and GPC3. We also included mouseGPC2 to determine whether any of the binders had cross-speciescapabilities. As shown in Fig. 1E, the five clones LH1, LH2, LH4,LH6, and LH7 specifically bound to human GPC2, but not humanGPC1 or GPC3. Interestingly, LH4 and LH6 are cross-reactiveagainst both human and mouse GPC2 proteins. LH3 showed thehighest binding signal to human GPC2 among all binders, but it wasslightly cross-reactive with GPC1 and GPC3. To determine bindingkinetics, we produced a LH7–Fc fusion protein and incubated itwith GPC2 protein in solution on the Octet platform. The Kd valueof the LH7–Fc fusion for GPC2 was 9.8 nM (Fig. 1F). Taken to-gether, we have successfully identified a group of high-affinity anti-GPC2 human single-domain antibodies by phage display.

GPC2 Is Highly Expressed in Neuroblastoma but Not in Normal HumanTissues. A microarray study showed that GPC2 mRNA wasoverexpressed in a panel of pediatric cancers, including neuro-blastoma (5). To examine the expression of GPC2 protein inneuroblastoma, we used anti-GPC2 antibodies as research toolsto examine established human cell lines and clinical tissue sam-ples. Our Western blotting data demonstrated that GPC2 washighly expressed in five neuroblastoma cell lines, includingLAN1, IMR5, LAN5, IMR32, and NBEB (Fig. 2A). GPC2 wasonly weakly expressed in the SKNSH, the sixth neuroblastomacell line that we evaluated. To assess the clinical relevance of ourobservation, GPC2 protein levels were measured in humanspecimens from patients with neuroblastoma or nonmalignantdisease by immunohistochemistry using the LH7 antibody. Asshown in Fig. 2B, GPC2 labeling was readily apparent in speci-mens derived from patients with neuroblastoma (i–iv), but es-sentially undetectable in normal peripheral nerves from patientswith nonmalignant disease (v and vi). Neuroblastoma tumortissues showed strong GPC2 staining in 13 of the 25 cases (52%)(SI Appendix, Fig. S1). To further analyze GPC2 expression innormal human tissues, we used a US Food and Drug Adminis-tration (FDA)-recommended human normal tissue array andprobed it with the LH7 antibody. As shown in Fig. 2C, no sig-nificant GPC2 staining was observed in the normal tissues, in-cluding essential organs such as the brain, heart, lung, andkidney. These results would suggest a tumor-specific expression

Fig. 1. Isolation of GPC2-specific human single-domain antibodies by phagedisplay. (A) Phage-displayed single-domain antibody fragments were se-lected against recombinant GPC2–hFc after four rounds of panning. Agradual increase in phage titers was observed during each round of panning.(B) Polyclonal phage ELISA from the output phage of each round of panning.BSA was used as an irrelevant antigen. (C) Monoclonal phage ELISA of theseven GPC2 binders. (D) Distribution of unique sequences of GPC2 binders in27 selected phage clones. (E) Monoclonal phage ELISA analysis of cross-reactivity of GPC2 binders to human GPC1 and GPC3 and mouse GPC2.(F) Octet association and dissociation kinetic analysis for the interactionbetween various concentrations of the LH7 antibody and human GPC2. Alldata are represented as mean ± SEM of three independent experiments.

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of GPC2. The complete panel of all 32 types of normal tissuesstained for GPC2 expression is shown in SI Appendix, Fig. S2. Inaddition, we also measured GPC2 mRNA levels in a humannormal tissue array by quantitative real-time PCR. GPC2 mRNAexpression was not found in any normal tissues except for amoderate mRNA expression in thymus and testis (SI Appendix,Fig. S3). However, our immunohistochemistry analysis showedno specific binding of the LH7 antibody for either testis (C1) orthymus (D2) (SI Appendix, Fig. S2). These data strongly supporttumor-specific expression of GPC2 and suggest it as a promisingneuroblastoma biomarker.There has been evidence that GPC3 expression or other gly-

picans (e.g., GPC1) has been correlated with poor prognosis inhepatocellular carcinoma or other types of cancer (30–33). Toanalyze a possible correlation between GPC2 mRNA levels andsurvival of patients with neuroblastoma, we used the R2 Geno-

mics Analysis and Visualization Platform. Patients with highGPC2 expression exhibited poorer overall survival and event-free survival compared with patients with low GPC2 expression(Fig. 2 D and E).We examined the ability of the LH7 antibody to bind GPC2 on

neuroblastoma cells by flow cytometry. LH7 showed specificbinding to IMR5, LAN1, IMR32, and LAN5 neuroblastoma cells(SI Appendix, Fig. S4A). In addition, LH7 exhibited no binding toSKNSH cells, which is consistent with the low expression ofGPC2 in this neuroblastoma cell line (Fig. 2A). Furthermore, wequantified the number of cell surface GPC2 sites per cell usingflow cytometry. LAN5 and IMR32 cells expressing nativeGPC2 contain between 104 and 105 sites per cell, whereasLAN1 and IMR5 cells contain between 103 and 104 GPC2 sitesper cell (SI Appendix, Fig. S4B). SKNSH cells showed an ex-tremely low number of cell surface GPC2 sites, with only433 sites per cell. Taken together, we have demonstrated thatGPC2 is a tumor-specific cell surface antigen in neuroblastoma.

Silencing of GPC2 Inhibits Neuroblastoma Cell Growth via Suppression ofWnt/β-Catenin Signaling. To analyze the role of GPC2 in neuroblas-toma cell growth, we used siRNA and CRISPR-Cas9 techniques tosilence GPC2 in two neuroblastoma cell models (IMR5 andLAN1). Three different GPC2 siRNAs were used to avoid potentialoff-target effects of siRNA. GPC2 knockdown efficiency was con-firmed by Western blotting, which showed substantial reductions ofGPC2 levels in both cell lines (Fig. 3A). As shown in Fig. 3B,GPC2 siRNAs suppressed the growth of neuroblastoma cells within3 d of transfection by ≈40–50% compared with cells transfectedwith scrambled siRNA. To validate the oncogenic effect of GPC2 inneuroblastoma, we generated GPC2 KO neuroblastoma cells byusing CRISPR-Cas9. Three single-guide RNAs (sgRNAs) targetingdifferent GPC2 exons (exons 1–3) were transfected into IMR5 cells.Expression of GPC2 protein was almost completely abolished in thesgRNA-transfected cells (Fig. 3C). As shown in SI Appendix, Fig.S5A, a 25–50% reduction of growth was observed in GPC2 KO cellscompared with vector control cells after 3 d of growth. In addition,KO of GPC2 induced apoptosis in neuroblastoma cells as measuredby elevated expression of cleaved-Poly (ADP ribose) polymerase(PARP) (SI Appendix, Fig. S5B) and increased activity of caspase-3and -7 (Fig. 3D).We hypothesized that GPC2 could be an extracellular modu-

lator of Wnt signaling in neuroblastoma cells. GPC3 has beenshown to interact with Wnt and suppress hepatocellular carci-noma cell proliferation (19). To determine whether GPC2 couldaffect Wnt signaling in neuroblastoma cells, active β-cateninlevels were measured. As shown in Fig. 3C, the expression ofactive β-catenin was lower in GPC2 KO IMR5 cells than invector control cells (Fig. 3C). Interestingly, we found the ex-pression of Wnt3a and Wnt11 in all of the neuroblastoma celllines expressing high levels of GPC2 (LAN1, IMR5, LAN5,IMR32, and NBEB). However, Wnt3a was undetectable andWnt11 expression was extremely low in the SKNSH cell line thathas low GPC2 expression (Fig. 3E). To determine the interactionof GPC2 and Wnt, we conducted a coimmunoprecipitation assayusing Wnt3a-conditioned media (CM) and demonstrated thatGPC2 could interact with Wnt3a (Fig. 3F). Furthermore, weused the luciferase-expressing HEK-293 SuperTopFlash cellmodel to analyze the function of GPC2. As shown in SI Ap-pendix, Fig. S6, GPC2 was expressed in HEK-293 cells. Treatingcells with the LH7 single-domain antibody decreased the Wnt3a-induced active β-catenin levels in a dose-dependent manner (Fig.3G). Lithium chloride (LiCl) is a GSK3β inhibitor and an in-tracellular β-catenin signaling inducer (19). As shown in Fig. 3H,a combination of Wnt3a and LiCl showed synergistic elevation ofβ-catenin expression. Interestingly, the elevated β-catenin ex-pression can be reduced by LH7 treatment, supporting the ideathat the Wnt/β-catenin pathway can be directly modulated by the

Fig. 2. GPC2 expression in human neuroblastoma tumors and normal hu-man tissues. (A) GPC2 protein levels in human neuroblastoma cell lines, in-cluding SKNSH, LAN1, IMR5, LAN5, IMR32, and NBEB as determined byWestern blotting. (B) Expression of GPC2 in neuroblastoma tumors (i–iv) andnormal nerve (v and vi) tissues as determined by immunohistochemistry.(C) Expression of GPC2 in human normal tissues, including nerve, brain, heart,lung, liver, stomach, small intestine, colon, pancreas, spleen, kidney, andthyroid as determined by immunohistochemistry. The tissues were labeledwith 1 μg/mL LH7–mFc antibody. The final magnification of all images was100×. (D) Kaplan–Meier analysis of overall survival in patients with neuro-blastoma with high GPC2 mRNA expression (n = 18) and low GPC2 mRNAexpression (n = 458) from the Kocak dataset in the R2 Genomics Analysisand Visualization Platform. (E) Kaplan–Meier analysis of event-free survivalin patients with neuroblastoma with high GPC2 mRNA expression (n = 20)and low GPC2 mRNA expression (n = 456) from the Kocak dataset.

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addition of the LH7 antibody. In addition to LH7, two other anti-GPC2 single-domain antibodies showed dose-dependent reductionof Wnt signaling, but to a lesser degree (Fig. 3I). Notably, LH7 at100 μg/mL resulted in a 90% reduction of β-catenin signalingcompared with control human IgG. The data indicate that theLH7 single-domain antibody has the most inhibitory effect onWnt signaling.MYCN amplification occurs in ≈25–33% of neuroblastoma cases

and results in N-Myc protein overexpression (1). Patients withMYCN-amplified tumors usually have a very poor prognosis. Inaddition, studies have shown that Wnt/β-catenin signaling actsupstream of N-Myc to regulate lung and limb development (34,35). As shown in Fig. 3J, N-Myc protein was found in GPC2 high-expressing neuroblastoma cells (LAN1, IMR5, LAN5, and IMR32)but not in GPC2 low-expressing SKNSH cells. Furthermore, wefound that silencing of GPC2 suppressed N-Myc expression (Fig.

3K). Taken together, our data show that GPC2 is involved in Wntsignaling, and that targeting GPC2 by single-domain antibodiessuch as LH7 can suppress neuroblastoma cell growth by inhibitingWnt signaling and down-regulating Wnt target genes includingN-Myc, an oncogenic driver of neuroblastoma pathogenesis. Fig. 3Lshows a working model based on our observations.

GPC2-Specific Immunotoxins Inhibit Neuroblastoma Growth. To de-termine whether GPC2 could be used as a target of immuno-toxins for the treatment of neuroblastoma, we constructed threeimmunotoxins using the LH1, LH4, and LH7 binding domains.All immunotoxins were expressed in Escherichia coli, refolded invitro, and isolated with over 90% purity (Fig. 4A). The bindingaffinities of all three immunotoxins on purified GPC2 proteinwere measured by ELISA. As shown in SI Appendix, Fig. S7, thecalculated EC50 values for the three immunotoxins were in the

Fig. 3. Genetic silencing of GPC2 inhibits neuroblastoma tumor cell growth and induces apoptosis by suppressing Wnt/β-catenin signaling. (A) GPC2 proteinexpression in LAN1 and IMR5 neuroblastoma cells after siRNA-mediated knockdown of GPC2. (B) Inhibition of tumor cell growth by GPC2 siRNAs in bothLAN1 and IMR5 cell lines. (C) GPC2 expression in IMR5 neuroblastoma cells after GPC2 KO using the CRISPR-Cas9 technique. GPC2 KO decreased activeβ-catenin protein levels at 72 h posttransfection. (D) Caspase 3/7 activity in IMR5 cells after treatment with GPC2 targeted sgRNA. (E) Protein expression ofWnt3a and Wnt11 in neuroblastoma cell lines. (F) Interaction between GPC2 and Wnt3a as determined by immunoprecipitation. (G) Reduction of activeβ-catenin levels by LH7 treatment after 6 h in HEK-293 SuperTopFlash cells that were stimulated with Wnt3a CM. (H) LH7 suppressed the expression ofβ-catenin in HEK-293 SuperTopFlash cells that were stimulated with LiCl and/or Wnt3a CM. Whole cell lysates were collected after 6 h of treatment. (I) Theanti-GPC2 antibodies decreased TopFlash activity in Wnt3a-activated HEK-293 SuperTopFlash cells after 6 h of treatment. (J) N-Myc protein level in neuro-blastoma cell lines as determined by Western blotting. (K) Inhibition of N-Myc expression by silencing GPC2 in neuroblastoma cells. (L) The proposedmechanism mediated by anti-GPC2 antibodies to inhibit neuroblastoma cell growth. Blockade of GPC2 suppresses the expression of β-catenin and its targetedgenes, including N-Myc. All data are represented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

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range of 4.6 nM to 43.9 nM. We found that the EC50 value(18 nM) for the LH7–PE38 monomeric immunotoxin in ELISAwas similar to the Kd value (9.8 nM) of the LH7–Fc fusionprotein (Fig. 1F), indicating the immunotoxin retained bindingproperties of the original single-domain antibody. To determinethe cytotoxicity of all immunotoxins in vitro, we examined theinhibition of cell proliferation on a panel of cell lines using theWST cell proliferation assay. All three immunotoxins potentlyand selectively inhibited the growth of GPC2+ cell linesLAN1 and IMR5 with similar IC50 values of 0.5–1.2 nM.LAN1 and IMR5 cells were poorly sensitive to an irrelevantimmunotoxin targeting mesothelin (IC50 for LAN1, 8 nM; IC50

for IMR5, 34 nM). None of the immunotoxins affected thegrowth of low GPC2-expressing SKNSH cells (Fig. 4 B–D).To evaluate the antitumor activity of LH7–PE38 in vivo, we s.c.

inoculated nude mice with LAN1 cells. When tumor reached

an average volume of 150 mm3, mice were treated with LH7–PE38 every other day for a total of 10 injections. The differentdose concentrations were used to determine the relative toxicity ofthe LH7–PE38 immunotoxin. As shown in Fig. 4E, the mice tol-erated 0.4 mg/kg LH7–PE38 well. However, mice treated with0.8 mg/kg died after 5 injections. Only two mice survived after10 injections at the 0.6 mg/kg dose. Notably, 0.4 mg/kg of LH7–PE38 inhibited tumor growth during treatment without affectingbody weight (Fig. 4 F and G). At the end of treatment, tumorvolumes in the LH7–PE38-treated group were significantly smallerthan those in the control group (Fig. 4H). In addition to measuringbody weight, we also performed toxicology studies to furtherevaluate any side effects of LH7–PE38 at 0.4 mg/kg. The LH7–PE38-treated mice had an increase in white blood cells, indicatingthat the immunotoxin could cause inflammatory effects in vivo(SI Appendix, Table S1). In addition, the LH7–PE38-treated groupshowed an increase in alanine aminotransferase; however, we didnot find any gross evidence of liver damage following mousenecropsy. All organ weights of the treated mice were statisticallysimilar to those of the control group, except for the spleen. Nosignificant differences were detected in any other parametersmeasured. In conclusion, we found that immunotoxins based onour anti-GPC2 human single domains inhibited neuroblastomacell proliferation both in vitro and in vivo.

GPC2 CAR T Cells Kill Neuroblastoma Cells. To explore other thera-peutic approaches, we constructed CARs containing anti-GPC2 antibody single domains linked to a CD8α hinge andtransmembrane region, followed by the 4-1BB costimulatory sig-naling moiety and the cytoplasmic component of CD3ζ signalingmolecule. The upstream GFP reporter was coexpressed with CARusing the “self-cleaving” T2A peptide (Fig. 5A). The geneticallymodified T cells began to expand after activation (Fig. 5B). Onday 9, the expression of GPC2 CARs in the transduced T cells wasdemonstrated through GFP expression. The transduction effi-ciencies of CARs were between 21% and 47% (Fig. 5C). To de-termine whether T cells targeting GPC2 could specificallyrecognize and kill GPC2+ neuroblastoma cells, a luminescent-based cytolytic assay was established using the neuroblastomacells engineered to express luciferase. As shown in Fig. 5D,IMR5 cells, which express high levels of GPC2, are resistant tomock-transduced T-cell–mediated killing. This resistance was trueeven at effector:target ratios as high as 8:1. Conversely, IMR5 cellswere efficiently lysed by the GPC2 CAR T cells in a dose-dependent manner. In addition, anti-GPC2 CAR T cells demon-strated equivalent lytic capacity against LAN1 neuroblastoma cells(SI Appendix, Fig. S8). As expected, the mock-transduced T cellsand GPC2 CAR T cells showed similarly low cytolytic activityagainst the low GPC2-expressing SKNSH cells (Fig. 5E). A cyto-kine production assay revealed that GPC2 CAR T cells producedsignificantly more IFN-γ and TNF-α after exposure to IMR5 cellsthan the mock T cells (Fig. 5 F and G). However, little to no in-duction of IFN-γ and TNF-α secretions were observed when mockor GPC2 CAR T cells were cocultured with SKNSH cells.We tested the killing ability of CAR T cells generated from eight

individual human donors. At an effector:target ratio of 8:1, GPC2-specific CAR T cells lytic activity against IMR5 neuroblastoma cellsranged from 44% to 71%, with an average of 56% (Fig. 6A).Minimal cell lysis was observed in IMR5 cells treated with mockT cells (Fig. 6B). Next, we assessed the antitumor activity of GPC2-targeting CAR T cells in nude mice i.v. engrafted with luciferase-expressing IMR5 cells. Although LH3 CAR T cells were the mostpotent in the cell killing assay (Fig. 5D), the LH3 phage binder wasalso cross-reactive with other glypican members (e.g., GPC3) (Fig.1E). Therefore, we chose LH7 for preclinical testing in neuroblas-toma models. Bioluminescence imaging using IVIS showed thatIMR5-bearing nude mice developed disseminated tumor lesionssurrounding spine and bones (Fig. 6C). Notably, LH7 CAR T cells

Fig. 4. Recombinant immunotoxins against GPC2 inhibit neuroblastomatumor growth in vitro and in vivo. (A) Purity of LH1–PE38 (molecular weightof 53 kDa), LH4–PE38 (molecular weight of 52 kDa), and LH7–PE38 (molec-ular weight of 52 kDa) as determined by SDS/PAGE. (B–D) Effectiveness ofanti-GPC2 immunotoxins on the growth of IMR5 (B), LAN1 (C), and SKNSH(D) cell lines, as measured by the WST-8 assay. An antimesothelin immu-notoxin was used as an irrelevant control immunotoxin. (E) Toxicity de-tection of LH7–PE38 in vivo. Athymic nu/nu nude mice were treated withindicated doses of immunotoxin i.v. every other day for a total of 10 injec-tions. Each arrow indicates an individual injection (n = 5 per group).(F) Antitumor activity of LH7–PE38. Athymic nu/nu nude mice were s.c. in-oculated with 1 × 107 LAN1 cells mixed with Matrigel. When tumors reachedan average volume of 150 mm3, mice were treated with a 0.4-mg/kg dose ofLH7–PE38 i.v. every other day for 10 injections. Each arrow indicates an in-dividual injection; n = 5 per group. *P < 0.05. (G) Body weight of the micetreated in F. (H) Representative pictures of tumors from control and treatedmice at the end of study. Values represent mean ± SEM. IT, immunotoxin.

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effectively suppressed metastatic tumors after 14 d of T-cell in-fusion, whereas mock T cells failed to reduce tumor burden (Fig. 6C and D). Four of eight (50%) of the mice treated with CART cells targeting GPC2 were tumor-free at the end of this study.Neither mock T cell nor LH7 CAR T cell treatment affected micebody weight (SI Appendix, Fig. S9). The efficacy of GPC2-targetingCAR T cells was also evaluated in a LAN1 xenograft mouse model.LH7 CAR T cells initially led to a reduction in tumor size andsignificantly suppressed tumor growth compared with the controlgroup at the end of the study (SI Appendix, Fig. S10). Taken to-gether, we have demonstrated that our single-domain antibodiescan be used to construct CAR T cells that are able to kill GPC2-expressing neuroblastoma cells in cell and mouse models.

DiscussionIn the present study, we reported that GPC2 protein expressionlevels were elevated in human neuroblastoma tumors compared withnormal human tissues. Additionally, silencing of GPC2 decreasedneuroblastoma cell viability and induced apoptosis. We also foundthat GPC2 modulated Wnt/β-catenin signaling and the expression ofthe key oncogenic driver gene N-Myc in neuroblastoma. By usingphage display, we succeeded in identifying a group of human single-domain antibodies specific for GPC2. The immunotoxins and CART cells based on these antibody binding domains significantlyinhibited neuroblastoma tumor cell growth. Our observations dem-

onstrate an important role for GPC2 as a target candidate ofantibody-based therapies for the treatment of neuroblastoma.One current approach to treating high-risk patients with neuro-

blastoma is immunotherapy targeting a tumor-associated antigen,such as, the disialoganglioside GD2. Anti-GD2 antibodies havebeen tested in clinical trials for neuroblastoma, with proven safetyand efficacy (3, 36). The FDA approved Unituxin, in combinationwith granulocyte-macrophage colony-stimulating factor (GM-CSF),interleukin-2 (IL-2), and 13-cis-retinoic acid (RA), for the treatmentof patients with high-risk neuroblastoma in 2015. However, in pa-tients with advanced disease, anti-GD2 antibodies show only limitedactivity. GD2 therapy is also associated with severe pain toxicity (37,38). These challenges emphasize the urgent need to discover ther-apeutic targets for neuroblastoma therapy.We found significant elevation of GPC2 protein levels in about

50% of neuroblastoma tumors obtained from clinical specimens

Fig. 5. The CAR T cells targeting GPC2 kill neuroblastoma cells. (A) Sche-matic diagram of bicistronic lentiviral constructs expressing CARs targetingGPC2 along with GFP using the T2A ribosomal skipping sequence.(B) Timeline of CAR T-cell production. (C) GPC2-specific CAR expression onhuman T cells transduced with lentiviral particles was analyzed using flowcytometry by detection of GFP fluorescence. (D–E) Cytolytic activities ofGPC2 targeting CAR T cells in cell assays. The luciferase-expressing IMR5 (D)and SKNSH (E) neuroblastoma cells were cocultured with mock or GPC2 CAR-transduced T cells at the indicated effector (E):target (T) ratios for 20 h, andspecific lysis was measured using a luminescent-based cytolytic assay. (F–G)The above culture supernatants at an E:T ratio of eight were harvested tomeasure IFN-γ (F) and TNF-α (G) secretions via ELISA. All data are representedas mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

Fig. 6. GPC2-specific CAR T cells demonstrate potent activity in mice bear-ing human neuroblastomas. (A–B) Cytotoxic activity of LH7 CAR T cells de-rived from multiple donors. PMBCs were isolated from eight healthy donors.The luciferase-expressing IMR5 cells were cocultured with LH7 CAR-transduced T cells (A) or mock T cells (B) at the indicated effector (E):target(T) ratios for 20 h, and specific lysis was measured using a luminescent-basedcytolytic assay. (C) LH7 CART demonstrated potent antitumor activity by sup-pressing metastatic IMR5 neuroblastoma cells as measured by bioluminescentimaging. Animals (n = 8 per group) were i.v. injected with a single infusion of30 × 106 mock T cells or LH7 CAR T cells. (D) Quantitation of bioluminescence inmice treated in C. Values represent mean ± SEM.

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(13 of 25). Immunohistochemistry confirmed that GPC2 proteinexpression was not detected in normal peripheral nerve tissues. Ourstudies also demonstrated that GPC2 protein was either present atlow levels or not expressed in most normal tissues in our compre-hensive array. This finding was especially true of the essential organtissues included in our array. In addition, using the data from a studycohort in the R2 Genomics Analysis and Visualization Platform, wefound that both overall survival and event-free survival of the patientswith neuroblastoma with high GPC2 expression were significantlyshorter than those with low GPC2 expression. Overall, we show thatGPC2 protein is uniquely overexpressed in neuroblastoma and thatits high expression may be correlated with poor survival. We alsofound an inhibitory effect of GPC2 in neuroblastoma cell growthusing siRNA and CRISPR-Cas9 approaches. Our results suggest thepossibility that the suppression of GPC2 expression in neuroblastomacells may induce apoptosis. Taken together, our data show thatGPC2 is a promising target candidate for neuroblastoma therapy.Given the importance of Wnt/β-catenin signaling in neuroblas-

toma (10–12), we decided to determine whether GPC2 inhibitioncould suppress this signaling pathway in neuroblastoma cells.GPC2 inhibition by our anti-GPC2 antibodies or KO by sgRNA wasfound to reduce the expression of active β-catenin and suppresstarget genes that may regulate neuroblastoma cell proliferation andsurvival. We notice the discrepancy between the IC50 (0.3–1.3 μM)of Wnt signaling inhibition and EC50 of binding activity (5–50 nM)of single domains (SI Appendix, Fig. S7). For example, the IC50 forthe best single domain (LH7) is 0.3 μM (25 μg/mL), 17-fold morethan its EC50 (18 nM), likely because in the luciferase reporterassay, a saturation level of the antibody is needed to functionallyblock the interaction of GPC2 and Wnt and/or other associatedproteins (e.g., Frizzled). We previously found that GPC3 could in-teract with Wnt3a to suppress hepatocellular carcinoma cell pro-liferation (19). Wnt11 is secreted in regions adjacent to the neuralcrest and could induce neural crest migration (39). It has beenshown that Wnt11 mRNA was highly expressed in neuroblastomaclinical samples (40). Thus, we particularly determined the expres-sion of Wnt3a and Wnt11 in neuroblastoma cell lines. Interestingly,Wnt3a and Wnt11 were expressed in GPC2 high-expressing neu-roblastoma cells (e.g., LAN1, IMR5, LAN5, and IMR32). Bycontrast, Wnt3a and Wnt11 proteins were either not detected orpoorly detected in GPC2 low-expressing SKNSH cells. The differ-ences in Wnt protein expression are in agreement with the sensi-tivity of GPC2-targeted immunotoxins and CAR T cells in LAN1/IMR5 and SKNSH cell lines. Furthermore, we demonstrated thatGPC2 could coimmunoprecipiate with Wnt3a. These observationsare consistent with previous reports showing that activation of Wnt/β-catenin signaling contributes to the aggressiveness of neuroblas-toma (41, 42) and indicate the role of GPC2 in modulation of Wntsignaling in neuroblastoma cells. It is possible that GPC2 can in-teract with other Wnt molecules beyond Wnt3a. Future studies arenecessary to understand the interactions of various Wnt moleculesand GPC2 in the tumor microenvironment to better understand therole of GPC2 in modulating Wnt signaling in cancer cells. N-Myc isa key driver for neuroblastoma tumorigenesis (43–45). Here wedemonstrated that N-Myc was expressed in MYCN-amplified neu-roblastoma cells, including LAN1, IMR5, LAN5, and IMR32 (Fig.3J). However, N-Myc protein was not found inMYCN-nonamplifiedSKNSH neuroblastoma cells. Interestingly, in the present study wefound genetic silencing of GPC2 significantly inhibited the expres-sion of N-Myc in neuroblastoma cells. It has been shown that Wntsignaling can regulate N-Myc expression level and β-catenin mayactivate the promoter of N-Myc during development (34, 35). Ourresult indicates that GPC2 can down-regulate N-Myc expression byinhibiting Wnt/β-catenin signaling. Protein surfaces contain cleftsthat are relatively inaccessible to conventional antibodies as a resultof steric hindrance. Interestingly, single-domain antibodies have theability to bind in protein clefts or hidden substrate pockets not ac-cessible to conventional antibodies (46, 47). We previously identi-

fied a human single-domain antibody that recognizes a crypticfunctional site on GPC3 and inhibits Wnt signaling in liver cancer(48). In the present study, we isolated a group of seven represen-tative binders specific for GPC2, and all of these single-domainantibodies significantly inhibited Wnt/β-catenin signaling in neuro-blastoma cells. Together, these studies indicate that single-domainantibodies are an emerging class of promising therapeutic candi-dates that can inhibit the signaling related to the growth of cancercells by blocking receptor–ligand interactions.The immunotoxins based on our anti-GPC2 antibodies demon-

strated highly specific and potent killing of neuroblastoma in both invitro and in vivo mouse models. In the present study, the irrelevantantimesothelin immunotoxin shows modest background cytotoxicityat high concentrations. It has been shown that the immunotoxins(e.g., mPE24) containing only the enzymatic domain (domain III)has significantly less nonspecific cytotoxicity than PE38, suggestingthat domain II may potentially contribute to nonspecific cytotoxicity(49). To improve therapeutic index and reduce potential side ef-fects, future reengineering of the anti-GPC2 immunotoxin by re-moving domain II may be useful for clinical development. In mousetesting, the optimal dose appears to be 0.4 mg/kg, which is similar tothe dose of other immunotoxins that are currently being evaluatedin preclinical and clinical stages (including phase III) (50). Despitenot observing any obvious side effects in mice treated with anti-GPC2 immunotoxins, we noted an increase in alanine amino-transferase. This increase could be indicative of liver damage, butwe did not note any gross evidence of liver damage upon mousenecropsy. Further comprehensive preclinical studies are needed tovalidate the effect of anti-GPC2 immunotoxins systematically, in-cluding pharmacodynamics, pharmacokinetics, and influence onliver function, before identifying the most promising candidate foruse in humans. Because PE38 is a bacterial protein, it is highlyimmunogenic in patients with solid tumors that have normal im-mune systems (51). Future efforts using immunosuppressive drugsor less immunogenic toxins should be pursued for clinical devel-opment of anti-GPC2 immunotoxins.CAR T cells have been shown to be a promising T-cell–based

immunotherapy in leukemia (25, 26, 29, 52, 53). CAR T cellstargeting CD19 have resulted in sustained complete responses andshown complete response rates of ≈90% in patients with relapsedor refractory acute lymphoblastic leukemia (54). However, CART-cell therapies have not been successful in treating solid tumors.In the present study, we sought to evaluate the use of CAR T cellsin treating neuroblastoma. We established an in vivo biolumi-nescent model of disseminated neuroblastoma in mice. Mostneuroblastomas begin in the abdomen in the adrenal gland or nextto the spinal cord, or in the chest. Neuroblastomas can spread tothe bones, such as in the face, skull, pelvis, and legs. They can alsospread to bone marrow, liver, lymph nodes, skin, and orbits. In ourstudy, we frequently found disseminated tumors near the spineand in the bones of face, skull, legs, and pelvis, indicating theclinical relevance of our animal model. We then demonstratedthat a single infusion of LH7 CAR T cells significantly suppressedthe growth of metastatic neuroblastoma cells in mice. Whereas theimmunotoxins can effectively inhibit neuroblastoma growth, CART cells targeting GPC2 can cause complete remission in 50% oftreated mice. The persistence of CAR T cells is a major challengein solid tumors. Preclinical and clinical studies investigatingcombinations of CAR T cells with immune checkpoint blockadetherapies would be pursued and examined (54). Future compre-hensive preclinical studies will be needed to validate GPC2-targeting CAR T-cell–based immunotherapy for neuroblastoma.In conclusion, we found that the GPC2 protein is highly

expressed in neuroblastoma and produced single-domain anti-bodies against GPC2. These antibodies can potentially be usedeither as immunotoxins or CARs in neuroblastoma treatment.This report establishes GPC2 as a potential therapeutic targetin cancer. The single-domain antibodies described here are

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promising candidates that should be further evaluated as cancertherapeutics for the treatment of neuroblastoma and otherGPC2+ cancers.

Materials and MethodsCell Culture. Human neuroblastoma cell lines, including SKNSH, LAN1, IMR5,IMR32, NBEB, and LAN5, were obtained from collaborators at the NationalCancer Institute (NCI). IMR5, LAN1, and SKNSH cell lines were also transducedwith lentiviruses expressing firefly luciferase, which were obtained from NCI-Frederick (55). Peripheral blood mononuclear cells (PBMCs) were isolatedfrom the blood of eight healthy donors using Ficoll (GE Healthcare)according to the manufacturer’s instructions. The aforementioned cell lineswere cultured in RPMI-1640 medium supplemented with 10% FBS, 1%L-glutamine, and 1% penicillin–streptomycin at 37 °C in a humidified at-mosphere with 5% CO2. The HEK-293T cell line was obtained from theAmerican Type Culture Collection. The HEK-293 SuperTopFlash stable cellline was a kind gift from Jeremy Nathans, Johns Hopkins University, Baltimore.These two cell lines were grown in DMEM supplemented with 10% FBS, 1%L-glutamine, and 1% penicillin–streptomycin at 37 °C in a humidified atmo-sphere with 5% CO2. All cell lines were authenticated by morphology andgrowth rate and were mycoplasma-free.

Phage Display and Biopanning. A combinational engineered human VH single-domain phage display library, with an estimated size of 2.5 × 1010, was usedfor the screening and has been previously described (56). The phage librarywas subjected to four rounds of panning on Nunc 96-well Maxisorp plate(Thermo Scientific) as described previously (57–59). Details are provided inthe SI Appendix, SI Materials and Methods.

Antibody Binding Assay. The binding kinetics of the LH7 antibody to GPC2 wasdetermined using the Octet RED96 system (FortéBio) as described previously(54). Briefly, all experiments were performed at 30 °C and reagents wereprepared in 0.1% BSA, 0.1% Tween20 PBS, pH 7.4 buffer. Biotinylated GPC2–hFc protein was immobilized onto streptavidin biosensors, which weresubsequently used in association and dissociation measurements for a timewindow of 600 s and 1,800 s, respectively. Data analysis was performed usingthe FortéBio analysis software provided with the instrument.

Immunohistochemistry. The human neuroblastoma tissue array and normaltissue array were purchased from US Biomax. The tissue arrays were sent toHistoserv Inc. for staining. Details are provided in the SI Appendix, SI Ma-terials and Methods.

Patient Samples Analysis. The R2: Genomics Analysis and Visualization Plat-form (r2.amc.nl) was used to analyze the correlation between GPC2 mRNAexpression and survival of patients with neuroblastoma. We chose thelargest tumor neuroblastoma Kocak dataset (n = 649) to perform analysis ofoverall survival and event-free survival by the Kaplan–Meier method.

Immunoblotting and Immunoprecipitation. Cells were harvested, vortexed inice-cold lysis buffer (Cell Signaling Technology), and clarified by centrifu-gation at 10,000 × g for 10 min at 4 °C. Protein concentration was measuredusing a Coomassie blue assay (Pierce) following the manufacturer’s specifi-cations. A total of 20 μg of cell lysates was loaded onto a 4–20% SDS/PAGEgel for electrophoresis. The anti-GPC2 antibody for Western blotting waspurchased from Santa Cruz Biotechnology. The anti-active β-catenin andN-Myc antibodies were obtained from Millipore. The anti-Wnt3a andWnt11 antibodies were purchased from Abcam. All other antibodies, in-cluding cleaved PARP, total β-catenin, β-actin, and GAPDH were obtainedfrom Cell Signaling Technology.

Fifty micrograms of GPC2–hFc protein was incubated withWnt3a CM for 4 hon ice. Protein A-Agarose beads (Roche) were added to the immune complexand incubate overnight at 4 °C. Immune complexes were washed three timeswith lysis buffer and subjected to immunoblotting with anti-Wnt3a antibody.

Production of Recombinant Immunotoxin. Anti-GPC2 single-domain anti-bodies were cloned into pRB98 expression plasmid containing PE38. Theexpression and purification of recombinant immunotoxins were performedfollowing our protocol as described previously (60).

Generation of GPC2-Specific CAR. The anti-GPC2 CAR included the isolatedanti-GPC2 single-domain antibody fragment, linked in-frame to the CD8αhinge and transmembrane regions, a 4-1BB costimulatory domain, and in-

tracellular CD3ζ. The construct was engineered to express an upstream GFPreporter separated from the CAR by a T2A sequence. The sequence encodingthe whole CAR construct was subcloned into the CMV promoter-containinglentiviral vector pLenti6.3/v5 (Invitrogen).

Lentivirus Production, T-Cell Transduction, and Expansion. To produce viral su-pernatant, HEK-293T cells were cotransfected with GPC2–CAR lentiviral vectorsand Mission viral packaging plasmids (Sigma-Aldrich) using Lipofectamine2000 (Invitrogen) per the manufacturer’s protocol. The supernatant was col-lected at 72 h posttransfection and then concentrated using Lenti-X concen-trator (Clontech) according to the manufacturer’s instructions.

PBMCs were purchased from Oklahoma Blood Institute and stimulated for24 h with anti-CD3/anti-CD28 antibody-coated beads (Invitrogen) at a2:1 bead-to-T cell ratio in growth medium supplemented with IL-2. ActivatedT cells were then transducedwith the lentivirus expressing GPC2-specific CARsat a multiplicity of infection of five. Cells were counted every other day andfed with fresh growth medium every 2–3 d. Once T cells appeared to becomequiescent, as determined by both decreased growth kinetics and cell size,they were used either for functional assays or cryopreserved.

T-Cell Effector Assays. Effector T cells were cocultured with luciferase ex-pressing neuroblastoma cells at different ratios for 20 h. At the end of thecoculture incubation period, supernatant was saved for IFN-γ and TNF-α levelsby ELISA (R&D Systems). The remaining tumor cells were lysed for 5 min. Theluciferase activity in the lysates was measured using the Steady Glo luciferaseassay system on Victor (PerkinElmer). Results were analyzed as percent kill-ing based on luciferase activity in wells with tumor cells alone: % killing =100 − [relative light units (RLU) from wells with effector and target cells]/(RLU from wells with target cells) × 100.

Animal Studies. All mice were housed and treated under the protocol ap-proved by the Institutional Animal Care and Use Committee at the NIH. Forxenograft tumor studies, 5-wk-old female athymic nu/nu nude mice (NCI-Frederick) were given s.c. injections of 10 × 106 LAN1 cells suspended inMatrigel (Corning). Tumor dimensions were determined using calipers, andtumor volume (in cubic millimeters) was calculated by the formula V =1/2 ab2, where a and b represent tumor length and width, respectively. ForLH7–PE38 treatment, when average tumor size reached around 150 mm3,mice were i.v. injected with indicated doses every other day for 10 injections.For T-cell treatment, when tumor burden was ≈120 mm3, mice were injectedi.p. with 200 mg/kg cyclophosphamide to deplete host lymphocyte com-partments. After 24 h, 10 × 106 of either mock T cells or LH7 CAR T cells werei.v. injected into mice on days 13, 20, and 27. Mice were given i.p. injection of2,000 units of IL-2 twice a week following T-cell infusion. Mice were killedwhen the tumor size reached 1,500 mm3.

For the disseminated tumor study, 5-wk-old female athymic nu/nu nudemice were i.v. injected with 7 × 106 luciferase-expressing IMR5 cells. Cyclo-phosphamide was injected i.p. at 200 mg/kg 24 h before any cell adminis-tration. Then animals were given single infusion of 30 × 106 mock T cells orLH7 CAR T cells by tail vein injection. Disease was detected using theXenogen IVIS Lumina (PerkinElmer). Nude mice were injected i.p. with 3 mgD-luciferin (PerkinElmer) and imaged 10 min later. Living Image softwarewas used to analyze the bioluminescence signal flux for each mouse asphotons per second per square centimeter per steradian (photons/s/cm2/sr).Mice were killed when they showed any sign of sickness or when bio-luminescence signal reached 1 × 109.

Statistics. All experiments were repeated a minimum of three times to de-termine the reproducibility of the results. All error bars represent SEM.Statistical analysis of differences between samples was performed using theStudent’s t test. A P value of <0.05 was considered statistically significant.

ACKNOWLEDGMENTS. We thank Dr. Javed Khan (NCI), Dr. Crystal Mackall(NCI; currently Stanford University), and Dr. Rimas J. Orentas (NCI; currentlyLentigen Technology, Inc.) for providing the neuroblastoma cell lines used inthe present study and for helpful discussions; Dr. Terry J. Fry (NCI) for pro-viding a laboratory protocol of CAR T-cell activation; Dr. Carol J. Thiele (NCI)for critical reading of the manuscript; Dr. Diana Haines and colleagues in thePathology/Histotechnology Laboratory (NCI) for pathological evaluation ofthe mice treated in the present study; and Dr. Bryan D. Fleming (NCI) and theNCI Editorial Board for editorial assistance. This research was supportedby the Intramural Research Program of NIH, NCI (Z01 BC010891 and ZIABC010891) (to M.H.). N.L. was a recipient of the NCI Directorʼs IntramuralInnovation Award/Career Development Award. H.F. was a recipient of afellowship from the China Scholarship Council.

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Li et al. PNAS | Published online July 24, 2017 | E6631

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