Therapeutically targeting glypican-2 via single-domain PNAS PLUS antibody-based chimeric antigen receptors and immunotoxins in neuroblastoma

Nan 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 of Immunology, 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, National Cancer 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 of in the nervous system (7). It is known to participate in cellular patients despite intense multimodality treatment. This cancer is adhesion and is thought to regulate the growth and guidance of derived from neuroendocrine tissue located in the sympathetic axons (8). The role of GPC2 in the pathogenesis of neuroblastoma nervous system. Glypican-2 (GPC2) is a cell surface or any other cancers has not been reported. that is important for neuronal cell adhesion and The Wnt/β-catenin signaling pathway is highly conserved and neurite outgrowth. In this study, we find that GPC2 protein is highly plays an essential role in various processes of embryonic devel- expressed in about half of neuroblastoma cases and that high opment (9). Additionally, it has been linked to pathogenesis of GPC2 expression correlates with poor overall survival compared numerous adult and pediatric tumors (10). Wnt/β-catenin signal- with patients with low GPC2 expression. We demonstrate that ing has been shown to mediate neural crest cell fate and neural silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of stem cell expansion. This signaling pathway may be of particular neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/ relevance to neuroblastoma cells because they arise from migra- β -catenin signaling and reduces the expression of the target gene tory neural crest-derived neuroblasts (11–13). Glypicans play an N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We important role in developmental morphogenesis and are known to have isolated human single-domain antibodies specific for GPC2 by be modulators for the (14–17). It has been phage display technology and found that the single-domain anti- shown that GPC3, another member of the glypican family, may bodies can inhibit active β-catenin signaling by disrupting the inter- function as a coreceptor for Wnt and facilitate the Wnt/Frizzled action of GPC2 and Wnt3a. To explore GPC2 as a potential target in binding in hepatocellular carcinoma cells (18, 19). neuroblastoma, we have developed two forms of antibody thera- Antibody-based therapeutics are of growing significance in the peutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma field of cancer therapy. Antibodies can be used as vehicles to growth in mice. CAR T cells targeting GPC2 eliminated tumors in a deliver a variety of effector molecules such as cytotoxic drugs or disseminated neuroblastoma mouse model where tumor metastasis toxins to tumor cells. Recombinant immunotoxins are chimeric had spread to multiple clinically relevant sites, including spine, skull, proteins composed of an antibody fragment fused to a toxin, such Pseudomonas legs, and pelvis. This study suggests GPC2 as a promising therapeutic as the 38-kDa truncated fragment of exotoxin target in neuroblastoma. (PE38). By combining the specificity of an antibody with the protein synthesis inhibitory domain from the exotoxin, it is pos- glypican | neuroblastoma | single-domain antibody | sible to directly target cancer cells (20–24). Another antibody- chimeric antigen receptor T-cell therapy | immunotoxin based therapy that is emerging with clinical applications involves MEDICAL SCIENCES euroblastoma is the most common extracranial solid tumor Significance Nof children and the most frequently diagnosed neoplasm during infancy (1). It accounts for 8–10% of all childhood can- Neuroblastoma is a childhood cancer that remains an important cers and 15% of all pediatric cancer mortality (2). This cancer is clinical challenge. It is fatal in almost half of the patients despite derived from neuroendocrine tissue located in the sympathetic advances in multimodal treatments. In this report, we show that nervous system. Neuroblastoma is a complex and heterogeneous the cell surface glypican-2 (GPC2) is overexpressed in disease, with nearly 50% of patients having a high-risk pheno- neuroblastomas when compared with normal tissues and that a type. Despite the use of intensive multimodal treatments, these high expression level is correlated with poor survival of neuro- high-risk phenotype cancers often present with widespread dis- blastoma. We also describe that GPC2 has proliferative effects in semination and poor long-term survival (3). Approximately 45% neuroblastoma via activating Wnt signaling and its downstream of patients receiving standard therapy relapse and ultimately target genes including N-Myc, a major driver for neuroblastoma succumb from metastatic disease (4). As such, there is an urgent tumorigenesis. We have produced the immunotoxins and chi- need for an effective neuroblastoma treatment. meric antigen receptor T cells that target GPC2 and exhibit One of the major challenges for the treatment of neuroblas- promising antitumor activities in cell and mouse models. This toma and other pediatric cancers is the lack of effective thera- study suggests GPC2 as a promising target in neuroblastoma. peutic targets. Interestingly, glypican-2 (GPC2) was recently found as one of several mRNA transcripts that were highly expressed in 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. multiple pediatric cancers, including neuroblastoma (5). Whereas and M.H. wrote the paper. these candidate markers have shown promise at the mRNA level, The authors declare no conflict of interest. they have yet to be validated at the protein level. GPC2 belongs to This article is a PNAS Direct Submission. a six member human glypican family of proteins (6). All of these 1To whom correspondence should be addressed. Email: [email protected]. proteins are attached to the cell surface by a GPI anchor (6). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. GPC2 is unique among the glypican family because it is expressed 1073/pnas.1706055114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1706055114 PNAS | Published online July 24, 2017 | E6623–E6631 Downloaded by guest on September 25, 2021 chimeric antigen receptor (CAR)-expressing T cells. CARs are composed of an antibody fragment fused to a transmembrane domain linked to a T-cell signaling moiety. T-cell expressing CARs (CAR T cells) have the ability to bind antigen directly, whereas normal T cells require antigen presented in MHC molecules. In recent years, CAR T-cell immunotherapy has emerged as one of the most promising approaches to treat leu- kemia (25–29). CAR T-cell immunotherapy has not been as successful in the treatment of solid tumors, in part due to the lack of tumor-specific targets. To improve engineered T-cell therapies in solid tumors, we will need to identify tumor antigens that can be safely and effectively targeted to discriminate cancers from normal tissues. In the present study, we found that GPC2 protein was specifically overexpressed in neuroblastoma compared with its expression in normal peripheral nerve tissues and its high expression correlated with a poor prognosis for patients with neuroblastoma. We also found 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 Wnt target gene and an oncogenic driver for neuroblastoma pathogen- esis. Furthermore, we identified a group of human single-domain antibodies (LH1–LH7) to GPC2 by phage display technology. To evaluate their potential use for the treatment of neuroblastoma, we constructed immunotoxins using these single-domain antibodies and demonstrated that the LH7–PE38 immunotoxin inhibited growth of neuroblastoma xenograft tumors in mice. In addition, we produced CARs targeting GPC2 and expressed them in T cells isolated from multiple healthy donors. Here we found that CAR Fig. 1. Isolation of GPC2-specific human single-domain antibodies by phage T cells could potently kill neuroblastoma cells. Notably, anti-GPC2 display. (A) Phage-displayed single-domain antibody fragments were se- CAR T cells were effective in suppressing the dissemination of lected against recombinant GPC2–hFc after four rounds of panning. A neuroblastomas in our mouse xenograft model. gradual increase in phage titers was observed during each round of panning. (B) Polyclonal phage ELISA from the output phage of each round of panning. Results BSA was used as an irrelevant antigen. (C) Monoclonal phage ELISA of the seven GPC2 binders. (D) Distribution of unique sequences of GPC2 binders in Generation of Anti-GPC2 Human Single Domain Antibodies. To identify 27 selected phage clones. (E) Monoclonal phage ELISA analysis of cross- the antibodies specific for GPC2, we decided to screen a phage- reactivity of GPC2 binders to human GPC1 and GPC3 and mouse GPC2. display engineered human VH single-domain antibody library. En- (F) Octet association and dissociation kinetic analysis for the interaction richment was determined by counting the number of phages between various concentrations of the LH7 antibody and human GPC2. All recaptured after each round of panning. Four rounds of panning data are represented as mean ± SEM of three independent experiments. resulted in an ≈1,000-fold enrichment of eluted phage (Fig. 1A). Phage pools after two rounds of panning exhibited enhanced binding to GPC2, whereas no binding to BSA was found with GPC2 Is Highly Expressed in Neuroblastoma but Not in Normal Human pooled phage from any of the four rounds of panning (Fig. 1B). At Tissues. A microarray study showed that GPC2 mRNA was the end of the fourth round of panning, 27 individual clones were overexpressed in a panel of pediatric cancers, including neuro- confirmed to be GPC2 binders by monoclonal phage ELISA. blastoma (5). To examine the expression of GPC2 protein in Subsequent sequencing analysis revealed seven unique binders neuroblastoma, we used anti-GPC2 antibodies as research tools to examine established human cell lines and clinical tissue sam- (LH1–LH7). The GPC2–hFc OD450 values of all seven clones were at least fivefold higher than that of BSA (Fig. 1C), further indicating ples. Our Western blotting data demonstrated that GPC2 was the specificity of the phages to GPC2. Notably, LH1 and LH7 were highly expressed in five neuroblastoma cell lines, including the two most abundant binders and combined for 14 of the LAN1, IMR5, LAN5, IMR32, and NBEB (Fig. 2A). GPC2 was 27 identified (Fig. 1D). The LH5 clone was excluded from further only weakly expressed in the SKNSH, the sixth neuroblastoma study due to its low affinity for GPC2. To determine whether the cell line that we evaluated. To assess the clinical relevance of our identified clones would bind to other members of the human gly- observation, GPC2 protein levels were measured in human pican family, we performed monoclonal phage ELISA using specimens from patients with neuroblastoma or nonmalignant recombinant human GPC1 and GPC3. We also included mouse disease by immunohistochemistry using the LH7 antibody. As GPC2 to determine whether any of the binders had cross-species shown in Fig. 2B, GPC2 labeling was readily apparent in speci- capabilities. As shown in Fig. 1E, the five clones LH1, LH2, LH4, mens derived from patients with neuroblastoma (i–iv), but es- LH6, and LH7 specifically bound to human GPC2, but not human sentially undetectable in normal peripheral nerves from patients GPC1 or GPC3. Interestingly, LH4 and LH6 are cross-reactive with nonmalignant disease (v and vi). Neuroblastoma tumor against both human and mouse GPC2 proteins. LH3 showed the tissues showed strong GPC2 staining in 13 of the 25 cases (52%) highest binding signal to human GPC2 among all binders, but it was (SI Appendix, Fig. S1). To further analyze GPC2 expression in slightly cross-reactive with GPC1 and GPC3. To determine binding normal human tissues, we used a US Food and Drug Adminis- kinetics, we produced a LH7–Fc fusion protein and incubated it tration (FDA)-recommended human normal tissue array and C with GPC2 protein in solution on the Octet platform. The Kd value probed it with the LH7 antibody. As shown in Fig. 2 , no sig- of the LH7–Fc fusion for GPC2 was 9.8 nM (Fig. 1F). Taken to- nificant GPC2 staining was observed in the normal tissues, in- gether, we have successfully identified a group of high-affinity anti- cluding essential organs such as the brain, heart, lung, and GPC2 human single-domain antibodies by phage display. kidney. These results would suggest a tumor-specific expression

E6624 | www.pnas.org/cgi/doi/10.1073/pnas.1706055114 Li et al. Downloaded by guest on September 25, 2021 mics Analysis and Visualization Platform. Patients with high PNAS PLUS GPC2 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 specific binding to IMR5, LAN1, IMR32, and LAN5 neuroblastoma cells (SI Appendix, Fig. S4A). In addition, LH7 exhibited no binding to SKNSH cells, which is consistent with the low expression of GPC2 in this neuroblastoma cell line (Fig. 2A). Furthermore, we quantified the number of cell surface GPC2 sites per cell using flow cytometry. LAN5 and IMR32 cells expressing native GPC2 contain between 104 and 105 sites per cell, whereas LAN1 and IMR5 cells contain between 103 and 104 GPC2 sites per cell (SI Appendix, Fig. S4B). SKNSH cells showed an ex- tremely low number of cell surface GPC2 sites, with only 433 sites per cell. Taken together, we have demonstrated that GPC2 is a tumor-specific cell surface antigen in neuroblastoma.

Silencing of GPC2 Inhibits Neuroblastoma Cell Growth via Suppression of Wnt/β-Catenin Signaling. To analyze the role of GPC2 in neuroblas- toma cell growth, we used siRNA and CRISPR-Cas9 techniques to silence GPC2 in two neuroblastoma cell models (IMR5 and LAN1). Three different GPC2 siRNAs were used to avoid potential off-target effects of siRNA. GPC2 knockdown efficiency was con- firmed by Western blotting, which showed substantial reductions of GPC2 levels in both cell lines (Fig. 3A). As shown in Fig. 3B, GPC2 siRNAs suppressed the growth of neuroblastoma cells within 3 d of transfection by ≈40–50% compared with cells transfected with scrambled siRNA. To validate the oncogenic effect of GPC2 in neuroblastoma, we generated GPC2 KO neuroblastoma cells by using CRISPR-Cas9. Three single-guide RNAs (sgRNAs) targeting different GPC2 exons (exons 1–3) were transfected into IMR5 cells. Expression of GPC2 protein was almost completely abolished in the sgRNA-transfected cells (Fig. 3C). As shown in SI Appendix,Fig. S5A,a25–50% reduction of growth was observed in GPC2 KO cells Fig. 2. GPC2 expression in human neuroblastoma tumors and normal hu- compared with vector control cells after 3 d of growth. In addition, man tissues. (A) GPC2 protein levels in human neuroblastoma cell lines, in- KO of GPC2 induced apoptosis in neuroblastoma cells as measured cluding SKNSH, LAN1, IMR5, LAN5, IMR32, and NBEB as determined by by elevated expression of cleaved-Poly (ADP ribose) polymerase Western blotting. (B) Expression of GPC2 in neuroblastoma tumors (i–iv) and (PARP) (SI Appendix,Fig.S5B) and increased activity of caspase-3 normal nerve (v and vi) tissues as determined by immunohistochemistry. D (C) Expression of GPC2 in human normal tissues, including nerve, brain, heart, and -7 (Fig. 3 ). lung, liver, stomach, small intestine, colon, pancreas, spleen, kidney, and We hypothesized that GPC2 could be an extracellular modu- thyroid as determined by immunohistochemistry. The tissues were labeled lator of Wnt signaling in neuroblastoma cells. GPC3 has been with 1 μg/mL LH7–mFc antibody. The final magnification of all images was shown to interact with Wnt and suppress hepatocellular carci- MEDICAL SCIENCES 100×.(D) Kaplan–Meier analysis of overall survival in patients with neuro- noma cell proliferation (19). To determine whether GPC2 could blastoma with high GPC2 mRNA expression (n = 18) and low GPC2 mRNA affect Wnt signaling in neuroblastoma cells, active β-catenin = expression (n 458) from the Kocak dataset in the R2 Genomics Analysis levels were measured. As shown in Fig. 3C, the expression of and Visualization Platform. (E) Kaplan–Meier analysis of event-free survival β = active -catenin was lower in GPC2 KO IMR5 cells than in in patients with neuroblastoma with high GPC2 mRNA expression (n 20) C and low GPC2 mRNA expression (n = 456) from the Kocak dataset. vector control cells (Fig. 3 ). Interestingly, we found the ex- pression of Wnt3a and Wnt11 in all of the neuroblastoma cell lines expressing high levels of GPC2 (LAN1, IMR5, LAN5, of GPC2. The complete panel of all 32 types of normal tissues IMR32, and NBEB). However, Wnt3a was undetectable and stained for GPC2 expression is shown in SI Appendix, Fig. S2.In Wnt11 expression was extremely low in the SKNSH cell line that addition, we also measured GPC2 mRNA levels in a human has low GPC2 expression (Fig. 3E). To determine the interaction normal tissue array by quantitative real-time PCR. GPC2 mRNA of GPC2 and Wnt, we conducted a coimmunoprecipitation assay using Wnt3a-conditioned media (CM) and demonstrated that expression was not found in any normal tissues except for a F moderate mRNA expression in thymus and testis (SI Appendix, GPC2 could interact with Wnt3a (Fig. 3 ). Furthermore, we used the luciferase-expressing HEK-293 SuperTopFlash cell Fig. S3). However, our immunohistochemistry analysis showed model to analyze the function of GPC2. As shown in SI Ap- no specific binding of the LH7 antibody for either testis (C1) or pendix SI Appendix , Fig. S6, GPC2 was expressed in HEK-293 cells. Treating thymus (D2) ( , Fig. S2). These data strongly support cells with the LH7 single-domain antibody decreased the Wnt3a- tumor-specific expression of GPC2 and suggest it as a promising induced active β-catenin levels in a dose-dependent manner (Fig. neuroblastoma biomarker. 3G). Lithium chloride (LiCl) is a GSK3β inhibitor and an in- There has been evidence that GPC3 expression or other gly- tracellular β-catenin signaling inducer (19). As shown in Fig. 3H, picans (e.g., GPC1) has been correlated with poor prognosis in a combination of Wnt3a and LiCl showed synergistic elevation of hepatocellular carcinoma or other types of cancer (30–33). To β-catenin expression. Interestingly, the elevated β-catenin ex- analyze a possible correlation between GPC2 mRNA levels and pression can be reduced by LH7 treatment, supporting the idea survival of patients with neuroblastoma, we used the R2 Geno- that the Wnt/β-catenin pathway can be directly modulated by the

Li et al. PNAS | Published online July 24, 2017 | E6625 Downloaded by guest on September 25, 2021 Fig. 3. Genetic silencing of GPC2 inhibits neuroblastoma tumor cell growth and induces apoptosis by suppressing Wnt/β-catenin signaling. (A) GPC2 protein expression in LAN1 and IMR5 neuroblastoma cells after siRNA-mediated knockdown of GPC2. (B) Inhibition of tumor cell growth by GPC2 siRNAs in both LAN1 and IMR5 cell lines. (C) GPC2 expression in IMR5 neuroblastoma cells after GPC2 KO using the CRISPR-Cas9 technique. GPC2 KO decreased active β-catenin protein levels at 72 h posttransfection. (D) Caspase 3/7 activity in IMR5 cells after treatment with GPC2 targeted sgRNA. (E) Protein expression of Wnt3a and Wnt11 in neuroblastoma cell lines. (F) Interaction between GPC2 and Wnt3a as determined by immunoprecipitation. (G) Reduction of active β-catenin levels by LH7 treatment after 6 h in HEK-293 SuperTopFlash cells that were stimulated with Wnt3a CM. (H) LH7 suppressed the expression of β-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)The anti-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 proposed mechanism mediated by anti-GPC2 antibodies to inhibit neuroblastoma cell growth. Blockade of GPC2 suppresses the expression of β-catenin and its targeted genes, including N-Myc. All data are represented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

addition of the LH7 antibody. In addition to LH7, two other anti- 3K). Taken together, our data show that GPC2 is involved in Wnt GPC2 single-domain antibodies showed dose-dependent reduction signaling, and that targeting GPC2 by single-domain antibodies of Wnt signaling, but to a lesser degree (Fig. 3I). Notably, LH7 at such as LH7 can suppress neuroblastoma cell growth by inhibiting 100 μg/mL resulted in a 90% reduction of β-catenin signaling Wnt signaling and down-regulating Wnt target genes including compared with control human IgG. The data indicate that the N-Myc, an oncogenic driver of neuroblastoma pathogenesis. Fig. 3L LH7 single-domain antibody has the most inhibitory effect on shows a working model based on our observations. Wnt signaling. MYCN amplification occurs in ≈25–33% of neuroblastoma cases GPC2-Specific Immunotoxins Inhibit Neuroblastoma Growth. To de- and results in N-Myc protein overexpression (1). Patients with termine whether GPC2 could be used as a target of immuno- MYCN-amplified tumors usually have a very poor prognosis. In toxins for the treatment of neuroblastoma, we constructed three addition, studies have shown that Wnt/β-catenin signaling acts immunotoxins using the LH1, LH4, and LH7 binding domains. upstream of N-Myc to regulate lung and limb development (34, All immunotoxins were expressed in Escherichia coli, refolded in 35). As shown in Fig. 3J, N-Myc protein was found in GPC2 high- vitro, and isolated with over 90% purity (Fig. 4A). The binding expressing neuroblastoma cells (LAN1, IMR5, LAN5, and IMR32) affinities of all three immunotoxins on purified GPC2 protein but not in GPC2 low-expressing SKNSH cells. Furthermore, we were measured by ELISA. As shown in SI Appendix, Fig. S7, the found that silencing of GPC2 suppressed N-Myc expression (Fig. calculated EC50 values for the three immunotoxins were in the

E6626 | www.pnas.org/cgi/doi/10.1073/pnas.1706055114 Li et al. Downloaded by guest on September 25, 2021 3 PNAS PLUS range of 4.6 nM to 43.9 nM. We found that the EC50 value an average volume of 150 mm , mice were treated with LH7– (18 nM) for the LH7–PE38 monomeric immunotoxin in ELISA PE38 every other day for a total of 10 injections. The different was similar to the Kd value (9.8 nM) of the LH7–Fc fusion dose concentrations were used to determine the relative toxicity of protein (Fig. 1F), indicating the immunotoxin retained binding the LH7–PE38 immunotoxin. As shown in Fig. 4E, the mice tol- – properties of the original single-domain antibody. To determine erated 0.4 mg/kg LH7 PE38 well. However, mice treated with the cytotoxicity of all immunotoxins in vitro, we examined the 0.8 mg/kg died after 5 injections. Only two mice survived after – inhibition of cell proliferation on a panel of cell lines using the 10 injections at the 0.6 mg/kg dose. Notably, 0.4 mg/kg of LH7 PE38 inhibited tumor growth during treatment without affecting WST cell proliferation assay. All three immunotoxins potently + body weight (Fig. 4 F and G). At the end of treatment, tumor and selectively inhibited the growth of GPC2 cell lines – – volumes in the LH7 PE38-treated group were significantly smaller LAN1 and IMR5 with similar IC50 values of 0.5 1.2 nM. than those in the control group (Fig. 4H). In addition to measuring LAN1 and IMR5 cells were poorly sensitive to an irrelevant body weight, we also performed toxicology studies to further immunotoxin targeting mesothelin (IC50 for LAN1, 8 nM; IC50 evaluate any side effects of LH7–PE38 at 0.4 mg/kg. The LH7– for IMR5, 34 nM). None of the immunotoxins affected the PE38-treated mice had an increase in white blood cells, indicating growth of low GPC2-expressing SKNSH cells (Fig. 4 B–D). that the immunotoxin could cause inflammatory effects in vivo To evaluate the antitumor activity of LH7–PE38 in vivo, we s.c. (SI Appendix,TableS1). In addition, the LH7–PE38-treated group inoculated nude mice with LAN1 cells. When tumor reached showed an increase in alanine aminotransferase; however, we did not find any gross evidence of liver damage following mouse necropsy. All organ weights of the treated mice were statistically similar to those of the control group, except for the spleen. No significant differences were detected in any other parameters measured. In conclusion, we found that immunotoxins based on our anti-GPC2 human single domains inhibited neuroblastoma cell 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 and transmembrane region, followed by the 4-1BB costimulatory sig- naling moiety and the cytoplasmic component of CD3ζ signaling molecule. The upstream GFP reporter was coexpressed with CAR using the “self-cleaving” T2A peptide (Fig. 5A). The genetically modified T cells began to expand after activation (Fig. 5B). On day 9, the expression of GPC2 CARs in the transduced T cells was demonstrated 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 specifically + recognize and kill GPC2 neuroblastoma cells, a luminescent- based cytolytic assay was established using the neuroblastoma cells engineered to express luciferase. As shown in Fig. 5D, IMR5 cells, which express high levels of GPC2, are resistant to mock-transduced T-cell–mediated killing. This resistance was true even at effector:target ratios as high as 8:1. Conversely, IMR5 cells

were efficiently lysed by the GPC2 CAR T cells in a dose- MEDICAL SCIENCES 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 cells and GPC2 CAR T cells showed similarly low cytolytic activity against the low GPC2-expressing SKNSH cells (Fig. 5E). A cyto- kine production assay revealed that GPC2 CAR T cells produced significantly more IFN-γ and TNF-α after exposure to IMR5 cells than 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 mock Fig. 4. Recombinant immunotoxins against GPC2 inhibit neuroblastoma – or GPC2 CAR T cells were cocultured with SKNSH cells. tumor growth in vitro and in vivo. (A) Purity of LH1 PE38 (molecular weight We tested the killing ability of CAR T cells generated from eight of 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 of individual human donors. At an effector:target ratio of 8:1, GPC2- anti-GPC2 immunotoxins on the growth of IMR5 (B), LAN1 (C), and SKNSH specific CAR T cells lytic activity against IMR5 neuroblastoma cells (D) cell lines, as measured by the WST-8 assay. An antimesothelin immu- rangedfrom44%to71%,withanaverageof56%(Fig.6A). notoxin was used as an irrelevant control immunotoxin. (E) Toxicity de- Minimal cell lysis was observed in IMR5 cells treated with mock tection of LH7–PE38 in vivo. Athymic nu/nu nude mice were treated with T cells (Fig. 6B). Next, we assessed the antitumor activity of GPC2- indicated doses of immunotoxin i.v. every other day for a total of 10 injec- targeting CAR T cells in nude mice i.v. engrafted with luciferase- tions. Each arrow indicates an individual injection (n = 5 per group). expressing IMR5 cells. Although LH3 CAR T cells were the most – (F) Antitumor activity of LH7 PE38. Athymic nu/nu nude mice were s.c. in- potent in the cell killing assay (Fig. 5D),theLH3phagebinderwas oculated with 1 × 107 LAN1 cells mixed with Matrigel. When tumors reached 3 also cross-reactive with other glypican members (e.g., GPC3) (Fig. an average volume of 150 mm , mice were treated with a 0.4-mg/kg dose of E LH7–PE38 i.v. every other day for 10 injections. Each arrow indicates an in- 1 ). Therefore, we chose LH7 for preclinical testing in neuroblas- dividual injection; n = 5 per group. *P < 0.05. (G) Body weight of the mice toma models. Bioluminescence imaging using IVIS showed that treated in F.(H) Representative pictures of tumors from control and treated IMR5-bearing nude mice developed disseminated tumor lesions mice at the end of study. Values represent mean ± SEM. IT, immunotoxin. surrounding spine and bones (Fig. 6C). Notably, LH7 CAR T cells

Li et al. PNAS | Published online July 24, 2017 | E6627 Downloaded by guest on September 25, 2021 onstrate an important role for GPC2 as a target candidate of antibody-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 have been tested in clinical trials for neuroblastoma, with proven safety and efficacy (3, 36). The FDA approved Unituxin, in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-2 (IL-2), and 13-cis-retinoic acid (RA), for the treatment of patients with high-risk neuroblastoma in 2015. However, in pa- tients with advanced disease, anti-GD2 antibodies show only limited activity. 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 targeting GPC2 along with GFP using the T2A ribosomal skipping sequence. (B) Timeline of CAR T-cell production. (C) GPC2-specific CAR expression on human T cells transduced with lentiviral particles was analyzed using flow cytometry by detection of GFP fluorescence. (D–E) Cytolytic activities of GPC2 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, and specific lysis was measured using a luminescent-based cytolytic assay. (F–G) The above culture supernatants at an E:T ratio of eight were harvested to measure IFN-γ (F) and TNF-α (G) secretions via ELISA. All data are represented as mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01.

effectively suppressed metastatic tumors after 14 d of T-cell in- fusion, whereas mock T cells failed to reduce tumor burden (Fig. 6 C and D). Four of eight (50%) of the mice treated with CAR T cells targeting GPC2 were tumor-free at the end of this study. Neither mock T cell nor LH7 CAR T cell treatment affected mice body weight (SI Appendix,Fig.S9). The efficacy of GPC2-targeting CAR T cells was also evaluated in a LAN1 xenograft mouse model. LH7 CAR T cells initially led to a reduction in tumor size and significantly suppressed tumor growth compared with the control group at the end of the study (SI Appendix,Fig.S10). Taken to- gether, we have demonstrated that our single-domain antibodies canbeusedtoconstructCARTcellsthatareabletokillGPC2- expressing neuroblastoma cells in cell and mouse models. Discussion In the present study, we reported that GPC2 protein expression Fig. 6. GPC2-specific CAR T cells demonstrate potent activity in mice bear- levels were elevated in human neuroblastoma tumors compared with ing human neuroblastomas. (A–B) Cytotoxic activity of LH7 CAR T cells de- normal human tissues. Additionally, silencing of GPC2 decreased rived from multiple donors. PMBCs were isolated from eight healthy donors. neuroblastoma cell viability and induced apoptosis. We also found The luciferase-expressing IMR5 cells were cocultured with LH7 CAR- that GPC2 modulated Wnt/β-catenin signaling and the expression of transduced T cells (A)ormockTcells(B) at the indicated effector (E):target the key oncogenic driver gene N-Myc in neuroblastoma. By using (T) ratios for 20 h, and specific lysis was measured using a luminescent-based cytolytic assay. (C) LH7 CART demonstrated potent antitumor activity by sup- phage display, we succeeded in identifying a group of human single- pressing metastatic IMR5 neuroblastoma cells as measured by bioluminescent domain antibodies specific for GPC2. The immunotoxins and CAR imaging. Animals (n = 8 per group) were i.v. injected with a single infusion of T cells based on these antibody binding domains significantly 30 × 106 mock T cells or LH7 CAR T cells. (D) Quantitation of bioluminescence in inhibited neuroblastoma tumor cell growth. Our observations dem- mice treated in C. Values represent mean ± SEM.

E6628 | www.pnas.org/cgi/doi/10.1073/pnas.1706055114 Li et al. Downloaded by guest on September 25, 2021 (13 of 25). Immunohistochemistry confirmed that GPC2 protein fied a human single-domain antibody that recognizes a cryptic PNAS PLUS expression was not detected in normal peripheral nerve tissues. Our functional site on GPC3 and inhibits Wnt signaling in liver cancer studies also demonstrated that GPC2 protein was either present at (48). In the present study, we isolated a group of seven represen- low levels or not expressed in most normal tissues in our compre- tative binders specific for GPC2, and all of these single-domain hensive array. This finding was especially true of the essential organ antibodies significantly inhibited Wnt/β-catenin signaling in neuro- tissues included in our array. In addition, using the data from a study blastoma cells. Together, these studies indicate that single-domain cohort in the R2 Genomics Analysis and Visualization Platform, we antibodies are an emerging class of promising therapeutic candi- found that both overall survival and event-free survival of the patients dates that can inhibit the signaling related to the growth of cancer with neuroblastoma with high GPC2 expression were significantly cells by blocking receptor–ligand interactions. shorter than those with low GPC2 expression. Overall, we show that The immunotoxins based on our anti-GPC2 antibodies demon- GPC2 protein is uniquely overexpressed in neuroblastoma and that strated highly specific and potent killing of neuroblastoma in both in its high expression may be correlated with poor survival. We also vitro and in vivo mouse models. In the present study, the irrelevant found an inhibitory effect of GPC2 in neuroblastoma cell growth antimesothelin immunotoxin shows modest background cytotoxicity using siRNA and CRISPR-Cas9 approaches. Our results suggest the at high concentrations. It has been shown that the immunotoxins possibility that the suppression of GPC2 expression in neuroblastoma (e.g., mPE24) containing only the enzymatic domain (domain III) cells may induce apoptosis. Taken together, our data show that has significantly less nonspecific cytotoxicity than PE38, suggesting GPC2 is a promising target candidate for neuroblastoma therapy. that domain II may potentially contribute to nonspecific cytotoxicity Given the importance of Wnt/β-catenin signaling in neuroblas- (49). To improve therapeutic index and reduce potential side ef- toma (10–12), we decided to determine whether GPC2 inhibition fects, future reengineering of the anti-GPC2 immunotoxin by re- could suppress this signaling pathway in neuroblastoma cells. moving domain II may be useful for clinical development. In mouse GPC2 inhibition by our anti-GPC2 antibodies or KO by sgRNA was testing, the optimal dose appears to be 0.4 mg/kg, which is similar to found to reduce the expression of active β-catenin and suppress the dose of other immunotoxins that are currently being evaluated target genes that may regulate neuroblastoma cell proliferation and in preclinical and clinical stages (including phase III) (50). Despite survival. We notice the discrepancy between the IC50 (0.3–1.3 μM) not observing any obvious side effects in mice treated with anti- of Wnt signaling inhibition and EC50 of binding activity (5–50 nM) GPC2 immunotoxins, we noted an increase in alanine amino- of single domains (SI Appendix,Fig.S7). For example, the IC50 for transferase. This increase could be indicative of liver damage, but the best single domain (LH7) is 0.3 μM(25μg/mL), 17-fold more we did not note any gross evidence of liver damage upon mouse than its EC50 (18 nM), likely because in the luciferase reporter necropsy. Further comprehensive preclinical studies are needed to assay, a saturation level of the antibody is needed to functionally validate the effect of anti-GPC2 immunotoxins systematically, in- block the interaction of GPC2 and Wnt and/or other associated cluding pharmacodynamics, pharmacokinetics, and influence on proteins (e.g., Frizzled). We previously found that GPC3 could in- liver function, before identifying the most promising candidate for teract with Wnt3a to suppress hepatocellular carcinoma cell pro- use in humans. Because PE38 is a bacterial protein, it is highly liferation (19). Wnt11 is secreted in regions adjacent to the neural immunogenic in patients with solid tumors that have normal im- crest and could induce neural crest migration (39). It has been mune systems (51). Future efforts using immunosuppressive drugs shown that Wnt11 mRNA was highly expressed in neuroblastoma or less immunogenic toxins should be pursued for clinical devel- clinical samples (40). Thus, we particularly determined the expres- opment of anti-GPC2 immunotoxins. sion of Wnt3a and Wnt11 in neuroblastoma cell lines. Interestingly, CAR T cells have been shown to be a promising T-cell–based Wnt3a and Wnt11 were expressed in GPC2 high-expressing neu- immunotherapy in leukemia (25, 26, 29, 52, 53). CAR T cells roblastoma cells (e.g., LAN1, IMR5, LAN5, and IMR32). By targeting CD19 have resulted in sustained complete responses and contrast, Wnt3a and Wnt11 proteins were either not detected or shown complete response rates of ≈90% in patients with relapsed poorly detected in GPC2 low-expressing SKNSH cells. The differ- or refractory acute lymphoblastic leukemia (54). However, CAR ences in Wnt protein expression are in agreement with the sensi- T-cell therapies have not been successful in treating solid tumors. tivity of GPC2-targeted immunotoxins and CAR T cells in LAN1/ In the present study, we sought to evaluate the use of CAR T cells

IMR5 and SKNSH cell lines. Furthermore, we demonstrated that in treating neuroblastoma. We established an in vivo biolumi- MEDICAL SCIENCES GPC2 could coimmunoprecipiate with Wnt3a. These observations nescent model of disseminated neuroblastoma in mice. Most are consistent with previous reports showing that activation of Wnt/ neuroblastomas begin in the abdomen in the adrenal gland or next β-catenin signaling contributes to the aggressiveness of neuroblas- to the spinal cord, or in the chest. Neuroblastomas can spread to toma (41, 42) and indicate the role of GPC2 in modulation of Wnt the bones, such as in the face, skull, pelvis, and legs. They can also signaling in neuroblastoma cells. It is possible that GPC2 can in- spread to bone marrow, liver, lymph nodes, skin, and orbits. In our teract with other Wnt molecules beyond Wnt3a. Future studies are study, we frequently found disseminated tumors near the spine necessary to understand the interactions of various Wnt molecules and in the bones of face, skull, legs, and pelvis, indicating the and GPC2 in the tumor microenvironment to better understand the clinical relevance of our animal model. We then demonstrated role of GPC2 in modulating Wnt signaling in cancer cells. N-Myc is that a single infusion of LH7 CAR T cells significantly suppressed a key driver for neuroblastoma tumorigenesis (43–45). Here we the growth of metastatic neuroblastoma cells in mice. Whereas the demonstrated that N-Myc was expressed in MYCN-amplified neu- immunotoxins can effectively inhibit neuroblastoma growth, CAR roblastoma cells, including LAN1, IMR5, LAN5, and IMR32 (Fig. T cells targeting GPC2 can cause complete remission in 50% of 3J). However, N-Myc protein was not found in MYCN-nonamplified treated mice. The persistence of CAR T cells is a major challenge SKNSH neuroblastoma cells. Interestingly, in the present study we in solid tumors. Preclinical and clinical studies investigating found genetic silencing of GPC2 significantly inhibited the expres- combinations of CAR T cells with immune checkpoint blockade sion of N-Myc in neuroblastoma cells. It has been shown that Wnt therapies would be pursued and examined (54). Future compre- signaling can regulate N-Myc expression level and β-catenin may hensive preclinical studies will be needed to validate GPC2- activate the promoter of N-Myc during development (34, 35). Our targeting CAR T-cell–based immunotherapy for neuroblastoma. result indicates that GPC2 can down-regulate N-Myc expression by In conclusion, we found that the GPC2 protein is highly inhibiting Wnt/β-catenin signaling. Protein surfaces contain clefts expressed in neuroblastoma and produced single-domain anti- that are relatively inaccessible to conventional antibodies as a result bodies against GPC2. These antibodies can potentially be used of steric hindrance. Interestingly, single-domain antibodies have the either as immunotoxins or CARs in neuroblastoma treatment. ability to bind in protein clefts or hidden substrate pockets not ac- This report establishes GPC2 as a potential therapeutic target cessible to conventional antibodies (46, 47). We previously identi- in cancer. The single-domain antibodies described here are

Li et al. PNAS | Published online July 24, 2017 | E6629 Downloaded by guest on September 25, 2021 promising candidates that should be further evaluated as cancer tracellular CD3ζ. The construct was engineered to express an upstream GFP therapeutics for the treatment of neuroblastoma and other reporter separated from the CAR by a T2A sequence. The sequence encoding + GPC2 cancers. the whole CAR construct was subcloned into the CMV promoter-containing lentiviral vector pLenti6.3/v5 (Invitrogen). Materials and Methods Lentivirus Production, T-Cell Transduction, and Expansion. To produce viral su- Cell Culture. Human neuroblastoma cell lines, including SKNSH, LAN1, IMR5, pernatant, HEK-293T cells were cotransfected with GPC2–CAR lentiviral vectors IMR32, NBEB, and LAN5, were obtained from collaborators at the National and Mission viral packaging plasmids (Sigma-Aldrich) using Lipofectamine Cancer Institute (NCI). IMR5, LAN1, and SKNSH cell lines were also transduced 2000 (Invitrogen) per the manufacturer’s protocol. The supernatant was col- with lentiviruses expressing firefly luciferase, which were obtained from NCI- lected at 72 h posttransfection and then concentrated using Lenti-X concen- Frederick (55). Peripheral blood mononuclear cells (PBMCs) were isolated trator (Clontech) according to the manufacturer’s instructions. from the blood of eight healthy donors using Ficoll (GE Healthcare) PBMCs were purchased from Oklahoma Blood Institute and stimulated for according to the manufacturer’s instructions. The aforementioned cell lines 24 h with anti-CD3/anti-CD28 antibody-coated beads (Invitrogen) at a were cultured in RPMI-1640 medium supplemented with 10% FBS, 1% 2:1 bead-to-T cell ratio in growth medium supplemented with IL-2. Activated L-glutamine, and 1% penicillin–streptomycin at 37 °C in a humidified at- T cells were then transduced with the lentivirus expressing GPC2-specific CARs mosphere with 5% CO . The HEK-293T cell line was obtained from the 2 at a multiplicity of infection of five. Cells were counted every other day and American Type Culture Collection. The HEK-293 SuperTopFlash stable cell fed with fresh growth medium every 2–3 d. Once T cells appeared to become line was a kind gift from Jeremy Nathans, Johns Hopkins University, Baltimore. quiescent, as determined by both decreased growth kinetics and cell size, These two cell lines were grown in DMEM supplemented with 10% FBS, 1% they were used either for functional assays or cryopreserved. L-glutamine, and 1% penicillin–streptomycin at 37 °C in a humidified atmo-

sphere with 5% CO2. All cell lines were authenticated by morphology and growth rate and were mycoplasma-free. 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 the coculture incubation period, supernatant was saved for IFN-γ and TNF-α levels Phage Display and Biopanning. A combinational engineered human VH single- by ELISA (R&D Systems). The remaining tumor cells were lysed for 5 min. The domain phage display library, with an estimated size of 2.5 × 1010, was used luciferase activity in the lysates was measured using the Steady Glo luciferase for the screening and has been previously described (56). The phage library assay system on Victor (PerkinElmer). Results were analyzed as percent kill- was subjected to four rounds of panning on Nunc 96-well Maxisorp plate ing based on luciferase activity in wells with tumor cells alone: % killing = (Thermo Scientific) as described previously (57–59). Details are provided in 100 − [relative light units (RLU) from wells with effector and target cells]/ the SI Appendix, SI Materials and Methods. (RLU from wells with target cells) × 100. Antibody Binding Assay. The binding kinetics of the LH7 antibody to GPC2 was Animal Studies. All mice were housed and treated under the protocol ap- determined using the Octet RED96 system (FortéBio) as described previously proved by the Institutional Animal Care and Use Committee at the NIH. For (54). Briefly, all experiments were performed at 30 °C and reagents were xenograft tumor studies, 5-wk-old female athymic nu/nu nude mice (NCI- prepared in 0.1% BSA, 0.1% Tween20 PBS, pH 7.4 buffer. Biotinylated GPC2– Frederick) were given s.c. injections of 10 × 106 LAN1 cells suspended in hFc protein was immobilized onto streptavidin biosensors, which were Matrigel (Corning). Tumor dimensions were determined using calipers, and subsequently used in association and dissociation measurements for a time tumor volume (in cubic millimeters) was calculated by the formula V = window of 600 s and 1,800 s, respectively. Data analysis was performed using 1/2 ab2, where a and b represent tumor length and width, respectively. For the FortéBio analysis software provided with the instrument. LH7–PE38 treatment, when average tumor size reached around 150 mm3, mice were i.v. injected with indicated doses every other day for 10 injections. Immunohistochemistry. The human neuroblastoma tissue array and normal For T-cell treatment, when tumor burden was ≈120 mm3, mice were injected tissue array were purchased from US Biomax. The tissue arrays were sent to i.p. with 200 mg/kg cyclophosphamide to deplete host lymphocyte com- Histoserv Inc. for staining. Details are provided in the SI Appendix, SI Ma- partments. After 24 h, 10 × 106 of either mock T cells or LH7 CAR T cells were terials and Methods. i.v. injected into mice on days 13, 20, and 27. Mice were given i.p. injection of 2,000 units of IL-2 twice a week following T-cell infusion. Mice were killed Patient Samples Analysis. The R2: Genomics Analysis and Visualization Plat- when the tumor size reached 1,500 mm3. form (r2.amc.nl) was used to analyze the correlation between GPC2 mRNA For the disseminated tumor study, 5-wk-old female athymic nu/nu nude expression and survival of patients with neuroblastoma. We chose the mice were i.v. injected with 7 × 106 luciferase-expressing IMR5 cells. Cyclo- largest tumor neuroblastoma Kocak dataset (n = 649) to perform analysis of phosphamide was injected i.p. at 200 mg/kg 24 h before any cell adminis- overall survival and event-free survival by the Kaplan–Meier method. tration. Then animals were given single infusion of 30 × 106 mock T cells or LH7 CAR T cells by tail vein injection. Disease was detected using the Immunoblotting and Immunoprecipitation. Cells were harvested, vortexed in Xenogen IVIS Lumina (PerkinElmer). Nude mice were injected i.p. with 3 mg ice-cold lysis buffer (Cell Signaling Technology), and clarified by centrifu- D-luciferin (PerkinElmer) and imaged 10 min later. Living Image software × gation at 10,000 g for 10 min at 4 °C. Protein concentration was measured was used to analyze the bioluminescence signal flux for each mouse as ’ using a Coomassie blue assay (Pierce) following the manufacturer s specifi- photons per second per square centimeter per steradian (photons/s/cm2/sr). μ – cations. A total of 20 g of cell lysates was loaded onto a 4 20% SDS/PAGE Mice were killed when they showed any sign of sickness or when bio- gel for electrophoresis. The anti-GPC2 antibody for Western blotting was luminescence signal reached 1 × 109. purchased from Santa Cruz Biotechnology. The anti-active β-catenin and N-Myc antibodies were obtained from Millipore. The anti-Wnt3a and Statistics. All experiments were repeated a minimum of three times to de- Wnt11 antibodies were purchased from Abcam. All other antibodies, in- termine the reproducibility of the results. All error bars represent SEM. cluding cleaved PARP, total β-catenin, β-actin, and GAPDH were obtained Statistical analysis of differences between samples was performed using the from Cell Signaling Technology. Student’s t test. A P value of <0.05 was considered statistically significant. Fifty micrograms of GPC2–hFc protein was incubated with Wnt3a CM for 4 h on ice. Protein A-Agarose beads (Roche) were added to the immune complex ACKNOWLEDGMENTS. We thank Dr. Javed Khan (NCI), Dr. Crystal Mackall and incubate overnight at 4 °C. Immune complexes were washed three times (NCI; currently Stanford University), and Dr. Rimas J. Orentas (NCI; currently with lysis buffer and subjected to immunoblotting with anti-Wnt3a antibody. Lentigen Technology, Inc.) for providing the neuroblastoma cell lines used in the present study and for helpful discussions; Dr. Terry J. Fry (NCI) for pro- Production of Recombinant Immunotoxin. Anti-GPC2 single-domain anti- viding a laboratory protocol of CAR T-cell activation; Dr. Carol J. Thiele (NCI) bodies were cloned into pRB98 expression plasmid containing PE38. The for critical reading of the manuscript; Dr. Diana Haines and colleagues in the expression and purification of recombinant immunotoxins were performed Pathology/Histotechnology Laboratory (NCI) for pathological evaluation of following our protocol as described previously (60). the mice treated in the present study; and Dr. Bryan D. Fleming (NCI) and the NCI Editorial Board for editorial assistance. This research was supported by the Intramural Research Program of NIH, NCI (Z01 BC010891 and ZIA Generation of GPC2-Specific CAR. The anti-GPC2 CAR included the isolated BC010891) (to M.H.). N.L. was a recipient of the NCI Directorʼs Intramural anti-GPC2 single-domain antibody fragment, linked in-frame to the CD8α Innovation Award/Career Development Award. H.F. was a recipient of a hinge and transmembrane regions, a 4-1BB costimulatory domain, and in- fellowship from the China Scholarship Council.

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