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Glypicans play a role in the growth and metastasis of several human cancers. However, little is known about its function in thyroid cancer. Therefore, this study aimed to investigate the specific functions of glypican 2 (GPC2) in thyroid cancer cells. The expression of six glypican family members (GPC1 to GPC6) was detected in different thyroid cancer cell lines. The abnormal expression of GPC2 was confirmed by immunohistochemistry in or around thyroid tumor tissues. It was overexpressed and silenced in highly (TPC1 and FTC-133) and poorly (ARO) differentiated thyroid cancer cell lines, respectively. Subsequently, its role in cell proliferation, apoptosis, migration, and invasion was investigated. We observed that almost all glypican family members were highly expressed in thyroid cancer cells, and GPC2 expression was higher in poorly differentiated thyroid cancer cell lines than highly differentiated cell lines. The knockdown of GPC2 inhibited cell proliferation, invasion, and migration and promoted apoptosis in the ARO cells, whereas GPC2 overexpression promoted cell proliferation, invasion, and migration and inhibited apoptosis in the TPC1 and FTC-133 cells. GPC2 is highly expressed in thyroid cancer tissues and regulates cell viability, apoptosis, migration, and invasion.

Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Here we show that the cell surface oncoprotein glypican 2 (GPC2) is highly expressed on multiple lethal pediatric brain tumors, including medulloblastomas, embryonal tumors with multi-layered rosettes, other CNS embryonal tumors, as well as definable subsets of highly malignant gliomas. To target GPC2 on these pediatric brain tumors with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we developed four mRNA chimeric antigen receptor (CAR) T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment. First, we validated and prioritized these CARs using in vitro cytotoxicity and T cell degranulation assays with GPC2-expressing neuroblastoma cells. Next, we expanded the testing of the two most potent GPC2-directed CAR constructs prioritized from these studies to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, locoregional delivery bi-weekly of two to four million GPC2-directed mRNA CAR T cells induced significant and sustained tumor regression in two orthotopic medulloblastoma models, and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed. Taken together, these data show that GPC2 is a highly differentially expressed cell surface protein on multiple malignant pediatric brain tumors that can be targeted safely with local delivery of mRNA CAR T cells.One Sentence SummaryGlypican 2 is expressed on the surface of multiple pediatric brain tumors and can be successfully targeted with mRNA chimeric antigen receptor T cells.

Summary Glypican 2 (GPC2) is a MYCN-regulated, differentially expressed cell-surface oncoprotein and target for immune-based therapies in neuroblastoma. Here, we build on GPC2’s immunotherapeutic attributes by finding that it is also a highly expressed, MYCN-driven oncoprotein on small-cell lung cancers (SCLCs), with significantly enriched expression in both the SCLC and neuroblastoma stem cell compartment.By solving the crystal structure of the D3-GPC2-Fab/GPC2 complex at 3.3 Å resolution, we further illustrate that the GPC2-directed antibody-drug conjugate (ADC; D3-GPC2-PBD), that links a human GPC2 antibody (D3) to DNA-damaging pyrrolobenzodiazepine (PBD) dimers, binds a tumor-specific, conformation-dependent epitope of the core GPC2 extracellular domain. We then show that this ADC induces durable neuroblastoma and SCLC tumor regression via induction of DNA damage, apoptosis, and bystander cell killing, notably with no signs of ADC-induced in vivo toxicity. These studies provide preclinical data to support the clinical translation of ADCs targeting GPC2., Graphical abstract, Highlights GPC2 is a MYCN transcriptionally regulated cell-surface oncoprotein on SCLCs GPC2 is enriched in the SCLC and neuroblastoma stem cell compartment The D3-GPC2 antibody binds a tumor-specific, GPC2 conformational epitope The D3-GPC2-PBD ADC evokes durable neuroblastoma and SCLC tumor regression, Raman et al. report that GPC2 is a MYCN transcriptionally regulated cell-surface oncoprotein that is robustly differentially expressed on neuroblastomas and SCLCs, with enriched expression in the tumor stem cell compartment. An ADC that targets a GPC2 conformational epitope induces sustained neuroblastoma and SCLC tumor regression without any apparent toxicity.

Cancer immunotherapies have revolutionized how we can treat adult malignancies and are being translated to pediatric oncology. Chimeric antigen receptor T-cell therapy and bispecific antibodies targeting CD19 have shown success for the treatment of pediatric patients with B-cell acute lymphoblastic leukemia. Anti-GD2 monoclonal antibody has demonstrated efficacy in neuroblastoma. In this review, we summarize the immunotherapeutic agents that have been approved for treating childhood cancers and provide an updated review of molecules expressed by pediatric cancers that are under study or are emerging candidates for future immunotherapies. Advances in our knowledge of tumor immunology and in genome profiling of cancers has led to the identification of new tumor-specific/associated antigens. While cell surface antigens are normally targeted in a major histocompatibility complex (MHC)-independent manner using antibody-based therapies, intracellular antigens are normally targeted with MHC-dependent T cell therapies. Glypican 2 (GPC2) and B7-H3 (CD276) are two cell surface antigens that are expressed by a variety of pediatric tumors such as neuroblastoma and potentially can have a positive impact on the treatment of pediatric cancers in the clinic.

Summary Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7–10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3-derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumor-associated exons that can be targeted by CAR T cell therapies., Graphical abstract, Highlights RNA-seq analysis identifies tumor-associated exons of glypican 2 (GPC2) The CT3 antibody specific for tumor-associated exons has been isolated The complex structure of CT3 with GPC2 is visualized by electron microscopy CT3-derived CAR T cells regress neuroblastoma in mice, Li et al. exploit RNA-seq data to identify tumor-associated exons of glypican 2 (GPC2) that can be targeted by a monoclonal antibody (CT3). The complex structure of CT3 with GPC2 is visualized by electron microscopy. CT3-derived CAR T cells regress neuroblastoma in mice.

Glypicans are a group of cell-surface glycoproteins in which heparan sulfate (HS) glycosaminoglycan chains are covalently linked to a protein core. The glypican gene family is broadly conserved across animal species and plays important roles in biological processes. Glypicans can function as coreceptors for multiple signaling molecules known for regulating cell growth, motility, and differentiation. Some members of the glypican family, including glypican 2 (GPC2) and glypican 3 (GPC3), are expressed in childhood cancers and liver cancers, respectively. Antibody-based therapies targeting glypicans are being investigated in preclinical and clinical studies, with the goal of treating solid tumors that do not respond to standard therapies. These studies may establish glypicans as a new class of therapeutic targets for treating cancer.

Neuroblastoma is a childhood cancer that is fatal in almost half of patients despite intense multimodality treatment. This cancer is derived from neuroendocrine tissue located in the sympathetic nervous system. Glypican-2 (GPC2) is a cell surface heparan sulfate proteoglycan that is important for neuronal cell adhesion and neurite outgrowth. In this study, we find that GPC2 protein is highly expressed in about half of neuroblastoma cases and that high GPC2 expression correlates with poor overall survival compared with patients with low GPC2 expression. We demonstrate that silencing of GPC2 by CRISPR-Cas9 or siRNA results in the inhibition of neuroblastoma tumor cell growth. GPC2 silencing inactivates Wnt/β-catenin signaling and reduces the expression of the target gene N-Myc, an oncogenic driver of neuroblastoma tumorigenesis. We have isolated human single-domain antibodies specific for GPC2 by phage display technology and found that the single-domain antibodies can inhibit active β-catenin signaling by disrupting the interaction of GPC2 and Wnt3a. To explore GPC2 as a potential target in neuroblastoma, we have developed two forms of antibody therapeutics, immunotoxins and chimeric antigen receptor (CAR) T cells. Immunotoxin treatment was demonstrated to inhibit neuroblastoma growth in mice. CAR T cells targeting GPC2 eliminated tumors in a disseminated neuroblastoma mouse model where tumor metastasis had spread to multiple clinically relevant sites, including spine, skull, legs, and pelvis. This study suggests GPC2 as a promising therapeutic target in neuroblastoma.