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Purpose:Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system.Experimental Design:We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models.Results:B7-H3 CAR T cells mediate significant antitumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3.Conclusions:B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials.

Purpose: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system. Experimental Design: We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models. Results: B7-H3 CAR T cells mediate significant antitumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3. Conclusions: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials.

High-grade gliomas harboring H3 G34R/V mutations exclusively occur in the cerebral hemispheres of adolescents and young adults, suggesting a distinct neurodevelopmental origin. Combining multimodal bulk and single-cell genomics with unbiased genome-scale CRISPR/Cas9 approaches, we here describe a GABAergic interneuron progenitor lineage as the most likely context from which these H3 G34R/V mutations drive gliomagenesis, conferring unique and tumor-selective gene targets essential for glioma cell survival, as validated genetically and pharmacologically. Phenotypically, we demonstrate that while H3 G34R/V glioma cells harbor the neurotransmitter GABA, they are developmentally stalled, and do not induce the neuronal hyperexcitability described in other glioma subtypes. These findings offer a striking counter-example to the prevailing view of glioma origins in glial precursor cells, resulting in distinct cellular, microenvironmental, and therapeutic consequences.

Gliomas are the most common solid tumor of childhood and while children with low grade gliomas (LGGs) are likely to survive their disease, their course is complicated by significant treatment-related neurocognitive and endocrinological sequelae. Few accurate preclinical LGG models exist, thereby limiting development and testing of novel targeted agents. We developed a 3D, hydrogel-based co-culture system utilizing mouse astrocytes to support growth of primary human pediatric LGG cells in vitro. This tunable hydrogel system recapitulates many features of brain extracellular matrix, while the co-cultured astrocytes provide growth factors and other components normally present in vivo. In this system, patients’ tumor cells are dissociated, and functionalized polyethylene glycol-based hydrogels are produced to surround tumor cells. These tumor-containing hydrogels are then placed adjacent to adherent layers of primary mouse astrocytes and the co-cultures grown for an extended period of time. Using this system, multiple types of human LGG cells and other pediatric tumor cells can be successfully cultured in hydrogels for up to one month. Moreover, these hydrogels can be enzymatically dissolved, releasing tumor cells to passage into new gels. Fluorescent 5-ethynyl-2’-deoxyuridine labeling followed by confocal microscopy of co-cultures reveals viable, proliferating cell clusters even one month later. While high grade gliomas in this setting proliferate rapidly, cultured LGG cells proliferate slowly, much like their original tumor counterparts. These methods can be used to provide patient-specific avatars to identify the best personalized targeted therapies. Moreover, the system enables investigations of the brain tumor microenvironment as a modulator of tumor growth.

Background Standard-of-care therapies for treating pediatric medulloblastoma have long-term side effects, even in children who are cured. One emerging modality of cancer therapy that could be equally effective without such side effects would be chimeric antigen receptor (CAR) T cells. Knowing that human epidermal growth factor receptor 2 (HER2) is overexpressed in many medulloblastomas and has been used as a CAR T target before, we sought to evaluate the efficacy of more sophisticated anti-HER2 CAR T cells, as well as the feasibility and efficacy of different routes of delivering these cells, for the treatment of pediatric medulloblastoma. Methods Daoy, D283 and D425 medulloblastoma cell lines were characterized by flow cytometry to evaluate HER2 expression. Anti-tumor efficacy of HER2-BBz-CAR T cells in vitro was performed using cytokine release and immune cytotoxicity assays compared to control CD19 CAR T cells. In vivo, Daoy and D283 tumor cells were orthotopically implanted in the posterior fossa of NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and treated with regional or intravenous HER2-BBz-CAR T cells or control CD19 CAR T cells. Non-human primates (NHPs) bearing ventricular and lumbar reservoirs were treated with target autologous cells bearing extracellular HER2 followed by autologous HER2-CAR T cells intraventricularly. Cerebrospinal fluid and blood were collected serially to measure the persistence of delivered cells and cytokines. Results HER2-BBz-CAR T cells effectively clear medulloblastoma orthotopically implanted in the posterior fossa of NSG mice via both regional and intravenous delivery in xenograft models. Intravenous delivery requires a log higher dose compared to regional delivery. NHPs tolerated intraventricular delivery of autologous cells bearing extracellular HER2 followed by HER2-BBz-CAR T cells without experiencing any systemic toxicity. Conclusions HER2-BBz-CAR T cells show excellent pre-clinical efficacy in vitro and in mouse medulloblastoma models, and their intraventricular delivery is feasible and safe in NHPs. A clinical trial of HER2-BBz-CAR T cells directly delivered into cerebrospinal fluid should be designed for patients with relapsed medulloblastoma. Electronic supplementary material The online version of this article (10.1186/s40425-018-0340-z) contains supplementary material, which is available to authorized users.

Human T-cells expressing chimeric antigen receptors (CARs) recognizing common cancer antigens have shown great promise in the clinic with the potential for significant anti-tumor responses. CSPG4 is a chondroitin sulfate proteoglycan previously shown to be overexpressed in melanoma and breast cancer, as well as glioblastoma stem cells. CARs directed against CSPG4 have recently shown promising pre-clinical efficacy against these tumors. We evaluated the feasibility of targeting CSPG4 using CAR T cell therapy in medulloblastoma and neuroblastoma. Response to standard therapies for advanced neuroblastoma and medulloblastoma is poor and carries significant toxicities, which may be ameliorated by immunotherapy. We found that multiple medulloblastoma and neuroblastoma cell lines (DAOY, D283 Med, D425 Med, UW-228, and SKNAS) express CSPG4 at high levels, as does 143b osteosarcoma as a positive control. By contrast, CSPG4 in normal tissue is expressed only on pericytes and in low levels on small bowel as previously assessed by protein array [Beard et al, J Immunother Cancer 2014]. To evaluate therapeutic efficacy, a second generation CAR with a CD3ζ signaling domain and CD28 co-stimulatory domain containing the TP41.2 anti-CSPG4 scFv was transduced using a γ-retrovirus into T cells. CAR T cells were found to specifically kill 20% of DAOY, 50% of D283 Med, and 50% of 143b cancer cells at a 20:1 effector-to-target ratio in a 4-hour chromium release cytotoxicity assay, while not affecting CSPG4 negative control SY5Y (neuroblastoma) and DND41 (T cell leukemia) cells. Both CD4 and CD8 CAR T cells were also shown to produce IFNγ after exposure to 143b cells. Cytokine production in response to other CSPG4+ lines is ongoing as are cytotoxicity studies with neuroblastomas. For our in vivo model, we have begun to evaluate the efficacy of CAR T cells delivered either intravenously or via intratumoral injection against DAOY and D283 Med cells that have been orthotopically implanted in the cerebellum of immunodeficient NOD/SCID/IL2Rγ-/- mice. These results, if successful, will inform the development of a new clinical protocol for the treatment of children and young adults with medulloblastoma and neuroblastoma. Citation Format: Christopher M. Rota, Nicholas Tschernia, Steven Feldman, Crystal Mackall, Daniel W. Lee. T cells engineered to express a chimeric antigen receptor targeting chondroitin sulfate proteoglycan 4 (CSPG4) specifically kill medulloblastoma and produce inflammatory cytokines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3151. doi:10.1158/1538-7445.AM2015-3151