Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors

Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Here we show that the neuroblastoma cell surface oncoprotein glypican 2 (GPC2) is also highly expressed on multiple lethal pediatric brain tumors, including medulloblastomas, embryonal tumors with multi-layered rosettes, other CNS embryonal tumors, as well as a subset of highly malignant gliomas including H3 G35 mutant gliomas and some H3 K28M diffuse midline gliomas. To target GPC2 on these pediatric brain tumors with adoptive cellular therapies and to mitigate potential inflammatory neurotoxicity, we developed four mRNA chimeric antigen receptor (CAR) T cell constructs using the highly GPC2-specific, fully human D3 scFv that targets a conformational epitope on human and murine GPC2. First, we validated and prioritized these GPC2-directed mRNA 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 (D3V3 and D3V4) prioritized from these studies to GPC2-expressing medulloblastoma and high-grade glioma models, showing significant GPC2-specific cell death in multiple medulloblastoma and HGG cellular models. Finally, locoregional delivery of both 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.

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Cell surface antigen profiling of pediatric brain tumors: B7-H3 is consistently expressed and can be targeted via local or systemic CAR T-cell delivery

Neuro-Oncology ◽

10.1093/neuonc/noaa278 ◽

2020 ◽

Author(s):

Dalia Haydar ◽

Haley Houke ◽

Jason Chiang ◽

Zhongzhen Yi ◽

Zelda Odé ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Brain Tumors ◽

Hla Class I ◽

Pediatric Brain Tumors ◽

Class I ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Pediatric Brain

Abstract Background Immunotherapy with CAR T-cells is actively being explored for pediatric brain tumors in preclinical models and early phase clinical studies. At present it is unclear which CAR target antigens are consistently expressed across different pediatric brain tumor types. In addition, the extent of HLA class-I expression is unknown, which is critical for tumor recognition by conventional αβTCR T-cells. Methods We profiled 49 low- and high-grade pediatric brain tumor patient-derived orthotopic xenografts (PDOX) by flow analysis for the expression of five CAR targets (B7-H3, GD2, IL13Rα2, EphA2, HER2), and HLA class-I. In addition, we generated B7-H3-CAR T-cells and evaluated their antitumor activity in vitro and in vivo. Results We established an expression hierarchy for the analyzed antigens (B7-H3 = GD2 >> IL13Rα2 > HER2 = EphA2) and demonstrated that antigen expression is heterogenous. All high-grade gliomas expressed HLA class-I, but only 57.1% of other tumor subtypes had detectable expression. We then selected B7-H3 as a target for CAR T-cell therapy. B7-H3-CAR T-cells recognized tumor cells in an antigen-dependent fashion. Local or systemic administration of B7-H3-CAR T-cells induced tumor regression in PDOX and immunocompetent murine glioma models resulting in a significant survival advantage. Conclusions Our study highlights the importance of studying target antigen and HLA class-I expression in PDOX samples for the future design of immunotherapies. In addition, our results support active preclinical and clinical exploration of B7-H3-targeted CAR T-cell therapies for a broad spectrum of pediatric brain tumors.

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IMMU-16. TARGETING GLYPICAN 2 (GPC2) ON PEDIATRIC MALIGNANT BRAIN TUMORS WITH MRNA CAR T CELLS

Neuro-Oncology ◽

10.1093/neuonc/noab090.123 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i30-i30

Author(s):

Jessica Foster ◽

Crystal Griffin ◽

Allison Stern ◽

Cameron Brimley ◽

Samantha Buongervino ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Brain Tumors ◽

Pediatric Brain Tumors ◽

Embryonal Tumors ◽

Car T Cells ◽

Group 4 ◽

Car T ◽

Pediatric Brain

Abstract Glypican 2 (GPC2) is a cell-surface oncoprotein initially identified in neuroblastoma, retinoblastoma, and medulloblastoma as an ideal target for immunotherapy (Cancer Cell, 2017). Here we evaluated GPC2 expression across the spectrum of pediatric brain tumors using RNA sequencing from specimens in the Children’s Brain Tumor Network (CBTN). High GPC2 expression, defined as >10 FPKM, was found in 100% of embryonal tumors with multilayered rosettes (ETMRs) (n=6), 95% of medulloblastomas (n=122), 86% of other embryonal tumors (n=21), 50% of choroid plexus carcinomas (n=4), 42% of high grade gliomas (HGG) (n=117), and 37% of diffuse midline gliomas (DMG) (n=65). Within medulloblastoma subtypes, group 4 tumors had the highest expression, and within the HGG tumor cohort H3.3 G34 mutated gliomas had the highest GPC2 expression. High GPC2 protein expression was validated with medulloblastoma and HGG/DMG primary tumors and cell lines using IHC, Western blot, and flow cytometry. We next developed two potent CAR T cell constructs using the D3 specific scFv directed against GPC2 for testing in brain tumor models. GPC2-directed CAR T cells were tested in vitro against medulloblastoma and HGG cells lines, and in vivo using two patient-derived medulloblastoma xenograft models: Rcmb28 (group 3) and 7316-4509 (group 4). GPC2-directed mRNA CAR T cells induced significant GPC2-specific cell death in medulloblastoma and HGG cellular models with concomitant T cell degranulation compared to CD19-directed mRNA CAR T cells. In vivo, GPC2-directed mRNA CAR T cells delivered locoregionally induced significant tumor regression measured by bioluminescence after 4–6 intratumoral infusions of 4 x 106 CAR T cells (p<0.0001 for Rcmb28, p<0.05 for 7316-4509). No GPC2-directed CAR T cell related toxicity was observed. 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.

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EXTH-73. HETEROGENEITY IN CELL SURFACE ANTIGEN AND HLA CLASS I EXPRESSION IN PEDIATRIC BRAIN TUMORS AND ITS IMPACT ON T-CELL IMMUNOTHERAPY

Neuro-Oncology ◽

10.1093/neuonc/noaa215.427 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii103-ii103

Author(s):

Haley Houke ◽

Xiaoyan Zhu ◽

Kimberly S Mercer ◽

Jennifer L Stripay ◽

Jason Chiang ◽

...

Keyword(s):

T Cells ◽

Brain Tumor ◽

T Cell ◽

Brain Tumors ◽

Cell Surface ◽

Hla Class I ◽

Pediatric Brain Tumors ◽

Class I ◽

Car T ◽

Pediatric Brain

Abstract Immunotherapy with tumor antigen-specific chimeric antigen receptor (CAR) and/or ab (T-cell receptor) TCR T-cells has the potential to improve clinical outcomes of patients with pediatric brain tumors. As a prerequisite for successful T-cell therapies, we must determine which cell surface antigens are expressed and targetable in these tumors, and if HLA Class I is present, which is necessary for ab TCR T-cell recognition. Therefore, in this study we systematically analyzed pediatric patient-derived orthotopic xenograft (PDOX) brain tumor samples for cell surface expression of five known CAR targets: IL13Ra2, HER2, EphA2, B7-H3, and GD2, as well as HLA Class I. We established and validated a flow cytometry-based method of profiling tumor-associated antigens. Fifty-three PDOX samples have been profiled to date, including medulloblastoma, high grade glioma (HGG), diffuse intrinsic pontine glioma (DIPG), atypical teratoid rhabdoid tumor (ATRT), and ependymoma, among others. Our results showed high variability within and between individual samples. B7-H3 was the most consistently expressed, seen in 98% of the samples tested. We validated these results by conventional immunohistochemistry staining for B7-H3 and found comparable RESULTS: HLA Class I was highly expressed on all HGG samples but was undetectable on 47.8% of other brain tumor samples. This suggests that down-regulation of HLA class I is one mechanism by which brain tumors evade conventional T-cells, and that HLA-independent CAR T-cells would be useful therapies. We also compared expression of antigens in fresh patient samples and corresponding PDOX tumors and saw that they were indeed similar. To our knowledge, this is the largest group of pediatric brain tumor PDOX samples methodically analyzed for potential CAR target antigens and HLA Class I. Taken together, our data demonstrate that elimination of tumors by CAR T-cell immunotherapies will require targeting multiple antigens, and our profiling method could inform how to circumvent antigen-negative relapse.

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Overcoming Heterogeneity of Antigen Expression for Effective CAR T Cell Targeting of Cancers

Cancers ◽

10.3390/cancers12051075 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1075 ◽

Cited By ~ 2

Author(s):

Sareetha Kailayangiri ◽

Bianca Altvater ◽

Malena Wiebel ◽

Silke Jamitzky ◽

Claudia Rossig

Keyword(s):

T Cells ◽

T Cell ◽

Cell Surface ◽

Antigen Expression ◽

T Cell Therapy ◽

Major Mechanism ◽

Car T Cells ◽

Car T Cell ◽

Combination Strategies ◽

Car T

Chimeric antigen receptor (CAR) gene-modified T cells (CAR T cells) can eradicate B cell malignancies via recognition of surface-expressed B lineage antigens. Antigen escape remains a major mechanism of relapse and is a key barrier for expanding the use of CAR T cells towards solid cancers with their more diverse surface antigen repertoires. In this review we discuss strategies by which cancers become amenable to effective CAR T cell therapy despite heterogeneous phenotypes. Pharmaceutical approaches have been reported that selectively upregulate individual target antigens on the cancer cell surface to sensitize antigen-negative subclones for recognition by CARs. In addition, advanced T cell engineering strategies now enable CAR T cells to interact with more than a single antigen simultaneously. Still, the choice of adequate targets reliably and selectively expressed on the cell surface of tumor cells but not normal cells, ideally by driving tumor growth, is limited, and even dual or triple antigen targeting is unlikely to cure most solid tumors. Innovative receptor designs and combination strategies now aim to recruit bystander cells and alternative cytolytic mechanisms that broaden the activity of CAR-engineered T cells beyond CAR antigen-dependent tumor cell recognition.

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NURS-02. CAR-T CELL THERAPY IN PEDIATRIC BRAIN TUMORS- EARLY SUPPORTIVE CARE LESSONS

Neuro-Oncology ◽

10.1093/neuonc/noy059.551 ◽

2018 ◽

Vol 20 (suppl_2) ◽

pp. i151-i151

Author(s):

Corrine Hoeppner ◽

Susan Holtzclaw

Keyword(s):

T Cell ◽

Brain Tumors ◽

Cell Therapy ◽

Supportive Care ◽

Pediatric Brain Tumors ◽

T Cell Therapy ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T ◽

Pediatric Brain

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Shed antigen-induced blocking effect on CAR-T cells targeting Glypican-3 in Hepatocellular Carcinoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-001875 ◽

2021 ◽

Vol 9 (4) ◽

pp. e001875

Author(s):

Luan Sun ◽

Fang Gao ◽

Zhanhui Gao ◽

Lei Ao ◽

Na Li ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

T Cells ◽

T Cell ◽

Cell Surface ◽

Negative Regulator ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundGlypican-3 (GPC3), a cell surface glycoprotein that is pathologically highly expressed in hepatocellular carcinoma (HCC), is an attractive target for immunotherapies, including chimeric antigen receptor (CAR) T cells. The serum GPC3 is frequently elevated in HCC patients due to the shedding effect of cell surface GPC3. The shed GPC3 (sGPC3) is reported to block the function of cell-surface GPC3 as a negative regulator. Therefore, it would be worth investigating the potential influence of antigen shedding in anti-GPC3 CAR-T therapy for HCC.MethodsIn this study, we constructed two types of CAR-T cells targeting distinct epitopes of GPC3 to examine how sGPC3 influences the activation and cytotoxicity of CAR-T cells in vitro and in vivo by introducing sGPC3 positive patient serum or recombinant sGPC3 proteins into HCC cells or by using sGPC3-overexpressing HCC cell lines.ResultsBoth humanized YP7 CAR-T cells and 32A9 CAR-T cells showed GPC3-specific antitumor functions in vitro and in vivo. The existence of sGPC3 significantly inhibited the release of cytokines and the cytotoxicity of anti-GPC3 CAR-T cells in vitro. In animal models, mice carrying Hep3B xenograft tumors expressing sGPC3 exhibited a worse response to the treatment with CAR-T cells under both a low and high tumor burden. sGPC3 bound to CAR-T cells but failed to induce the effective activation of CAR-T cells. Therefore, sGPC3 acted as dominant negative regulators when competed with cell surface GPC3 to bind anti-GPC3 CAR-T cells, leading to an inhibitory effect on CAR-T cells in HCC.ConclusionsWe provide a proof-of-concept study demonstrating that GPC3 shedding might cause worse response to CAR-T cell treatment by competing with cell surface GPC3 for CAR-T cell binding, which revealed a new mechanism of tumor immune escape in HCC, providing a novel biomarker for patient enrolment in future clinical trials and/or treatments with GPC3-targeted CAR-T cells.

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New Era of Immunotherapy in Pediatric Brain Tumors: Chimeric Antigen Receptor T-Cell Therapy

International Journal of Molecular Sciences ◽

10.3390/ijms22052404 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2404

Author(s):

Wan-Tai Wu ◽

Wen-Ying Lin ◽

Yi-Wei Chen ◽

Chun-Fu Lin ◽

Hsin-Hui Wang ◽

...

Keyword(s):

T Cell ◽

Brain Tumors ◽

Cell Therapy ◽

Pediatric Brain Tumors ◽

T Cell Therapy ◽

New Era ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T ◽

Pediatric Brain

Immunotherapy, including chimeric antigen receptor (CAR) T-cell therapy, immune checkpoint inhibitors, cancer vaccines, and dendritic cell therapy, has been incorporated as a fifth modality of modern cancer care, along with surgery, radiation, chemotherapy, and target therapy. Among them, CAR T-cell therapy emerges as one of the most promising treatments. In 2017, the first two CAR T-cell drugs, tisagenlecleucel and axicabtagene ciloleucel for B-cell acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL), respectively, were approved by the Food and Drug Administration (FDA). In addition to the successful applications to hematological malignancies, CAR T-cell therapy has been investigated to potentially treat solid tumors, including pediatric brain tumor, which serves as the leading cause of cancer-associated death for children and adolescents. However, the employment of CAR T-cell therapy in pediatric brain tumors still faces multiple challenges, such as CAR T-cell transportation and expansion through the blood–brain barrier, and identification of the specific target antigen on the tumor surface and immunosuppressive tumor microenvironment. Nevertheless, encouraging outcomes in both clinical and preclinical trials are coming to light. In this article, we outline the current propitious progress and discuss the obstacles needed to be overcome in order to unveil a new era of treatment in pediatric brain tumors.

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