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Updated: Aug 27, 2025
Intracranial Cannula Implantation for Serial Locoregional Chimeric Antigen Receptor CAR T Cell Infusions in Mice
Published on: February 24, 2023
Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors
Jessica B Foster1,2,3, Crystal Griffin3, Jo Lynne Rokita3,4,5
1Division of Oncology, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA fosterjb@chop.edu.
Background:
Pediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma for which targeted immunotherapies have been developed. This work aimed to characterize GPC2 expression in pediatric brain tumors and develop an mRNA CAR T cell approach against this target.
Methods:
We investigated GPC2 expression across a cohort of primary pediatric brain tumor samples and cell lines using RNA sequencing, immunohistochemistry, and flow cytometry. To target GPC2 in the brain with adoptive cellular therapies and mitigate potential inflammatory neurotoxicity, we used optimized mRNA to create transient chimeric antigen receptor (CAR) T cells. We developed four mRNA CAR T cell constructs using the highly GPC2-specific fully human D3 single chain variable fragment for preclinical testing.
Results:
We identified high GPC2 expression across multiple pediatric brain tumor types including medulloblastomas, embryonal tumors with multilayered rosettes, other central nervous system embryonal tumors, as well as definable subsets of highly malignant gliomas. We next validated and prioritized CAR configurations using in vitro cytotoxicity assays with GPC2-expressing neuroblastoma cells, where the light-to-heavy single chain variable fragment configurations proved to be superior. We expanded the testing of the two most potent GPC2-directed CAR constructs to GPC2-expressing medulloblastoma and high-grade glioma cell lines, showing significant GPC2-specific cell death in multiple models. Finally, biweekly locoregional delivery of 2-4 million GPC2-directed mRNA CAR T cells induced significant tumor regression in an orthotopic medulloblastoma model and significantly prolonged survival in an aggressive orthotopic thalamic diffuse midline glioma xenograft model. No GPC2-directed CAR T cell related neurologic or systemic toxicity was observed.
Conclusion:
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, laying the framework for the clinical translation of GPC2-directed immunotherapies for pediatric brain tumors.
Insights
Glypican 2 (GPC2) is a targetable oncoprotein on pediatric brain tumors. Local delivery of mRNA chimeric antigen receptor (CAR) T cells targeting GPC2 demonstrated significant tumor regression and survival extension without observed toxicity.
Area of Science:
- Oncology
- Immunotherapy
- Pediatric Cancer Research
Background:
- Pediatric brain tumors are a leading cause of childhood cancer mortality, necessitating novel therapeutic strategies.
- Glypican 2 (GPC2) is a cell surface oncoprotein identified in neuroblastoma, making it a potential target for immunotherapies.
- This study investigates GPC2 expression in pediatric brain tumors and explores an mRNA chimeric antigen receptor (CAR) T cell therapy approach.
Purpose of the Study:
- To characterize Glypican 2 (GPC2) expression across various pediatric brain tumors.
- To develop and evaluate transient mRNA CAR T cell therapy targeting GPC2 for pediatric brain tumors.
- To assess the safety and efficacy of GPC2-directed mRNA CAR T cells in preclinical models.
Main Methods:
- Investigated GPC2 expression using RNA sequencing, immunohistochemistry, and flow cytometry in pediatric brain tumor samples and cell lines.
- Developed four mRNA CAR T cell constructs utilizing a GPC2-specific single chain variable fragment (scFv) for preclinical testing.
- Assessed in vitro cytotoxicity and in vivo efficacy and toxicity in orthotopic xenograft models of medulloblastoma and diffuse midline glioma.
Main Results:
- High GPC2 expression was identified in medulloblastomas, embryonal tumors, and subsets of high-grade gliomas.
- GPC2-directed mRNA CAR T cells demonstrated significant GPC2-specific cell death in vitro and induced substantial tumor regression and prolonged survival in vivo.
- Locoregional delivery of GPC2-directed mRNA CAR T cells showed no observed neurologic or systemic toxicity in preclinical models.
Conclusions:
- Glypican 2 (GPC2) is a differentially expressed cell surface target on multiple malignant pediatric brain tumors.
- Local delivery of mRNA CAR T cells targeting GPC2 offers a safe and effective therapeutic strategy.
- These findings provide a framework for the clinical translation of GPC2-directed immunotherapies for pediatric brain tumors.
