Poster BB17 - Genetically Engineered Extracellular Vesicles with Chimeric Antigen Receptor of Human Stem Cell-Derived Neutrophils for Treating Glioblastoma.

Introduction: Glioblastoma multiforme (GBM) is the most commonly occurring tumor in the central nervous system, accounting for 80% of CNS tumors. Current treatments for GBM include surgical resection, radiotherapy, and/or chemotherapy. These prove ineffective at treating GBM, with the median survival of 15 months post diagnosis. Work in gene editing and immunology has led to the development of chimeric antigen receptors (CARs), artificial proteins designed to illicit a strong immune response in the presence of specific antigens. CARs employing a chlorotoxin receptor domain have recently been incorporated into neutrophils and used for the lysis of glioblastoma cell lines. However, such cells are liable for inducing body wide off- target effects that can be deadly. Extracellular vesicles (EVs), natural products of cells, share properties with their parent cells including CAR expression and tumor-lytic effects when derived from CAR-T cells. However, this has never been investigated in CAR Neutrophils.

Materials and

Methods: DNA for CAR-CLTX chimeric antigen receptors were inserted into the AAV1 safe locus of induced pluripotent stem cells (iPSCs), which were induced to differentiate into neutrophils. Conditioned media was collected along differentiation and went through the extraPEG EV isolation protocol. Isolated EVs were quantified by nanoparticle tracking analysis (NTA), electron microscopy, and western blot assay. Proteomics analysis and microRNA (miRNA) sequencing were performed for day 12 and 21 EV samples. EV cytotoxicity was tested through viability and proliferation assays performed with U87MG and LN229 glioblastoma cells.

Results, Conclusions, and Discussions: Results and

Discussion: NTA analysis shows that EVs are secreted in high quantities in the size range of 100-200 nm. Proteomics analysis indicate that neutrophil markers and proteins related to regulation of neutrophil migration, chemotaxis, and degranulation were identified in EVs. miRNA profiling of reveals high levels of miRNAs active in apoptotic and tumor suppressive pathways, including T cell receptor signaling pathway, cytokine-cytokine receptor interactions, Jak-STAT, Notch, Hedgehog signaling pathways, and natural killer cell-mediated cytotoxicity. Uptake analysis reveals successful EV uptake in a glioblastoma model. Further testing should reveal that CAR Neutrophil EVs are cytotoxic to multiple glioblastoma cell lines and organoids.

Conclusions: EVs of CAR Neutrophils contain anti-tumor cargo. EVs derived from these cells have shown uptake in glioma cells. These results demonstrate the feasibility to establish correlation between protein and miRNA cargo of CAR Neutrophil EVs and the function of EVs in GBM treatment, with the expected results of tumor cell cytotoxicity.

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