(653f) Genetically Engineered Extracellular Vesicles with Chimeric Antigen Receptor of Human Stem Cell-Derived Neutrophils for Treating Glioblastoma

Glioblastoma multiforme (GBM) is the most commonly occurring tumor in the central nervous system, accounting for 80% of brain tumors. Work in gene editing and immunology has led to the development of chimeric antigen receptors (CARs). CARs employing a chlorotoxin receptor domain have recently been incorporated into neutrophils for GBM treatment. However, such cells are liable for inducing body wide off- target effects that can be deadly. Extracellular vesicles (EVs) secreted by the cells have been shown to share properties with their parent cells including the expression of CARs and tumor-lytic effects. However, this has never been investigated in CAR Neutrophils.

The DNA for the CAR-CLTX was inserted into the AAV1 safe locus of induced pluripotent stem cells, which were differentiated into neutrophils. Conditioned media were collected along the differentiation and went through the extraPEG EV isolation. EVs were quantified by nanoparticle tracking analysis, electron microscopy, and western blot. Proteomics analysis of protein cargo and microRNA (miRNA) sequencing of the EVs were performed for day 12 and 21 samples. The cytotoxicity of the EVs was performed with U87MG and LN229 glioblastoma cells in 2D culture and 3D organoids, as well as in vivo.

EVs are secreted in high quantities in the size range of 100-200 nm. The EVs expressed exosomal markers and displayed exosomal morphology. Proteomics analysis indicate that neutrophil markers and proteins related to regulation of neutrophil migration, chemotaxis, and degranulation were identified in EVs. miRNA profiling reveals high levels of miRNAs (e.g., miR-182) active in apoptotic and tumor suppressive pathways, including T cell receptor signaling pathway, cytokine-cytokine receptor interactions, Jak-STAT, Hedgehog signaling pathways, and natural killer cell-mediated cytotoxicity. EV uptake in an in vitro glioblastoma model was demonstrated. CAR Neutrophil EVs are cytotoxic to multiple glioblastoma cell lines and organoids, and in vivo.

In vitro testing confirmed the anti- tumor properties of CLTX- CAR EVs. 2D assays revealed that CLTX EVs were taken up effectively by glioblastoma cells and induced cell death in multiple glioblastoma lines. EV uptake and anti- tumor cytotoxicity was also confirmed in 3D tumor spheroids. RT- PCR revealed that CLTX- CAR EVs act, at least in part, by upregulating the transcription of the p53 and PTEN tumor suppression genes, suggesting that CLTX CAR EVs exert their effects through the induction of apoptosis and by inhibiting cell division by suppressing the PI3K/AKT pathway. We conclude that CLTX CAR EVs are potent candidates for cancer treatment based upon the proteomic, genomic, and in vitro investigations presented here.