Collaborative Research: Chimeric Antigen Receptor Engineered Neutrophil Extracellular Vesicles Targeting Glioblastoma

About This Grant

CAR T-cells exhibit some success in the treatment of some cancer types. The acronym CAR refers the fact that the T-cell has been engineered to express a novel receptor protein on its surface. This receptor protein allows the cell to recognize tumor cells and activate the T-cell to attack it. Neutrophils are another type of immune cell that may possess enhanced targeting to glioblastoma. Neutrophils also produce extracellular vesicles (EVs). EVs are membrane-enclosed particles that can carry some material found inside the cell to be delivered elsewhere. If EVs from neutrophils can be engineered with the CAR receptors that can target specific cancers, then the EVs could deliver a toxic payload to cancer cells. This project will develop CAR-engineered EVs, introduce potentially toxic cargos, and test their effectiveness. They will test the CAR EVs against two targets. The first is glioblastoma cells. The second involves brain organoids, which are clumps of neural cells that exhibit features of a fully developed brain. Seeding these organoids with glioblastoma cells will be used to approximate a brain tumor. The effectiveness of the CAR EVs against the "brain tumor organoid" will also be evaluated. Educational outreach activities will complement the research efforts. These will include outreach to high schools, involvement of undergraduates in research, and developing digital platforms targeting the training of industrial employees. The objective is to engineer chimeric antigen receptor (CAR)-associated extracellular vesicles (EVs) of induced pluripotent stem cell (iPSC)-derived neutrophils (Neu). CAR-Neu may possess enhanced targeting ability to glioblastoma using iPSCs engineered with chlorotoxin (CLTX), a glioblastoma-targeting peptide. EVs derived from CAR-neutrophils, i.e., Neu EVs, express a high level of cytotoxic molecules and better inhibit tumor growth, being safer than the CAR cells. The antitumor properties of EVs secreted by CAR-Neu with loaded microRNAs have not been investigated. The central hypothesis of this proposal is that CAR-Neu EVs carrying PDL1 and LMNB2 microRNA deliver their payload to glioblastoma and are able to reduce tumor significantly. Specifically, the following three sub-objectives are proposed: (1) To test the hypothesis that CAR engineered iPSC-Neu EVs from a scalable bioreactor contain anti-tumor cargo; (2) To test the hypothesis that CAR-iPSC-Neu EVs containing PDL1 and LMNB2 microRNAs exhibit strong cytotoxicity again glioblastoma cells; (3) To test the hypothesis that the dual microRNA CAR-iPSC-Neu EVs have strong anti-tumor ability in 3D human brain organoids bearing glioblastoma. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.