CAR neutrophils produced in vivo to remodel tumor microenvironment and treat glioblastoma

Project Summary Glioblastoma (GBM) is the most aggressive type of cancer that occurs in the brain. While functional anti- cancer therapeutics, such as emerging chimeric antigen receptor (CAR)-T and natural killer (NK) cell therapies, have been developed to treat various cancers, their therapeutic applications for brain cancers have been impeded by the blood-brain barrier (BBB) and immunosuppressive tumor microenvironment. Due to their native ability to cross BBB and penetrate the brain parenchyma, neutrophils have been recently used as carrier cells to deliver imaging and therapeutic drugs into brain tumors. Recently, the investigators engineered a GBM-targeted CAR-neutrophils from human pluripotent stem cells (hPSCs) for the first time and demonstrated their superior antitumor activities in animal models of GBM as compared to CAR-NK cells. However, the limitation of such approaches is the expensive and strenuous in vitro process required for generating the engineered cells. Currently, a significant gap remains in our understanding and programming of tumor-associated neutrophils within the tumor microenvironment (TME) in vivo. In this proposal, the investigators will harness the power of synthetic biology, unbiased machine learning, next-generation sequencing, murine and canine models to interrogate tumor-associated neutrophils and develop new strategies to program them towards antitumor effector cells. Our preliminary data shows that synthetic nucleoside-modified messenger RNA (modRNA) could be specifically delivered to circulating neutrophils via lipid nanoparticles (LNPs) or exosomes and produce effective antitumor CAR-neutrophils directly in vivo. The central hypothesis of this proposal is that modRNA CARs specific for glioma cells will direct neutrophils to remodel TME and extend the lifespan of tumor-bearing animals. To test this hypothesis, we will first develop and optimize neutrophil-specific CAR constructs with machine learning algorithms in Aim 1. Then in Aim 2, we will determine the antitumor activities of combinatory CAR-neutrophils and radiation or CAR-T cell therapy in murine models. In Aim 3, we will evaluate the safety and therapeutic efficacy of in vivo produced CAR-neutrophils in pet dog patients with spontaneous glioma. It's expected that this study will lead to the establishment of a novel in vivo neutrophil programming platform, providing proof-of-concept for modRNA CAR-neutrophil immunotherapy.