Chimeric antigen receptor (CAR) T cell therapy faces challenges including intratumor heterogeneity, dynamic expression of target receptors, and the inability of T cells to traffic to the tumor. In contrast to the lack of T cells, many tumors are abundant in macrophages. CAR macrophage has emerged as a new and promising cell therapy for cancer treatment. However, the preparation of autologous CAR macrophages is complex, time-consuming, and inefficient due to their non-dividing nature. Here, we propose a revolutionary strategy to generate CAR macrophages (ChARMs) in vivo against glioblastoma (GBM) using engineered exosomes loaded with mRNA encoding CAR targeting the mutant receptor EGFRvIII (C...

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Chimeric antigen receptor (CAR) T cell therapy faces challenges including intratumor heterogeneity, dynamic expression of target receptors, and the inability of T cells to traffic to the tumor. In contrast to the lack of T cells, many tumors are abundant in macrophages. CAR macrophage has emerged as a new and promising cell therapy for cancer treatment. However, the preparation of autologous CAR macrophages is complex, time-consuming, and inefficient due to their non-dividing nature. Here, we propose a revolutionary strategy to generate CAR macrophages (ChARMs) in vivo against glioblastoma (GBM) using engineered exosomes loaded with mRNA encoding CAR targeting the mutant receptor EGFRvIII (CARvIII), which is a tumor-specific receptor that is expressed in up to 40% of GBM. We previously showed that mRNA-loaded exosomes readily crossed the blood-brain barrier and restored functional protein expressions in GBM cells and tumor-associated macrophages. In this proposal, we will engineer exosomes containing mRNA for a second-generation CARvIII to produce ChARMs in vivo for treating GBM. When mouse macrophages were treated with CARvIII-mRNA exosomes, they expressed the CAR on their surfaces and exhibited enhanced phagocytosis against EGFRvIII+ GBM cells. Therefore, based on these preliminary results, we hypothesize that mRNA exosomes enable in vivo production of second-generation CAR macrophages and promote potent antitumor responses against GBM. To test this hypothesis, we will evaluate the persistence and specificity of in vivo-produced ChARMs. Next, we will perform transcriptomic and functional analyses of ChARMs and evaluate potential toxicities from the treatment. Finally, we will investigate the antitumor efficacy of CARvIII macrophages against GBM in mouse models. Our proposed study would be the first to investigate a strategy to generate ChARMs in vivo. The use of mRNA to generate CAR cells would greatly expand the utility of cell therapies for cancer treatment.

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