(L-456) Paving the Way for CAR Macrophages: A Novel Lipid-Based Therapy for Enhanced Glioblastoma Treatment

Introduction: Glioblastoma (GBM), the most common and deadly primary brain cancer, accounts for approximately 45% of such tumors and presents a daunting median survival of 15 months post-diagnosis. Conventional treatments, namely surgery, radiation, and chemotherapy, are invasive and induce severe off-target toxicities. Chimeric Antigen Receptor (CAR) T-cell therapy has emerged as a promising alternative for GBM treatment. However, the therapy's effectiveness is compromised by the tumor microenvironment's (TME) immunosuppressive nature, densely populated by M2-phenotype tumor-associated macrophages (TAMs) contributing significantly to immunosuppression, thus establishing an 'immunological cold site'. Our proposed innovative approach is the delivery of CAR mRNA to TAMs within the GBM TME. This strategy aims to transform these TAMs to adopt an M1- or pro-inflammatory phenotype, consequently generating CAR macrophages and creating an 'immunological hot spot'. This conversion could significantly enhance the efficacy of CAR T-cell therapy for GBM.

Yet, the actualization of this innovative approach is contingent upon overcoming a principal biomedical challenge: the non-invasive delivery of CAR mRNA across the formidable blood-brain barrier (BBB). Lipid nanoparticles (LNPs), due to their small size, hydrophilic surface, and ability to encapsulate and protect mRNA, show promise in surmounting this barrier, facilitating the delivery of their mRNA cargo within the brain tissue. Traditional intravenous delivery of LNPs may not ensure optimal BBB penetration. Hence, we aim to explore intranasal administration. Given the nasal cavity's close proximity to the brain, intranasal administration offers a unique opportunity to bypass the BBB entirely, traversing the olfactory epithelium, thereby proposing an alternative efficient drug delivery route.

Materials and Methods: We began by generating GBM mouse models. U87 cells, which were GFP-labeled to indicate tumor cells within the GBM TME, were cultured in DMEM, supplemented with 10% FBS and 2% pen-strep, in a controlled environment at 37°C and 5% CO2. Upon reaching optimal confluence, one million cells were intracranially injected into NSG mice (n=12). Post-operative care was administered, allowing mice to recover for one week.

Following model preparation, our research compared two lipid nanoparticles (LNPs): one formulated with the C14i-494 ionizable lipid - a uniquely structured lipid showcasing promising transfection capabilities in other cancers, and the other with the C12-200 ionizable lipid, widely recognized as the gold standard for mRNA delivery. Both LNPs were formulated with mCherry mRNA to track mRNA translation and localization relative to the GFP-labeled tumor sites.

We delivered LNPs to our GBM mouse models via intranasal (3 mice), intravenous (IV) (3 mice), and intraperitoneal (IP) injections (3 mice), each at a dosage of 0.3 mg mRNA/kg. Biodistribution and therapeutic action of LNPs were monitored at 6 hours and 24 hours post-injection - crucial time points aligning with the known peak activity and degradation periods for mRNA, respectively. Fluorescence signals were quantified using an IVIS system, allowing real-time monitoring.

To confirm mRNA translation within the tumor cells of the GBM TME, we utilized confocal microscopy. Using a liv-dead stain, we were able to differentiate between live and dead cells. Our results displayed mCherry translation predominantly located within or near GFP-labeled tumor cells, suggesting a specific targeting mechanism within the GBM TME.

Concurrently, we are undertaking a second biodistribution study to map the LNPs' journey throughout the body, aiming to optimize LNP formulation and delivery while assessing potential off-target toxicities. The findings of these investigations will guide the further development and refinement of this promising approach for GBM treatment.

Results, Conclusions, and Discussions: In this study, we aimed to investigate the potential of lipid nanoparticles (LNPs) as vehicles for mRNA delivery to tumor-associated macrophages (TAMs) within the glioblastoma (GBM) tumor microenvironment (TME). Confocal microscopy results confirmed successful mRNA translation, with notable mCherry translation primarily located within or near GFP-labeled tumor cells. Regions of the brain devoid of tumor cells exhibited no mCherry translation, underscoring the specificity of the LNPs in targeting the tumor cells within the GBM TME. Our present findings, however, do not differentiate LNP uptake between M2 phenotype TAMs and other macrophage phenotypes present within the GBM TME. To interrogate this, we are inducing differentiation of primary monocytes into M1-, M2-, and tumor-associated macrophages. These cells will subsequently be exposed to C14i-494 and C12-200 LNPs carrying CAR mRNA. This investigation should yield key insights into the roles of these cells in immune response downregulation, which remain largely undefined.

We demonstrated a notable fluorescence signal enhancement using C14i-494 LNP relative to the established C12-200 LNP. Delivery methods - intravenous (IV), intranasal (IN), and intraperitoneal (IP) - were evaluated, with IN administration generating the highest fluorescence signal within the brain. This suggests that IN administration, circumventing the blood-brain barrier, may provide a superior method for GBM treatment over conventional IV LNP administration. Notably, diminished fluorescence signals were observed in other organs, indicating a potential decrease in off-target toxicities with this lipid-based therapy.

Our upcoming investigations aim to further corroborate the potential of IN LNP administration for GBM treatment. This involves comprehensive biodistribution studies with an increased number of replicates, initially using a reporter gene to conserve the therapeutic CAR mRNA. Following biodistribution validation, we will transition to assessing the therapeutic efficacy of LNPs encapsulating CAR mRNA in vivo. This systematic approach intends to facilitate a smooth transition of our novel therapy from preclinical stages to clinical trials, supported by robust data.