Engineering PBAE nanoparticles for immunological reprogramming in clinically relevant humanized mouse models.

Abstract

e14042 Background: Standard of care for high-grade brain tumors involves surgical resection, followed by radiotherapy, leading to long recovery times. Nanoparticle (NP) gene therapy may provide a less invasive and targeted immune response to eliminate brain tumors. Antigen-presenting cells (APCs) present T cells with antigens alongside costimulatory proteins (signal 2) and cytokines (signal 3), activating naive T cells. Plasmids encoding Signal 2 (4-1BBL) and Signal 3 (IL-12) can be delivered via poly(beta-amino ester) (PBAE) nanoparticles (NPs) into tumor cells, reprogramming them into tumor-associated APCs (tAPCs). This results in increased T-cell affinity to tumors and enhanced anti-tumor immune responses without prior knowledge of tumor antigens. Methods: PBAE NPs were delivered to IOMM-Lee (human meningioma) and CT-2A (mouse glioma) cells in vitro and in vivo. Delivery of GFP/luciferase genes confirmed gene expression, and delivery of 4-1BBL/IL-12 confirmed immune responses. In mouse models, CT-2A cells were implanted in C57BL/6 mice and IOMM-Lee in MHC I/II double knockout mice humanized with peripheral blood mononuclear cells (PBMCs). Results: PBAE NP delivery of GFP DNA successfully transfected CT-2A and IOMM-Lee cells across varying dosages and mass ratios. Upon co-culturing IOMM-Lee cells with healthy human astrocytes, we found that the PBAE NPs transfected the cancer cells at a higher rate than the healthy brain cells. Next, tumor reprogramming was evaluated in vitro . 4-1BBL and IL-12 were delivered into IOMM-Lee or CT-2A cells, which were then co-cultured with PBMCs or splenocytes, respectively. These co-cultures demonstrated that the 4-1BBL/IL12 (tAPC) NPs elicited an increase in IFNγ, tumor cell killing, and MHC II presentation, indicating T cell activation and tumor reprogramming. Prior to survival studies, we performed both subcutaneous and orthotopic implantations of IOMM-Lee and CT-2A cells into mice and confirmed reporter gene expression. Finally, IOMM-Lee cells were implanted subcutaneously in humanized mice, while CT-2A cells were implanted orthotopically in C57BL/6 mice, and both were treated with tAPC NPs vs control (luciferase/GFP) NPs. In both models, we observed extended survival in mice receiving the tAPC NPs. Conclusions: These results demonstrate that PBAE tAPC NPs can transfect human and rodent brain tumor cells in vitro and in vivo , generating an anti-tumor effect.