Lipid Nanoparticle Delivery of CAR276 mRNA Enables Antigen-Specific Cytotoxicity in T Cells Against Glioblastoma Cells

Background/Objectives: Glioblastoma multiforme (GBM) remains the most aggressive and treatment-refractory primary brain tumor, with current therapeutic modalities providing minimal survival improvement. Chimeric antigen receptor (CAR)-based immunotherapies succeeded in hematologic malignancies but are constrained in solid tumors by tumor heterogeneity, immunosuppressive microenvironment, and delivery challenges. CD276 (B7-H3), a pan-cancer antigen overexpressed in gliomas and correlated with poor patient outcomes, represents a compelling immunotherapeutic target. Methods: Here, we developed a non-viral lipid nanoparticle (LNP)-based platform for transient CAR mRNA delivery to T cells and evaluated its application for generating CD276 (B7-H3)-targeted CAR-T cells against GBM. An in-house-assembled T-mixer incorporating polyether ether ketone (PEEK) tubing was integrated with a dual-syringe-pump system for controlled LNP production. Results: LNPs formulated with clinically relevant ionizable lipids, SM-102 and DLin-MC3-DMA, demonstrated high encapsulation efficiency and effective mRNA delivery. Cell-type-dependent expression kinetics were further characterized following LNP-mediated reporter mRNA delivery in K562, NK, NK92, and T cells, revealing differences in transgene expression across cell populations. LNP-mediated delivery of CD276 CAR mRNA enabled generation of CAR-expressing T cells that exhibited antigen-specific cytotoxicity against CD276-positive LN229 GBM cells in vitro. Conclusions: Collectively, this study demonstrates the feasibility of an adaptable LNP-based platform for non-viral CAR mRNA engineering of T cells and CD276-targeted cytotoxicity against GBM cells in vitro. The combination of transient CAR expression and LNP-mediated mRNA delivery provides a foundation for further optimization and evaluation in preclinical in vivo models.