Targeted Lipid Nanoparticle Delivery of FAP-CAR mRNA Enables Potent <i>In Vivo</i> T-cell Engineering against Pancreatic Tumors

Fibroblast activation protein (FAP), which is highly expressed on cancer-associated fibroblasts (CAFs), is a promising therapeutic target to achieve normalization of the tumor microenvironment. We previously established an ex vivo retroviral-transduced FAP-specific chimeric antigen receptor (FAP-CAR) T-cell approach to deplete FAP+ CAFs that resulted in delayed tumor growth associated with disruption of desmoplastic matrix, enhanced immune-cell infiltration, and reversed immune exclusion and immunosuppression. Herein, we describe an in vivo strategy for generating FAP-CAR T cells using anti-CD5-conjugated targeted lipid nanoparticles (tLNPs) encapsulating FAP-CAR mRNA and assessed the efficacy of this approach compared to adoptive transfer of retrovirus-transduced CAR T cells in a preclinical model of pancreatic ductal adenocarcinoma. With transient CAR expression in >45% of splenic, >69% of circulating, and >35% of tumor-infiltrating T cells, the abundance of peripheral and intratumoral FAP-CAR+ T cells detected following a single intravenous dose of FAP-CAR mRNA tLNPs was greater than that detected following administration of 1x107 ex vivo retrovirally-transduced FAP-CAR T cells. Furthermore, in vivo mRNA CAR T-cell engineering resulted in as good or greater inhibition of tumor growth as compared to adoptive transfer of ex vivo retroviral-engineered T cells. Given that in vivo generation of CAR T cells resulted in transient CAR expression and circumvented the need for autologous T-cell isolation, viral vectors, and lymphodepletion, this platform represents a potentially safer, more accessible, and cost-effective method for targeting stromal cells to normalize the tumor microenvironment in desmoplastic tumors and has potential implications for tumor antigen-targeted CAR T cells.