In Vitro and In Vivo Activity of a Lipid Nanoparticle System for the In Vivo Generation of CAR T Cells

Background: CAR T-cells have shown impressive clinical responses in patients with hematologic malignancies, but barriers exist to broader patient access, these include personalized manufacturing, toxicity, and preconditioning chemotherapy. The current methods used to generate a CAR-T cell dose ex vivo are complex and hinder widespread application. Lipid nanoparticle technology has demonstrated the ability to deliver mRNA payloads to cells. A therapeutic platform based on that technology could provide an off-the-shelf option for generating therapeutic T cells in vivo, enabling broader patient access without costly personalized manufacturing. Here we share the key capabilities and data for a targeted lipid nanoparticle (LNP) encapsulating mRNA to reprogram circulating human T-cells in vivo, designed to overcome the significant limitations of current CAR T therapy. Methods: The LNP consists of a proprietary ionizable lipid formulation containing an mRNA encoding a second-generation CAR against CD22. The ionizable lipid has been selected from a panel of candidate lipids for the efficient transfection of T cells. Transfection of T cells is then enabled by a CD8 targeting moiety attached to the surface. The LNPs were screened in vitro using cell lines and human primary T cells, and in vivo using PBMC humanized mice. Transfection specificity was examined in human whole blood assays and in PBMC humanized mice. Results: LNPs made from a series of ionizable lipids were capable of in vitro transfection of human T cells to express reporter proteins (GFP, mCherry). LNP performance was dependent on ionizable lipid chemistry, including; chain length, branching, ester linkages, and head group. Untargeted lipid nanoparticles were not capable of transfecting T cells, indicating that the inclusion of a T cell targeting moiety was essential for transfection. Our targeted LNPs transfect up to 80% of CD8+ T cells in culture. CAR expression lasts for multiple days, with minimal loss of viability and no nonspecific activation. In a whole-blood assay, CAR expression was limited to CD8+ cells (T cells and NK cells), with no CAR expression observed in CD4+ T cells, B cells, or granulocytes. CAR transfection resulted in robust antigen specific killing in vitro. Against a Nalm-6 cell line, CAR-transfected human T cells induced cytotoxity at multiple E:T ratios, killing over 90% of the target cells. In a repeat transfection experiment, we maintained the same population of T cells in culture under repeat challenge from nalm-6 cells. The cultured T cells maintained cytotoxicity against the target cells under repeated transfection with the LNPs. This demonstrates both the cytotoxicity of the CAR T cells, and that repeated transfection in culture does not induce disfunction. The capabilities of the LNP system have also been demonstrated in multiple in vivo models. In PBMC humanized mice, LNP dosing resulted in serial expression of CAR in over 80% of circulating human CD8 T cells. Expression of a CAR resulted elimination of any remaining B cells from the PBMC population without severe systemic toxicities. Both CAR expression and B cell aplasia were maintained over multiple doses in the same model. Anti-tumor activity was also observed against a B cell tumor model in the same humanized mouse model. Conclusions: Here we have demonstrated a targeted LNP system optimized for human T cell transfection. We have also shown that mRNA payload expression depends on multiple properties of the ionizable lipid within the LNP formulation. Our targeted LNP is capable of specific expression of a CAR on human T cells that induces antigen specific cell killing. These capabilities offer the potential to expand the population of patients addressable with CAR-T therapies, and fundamentally alter how patients experience cellular therapies.