Background/Purpose: Chimeric Antigen Receptor (CAR) T-cell therapy has revolutionized cancer treatment and shown promise in addressing autoimmune diseases. However, current ex vivo CAR T-cell therapies encounter challenges such as complex manufacturing processes, scalability issues, reliance on viral vectors, and the necessity for lymphodepleting chemotherapy, which limit broader clinical applications. To address these limitations, there is a critical need for an off-the-shelf in vivo CAR technology that can simplify delivery and expand therapeutic potential. We have developed tLNP-circCAR, a novel in vivo anti-CD19-CAR circular RNA product delivered via targeted lipid nanoparticles (tLNPs). Preclinical studies in animal models demonstrate its efficacy in B cell depletion, marking a significant advancement towards overcoming existing barriers in CAR T-cell therapy and expanding its applicability to a broader range of diseases.

Methods: tLNP-circCAR encapsulates a circular RNA encoding an anti-CD19-CAR within a tLNP containing a novel ionizable lipid and a T cell targeting domain, enabling efficient transfection of primary human T cells. The functional activity of tLNP-circCAR-mediated CAR T cells was evaluated using multiple in vitro and in vivo systems.

Results: In activated primary human T cells, circCAR mediated CAR expression lasted up to 9 days. Notably, tLNP-circCAR efficiently engineered non-activated primary human T cells into functional CAR T cells. In vitro, these engineered CAR T cells exhibited robust cytotoxicity against autologous B cells and the CD19+ Raji cell line. In vivo, utilizing a humanized NOG-PBMC mouse model, a single intravenous dose of tLNP-circCAR resulted in efficient CAR engineering of T cells, leading to complete B cell depletion in the spleen. In humanized NOG-PBMC mice bearing CD19+ NALM-6-Luc lymphoma xenografts, tLNP-circCAR treatment induced complete tumor remission at the dose of sub-microgram per mouse. In cynomolgus monkeys, single dose of tLNP-circCAR treatment achieved remarkable B cell reduction in circulating whole blood, lymph node, bone marrow and spleen.

Conclusion: tLNP-circCAR represents a novel in vivo CAR therapy that delivers therapeutic anti-CD19 CAR circular RNA efficiently to human T cells, generating highly functional CAR T cells both in vitro and in vivo. These results support the further development of tLNP-circCAR for treating B cell disorders, including B cell lymphoma and autoimmune diseases. This innovative approach promises to enhance the accessibility and efficacy of CAR T-cell therapy.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/in-vivo-generation-of-anti-cd19-car-t-cells-utilizing-circular-rna-encapsulated-in-targeted-lipid-nanoparticles/