Background/Purpose: Ex vivo chimeric antigen receptor (CAR) T cell therapies have revolutionized cancer treatment and are demonstrating clinical efficacy in various autoimmune disease indications. Despite the success of current CAR T cell therapies, challenges in cell manufacturing, scalability, utilization of integrating viral vectors, and the need for lymphodepleting chemotherapy highlight the necessity for an off-the-shelf in vivo CAR technology applicable to broader indications. To that aim, we developed a novel in vivo anti-CD19 CAR mRNA product delivered by a CD8-targeted lipid nanoparticle (tLNP) (CPTX2309) optimized for performance in the preclinical setting with a goal to advance CPTX2309 to clinical development.

Methods: CPTX2309 comprises a fully human anti-CD19 CAR mRNA payload encompassed by a tLNP containing a novel ionizable lipid and a CD8 targeting antibody enabling effective CAR transfection of CD8+ T cells, designed for transient engineering and tunability of the dose-regimen. We evaluated the tLNP-mediated CAR engineering and functional activity of CAR T cells produced from the peripheral blood of healthy donors in vitro and in vivo.

Results: CPTX2309 selectively and efficiently engineered non-activated primary human CD8+ T cells into functional CAR T cells in vitro. These engineered CAR T cells demonstrated reproducibly high CAR expression levels and robust cytotoxicity against both autologous B cells and CD19+ cell lines. This was coupled with antigen-specific T cell activation, proliferation, and polyfunctionality manifested through cytokine production. When compared to CAR T cells produced via viral transduction, CPTX2309-engineered CAR T cells demonstrated superior CAR expression and more rapid activation and killing of CD19+ target cells. To determine if this rapid pharmacological activity translated in vivo, we utilized the humanized NSG-PBMC mouse model. Animals administered CPTX2309 by intravenous injection exhibited specific and efficient CAR engineering of CD8+ T cells, leading to rapid and profound B cell depletion in blood and tissues by 3 hours post-treatment. Single dose studies in the same model revealed a dose-effect relationship, with a dose as low as 0.075 mg/kg depleting the majority of human B cells in the spleen. To evaluate the efficacy of CPTX2309 in a model with reconstitutive capacity for human B cells, CPTX2309 was dosed into NSG-CD34+ mice. Repeat dosing every three days at 1.5 mg/kg for three infusions was well tolerated, showed selective CD8+ T cell engineering, and led to effective elimination of all human B cells in the blood.

Conclusion: CPTX2309 is a novel in vivo CAR therapy aimed to bring together the unprecedented potency of CAR constructs with scalability and feasibility of this novel tLNP-mRNA platform. CPTX2309 rapidly, efficiently, and specifically delivers a therapeutic anti-CD19 CAR mRNA payload to human T cells resulting in highly functional CD8+ anti-CD19 CAR T cells in vitro and in vivo. These data support the development of CPTX2309 for a broad range of B cell disorders including autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/a-novel-product-candidate-cptx2309-for-in-vivo-mrna-engineering-of-anti-cd19-car-t-cells-utilizing-novel-cd8-targeted-lipid-nanoparticles/