Background/Purpose: A major objective in autoimmunity is immune reset, requiring autoreactive B cell depletion in tissues. We designed an in vivo CAR product candidate (CPTX2309) utilizing a targeted lipid nanoparticle (tLNP) technology (CellSeekerTM), comprising proprietary ionizable lipid-based LNP targeted to engineer CD8+ T cells from normal subjects or autoimmune disease patients, by delivering encapsulated mRNA CD19 CAR (ACR Convergence, 2024). Utilizing NHPs and a surrogate product, we defined a compact dose regimen of two infusions 72 hours apart that resulted in rapid engineering of CD8+ T cells and profound B cell depletion in peripheral blood and tissues (ASGCT, 2025). Herein, we report mechanistic data generated on human T cells in vitro, in support of clinical translation of CPTX2309.

Methods: CPTX2309 contains an mRNA encoding for an all human anti-human CD19 CAR, in a CD8-targeted tLNP. Transcriptomic analysis was performed on T cells upon incubating CPTX2309 with human peripheral blood mononuclear cells (PBMC). Additional analysis was performed by depletion of cell subsets from PBMC utilizing immunomagnetic beads, followed by cytotoxicity and cytokine production analysis.

Results: CPTX2309 incubation with human PBMCs in vitro resulted in rapid (< 24 hr) and selective engineering of CD8+ T cells to express a CD19 CAR. This resulted in rapid depletion of autologous B cells (24-72hr timeframe). The interaction with autologous B cells also resulted in activation of CAR+ CD8+ T cells, namely augmentation of cellular metabolic programs including cellular proliferation and ribosomal translational activity. Transcriptomic analysis demonstrated the activation of several immune programs known to induce immune cell effector polyfunctionality – including cytotoxicity and the type II IFN pathway. In contrast, mCherry mRNA-loaded tLNPs showed minimal effect. Depletion of CD8+ T cells but not of any other cell subset (CD4+ T cells, NK cells or myeloid cells) within the PBMC population, resulted in profound diminution of B cell depletion as well as IFNγ production in culture, demonstrating that the tLNP-engineered CD8+ CAR T cells are the primary driver of efficacy and on-target activity of CPTX2309.

Conclusion: CellSeekerTM technology affords a tunable CAR exposure without the risk of prolonged on target toxicities or genotoxicity. These novel data including transcriptomic analysis shed light on the cellular and molecular mechanism of action of CPTX2309, explaining the high engineering rate and pharmacodynamic activity afforded by this platform technology. Together with preclinical data generated in mouse and NHPs, this supports the translation of CPTX2309 to clinic.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/potent-engineering-of-polyfunctional-cd8-t-cells-by-a-novel-in-vivo-car-mrna-product-candidate-cptx2309-in-a-targeted-lipid-nanoparticle-tlnp-utilizing-cellseekertm-technology/