(W1230-09-55) Formulation Development of Novel T-Cell Targeting Ligand-Conjugated Lipid Nanoparticles for In Situ CAR-T Cell Therapy Using Model mRNAs

Purpose: In-situ generation of CAR-T cells via systemic delivery of mRNA-loaded lipid nanoparticles (LNPs) represents a transformative approach to immunotherapy. Unlike conventional ex vivo CAR-T therapies that require complex and costly cell manufacturing processes, in-situ strategies enable direct programming of T-cells within the body, offering the potential to significantly reduce cost of goods (COGs), simplify logistics, and broaden patient accessibility. However, a major challenge in realizing this approach is the efficient and selective transduction of T-cells in vivo. T-cells are notoriously difficult to transfect due to their low endocytic activity. To address this, we developed a novel LNP formulation conjugated with T-cell targeting ligands, including CD3 and CD8 to enhance T-cell specific delivery of mRNA. This study aimed to evaluate ligand type and density, and drug product (DP) buffer composition on the transduction efficiency of our novel LNPs using model mRNAs encoding luciferase (Luc-mRNA).

Methods: mRNA encapsulated T-cell specific ligand conjugated LNPs were formulated using a proprietary ionizable lipid (IL) and post-conjugation of T-cell specific Fab fragments via Anchor-PEG lipids on the LNP surface. In brief, LNPs were prepared using the Ignite microfluidic mixing system from Precision Nanosystems Inc. (PNI), employing Luc-mRNA in buffer and lipids composition in ethanol including IL, helper lipids and PEG lipids. Following formulation, buffer exchange was performed via dialysis to remove ethanol and adjust the formulation to the desired DP buffer. T-cell specific Fab fragments were conjugated to the LNPs surface as post-conjugation using Anchor-PEG lipids through Copper-free click chemistry. Finally LNPs were stored at -80C° condition before use. LNPs size and mRNA encapsulation efficiency were evaluated by DLS and Ribogreen assay, respectively. The ligand conjugation efficiency on the LNP surface was evaluated Reverse Phase (RP)-UV/Charged Aerosol Detector (CAD) HPLC method. The binding affinity was assessed using ELISA assays. In vitro T-cell transduction potency and cytokine assay was evaluated by luciferase activity and INF-γ expression using Pan Tcell and human Peripheral Blood Mononuclear Cells (PBMCs). DP buffer composition was screened to assess its influence on LNPs stability and mRNA delivery efficiency.

Results: Luc-mRNA encapsulated in T-cell targeting ligand conjugated LNPs exhibited a monodisperse particle size of less than 100 nm, demonstrated high mRNA encapsulation efficiency and ligand conjugation efficiency, and showed higher binding affinity compared to the ligand alone (Figure 1). The activity of T-cell transduction significantly improved by ligand attachment on the LNPs surface and changed depending on the ligand type (Figure 2). Furthermore, the density of the CD3 Fab ligand significantly influenced transduction efficiency, with optimized Fab density yielding the highest expression levels (Figure 2). LNPs lacking Anchor-PEG lipids, and consequently not having Fab attached to the surface, showed negligible T-cell transduction, underscoring the importance of proper ligand attachment. DP buffer composition also played a pivotal role, with certain buffers enhancing mRNA transduction efficiency.

Conclusion: This study successfully established a ligand-conjugated LNP platform for in-situ CAR expression using model mRNAs. The evaluation system for Fab conjugation was identified as a critical factor in formulation design. Ligand type and density, as well as DP buffer composition, were shown to significantly affect transduction outcomes. These findings support the feasibility of in-situ CAR-T generation via systemic LNPs delivery and provide a foundation for further clinical translation.

Figure 1. ELISA-based competitive binding assay results for CD3 Fab ligand and ligand conjugated LNPs

Figure 2. Screening results of Fab type and density on cell potency and cytokine expression in Pan Tcell and PBMCs.