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Drug Delivery

Drug Delivery - Poster Session E

Poster X7 - Lipid Nanoparticle-Mediated Delivery of Synthetic Co-Stimulatory Molecules to Enhance Anti-Tumor Immune Response

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Immunotherapies based on Chimeric Antigen Receptor (CAR) T-cells and Immune Checkpoint Blockade (ICB) have revolutionized cancer treatment. Ex vivo engineered CAR T-cells enhance immune activity, while ICB blocks inhibitory molecules such as PD-1 and CTLA-4 to boost endogenous immune responses. Despite clinical successes, therapeutic resistance often develops due to antigen escape, lack of functional T-cell persistence, and immune suppression in the hostile tumor environment. To improve T-cell persistence, the Hamieh lab engineered a synthetic co-stimulatory molecule by fusing the ectodomain of CD80 with the endodomain of 4-1BB, termed 80BB (Fig.1). Co-expression of 80BB with an HLA Independent TCR, an antigen-specific TCR, and most importantly patient-derived TILs relying only on endogenous TCRs, extended survival and sustained effective anti-tumor response in mice. These findings prompted us to hypothesize that in situ delivery of 80BB to T-cells would improve anti-tumor function without the need for cumbersome ex vivo processing. To this end, we leveraged versatile mRNA-encapsulating lipid nanoparticles (LNPs) to target T-cells and provide safe delivery of synthetic co-stimulatory molecules in vivo. A significant issue in the LNP field is cell-type specific targeting, requiring extensive ionizable lipid screening or cumbersome antibody conjugation. In an effort to achieve targeting without extra processing steps, we incorporated glycolipids into our novel LNP formulation and improved T cell transfection. We aim to further test and optimize this formulation to determine the potential for specific in vivo targeting, with the goal to deliver mRNA encoding 80BB to provide a boost in T cell anti-tumor efficacy in situ.

Materials and

Methods:  To formulate LNPs and test transfection, we generated mRNA for mScarlet using an in vitro transcription kit and a pUC19 vector with a T7 promoter. For all luminescence experiments, we purchased luciferase mRNA from ApexBio. All LNPs were formulated using microfluidic mixing and subsequent buffer exchange using dialysis. For antibody conjugated particles, anti-mouse or anti-human CD5 antibodies were purchased from BioLegend and conjugated to LNPs using TCEP. For in vitro experiments, primary human T cells were isolated from PBMCs using negative selection with an antibody cocktail and magnetic beads. For in vivo experiments, C57BL/6J mice were obtained from Jackson Laboratories and injected with PBS, gLNPs, LNPs, or Ab-LNPs via tail vein injection. Mice were then injected 6 hours later with luciferin and imaged with IVIS. Bioluminescence was quantified using LivingImage software.

Results, Conclusions, and Discussions: To achieve T cell targeting, we elected a glycolipid that is a ligand L-selectin, which is expressed highly on naïve and memory T cells. To test whether we could incorporate the glycolipid into the LNPs (gLNPs) without disrupting the structure, assembly, or transfection process, we first tested a range of different percentages of glycolipid. We then tested these particles on primary human T cells and identified the optimal molar concentration of glycolipid along with the optimal ionizable lipid for T cell transfection in vitro. The current most selective LNPs for T cell targeting in the literature are anti-CD5 antibody-conjugated LNPs. We tested our gLNPs compared to these and a standard LNP in vitro and observed the highest transfection from gLNPs in both cases (Fig. 2). These data suggest that our gLNPs have improved transfection efficiency compared to other industry standard formulations and may exhibit targeting specificity of L-selectin expressing T cells. These results show promise for the eventual delivery of 80BB with our novel gLNP formulation, and we plan to perform further in vivo biodistribution experiments to assess the targeting capability and transfection efficiency of the particles in mice.

Acknowledgements (Optional):