R39 - Lipid nanoparticle delivery of GPI-anchored T cell engagers to potentiate anti-tumor response

Saturday, October 11, 2025

10:00 AM - 11:00 AM PDT

Location: Exhibit Hall F, G & H

Presenting Author(s)

Sydney K. Fabrega

Undergraduate Student
Georgia Institute of Technology
Atlanta, Georgia, United States

Co-Author(s)

Ali Zamat, PhD

Research Engineer II
Georgia Institute of Technology, United States
RS

Riya Sen

Graduate Student
Georgia Institute of Technology, United States
MB

Meredith Brienen

Undergraduate Student
Georgia Institute of Technology, United States
CT

Chloé Thiveaud

Graduate Student
Georgia Institute of Technology
Atlanta, Georgia, United States
CC

Ching Shen Chan

Graduate Research Assistant
Georgia Institute of Technology, United States

Last Author(s)

Gabriel Kwong, PhD

Associate Professor of Biomedical Engineering
Georgia Institute of Technology, United States

Primary Investigator(s)

Gabriel Kwong, PhD

Associate Professor of Biomedical Engineering
Georgia Institute of Technology, United States

Introduction: : Head and neck cancers (HNC), including head and neck squamous cell carcinoma (HNSCC), are a highly aggressive and genetically heterogeneous group of tumors that remain difficult to treat. Despite the availability of surgery, radiation, and immunotherapy, treatment outcomes are poor, with a high rate of locoregional recurrence due to minimal residual disease and the emergence of radiation-resistant subclones. Though CAR T cell therapies are highly efficacious at targeting liquid tumors, the lack of widely expressed tumor associated antigens (TAAs), high costs associated with ex vivo engineering, and side effects of systemic delivery pose challenges to effectively treating solid tumors. Despite these roadblocks, HNSCC’s focality provides the opportunity for an intratumoral (i.t.) strategy to redirect endogenous T cells against a wide variety of HNC. We developed lipid nanoparticles (LNP) containing mRNA encoding a GPI-anchored T cell engager (GATE). Once injected intratumorally, GATE LNPs induce transient expression of an α-CD3e single chain variable fragment (scFv) in tumor cells and potentiate an anti-tumor response through engaging the T cell receptor (TCR) on endogenous T cells. GATEs offer a local, antigen-independent therapy that reduces economic burdens on patients.

Materials and

Methods: : We designed a DNA construct encoding the GATE with a poly-A tail that we linearized before synthesizing GATE mRNA through in vitro transcription (IVT) with T7 polymerase. We then formulated LNP with the ionizable lipid cKK-E12, cholesterol, PEGylated lipid, and phospholipid at a 35:46.5:2.5:16 molar ratio and a lipid-to-GATE mRNA mass ratio of 20:1.
FaDu cells were transfected in vitro with GATE LNP at 100 ng and 500 ng doses, and transfected cells were then stained with protein L every day for 7 days to characterize GATE expression kinetics. To validate T cell-mediated killing, 24 hours after LNP transfection, FaDu cells were cocultured with WT human T cells, and another 24 hours after coincubation, cytotoxicity was measured through quantification of lactate dehydrogenase (LDH) in spent media.
To validate GATE targeting in vivo, NSG mice were inoculated in the flank with GATE+ or WT FaDu cells and once tumors reached ~100mm3, 5x106 WT human T cells or saline were delivered intravenously (i.v). To assess GATE LNP therapy in vivo, NSG mice were inoculated with WT FaDu flank tumors. Once tumors reached ~100mm3, mice received 2 ug i.t. injections of GATE LNP or a control Fluc LNP and 5e6 WT human T cells or PBS i.v..

Results, Conclusions, and Discussions:: In vitro, transfected FaDu cells showed sustained expression on the tumor cell surface for three days at 100 ng and 500 ng doses (Figure 1A). 24 hours after LNP transfection, FaDu cells that received WT human T cells showed >70% cytotoxicity regardless of GATE LNP dose (20-500 ng) (Figure 1B). Though differences in cytolysis between LNP doses were negligible, expression of GATEs on the cell surface exhibited a dose response. In vivo, almost all mice bearing GATE+ FaDu flank tumors that received WT human T cells displayed complete tumor clearance 13 days after ACT (Figure 1C), whereas all mice in control groups reached euthanasia criteria 27 days post ACT. In mice bearing WT FaDu flank tumors treated with GATE LNP and WT human T cells, all mice showed significantly delayed tumor growth over control groups (Figure 1D), demonstrating that GATE therapy can redirect WT T cells to target tumors, independent of endogenous antigen expression.
We have demonstrated that induced GATE expression can redirect T cells to potentiate tumor cytotoxicity in vivo in a transgenic model. Moving forward, we will characterize the kinetics of in vivo delivery of GATE LNP to optimize the dosing regimen and improve therapeutic outcomes. Additionally, we will develop a murine GATE to engage endogenous T cells in an immunocompetent model. Future iterations will employ immunostimulatory molecules like decoy-resistant IL-18 (DR-18) to overcome immunosuppression in the tumor microenvironment and induce pyroptosis. GATE therapy shows promise as a novel, off-the-shelf therapy that is applicable across many tumor types, reduces patient costs, and utilizes a form of drug delivery already FDA-approved and widely administered.

Acknowledgements and/or References (Optional): :