AbstractBackground:T cell engagers (TCEs) have been clinically validated but are mostly limited to hematological cancers. Developing BiTEs for solid tumors faces hurdles such as T cell penetration into tissue, suppressive nature of the tumor microenvironment and systemic toxicity, etc. This study aims to tackle these challenges by delivering an mRNA-encoded TCE targeting the hepatocellular carcinoma antigen Glypican-3 (GPC3) directly to liver tissue, thus enhancing the local TCE concentration to promote anti-tumor activity and exhibiting a favorable pharmacokinetics (PK) profile.Methods:The BiTE was engineered by fusing single-chain variable fragments (scFvs) targeting GPC3 and CD3, separated by a flexible peptide linker. The mRNA encoding the BiTE was engineered using a combinatorial approach to optimize codon usage and enhance translational efficiency and stability, balancing the Codon Adaptation Index (CAI) and Minimal Free Energy (MFE). The mRNA was encapsulated in a novel lipid nanoparticle (LNP) for efficient liver-tropic delivery, forming the complex named MTS105. BiTE’s cell-killing activity was tested using human GPC3-expressing cell lines and human PBMC. Anti-tumor efficacy was evaluated in liver orthotopic tumor-bearing mouse models. GLP Tox studies including PK and biodistribution were assessed in rodents and cynomolgus monkeys.Results:Mechanistic studies demonstrated that MTS105-translated TCE mediated specific killing of GPC3-positive cells, T cell activation and proliferation in vitro and ex vivo. In the two liver orthotopic tumor-bearing mouse models, MTS105 achieved complete tumor regression in a dose-dependent manner, accompanied with intra-tumoral T cell activation. In mice, rats and cynomolgus monkeys, MTS105-translated TCE consistently exhibited higher exposure levels in the liver than the plasma. The sustained release of MTS105-translated TCE resulted in higher functional protein exposure in liver/tumor, while showing lower peripheral TCE Cmax (maximum concentration) and AUC (area under the curve) than the Fc-domain containing antibody-based TCE. In the cynomolgus monkeys, MTS105 displayed favorable and linear plasma PK profiles including mRNA, lipid and translated TCE in the up to 100 μg/kg following a single and 4-time repeat dosing weekly. The time to peak (Tmax) was 6 to 12 h in both periphery and targeting tissues, which correlated with the observation of fast Intra-tumoral T cell activity in mice. All monkeys tolerated well to a dose up to 100 μg/kg, with no sever adverse effects and gross pathology observed.Conclusion:The mRNA-encoded TCE demonstrated optimal tissue/tumor-specific PK, safety and potent anti-tumor activity in preclinical models. These preclinical studies enabled a first-in-human study to assess its safety and preliminary efficacy which is currently ongoing.Citation Format:Yan Huang, Shaoli Liu, Xiaoju Zhang, Bingxu Zhang, Hongya Han, Andong Liu, Xiaoyun Ma, Wei Xu. Preclinical development of an mRNA-encoded T cell engager targeting Glypican-3 with tissue- and tumor-specific enrichment in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3502.