Lipid Nanoparticle Delivery System for mRNA Encoding B7H3-redirected Bispecific Antibody Displays Potent Antitumor Effects on Malignant Tumors

Purpose: Bispecific T-cell engagers (BiTEs) are promising but require large amounts of purified protein, have high manufacturing costs, poor in vivo stability, and short serum half-lives. mRNA delivery could enable continuous endogenous production of BiTEs, but efficient and safe delivery systems are needed. The study developed a novel ionizable lipid nanoparticle (LNP) for mRNA encoding B7H3×CD3 BiTE to achieve prolonged half-life and potent antitumor efficacy.

Hypothesis: If mRNA encoding B7H3×CD3 BiTE is encapsulated in novel IC8-based LNPs and administered intravenously, then the liver will produce high concentrations of functional BiTE, the BiTE half-life will be prolonged compared with recombinant BiTE, and robust antitumor effects will be achieved in both hematologic and solid tumor models.

Aims: Analyze B7H3 expression in AML and melanoma patient samples, databases, and tumor cell lines to support B7H3 as a target. - Synthesize and characterize a novel ionizable lipid IC8 and formulate LNP@mRNA; evaluate transfection, endosomal escape, and stability. - Evaluate in vivo biodistribution, pharmacokinetics, and endogenous BiTE production after intravenous LNP@BiTE-mRNA. - Assess antitumor efficacy, T-cell infiltration, anti-angiogenic/apoptotic effects, and safety in MV411 hematologic and A375 melanoma xenograft models.

Delivery system: Platform: Novel ionizable lipid nanoparticle (LNP) for mRNA delivery. - Key material: IC8, a pH-sensitive ionizable lipid synthesized by a simple one-step reaction; contains four ionizable N atoms and hydroxyl groups. - LNP composition: IC8, DSPC, cholesterol, and DMG-PEG2000 at molar ratios of 35%, 16%, 46.5%, and 2.5%, respectively. - Payload: Nucleoside-modified mRNA (N1-methylpseudouridine) encoding B7H3×CD3 BiTE, GFP, or luciferase. - Targeting: Liver-targeted after intravenous administration; no active targeting ligand reported. - Physicochemical properties: ~118 nm diameter, PDI 0.234, zeta potential ~+10 mV; stable at 4°C for at least 1 month.

Approach: In vitro: 293T, AML12, and LO2 cells for transfection; A375, MV411, THP-1, and SKOV3 tumor cells for BiTE-mediated cytotoxicity; human PBMC-derived T cells as effectors. - In vivo: NSG mice bearing MV411-Luc hematologic tumors or A375 melanoma subcutaneous xenografts. - Treatment groups: (a) normal saline + T cells, (b) 22 mg/kg IC8-LNP + T cells, (c) 1.5 mg/kg BiTE mRNA + T cells, (d) 6 mg/kg recombinant BiTE + T cells, (e) 1.5 mg/kg LNP@BiTE-mRNA + T cells; n = 5. - Dosing: Intravenous injection; T cells and IL-2 co-administered; tumor growth monitored by IVIS and caliper. - Safety: Body weight, H&E staining of major organs.

Key methods: NMR for IC8 structure; DLS and TEM for LNP size/morphology; gel retardation for mRNA loading. - CLSM for endosomal/lysosomal escape of LNP@CY5-mRNA. - FACS and fluorescence microscopy for GFP transfection efficiency. - IVIS for in vivo biodistribution of LNP@Luc-mRNA and tumor bioluminescence. - ELISA for serum BiTE concentration and pharmacokinetics. - xCELLigence real-time cytotoxicity assay for BiTE function. - FACS and IHC for tumor-infiltrating CD3+ T cells, CD31 (MVD), Ki-67, TUNEL, and B7H3 expression.

Key results: LNP@GFP-mRNA showed transfection efficiency comparable to Lipofectamine 8000 in 293T, AML12, and LO2 cells; serum presence did not affect transfection. - After IV LNP@Luc-mRNA, strongest luciferase signal was in liver at 6 h. - LNP@BiTE-mRNA produced serum BiTE peak of 6.455 ± 0.824 µg/mL at 6 h; half-life ~73 h vs ~2 h for recombinant BiTE; AUC ~146.6 h·µg/mL. - In MV411 model, LNP@BiTE-mRNA group had lowest tumor bioluminescence, no deaths, reduced peripheral B7H3+ cells (10.2 ± 2.14% vs 21.2 ± 2.23% for BiTE), and minimal liver metastasis. - In A375 melanoma model, tumor inhibition rate was 70% for LNP@BiTE-mRNA vs 50% for BiTE; MVD 17.2 ± 2.03% vs 29.2 ± 2.43%; Ki-67 LI 26.5 ± 2.6% vs 38.2 ± 2.2%; apoptosis index 60.4 ± 3.2% vs 43.3 ± 3.4%; CD3+ TILs 13.44% vs 8.86%. - No significant body weight loss or major organ lesions observed.

Interpretation: The authors conclude that this is the first study to use an mRNA-LNP platform to generate therapeutic B7H3×CD3 BiTE in the host, achieving prolonged antibody half-life and potent antitumor effects against hematologic and solid tumors. They propose IC8-LNP as a simple, efficient, and stable mRNA delivery system with potential for other therapeutic antibodies or proteins.

Limitations: Only immunodeficient NSG mouse xenograft models with human T cells were used; no immunocompetent or syngeneic tumor models. - Solid tumor regression was incomplete, possibly due to the acidic tumor microenvironment. - No large-animal validation or chronic toxicity evaluation. - Long-term follow-up beyond the 60-day trial was limited. - The study did not directly compare IC8-LNP with other clinically advanced ionizable lipids in vivo beyond Lipofectamine 8000 in vitro.