(L-476) Antibody Targeted Lipid Nanoparticles for Selective Organ Delivery

Ionizable lipid nanoparticles (LNPs) have emerged as the most clinically advanced platform for therapeutic delivery of nucleic acids. Despite their popularity, many LNP therapies have failed in clinical trials, with persisting challenges including insufficient drug delivery at the region of interest and off-target delivery in distal tissues. Specifically, off-target delivery is a major concern for pregnant patients, as widespread delivery of a payload could have detrimental effects for the pregnancy itself, the mother’s health, and the health of the fetus. The development of antibody targeted LNPs via modulation of the LNP surface with affinity ligands serves as a promising platform to promote selective LNP delivery to a specific tissue or cell type of interest. During many obstetric complications, such as preeclampsia, enhanced expression of the epidermal growth factor receptor (EGFR) is observed, motivating its use as a target for selective delivery to the placenta during pregnancy. In this work, antibody targeted LNPs are developed and characterized against standard non-targeted formulations. EGFR-targeted LNPs are then screened in vitro for enhanced mRNA delivery in placental cells, validating their potential as a potent and targeted drug delivery vehicle during pregnancy.

A library of 6 LNPs was formulated at a weight ratio of 10:1 ionizable lipid: mRNA. For all formulations, the ionizable lipid C12-494, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (Lipid-PEG) were used in the ethanol phase. Control LNPs included a standard, non-targeted formulation S1 and a standard azide LNP formulation A1 (azide, no antibody). For particles to be conjugated with antibody, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG-Azide) was also added to the ethanol phase with varying ratios of DSPE-PEG-Azide to Lipid-PEG (1:7, 1:5, 1:3, 1:2). LNPs encapsulating luciferase mRNA were synthesized via microfluidic mixing. EGFR IgG1 and CD3 IgG2 antibodies were labeled with dibenzocyclooctyne (DBCO) and purified. LNPs were then incubated with DBCO-labeled antibodies using a click chemistry reaction (Fig. 1A). LNPs were characterized for size, surface charge, encapsulation efficiency, relative pK_a measurements, and 48-hour stability. Luciferase mRNA LNP delivery and cytotoxicity were assessed in vitro 24 hours after treatment in the JEG-3 placental trophoblast cell line.

Utilizing click chemistry, we synthesized LNPs with varying ratios of PEG-Azide: PEG, thereby increasing available sites for antibody conjugation. When compared to control formulations, antibody-conjugated LNPs displayed a significant size increase (Fig. 1B), confirming the presence of antibody on the LNP surface. Surface charge measurements remained overall neutral, but trended downward at higher antibody concentrations, further confirming successful antibody conjugation. Encapsulation efficiencies remained consistent in all formulations ( >80%) (Fig. 1C). Relative pK_ameasurements were consistent, ranging from 5.7-6.8 (Fig. 1D). All LNP formulations remained stable in PBS for 48 hours (Fig. 1E). Together, this data suggests that modulation of LNPs via antibody conjugation does not negatively influence properties characteristic of potent nucleic acid delivery. To validate this platform, LNPs were screened in vitro in placental cells. Both EGFR and CD3 antibody conjugation was confirmed with size measurements (Fig. 1F). No changes were observed in placental cells dosed with CD3-targeted LNPs; on the contrary, EGFR-targeted LNPs demonstrated enhanced mRNA delivery in placental cells, with three out of four EGFR-LNP groups outperforming S1 and A1 formulations in vitro (Fig. 1G). Specifically, the 1:2 and 1:3 LNP formulations resulted in approximately 9- and 8- fold increases in luciferase delivery, respectively. No cytotoxicity was observed in vitro across all LNP treatments. Overall, this work demonstrates the use of click chemistry to create antibody-targeted LNPs capable of selective mRNA delivery. Given the consistency in characterization data between antibody-conjugated LNPs with previously accepted LNP formulations, enhanced mRNA delivery observed in placental cells is likely a result of antibody targeting rather than modulation of LNP surface properties. Taken together, we have developed a targeted LNP platform that holds great potential to be used for organ-specific delivery of nucleic acid therapies for a broad range of diseases. Specifically, EGFR-targeted LNPs are a promising vehicle for drug delivery during pregnancy and could be employed to treat a variety of obstetric complications, such as preeclampsia and fetal growth restriction, allowing for selective delivery in the placenta and minimized fetal exposure.

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