Targeted mRNA delivery with bispecific antibodies that tether LNPs to cell surface markers

Abstract

Efficient delivery of mRNA-lipid nanoparticles (LNPs) to specific cell types remains a major challenge for mRNA therapeutics. Conventional targeting approaches involve modifying the lipid composition or functionalizing the surface of LNPs, which complicates manufacturing and alters nanoparticle size, charge, and stealth, impacting their delivery and immunogenicity. Here, we present a generalizable method for targeted mRNA-LNP delivery that uses bispecific antibodies (BsAbs) to form a bridge between LNPs and cell surface markers. BsAbs can be combined with LNPs or administered first, binding to surface proteins on target cells and later retaining unmodified LNPs in affected tissues. We demonstrate the efficient and cell-type-specific delivery of mRNA-LNPs beyond the liver, targeting epidermal growth factor receptor (EGFR)- and folate hydrolase 1 (PSMA)-positive cells in vitro and in vivo. The flexibility of this technology, achieved by substituting the cell-targeting region of the BsAbs, enables the rapid development of next-generation targeted mRNA drugs.

Keywords: LNP; MT: Delivery Strategies; bispecific antibodies; cancer; drug delivery; lipid nanoparticles; mRNA; targeted delivery.

Graphical abstract

Figure 1

Pre-mixing with bispecific antibodies alters physicochemical properties and delivery of mRNA-LNPs (A) During pre-mixing, bispecific antibodies (BsAbs) bind to polyethylene glycol (PEG) on the surface of mRNA-loaded lipid nanoparticles (LNPs). The second binding region of the BsAb can bind to the surface protein on the target cell. (B) For pre-targeting, cells are exposed to BsAbs that can specifically bind to surface proteins. mRNA-carrying LNPs can then bind to the PEG-specific binding region of the BsAb. (C) Triplicate dynamic light scattering measurements of size distribution of EGFP-mRNA LNPs without BsAbs, after pre-mixing with PSMA-PEG BsAbs, and after pre-mixing with EGFR-PEG BsAbs. (D) Cryogenic transmission electron microscopy images of EGFP-mRNA LNPs without BsAbs, after pre-mixing with PSMA-PEG BsAbs, and after pre-mixing with EGFR-PEG BsAbs. Scale bar: 100 nm. (E and F) Mean EGFP fluorescence intensity of cells transfected with untargeted EGFP-mRNA LNPs, with LNPs pre-mixed with PSMA-PEG BsAbs, or LNPs pre-mixed with EGFR-PEG BsAbs, respectively, for (E) MDA-MB-468 breast cancer cells (PSMA−ve and EGFR+ve) and (F) LNCaP prostate cancer cells (PSMA+ve and EGFR+ve). Mean EGFP fluorescence intensity was measured using flow cytometry. Statistical analysis was performed using two-tailed t tests assuming equal variance. Bars represent the mean value, and error bars indicate standard deviation (n = 3). ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.

Figure 2

Pre-targeting of cells with bispecific antibodies improves cell-specific delivery of mRNA-LNPs (A) Mean EGFP fluorescence intensity of MDA-MB-468 breast cancer cells (PSMA−ve and EGFR+ve) transfected with EGFP-mRNA LNPs. PSMA-PEG BsAbs or EGFR-PEG BsAbs were pre-mixed with LNPs or pre-targeted to MDA-MB-468 cells, respectively. (B) Confocal microscopy images of MDA-MB-468 after addition of EGFP mRNA-LNPs, PSMA-PEG, or EGFR-PEG BsAbs, BsAbs pre-mixed with LNPs, or pre-targeting with BsAbs followed by addition of LNPs. (C) Mean EGFP fluorescence intensity of LNCaP prostate cancer cells (PSMA+ve and EGFR+ve) transfected with EGFP-mRNA LNPs. PSMA-PEG BsAbs or EGFR-PEG BsAbs were pre-mixed with LNPs or pre-targeted to LNCaP cells, respectively. (D) Confocal microscopy images of LNCaP after addition of EGFP mRNA-LNPs, PSMA-PEG, or EGFR-PEG BsAbs, BsAbs pre-mixed with LNPs, or pre-targeting with BsAbs followed by addition of LNPs. Confocal microscopy images were taken at 63× magnification and show EGFP expression (yellow), BsAb localization (protein L-phycoerythrin conjugate labeled; magenta), and 4′,6-diamidino-2-phenylindole DNA stain (DAPI; blue) 4 h after addition of LNPs. Scale bar: 10 μm. Mean EGFP fluorescence intensity was measured using flow cytometry. Statistical analysis was performed using two-tailed t tests assuming equal variance. Bars represent the mean value, and error bars indicate standard deviation (n = 3). ∗∗∗p < 0.001 and ∗∗∗∗p < 0.0001.

Figure 3

Pre-targeting with bispecific antibodies improves targeted delivery of mRNA-LNPs in vivo (A) In vivo bioluminescence images of MDA-MB-468 tumor-bearing mice injected with untargeted luciferase mRNA-LNPs, LNPs pre-mixed with EGFR-PEG BsAbs, or mice pre-injected with EGFR-PEG BsAbs followed by administration of mRNA-LNPs. White arrows indicate posterior tumor localization. (B and C) In vivo bioluminescence in the liver compared to tumor for different targeting approaches (B) 8 and (C) 48 h after luciferase mRNA-LNP administration. (D–G) Ex vivo bioluminescence imaging of (D) tumor, (E) liver, (F) spleen, and (G) kidney tissue at 48 h post-injection. Background was subtracted based on a saline-injected mouse. Statistical analysis was performed using two-tailed t tests assuming equal variance. Bars represent the mean value, and error bars indicate standard deviation (n = 3).