To generate functional anti-protease-activated receptor-4 (PAR4), a G protein-coupled receptor, antibodies through PAR4-mRNA-LNP immunization

G protein-coupled receptors (GPCRs) are key therapeutic targets for various diseases, such as the thrombin receptor protease-activated receptor 4 (PAR4) in thrombotic cardiovascular disorders. The structural complexity of native GPCRs limits recombinant protein production. Traditional GPCR antibody development relies on GPCR peptide fragments for animal immunization. These peptides poorly mimic the native structure and affect the quality of the antibodies. Therefore, it is necessary to develop a strategy for immunizing native GPCRs to generate potent anti-GPCR antibodies. Here, we developed a PAR4-mRNA-LNP to immunize mice, which promotes the surface expression of the native PAR4 structure in vivo to induce highly specific and functional anti-PAR4 antibodies. These anti-PAR4 antibodies effectively inhibit platelet aggregation and provide a long-term therapeutic approach for cardiovascular disease. In result, PAR4-mRNA-LNP was synthesized with an encapsulation efficiency of 93.44%, mean size of 127.5 nm, and polydispersity index (PDI) of 0.1033. We confirmed the 55-kDa native PAR4 structure with complete glycosylation expressing on cell surface. We immunized mice with PAR4-mRNA-LNP and detected high anti-PAR4 antibody titers with ELISA. This assay used 293T cells that stably express the native PAR4 structure, enabling us to screen for high-quality anti-PAR4 antibodies. We established 15 hybridoma cell lines, which resulted in 13 including 10 IgG and 3 IgM anti-PAR4 antibodies. Five clones significantly inhibited PAR4-mediated platelet aggregation. We confirmed that PAR4-mRNA-LNP expresses the native PAR4 structure on the cell surface and the immunization produced highly functional anti-PAR4 antibodies. mRNA-LNP technology may be widely used to produce anti-GPCR antagonists and agonists for therapy.