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<p>G protein-coupled receptors (GPCRs) are key therapeutic targets for various diseases, such as the thrombin receptor protease-activated receptor 4 (PAR4) involved in thrombotic cardiovascular disorders. However, the structural complexity of native GPCRs hinders recombinant protein production, making peptide-based immunization insufficient for generating high-quality antibodies. Here, we developed a PAR4-mRNA-LNP vaccine to express native PAR4 in vivo, thereby inducing potent and specific anti-PAR4 antibodies. The PAR4-mRNA-LNP formulation showed 93.44% encapsulation efficiency, an average size of 127.5 nm, and a polydispersity index (PDI) of 0.1033. Native 55-kDa PAR4 protein with complete glycosylation was confirmed on the cell surface using western blotting and flow cytometry. Mice immunized with PAR4-mRNA-LNP produced strong anti-PAR4 antibody responses, as detected by cell-based ELISA. Finally, we established hybridoma cell lines and identified five clones that significantly inhibited PAR4-mediated platelet aggregation. These findings suggest that mRNA-LNP technology can be broadly applied to generate functional anti-GPCR antibodies for therapy.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年11月20日

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  • En-Shuo Liu,
  • Kai-Wen Ho,
  • Chin-Chung Wu,
  • Hsiao-Li Fan,
  • Ting-Yu Wang,
  • Yuan-Chin Hsieh,
  • Bo-Cheng Huang,
  • Shih-Ting Hong,
  • Tzu-Yi Liao,
  • Yen-Ling Liu,
  • Yu-Tung Chen,
  • Chia-Ching Lee,
  • Chiao-Yun Chen,
  • Chih-Lung Lin,
  • Tian-Lu Cheng

摘要

G protein-coupled receptors (GPCRs) are key therapeutic targets for various diseases, such as the thrombin receptor protease-activated receptor 4 (PAR4) involved in thrombotic cardiovascular disorders. However, the structural complexity of native GPCRs hinders recombinant protein production, making peptide-based immunization insufficient for generating high-quality antibodies. Here, we developed a PAR4-mRNA-LNP vaccine to express native PAR4 in vivo, thereby inducing potent and specific anti-PAR4 antibodies. The PAR4-mRNA-LNP formulation showed 93.44% encapsulation efficiency, an average size of 127.5 nm, and a polydispersity index (PDI) of 0.1033. Native 55-kDa PAR4 protein with complete glycosylation was confirmed on the cell surface using western blotting and flow cytometry. Mice immunized with PAR4-mRNA-LNP produced strong anti-PAR4 antibody responses, as detected by cell-based ELISA. Finally, we established hybridoma cell lines and identified five clones that significantly inhibited PAR4-mediated platelet aggregation. These findings suggest that mRNA-LNP technology can be broadly applied to generate functional anti-GPCR antibodies for therapy.