Bacterial CPP-mediated internalization of antibodies and proteins

2022 Fiscal Year Final Research Report

• Project/Area Number: 20H02918
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 38030:Applied biochemistry-related
• Research Institution: Fukui Prefectural University
• Principal Investigator: Hamano Yoshimitsu 福井県立大学, 生物資源学部, 教授 (50372834)
• Co-Investigator(Kenkyū-buntansha): 丸山 千登勢 福井県立大学, 生物資源学部, 准教授 (20452120)
• Project Period (FY): 2020-04-01 – 2023-03-31
• Keywords: 細胞膜透過性ペプチド / ポリリジン / CPP

Outline of Final Research Achievements

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. Here, for the first time, we provide direct evidence that two representative bacterial polycationic isopeptides, ε-poly-L-α-lysine (ε-PαL) and ε-oligo-L-β-lysine (ε-OβL), were internalized into mammalian cells by direct cell-membrane penetration and then diffused throughout the cytosol. In this study, we used clickable ε-PαL and ε-OβL derivatives carrying a C-terminal azide group, which were enzymatically produced and then conjugated with a fluorescent dye to analyze subcellular localization. Interestingly, fluorescent proteins conjugated with the clickable ε-PαL or ε-OβL were also internalized into cells and diffused throughout the cytosol. Notably, a Cre recombinase conjugate with ε-PαL entered cells and mediated the Cre/loxP recombination, and ε-PαL was found to deliver a full-length IgG antibody to the cytosol and nucleus.

Free Research Field

応用微生物学

Academic Significance and Societal Importance of the Research Achievements

抗体医薬などの高分子医薬は分子標的への特異性が高く、がんやリウマチなど難病とされてきた疾患に対しても優れた治療効果を発揮する。また、近年のバイオテクノロジー技術の革新により高分子医薬の製造法も確立され、現在の医薬品ベストセラー上位のほとんどが高分子医薬になっている。ただし、高分子医薬には弱点もあり、高分子であるため基本的に細胞内に入らない。したがって、高分子医薬の分子標的は細胞表面の受容体などに限定され、治療可能な疾患も限定的である。本研究では、ε-PαLとε-OβLの優れた細胞膜透過性を明らかにするとともに、これらを利用することで高分子医薬を細胞内分子標的にも応用できる基盤技術を構築した。