Novel Anti-Mesothelin Nanobodies and Recombinant Immunotoxins with Pseudomonas Exotoxin Catalytic Domain for Cancer Therapeutics

• Published in: Molecules and cells Vol. 46; no. 12; pp. 764 - 777
• Main Authors: Nguyen, Minh Quan, Kim, Do Hyung, Shim, Hye Ji, Ta, Huynh Kim Khanh, Vu, Thi Luong, Nguyen, Thi Kieu Oanh, Lim, Jung Chae, Choe, Han
• Format: Journal Article
• Language: English
• Published: Korea (South) 한국분자세포생물학회 01.12.2023
• Subjects: ADP Ribose Transferases - chemistry; ADP Ribose Transferases - genetics; ADP Ribose Transferases - metabolism; Animals; Antineoplastic Agents; Bacterial Toxins - chemistry; Bacterial Toxins - genetics; Bacterial Toxins - metabolism; Catalytic Domain; Cell Line, Tumor; Exotoxins - chemistry; Exotoxins - genetics; Exotoxins - pharmacology; Humans; Immunotoxins - chemistry; Immunotoxins - genetics; Immunotoxins - pharmacology; Mesothelin; Mice; Neoplasms - drug therapy; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Single-Domain Antibodies - genetics; Single-Domain Antibodies - pharmacology; 생물학; cancer therapeutics; mesothelin-targeting; nanobody-based recombinant immunotoxin; PE24B; cytotoxic efficacy
• ISSN: 1016-8478; 0219-1032
• DOI: 10.14348/molcells.2023.0155

Recombinant immunotoxins (RITs) are fusion proteins consisting of a targeting domain linked to a toxin, offering a highly specific therapeutic strategy for cancer treatment. In this study, we engineered and characterized RITs aimed at mesothelin, a cell surface glycoprotein overexpressed in various malignancies. Through an extensive screening of a large nanobody library, four mesothelin-specific nanobodies were selected and genetically fused to a truncated (PE24B). Various optimizations, including the incorporation of furin cleavage sites, maltose-binding protein tags, and tobacco etch virus protease cleavage sites, were implemented to improve protein expression, solubility, and purification. The RITs were successfully overexpressed in , achieving high solubility and purity post-purification. cytotoxicity assays on gastric carcinoma cell lines NCI-N87 and AGS revealed that Meso(Nb2)-PE24B demonstrated the highest cytotoxic efficacy, warranting further characterization. This RIT also displayed selective binding to human and monkey mesothelins but not to mouse mesothelin. The competitive binding assays between different RIT constructs revealed significant alterations in IC values, emphasizing the importance of nanobody specificity. Finally, a modification in the endoplasmic reticulum retention signal at the C-terminus further augmented its cytotoxic activity. Our findings offer valuable insights into the design and optimization of RITs, showcasing the potential of Meso(Nb2)-PE24B as a promising therapeutic candidate for targeted cancer treatment.