Site-Specific PEGylation of Anti-Mesothelin Recombinant Immunotoxins Increases Half-life and Antitumor Activity.

Abstract

Recombinant immunotoxins (RIT) are chimeric proteins containing an Fv that binds to tumor cells, fused to a fragment of Pseudomonas exotoxin (PE) that kills the cell. Their efficacy is limited by their short half-life in the circulation. Chemical modification with polyethylene glycol (PEG) is a well-established method to extend the half-lives of biologics. Our goal was to engineer RITs with an increase in half-life and high cytotoxic activity. We introduced single cysteines at different locations in five anti-mesothelin RITs and employed site-specific PEGylation to conjugate them to 20-kDa PEG. Because our previous PEGylation method using β-mercaptoethanol reduction gave poor yields of PEG-modified protein, we employed a new method using tris(2-carboxyethyl)phosphine to reduce the protein and could PEGylate RITs at approximately 90% efficiency. The new proteins retained 19% to 65% of cytotoxic activity. Although all proteins are modified with the same PEG, the radius of hydration varies from 5.2 to 7.1, showing PEG location has a large effect on protein shape. The RIT with the smallest radius of hydration has the highest cytotoxic activity. The PEGylated RITs have a 10- to 30-fold increase in half-life that is related to the increase in hydrodynamic size. Biodistribution experiments indicate that the long half-life is due to delayed uptake by the kidney. Antitumor experiments show that several PEG-RITs are much more active than unmodified RIT, and the PEG location greatly affects antitumor activity. We conclude that PEGylation is a useful approach to improve the half-life and antitumor activity of RITs.

• Authors: Zeliang Zheng, Ryuhei Okada, Hisataka Kobayashi, Tadanobu Nagaya, Junxia Wei, Qi Zhou, Fred Lee, Tapan K Bera, Yun Gao, William Kuhlman, Chin-Hsien Tai, Ira Pastan
• Journal: Molecular cancer therapeutics (Mol Cancer Ther) Vol. 19 Issue 3 Pg. 812-821 (03 2020) ISSN: 1538-8514 [Electronic] United States
• PMID: 31871266 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, N.I.H., Intramural)
• Copyright: ©2019 American Association for Cancer Research.

Chemical References

• Antineoplastic Agents
• GPI-Linked Proteins
• Immunotoxins
• Msln protein, mouse
• Recombinant Proteins
• Polyethylene Glycols
• Mesothelin

Topics

• Animals
• Antineoplastic Agents (chemistry, pharmacology)
• Apoptosis
• Cell Proliferation
• Female
• GPI-Linked Proteins (antagonists & inhibitors)
• Half-Life
• Humans
• Immunotoxins (chemistry, pharmacology)
• Mesothelin
• Mice
• Mice, Nude
• Pancreatic Neoplasms (drug therapy, metabolism, pathology)
• Polyethylene Glycols (chemistry)
• Recombinant Proteins (chemistry, pharmacology)
• Tumor Cells, Cultured
• Xenograft Model Antitumor Assays