Site-Specific PEGylation of Anti-Mesothelin Recombinant Immunotoxins Increases Half-life and Antitumor Activity.
Mol Cancer Ther
; 19(3): 812-821, 2020 03.
Article
en En
| MEDLINE
| ID: mdl-31871266
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 ß-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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Neoplasias Pancreáticas
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Polietilenglicoles
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Proteínas Recombinantes
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Inmunotoxinas
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Proteínas Ligadas a GPI
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Antineoplásicos
Tipo de estudio:
Prognostic_studies
Límite:
Animals
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Female
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Humans
Idioma:
En
Revista:
Mol Cancer Ther
Asunto de la revista:
ANTINEOPLASICOS
Año:
2020
Tipo del documento:
Article