Site-specific pegylation crosslinking of L-asparaginase subunits to improve its therapeutic efficiency

Ramirez-Paz, Josell
Saxena, Manoj
Delinois, Louis J.
Joaquín-Ovalle, Freisa
Lin, Shiru
Chen, Zhongfang
Rojas-Nieves, Virginia A.
Griebenow, Kai
Journal Title
Journal ISSN
Volume Title
Cold Spring Harbor Laboratory

L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin’s lymphoma. However, some disadvantages still limit its full application potential, e.g., allergic reactions, pancreatitis, and blood clotting impairment. Therefore, much effort has been directed at improving its performance. A popular strategy is to randomly conjugate L-asparaginase with mono-methoxy polyethylene glycol, which became a commercial FDA approved formulation widely used in recent years. To improve this formulation by PEGylation, herein we performed cysteine-directed site-specific conjugation of the four L-asparaginase subunits to prevent dissociation-induced loss of activity. The conjugation sites were selected at surface-exposed positions on the protein to avoid affecting the catalytic activity. Three conjugates were obtained using different linear PEGs of 1000, 2000, and 5000 g/mol, with physical properties ranging from a semi-solid gel to a fully soluble state. The soluble-conjugate exhibited higher catalytic activity than the non-conjugated mutant, and the same activity than the native enzyme. Site-specific crosslinking of the L-asparaginase subunits produced a higher molecular weight conjugate compared to the native tetrameric enzyme. This strategy might improve L-asparaginase efficiency for leukemia treatment by reducing glomerular filtration due to the increase in hydrodynamic size thus extending half-live, while at the same time retaining full catalytic activity.

Article originally published by BioRxiv. Published online 2018.
Acute lymphoblastic leukemia, Catalytic activity, Intramolecular, Tetramer, Thiol-maleimide
This is a pre-print version of an article that is available at Recommended citation: Ramirez-Paz, J., Saxena, M., Delinois, L. J., Joaquín-Ovalle, F. M., Lin, S., Chen, Z., Rojas-Nieves, V. A., & Griebenow, K. (2018). Site-specific pegylation crosslinking of L-asparaginase subunits to improve its therapeutic efficiency. BioRxiv. This item has been deposited in accordance with publisher copyright and licensing terms and with the author’s permission.