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bioRxiv · 10.1101/317040

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

Ramirez-Paz, J.·Saxena, M.·Delinois, L. J.·Joaquin-Ovalle, F. M.·Lin, S.·Chen, Z.·Rojas-Nieves, V. A.·Griebenow, K.
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Abstract

L-Asparaginase is an enzyme successfully being used in the treatment of acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkins 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.

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Ramirez-Paz, J., Saxena, M., Delinois, L. J., Joaquin-Ovalle, F. M., Lin, S., Chen, Z., Rojas-Nieves, V. A., Griebenow, K.. 2018-05-08. Site-specific PEGylation crosslinking of L-asparaginase subunits to improve its therapeutic efficiency. https://doi.org/10.1101/317040

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