Thiol-maleimide poly(ethylene glycol) crosslinking of L-asparaginase subunits at recombinant cysteine residues introduced by mutagenesis

Abstract

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 conjugation of the L-asparaginase subunits to prevent dissociation-induced loss of activity. The recombinant cysteine conjugation sites were introduced by mutagenesis 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. The cysteine-directed 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.

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles

“…Unfortunately, a new ASNase containing all these characteristics has not yet been developed. Using genetic engineering, changes in residues not involved in the catalytic site, as the introduction of recombinant cysteine residues in the protein surface, are possible, which could, for example, improve ASNase PEGylation, and prevent dissociation-induced loss of activity (Ramirez-paz et al, 2018). Another strategy with great potential is the use of expression systems capable of expressing glycosylation patterns similar to those of mammals or even human-like (Sajitha et al, 2015; Nadeem et al, 2018).…”

L-Asparaginase Conjugates from the Hyperthermophilic Archaea Thermococcus sibiricus with Improved Biocatalytic Properties

“…PEG is known to assist in stabilizing the enzyme's secondary structure and preserving its natural conformation. The polymer is used for the modification of L-ASNase, and PEGylated L-ASNase preparations are already commercially available [25][26][27].…”

Engineering protein-based therapeutics through structural and chemical design

“…This presents a challenge as the resultant heterogeneous pool of products can have vastly differing therapeutic properties. In this regard, later efforts focused on the sitespecific modification of proteins towards overcoming these drawbacks 46 . One example is certolizumab pegol (Cimzia), containing an engineered unpaired cysteine for conjugation to PEG 47 .…”