Assessment of immunogenic and catalytic properties of mutant proteoforms of Escherichia coli L-asparaginase in murine models

Biopharmaceuticals represent an important and expanding industrial sector within the pharmaceutical world, encompassing a wide range of products and drugs produced by living organisms. Through recombinant DNA technology (rDNA) under specific biotechnological conditions and protocols, they include therapies based on recombinant proteins, vaccines, and cellular and gene therapies. Like many other clinically approved biological drugs, native Escherichia coli L-asparaginase (ASNase) (also known as EcAII), successfully applied since 1978 in the treatment of lymphoma and leukemia, has been the subject of continuous research aimed at improving its thermostability, half-life, and resistance to human proteases, as well as reducing adverse effects such as allergenicity. To overcome these limitations, a new enzymatic source or an improved version (biobetter) of the currently available ASNase may provide enhanced therapeutic efficacy with fewer adverse reactions. Previously, we observed a low production of anti-asparaginase antibodies and a prolonged serum half-life for the double mutant enzyme P40S/S206C in mice; however, its specific activity was significantly reduced. Thus, our objective was to evaluate the single mutants S206C and P40S through in vitro and in vivo assays. Furthermore, we tested whether the double mutant P40S/S206C, in combination with the N24S mutation (generating triple mutant TM), could retain specific activity comparable to the WT enzyme while improving its biochemical properties, thermostability, and proteolytic resistance. Our results demonstrated that the decrease in specific activity was caused by the P40S substitution. Moreover, the single mutants exhibited high stability under simulated biological conditions, in contrast to the double mutant P40S/S206C. The in vitro cell viability assay showed that the mutant enzymes had greater cytotoxic effects than the wild-type (WT) enzyme against T-cellderived ALL and several solid tumor cell lines. In vivo experiments were conducted in mice to identify toxicological effects, assess immunological responses, and evaluate the pharmacokinetic profiles of the enzymes. From these analyses, the S206C mutant produced milder physiological alterations and lower immune/allergenic responses. Regarding the pharmacokinetic profile, S206C exhibited twice the enzymatic activity of the WT and P40S two hours after injection. The triple mutant (TM) enzyme was biochemically characterized, and its cytotoxicity toward leukemic cell lines as well as its antigenic properties in SPF Balb/c mice were evaluated. The TM displayed robust asparaginase activity, a >3-fold reduction in Km for asparagine, superior thermostability, increased proteolytic resistance, and a lower in silico immunogenicity score compared with the wild-type enzyme. In vivo, the TM showed no apparent toxicity, caused only a mild decrease in platelet count, reduced induction of antiasparaginase IgE antibodies, and preserved the pharmacokinetic profile. In conclusion, we report E. coli bioengineered asparaginases with enhanced biochemical properties and biological performance, as well as reduced adverse reactions, supporting the strategy of targeted amino acid substitutions as a promising approach for the development of next-generation asparaginase biopharmaceuticals.