Asparaginase with Combined Mutations: Optimized Biochemistry and Lowered Allergic Risk

Biotechnology and biomedical advances have driven the development of novel biopharmaceuticals to meet growing clinical demands. Among approved biologics, native <i>Escherichia coli</i> asparaginase has been under continuous optimization to improve thermostability, half-life, resistance to human proteases, and reduce adverse effects, particularly allergenicity. Here, we engineered an antileukemic biobetter by combining the substitutions P40S/S206Cpreviously identified by our group as less immunogenic and with extended bloodstream activity in micewith N24S, reported to enhance in vitro stability. The purified triple mutant enzyme was biochemically characterized, and its cytotoxicity against leukemic cell lines and antigenic properties in Balb/c SPF mice were evaluated. TM displayed robust asparaginase activity, a >3-fold reduction in <i>K</i> _M for asparagine, superior thermostability, enhanced proteolytic resistance, and a lower in silico immunogenicity score compared to wild-type. In vivo, compared to wild-type, TM showed no apparent toxicity, a lower decrease in platelet counts, reduced induction of antiasparaginase IgE antibodies, and a preserved pharmacokinetic profile. In conclusion, combined mutations conferred substantial biochemical and immunological improvements, supporting the strategy of targeted amino acid substitutions to advance next-generation asparaginase biopharmaceuticals.