Engineered L-asparaginase variants with enhanced therapeutic properties to improve treatment of childhood acute lymphatic leukemia.

Escherichia coli L-asparaginase (EcA), a key component of a multi-drug acute lymphatic leukemia (ALL) treatment regimen, has several limitations that reduce its therapeutic efficacy. The major disadvantages include immunogenicity, serum instability, shorter half-life, and accompanying glutaminase activity that causes neurotoxicity and pancreatitis. Pegylated asparaginase and Erwinase have better therapeutic potential, but they are expensive. Using site-directed mutagenesis, we created several EcA variants by substituting specific amino acid residues at the dimer-dimer interface and B-cell epitope regions. After several rounds of screening and selection, we identified two EcA variants viz. K288S/Y176F (KSY-17) and K288S/Y176F/W66Y (KSYW-17), which showed comparable asparaginase activity to wild-type (WT) and significantly less glutaminase activity (30.36 U/mg for WT vs 1.54 and 0.99 U/mg for KSY-17 and KSYW-17). KSYW-17 was less immunogenic than WT, eliciting 4.8-5.3-fold and 2.4-3.8-fold less IgG and IgM responses, respectively. Compared to WT EcA, we also observed significantly less (~1.5-2-fold) binding of these variants to pre-existing antibodies in ALL patients' serum. Pharmacokinetic studies showed that KSY-17 (213.3 ± 6.5 min) and KSYW-17 (244.8 ± 35.5 min) had longer plasma half-lives than WT (101.1 ± 5.1 min). Both variants showed no toxicity up to 5000 IU/kg (single dose) and 1600 IU/kg (repeat dose) in mice. ALL xenograft mice studies showed a 90% and 70% reduction in leukemia burden in KSY-17 and KSYW-17 administered mice, respectively, as compared to 30% for WT after repeat dose administration, accompanied by significantly higher mice survival (100% vs. 70% vs. 10% for KSY-17 vs. KSYW-17 vs. WT). Overall, the engineered EcA variants' showed improved therapeutic efficacy, thus making them promising candidates for primary and relapsed ALL treatment.