Background:E. coli derived L‐asparaginase (EcA) is an essential medicine in the combination chemotherapy for Acute Lymphoblastic Leukemia (ALL). Since it is exogenously derived, silent inactivation and hypersensitivity due to anti‐drug antibody have often necessitated discontinuation of the drug.Aims:Using a rational protein engineering approach, we created four EcA mutants with improved thermal stability, better serum stability, less antigenicity and less glutaminase activity. The mutant with most favourable pharmacological profile will be developed as a novel asparaginase for therapy of ALL.Methods:We identified and mutated several B‐cell epitopes and amino acid residues at the EcA interface that are responsible for activity, stability and antigenicity. Steady state enzyme kinetics was measured at 37oC (optimum temperature for EcA) and 62oC (EcA degrades at 65oC). The kinetic parameters, such as Km and Vmax, were calculated from linear fits of Michaelis‐Menten equation. Glutaminase activity of the mutants was measured and compared to the wild type EcA. The cytotoxicity of the EcA mutants was verified by performing MTT assay in ALL sensitive REH cell lines. Further indirect ELISA was performed to check the antigenicity of the mutants. The binding of the mutants to the commercially available l‐asparaginase antibody was analysed. Moreover, to evaluate the in vivo immunogenicity, mice were immunized with primary and booster doses of EcA mutants over 66 days followed by the measurement of IgG and IgM titers. In addition, the binding of wild‐type EcA and mutants to pre‐existing anti‐asparaginase antibodies present in ALL patient serum receiving asparaginase therapy was studied by indirect ELISA. Finally the activity‐time profile of the EcA mutants was plotted and was used to calculate the pharmacokinetic parameters, which were further compared with the commercially available l‐asparaginase drug.Results:At 37oC catalytic activity of the mutants was comparable to wild type. At 62oC, the catalytic activity of Mutant A, B and C was 155%, 96.5% and 98.2% respectively, compared to 56.9% for the wild type. All four mutants showed significantly lower glutaminase activity as compared to wild type EcA. In MTT assay, the wild type EcA showed 25.31% of cell kill, whereas mutant B showed highest cytotoxicity with 60% cell kill in Reh ALL cell lines. In terms of antigenicity, Mutant D was found to be 48% less antigenic compared to wild EcA, followed by Mutant B (26.4% less) and Mutant C (23.7% less). Pharmacokinetic profiling showed that all mutants had higher AUC and lower elimination rate constant, with Mutant B having highest AUC (1127.7 ± 363.6 vs. 72.12 ± 28.03 min∗ug/ml) and lowest elimination rate constant (0.048 ± 0.017 vs. 0.79 ± 0.42 ml/min) compared to commercially available L‐asparaginase. Mice immunized with Mutant B variant showed approximately 10‐fold lower IgG and IgM titers as compared to antibody responses induced by wild‐type EcA. Further, the binding of mutant B to serum antibody from the ALL patients was approximately 2‐fold less compared to wild‐type EcA.Summary/Conclusion:Considering all parameters, Mutant B was found to have the most favorable characteristics and emerged as the lead candidate for future development. This formulation is expected to overcome the existing deficiencies and clinical challenges encountered with the EcA formulations. Clinical development of this drug candidate is envisaged.