[0001] The present invention relates to non-naturally occurring L-Asparaginases mutants particularly Type II Escherichia coli L- asparaginase (EcA) mutants comprising at least one mutation in the amino-acid sequence of wild-type EcA, wherein such mutation causes the mutants to induce significantly lesser immunogenicity, higher stability and higher cytotoxicity against leukemic cells as compared to wild-type EcA. The present invention also provides use of the EcA mutants as anti-leukemic agents for treatment of Acute Lymphoblastic Leukemia (ALL) in a subject in need thereof.