Introduction: Abstract: L-Asparaginase is a key enzyme in the treatment of pediatric acute lymphoblastic leukemia (ALL), a common cancer in children. Asparaginase works by depleting asparagine, an amino acid crucial for the survival and proliferation of leukemia cells. For over 40 years, L-Asparaginase has been used as a first-line therapy for ALL, contributing significantly to remission rates. However, its clinical application is limited by several challenges, including in vivo instability, a short half-life, and sensitivity to proteases such as AEP (asparagine endopeptidase) and cathepsin B, which can reduce its therapeutic effectiveness and lead to hypersensitivity reactions. To overcome these limitations, PEGylated forms of L-Asparaginase, Oncaspar, have been developed, which extend its half-life and reduce proteolytic degradation. In addition to PEGylation, recent advances in enzyme engineering have focused on enhancing protease resistance while preserving enzymatic activity. These engineering efforts aim to improve the enzyme's stability and reduce side effects, optimizing its use in ALL treatment. This review examines these innovations, focusing on the advancements in L-Asparaginase to improve its clinical efficacy, stability, and safety profile for pediatric patients.
Methods: Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, and L-asparaginase has been a central component of its treatment for more than four decades (Chan et al., 2021). Its therapeutic principle is based on the depletion of circulating asparagine, an amino acid essential for leukemic blasts, which lack sufficient asparagine synthetase to compensate for its loss. This selective metabolic vulnerability has made L-asparaginase uniquely effective in pediatric ALL.
The anticancer potential of L-asparaginase was first recognized in the 1950s, when guinea pig serum induced regression of mouse lymphomas (Kidd, 1953). Clinical use followed in the 1960s, with purified bacterial enzymes from Escherichia coli and Erwinia chrysanthemi eventually establishing the drug as a standard component of multi-agent chemotherapy regimens (Dolowy, 1966; Hill, 1967).
Despite its clinical success, limitations such as rapid clearance, short half-life, protease sensitivity, and treatment-related hypersensitivity reactions continue to restrict its utility. To address these challenges, efforts have focused on optimizing enzyme formulations and engineering variants with improved pharmacological and safety profiles.
This review highlights advances in understanding L-asparaginase activity and stability, with emphasis on strategies—such as PEGylation and targeted mutagenesis—that enhance its therapeutic performance in pediatric ALL.
Results: Results:
Enzymatic Mechanism
Advances in structural biology have clarified how E. coli asparaginase II (EcAII), the most widely used therapeutic enzyme, catalyzes asparagine hydrolysis. The reaction proceeds through a double-displacement (ping-pong) mechanism in which a threonine residue initiates nucleophilic attack, forming a covalent acyl-enzyme intermediate that is subsequently resolved to generate aspartate and ammonia (Unno et al., 2020). Stabilization of tetrahedral intermediates relies on motifs that are unique among hydrolytic enzymes, highlighting the enzyme’s distinct evolutionary adaptation. This mechanistic insight has provided a foundation for rational design of variants with improved activity and stability.
PEGylated Clinical Formulations
Native bacterial asparaginases are limited by rapid clearance and high immunogenicity. PEGylation, the covalent attachment of polyethylene glycol chains, has addressed some of these drawbacks by reducing immune recognition and shielding the enzyme from proteolysis. The first PEGylated product, Oncaspar®, was generated by conjugating EcAII with succinimidyl succinate PEG, leading to an extended half-life and reduced hypersensitivity. More recently, calaspargase pegol, synthesized with succinimidyl carbonate PEG, has demonstrated an even longer half-life, thereby allowing less frequent dosing (Marini et al., 2017). Together, these PEGylated formulations represent a significant advance in improving pharmacokinetics and tolerability.
Protease Sensitivity and Cleavage Sites
A persistent limitation of current formulations is their susceptibility to proteolytic inactivation by cathepsin B and asparagine endopeptidase (AEP), proteases that are often upregulated in resistant leukemic cells (Cachumba et al., 2019). Cleavage at specific residues, particularly N24, D124, and N143, disrupts enzyme integrity and reduces catalytic function (Patel et al., 2009). N24, located on a loop adjacent to the active site, is especially critical, as cleavage here destabilizes the catalytic pocket. D124 contributes to structural stabilization through hydrogen bonding, and its loss compromises enzymatic stability. N143, although more peripheral, may contribute indirectly to immunogenicity through enhanced antigen presentation. Comparative studies with Erwinia-derived enzymes, which show differential susceptibility, further underscore the sequence-specific nature of protease sensitivity.
Engineered Variants
Protein engineering has sought to address protease sensitivity while preserving activity. Among AEP-sensitive residues, the N24 position has been most extensively studied. The N24G substitution confers resistance to cleavage but significantly reduces catalytic efficiency, limiting clinical applicability (Patel et al., 2009). By contrast, the N24S mutant retains enzymatic activity and demonstrates enhanced stability, representing a more favorable therapeutic candidate (Nguyen et al., 2017). Other substitutions, including N24A and N24T, also improve protease resistance without major loss of function (De Groot et al., 2011). Together, these variants illustrate the potential of targeted mutagenesis in generating next-generation enzymes with optimized stability, reduced immunogenicity, and sustained therapeutic efficacy.
Conclusion: Conclusion: While L-Asparaginase remains a cornerstone in the treatment of ALL, its clinical application is limited by protease sensitivity and instability. The enzyme’s homotetrameric structure and unique double-displacement catalytic mechanism play crucial roles in its function, but they also make it vulnerable to proteolytic cleavage. Advances in PEGylation, such as the development of Oncaspar® and Calaspargase pegol, have significantly improved its pharmacokinetics. Moreover, engineering mutations like N24S, N24G, and N24T offer promising strategies to enhance protease resistance while retaining enzymatic activity. These innovations represent important steps toward developing a more stable and effective version of L-Asparaginase, which could ultimately lead to better therapeutic outcomes for pediatric ALL patients.