Protein Engineering of L-Asparaginase II to Reduce Immunogenicity for Acute Lymphoblastic Leukemia Therapy

L-asparaginase II remains essential for ALL treatment, yet its clinical utility is hindered by immune-mediated adverse effects. Our investigation focused on developing a modified version with reduced antigenicity through structural optimization. Initial computational analysis identified tyrosine-189 within residues 185-211 as a dominant immunogenic hotspot. Computational prediction tools (IEDB, Ellipro) guided the substitution of 189Y with methionine and alanine. Structural modeling (SWISS-MODEL) and validation (ProTSAV) confirmed the preservation of the enzyme's architecture post-modification. Molecular dynamics analysis (GROMACS 2020, 100ns simulation) revealed maintained stability under physiological conditions despite altered surface characteristics. The modified constructs were successfully generated via SOE-PCR and cloned recombinantly. In silico analysis suggested that both 189M and 189A variants may exhibit decreased antibody binding capacity, while computational models predicted retained catalytic functionality. The experimental verification confirmed successful gene cloning and construction. This structure-based engineering approach yielded an L-asparaginase II derivative with a potentially improved immunological profile, suggesting safer therapeutic application for ALL patients after further experimental validation. The combined computational and experimental strategy provides a framework for developing next-generation biologics with reduced immunogenicity.