Hypersensitivity and L-Asparaginase: A structural analysis of the interaction with HLA-DRB1*07:01

L-Asparaginase derived from Escherichia coli (EcA) is extensively employed in the treatment of acute lymphoblastic leukemia (ALL); however, immunogenic responses frequently result in therapeutic failure. This study aims to identify residues involved in EcA immunogenicity and to propose structural modifications that may reduce antigenic potential or facilitate the development of targeted therapeutic strategies to inhibit such interactions. To achieve this, we investigate the sites of interaction of HLA-DRB1*07:01 allele and EcA by crosslinking mass spectrometry (XL-MS) using recombinantly expressed proteins. Circular dichroism (CD) spectroscopy confirmed the proper folding of the expressed enzymes, ensuring their structural integrity. Additionally, XL-MS allowed us to experimentally determine the structure of HLA-DRB1*07:01, which was previously unknown. Structural analysis and sequence alignment revealed immunogenic epitopes on the enzyme surface, particularly near the active site and at K288, a highly reactive residue. Our findings highlight key immunogenic sites on EcA, particularly residues 53-58, 283-289, and K288, which represent promising targets for reducing immunogenicity while preserving enzymatic function. These proposed modifications should be experimentally validated to ensure enzyme activity is maintained while effectively mitigating immune responses. SIGNIFICANCE: Hypersensitivity to Escherichia coli-derived L-asparaginase (EcA) remains one of the main challenges in the treatment of acute lymphoblastic leukemia (ALL), often forcing patients to interrupt a therapy that could be lifesaving. In this study, we mapped at the structural level how EcA interacts with the HLA-DRB1*07:01 allele, one of the main genetic factors associated with these adverse reactions. By identifying specific regions of the enzyme that trigger the immune response, especially residue K288, we offer a foundation for targeted modifications that may reduce immunogenicity without affecting enzymatic activity. These findings provide a concrete path toward developing safer EcA variants and bring us closer to more effective and personalized treatments.