Enhanced immune clearance of L-Asparaginase and its genetic predisposition among children with acute Lymphoblastic Leukaemia

Abstract

Pegylated L-asparaginase (peg-ASNase) is the cornerstone of childhood acute lymphoblastic leukaemia (ALL) chemotherapy. Its immunogenicity often prompts the production of antibodies, rendering the post-administration drug activity suboptimal in patients. Potential genetic variants may increase the risk of the drug's immune- mediated sequelae, with their allelic prevalences in the Southeast Asian patient population remaining obscure. The absence of validated commercial assays for quantifying serum peg-ASNase activity levels and anti-ASNase antibody titres further complicates the assessment of the drug effectiveness in patients. This study aimed (a) to develop and validate biochemical assays for measuring peg-ASNase activity levels and anti-ASNase IgG titres in human serum, (b) to determine the correlation between serum peg-ASNase activity and anti-ASNase IgG titres, (c) to investigate the association of seven genetic variants with serum anti-ASNase IgG titres, and (d) to determine the association between serum peg-ASNase activity and childhood ALL induction outcomes, mainly the marrow morphology status and minimal residual disease. A reversed-phase high- performance liquid chromatography assay with automated sample derivatisation was optimised and validated for measuring serum peg-ASNase activity (%CV range: 3.05%- 4.16%; %error range: -3.03% -0.90%; LLOQ: 0.03 IU/mL). An indirect enzyme-linked immunosorbent assay was developed and validated to quantify human serum anti- ASNase IgG titres (%CV range: 6.54%-14.81%; %recovery range: 95.89%-100.77%; cut-off: ≥1.60 μg/mL). For genetic variants genotyping, conventional PCR and Sanger sequencing were performed. Statistical analysis was conducted using SPSS v29.0.1.0. Mean serum trough peg-ASNase activity levels were 0.11 IU/mL and 0.07 IU/mL for the newly diagnosed and relapsed patients, respectively, with 47.5% (29/61) and 20% (1/5) samples achieving 20.1 IU/mL in the respective cohorts after first drug dosing. There was an inverse correlation between serum peg-ASNase activity levels and anti- ASNase IgG titres (p=0.006). Patients with anti-ASNase IgG were found with lower serum peg-ASNase activity than those without antibody (p=0.002). GRIA1 rs4958351 was inversely associated with post-administration anti-ASNase IgG titres, whereas IL16 rs11556218 was associated with baseline antibody levels. Serum peg-ASNase activity levels were not associated with childhood ALL induction outcomes. This is the first local study on generic peg-ASNase, paving the way for future studies and improvements in drug administration for children using the developed laboratory assays.