A human-like glutaminase-free asparaginase is highly efficacious in ASNS^low leukemia and solid cancer mouse xenograft...

L-asparaginase (L-ASNase) is crucial in treating pediatric acute lymphoblastic leukemia (ALL), but its use is hampered by side effects from the immunogenicity and L-glutaminase (L-GLNase) co-activity of FDA-approved bacterial L-ASNases, often leading to treatment discontinuation and poor outcomes. The toxicity of these L-ASNases makes them especially challenging to use in adult cancer patients. To overcome these issues, we developed EBD-200, a humanized guinea pig L-ASNase with low Km and no L-GLNase activity, eliminating glutamine-related toxicity. EBD-200 showed comparable anti-cancer effects to PEGylated L-ASNase in ASNS^low ALL, melanoma and liver cancer models, with improved tolerability. Its potent anti-cancer efficacy and enhanced safety profile suggest that EBD-200 could benefit ALL patients and broaden treatment options for ASNS^low solid cancers.