Discovery of human-like L-asparaginases with potential clinical use by directed evolution

• Published in: Scientific reports 2017-08, Vol.7 (1), p.10224-10224, Article 10224
• Main Authors: Rigouin, Coraline, Nguyen, Hien Anh, Schalk, Amanda M., Lavie, Arnon
• Format: Article
• Language: English
• Subjects: 631/61/51/2314; 692/4028/67/1990/283/2125; 82/80; 82/81; 82/83; Acute lymphoblastic leukemia; Amino acids; Asparaginase; Asparagine; Brand names; Chemotherapy; Chimeras; Cloning; Directed evolution; DNA shuffling; E coli; Enzymes; FDA approval; Genes; Humanities and Social Sciences; Immunogenicity; L-asparaginase; Laboratories; Leukemia; Lymphatic leukemia; Medical research; multidisciplinary; Nitrogen; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-10758-4

L-asparaginase is a chemotherapy drug used to treat acute lymphoblastic leukemia (ALL). The main prerequisite for clinical efficacy of L-asparaginases is micromolar K M for asparagine to allow for complete depletion of this amino acid in the blood. Since currently approved L-asparaginases are of bacterial origin, immunogenicity is a challenge, which would be mitigated by a human enzyme. However, all human L-asparaginases have millimolar K M for asparagine. We recently identified the low K M guinea pig L-asparaginase (gpASNase1). Because gpASNase1 and human L-asparaginase 1 (hASNase1) share ~70% amino-acid identity, we decided to humanize gpASNase1 by generating chimeras with hASNase1 through DNA shuffling. To identify low K M chimeras we developed a suitable bacterial selection system ( E. coli strain BW5Δ). Transforming BW5Δ with the shuffling libraries allowed for the identification of several low K M clones. To further humanize these clones, the C-terminal domain of gpASNase1 was replaced with that of hASNase1. Two of the identified clones, 63 N -h C and 65 N -h C , share respectively 85.7% and 87.1% identity with the hASNase1 but have a K M similar to gpASNase1. These clones possess 100–140 fold enhanced catalytic efficiency compared to hASNase1. Notably, we also show that these highly human-like L-asparaginases maintain their in vitro ALL killing potential.