Biosynthesized uricase with dual-terminus zwitterionic EKylation for prolonged elimination half-life and reduced immunogenicity.

Zwitterionic EKylation of exogenous enzymes has emerged as a safe and effective method to enhance pharmacokinetics (PK) and mitigate immunogenicity by increasing size and providing steric shielding. However, the conventional approach of fusing zwitterionic peptides to the C-terminus is insufficient to cover large enzymes like uricase due to the rigid alpha helix conformation of the Glu-Lys (EK) peptide. In this work, uricases were fused with 60 pairs of EKs at both the C- and N-termini of each subunit, using 2 His-Gly-His (HGH) hinges to increase surface coverage through structure fitting. The results showed that EKylation significantly improved solubility over 100-fold, maintained stable monodispersity, and increased catalytic activity approximately 4.06 fold. The 2 HGH hinges increased the steric shielding of the EK peptide by fitting the uricase surface, leading to a 2.5-fold increase in the elimination half-life compared to the EKylated uricase without hinges via intravenous injection (IV), with a slight decrease after three repeated subcutaneous injections (SC). These findings suggest that it is feasible to reduce immunogenicity and extend the elimination half-life by minimally disrupting the integrity of non-immunogenic EK peptides when fusing them at both the C- and N-termini. Further optimization is anticipated to yield a long-acting uricase suitable for SC.