Albumin fusion/conjugation (albumination) has been an effective method to prolong in vivo half-life of therapeutic
proteins. However, its broader application to
proteins with complex folding pathway or multi-subunit is restricted by incorrect folding, poor expression, heterogeneity, and loss of native activity of the
proteins linked to
albumin. We hypothesized that the site-specific conjugation of
albumin to a permissive site of a target
protein will expand the utilities of
albumin as a therapeutic activity extender to
proteins with a complex structure. We show here the genetic incorporation of a non-natural
amino acid (NNAA) followed by chemoselective
albumin conjugation to prolong therapeutic activity in vivo.
Urate oxidase (Uox), a therapeutic
enzyme for treatment of
hyperuricemia, is a homotetramer with multiple surface lysines, limiting conventional approaches for albumination. Incorporation of p-azido-
l-phenylalanine into two predetermined positions of Uox allowed site-specific linkage of dibenzocyclooctyne-derivatized
human serum albumin (HSA) through strain-promoted
azide-
alkyne cycloaddition (SPAAC). The bio-orthogonality of SPAAC resulted in the production of a chemically well-defined conjugate, Uox-HSA, with a retained enzymatic activity. Uox-HSA had a half-life of 8.8 h in mice, while wild-type Uox had a half-life of 1.3 h. The AUC increased 5.5-fold (1657 vs. 303 mU/mL x h). These results clearly demonstrated that site-specific albumination led to the prolonged enzymatic activity of Uox in vivo. Site-specific albumination enabled by NNAA incorporation and orthogonal chemistry demonstrates its promise for the development of long-acting
protein therapeutics with high potency and safety.