title: Site-specific albumination of a therapeutic protein with multi-subunit to prolong activity in vivo.
abstract: 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.
authors: Department of Chemical Engineering, University of Virginia, VA 22904, United States.
authors: Sung In
authors: SI
authors: Lim
authors: Department of Microbiology, Immunology, and Cancer Biology, University of Virginia, VA 22908, United States.
authors: Young S
authors: YS
authors: Hahn
authors: Department of Chemical Engineering, University of Virginia, VA 22904, United States; School of Materials Science and Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea. Electronic address: inchan@gist.ac.kr.
authors: Inchan
authors: I
authors: Kwon
mesh_terms: D000818:Animals; D019540:Area Under Curve; D001231:Aspergillus flavus / Q000201:enzymology* / Q000235:genetics; D004355:Drug Stability; D004795:Enzyme Stability; D005260:Female; D005656:Fungal Proteins / Q000008:administration & dosage / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D006207:Half-Life; D007275:Injections, Intravenous; D008810:Mice, Inbred C57BL; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012709:Serum Albumin / Q000008:administration & dosage / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics; D000075462:Serum Albumin, Human; D014503:Urate Oxidase / Q000008:administration & dosage / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000493:pharmacokinetics
chemical_list: C422455:ALB protein, human; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D014503:Urate Oxidase; D000075462:Serum Albumin, Human
keywords: Non-natural amino acid; Site-specific albumination; Strain-promoted azide-alkyne cycloaddition; Therapeutic protein; Urate oxidase