Urate oxidase-albumin conjugate, preparation method thereof, and use thereof

A method for manufacturing a urate oxidase-albumin conjugate, the method comprising:

• reacting an albumin and a linker, wherein the linker comprises a dienophile functional group, an anchor, and a thiol reactive moiety, wherein the dienophile functional group is a trans-cyclooctene or a derivative of trans-cyclooctene, and the thiol reactive moiety is selected from a maleimide or a derivative of maleimide, and a 3-arylpropiolonitriles or a derivative of 3-arylpropiolonitriles, wherein the thiol reactive moiety of the linker is bound with thiol moiety of albumin through reaction to form an albumin-linker conjugate;
• and
• reacting the albumin-linker conjugate and the urate oxidase variant, wherein the urate oxidase is a tetramer in which four urate oxidase subunits are oligomerized,
• wherein each of the urate oxidase subunit is represented by a sequence independently selected from SEQ ID NOs:2 to 50, or a sequence that is 90% or more identical to the sequence selected from SEQ ID NOs:2 to 50,
• wherein the X of a sequence of SEQ ID NOs:2 to 50 is nonnatural amino acid which is 4-(1,2,3,4-tetrazine-3-yl) phenylalanine (frTet),
• wherein the urate oxidase variant comprises four frTets,
• wherein a tetrazine functional group of a residue of the frTet is bound with the dienophile functional group of the linker through Inverse Electron Demand Diels-Alder (IEDDA) reaction to form a urate oxidase-albumin conjugate, and
• wherein the urate oxidase-albumin conjugate is characterized in that three or more albumins are conjugated to the urate oxidase variant through the linkers.