Urate oxidase-albumin conjugate, preparation method thereof, and use thereof
The present specification discloses a urate oxidase-albumin conjugate, a preparation method thereof, a urate oxidase variant contained in the urate oxidase-albumin conjugate, and a preparation method thereof. The urate oxidase-albumin conjugate is characterized in that three or more albumins are conjugated to the urate oxidase variant through a linker, thereby improving half-life and reducing immunogenicity. In addition, the urate oxidase-albumin conjugate can be used to prevent or treat various diseases, disorders and/or indications caused by uric acid.
1 . A urate oxidase-albumin conjugate represented by [Formula 1]:
Uox-[J1-A-J2-HSA] n [Formula 1]
wherein Uox is a urate oxidase variant, J1 is a urate oxidase-linker junction, A is an anchor, J2 is an albumin-linker junction, and HSA is Human Serum Albumin,
n is 3 or 4,
the urate oxidase variant is a tetramer formed by oligomerization of four urate oxidase variant subunits,
each of the urate oxidase variant subunit is a peptide independently selected from SEQ ID NOs:2 to 50, or a peptide which is 90% or more identical to the peptide selected from SEQ ID NOs:2 to 50,
the peptide selected from SEQ ID NOs:2 to 50 comprises a nonnatural amino acid X which is 4-(1,2,3,4-tetrazine-3-yl) phenylalanine (frTet),
the urate oxidase-linker junction is a structure formed through Inverse Electron Demand Diels-Alder (IEDDA) reaction between a tetrazine moiety of the nonnatural amino acid of the urate oxidase variant and a trans-cyclooctene moiety linked to the anchor,
the urate oxidase-linker junction is represented by following,
wherein the (1) is linked to the residue of the nonnatural amino acid, and the (2) is linked to the anchor,
wherein the anchor is selected from the following:
wherein J 1 is urate oxidase-linker junction, and J 2 is albumin-linker junction,
wherein the albumin-linker junction is a structure formed through a reaction between a thiol moiety of the albumin and a thiol reactive moiety of the anchor,
wherein the albumin-linker junction is selected from the following:
wherein (1) is linked to the albumin and (2) is linked to the anchor.
2 . The urate oxidase-albumin conjugate of claim 1 , wherein the urate oxidase variant comprises four urate oxidase variant subunit of SEQ ID NO: 49, wherein a nonnatural amino acid X of the SEQ ID NO: 49 is frTet.
3 . The urate oxidase-albumin conjugate of claim 1 , wherein the albumin is a sequence selected from SEQ ID NOs:133 to 144, or a sequence that is 90% or more identical to the sequence selected from SEQ ID NOs:133 to 144.
4 . 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.
5 . The method of claim 4 , the linker is selected from the following:
6 . The method of claim 4 , wherein the urate oxidase variant is a tetramer that four urate oxidase variant subunits represented by SEQ ID NO: 29 are oligomerized, wherein the X of SEQ ID NO: 29 is frTet.
7 . The method of claim 4 , wherein the albumin is represented by a sequence selected from SEQ ID NOs: 133 to 144 or a sequence 90% or more identical with the sequence selected from SEQ ID NOs: 133 to 144, wherein the thiol reactive moiety of the linker is bound with thiol group of 34th cysteine of the sequence of the albumin through reaction.
8 . The method of claim 4 , wherein the reacting of the urate oxidase variant with the linker is performed at pH of 6 to 8.
9 . A method for treating uric acid-related disease, the method comprising:
administering the urate oxidase-albumin conjugate of claim 1 into a subject.
10 . The method of claim 9 , wherein the uric acid-related disease is any one of hyperuricemia, acute gouty arthritis, intermittent gout, chronic nodular gout, Chronic Kidney Disease, and Tumor Lysis Syndrome (TLS).
11 . The method of claim 9 , wherein the administering the pharmaceutical composition into a subject is selected from oral administration, parenteral administration, intravenous administration, intravenous infusion, intraperitoneal administration, intramuscular administration, transdermal administration, and subcutaneous administration.
12 . The method of claim 9 , wherein the dosage of the pharmaceutical composition is 1 mg/kg to 10 mg/kg, based on the mass of the administered urate oxidase-albumin conjugate relative to the mass of the subject.