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US-12622951-B2 - Urate oxidase-albumin conjugate, preparation method thereof, and use thereof

US12622951B2US 12622951 B2US12622951 B2US 12622951B2US-12622951-B2

Abstract

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.

Inventors

  • Jeong Haeng Cho
  • Sun Oh Shin
  • Hyun Woo Kim
  • Hyeongseok KIM
  • Dong HO BAK
  • Inchan Kwon
  • Byungseop Yang

Assignees

  • PROABTECH INC.

Dates

Publication Date
20260512
Application Date
20210924
Priority Date
20200925

Claims (12)

  1. 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. 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. 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. 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. 5 . The method of claim 4 , the linker is selected from the following:
  6. 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. 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. 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. 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. 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. 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. 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.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is a National Stage Application of International Application No. PCT/KR2021/013037, filed Sep. 24, 2021, which claims the benefit of Korean Application Nos. 10-2020-0125215, filed Sep. 25, 2020, and 10-2021-0013537, filed Jan. 29, 2021, the entire contents of which are incorporated herein by reference. INCORPORATION BY REFERENCE OF SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 22, 2022, is named “PYH-01101_Sequence Listing” and is 453,880 bytes in size. TECHNICAL FIELD The present description discloses a urate oxidase variant into which a nonnatural amino acid is introduced site-specifically a preparation method thereof, a conjugate in which the urate oxidase variant and albumin are conjugated by a linker, and a preparation method thereof. In addition, the present description discloses the use of the urate oxidase-albumin conjugate. BACKGROUND ART Urate oxidase (Uricase) is a type of enzyme that cannot be synthesized in primates including humans, and it functions to break down uric acid into allantoin. Urate oxidase has a direct therapeutic mechanism of decomposing uric acid, which is the main cause of gout, into an excretable form, and thus has the advantage of a strong uric acid lowering effect. However, urate oxidase has limited use for treatment of gout because 1) it can only be used as an injection due to a short half-life in the body, and 2) an immune response occurs when administered to the body because it is an externally derived protein, resulting in side effects. On the other hand, therapeutic protein has been reported to be effective in the treatment of various diseases, and it is one of the important growth motives in the pharmaceutical industry. However, there is a problem in that therapeutic protein is continuously removed by glomerular filtration, pinocytosis, and immune response in a patient's body. Therefore, when developing a therapeutic protein, it is very important to extend the duration of the drug effect by lowering the rate at which it is removed from the patient's body due to such a phenomenon. A technique for improving the half-life to solve the problem by physically or chemically binding albumin to a therapeutic protein is called albumination. In the present description, in order to solve the above-described problems that may occur when urate oxidase is used as a therapeutic agent, a urate oxidase-albumin conjugate produced through albumination of urate oxidase is disclosed. DISCLOSURE Technical Problem The present description is intended to provide a urate oxidase-albumin conjugate. The present description is intended to provide a method of preparing the urate oxidase-albumin conjugate. The present description is intended to provide a urate oxidase variant included in the urate oxidase-albumin conjugate. The present description is intended to provide a method of preparing the urate oxidase variant. The present description is intended to provide a pharmaceutical composition including the urate oxidase-albumin conjugate. The present description is intended to provide a use of the urate oxidase-albumin conjugate. Technical Solution In the present description, a urate oxidase-albumin conjugate, which is represented by Formula 1: [Formula 1] Uox-[J1-A-J2-HSA]n in which Uox is a urate oxidase variant, J1 is a urate oxidase-linker junction, A is an anchor, J2 is an albumin-linker junction, HSA is human serum albumin, the urate oxidase variant includes three or more nonnatural amino acids having a diene functional group, the urate oxidase-linker junction is formed by Inverse Electron Demand Diels-Alder (IEDDA) reaction of a diene functional group of the nonnatural amino acid and a dienophile functional group connected to the anchor, and n is 3 or 4. Provided herein is a urate oxidase-albumin conjugate including: 3 or 4 albumin-subunit conjugates represented by Formula 2: [Formula 2]p′-J1-A-J2-HSA, where p′ is a urate oxidase variant subunit, J1 is a urate oxidase-linker junction, A is an anchor, J2 is an albumin-linker junction, and HSA is human serum albumin. The urate oxidase variant subunit is formed by substituting at least one amino acid in the sequence of a wild-type urate oxidase subunit with a nonnatural amino acid containing a tetrazine functional group or a triazine functional group. The urate oxidase-linker junction is formed by an inverse electron demand Diels-Alder (IEDDA) reaction between a tetrazine or triazine functional group of the nonnatural amino acid and a trans-cyclooctene functional group connected to the anchor; and Optionally, one urate oxidase variant subunit, when the urate oxidase-albumin conjugate includes three albumin-subunit complexes, and the urate oxidase-albumin conjugate includes one urate oxidase variant subunit, the urate oxidase