CN115197923A

CN115197923A - Uricase, pharmaceutical composition thereof and application thereof

Info

Publication number: CN115197923A
Application number: CN202210378688.1A
Authority: CN
Inventors: 刘洪川; 刘沛想; 黄国锋; 张静; 冯辉; 姚盛
Original assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Current assignee: Shanghai Junshi Biosciences Co Ltd; Suzhou Junmeng Biosciences Co Ltd
Priority date: 2021-04-09
Filing date: 2022-04-08
Publication date: 2022-10-18
Also published as: WO2022214086A1

Abstract

The invention provides uricase, a pharmaceutical composition thereof and application thereof. The uricase comprises the amino acid sequence shown as SEQ ID NO:9 or a variant thereof, a pharmaceutical composition comprising a buffer and uricase, said pharmaceutical composition having a pH of about 6.0 to about 8.5, and preferably about 7.5 to about 8.1.

Description

Uricase, pharmaceutical composition thereof and application thereof

Technical Field

The invention relates to the field of therapeutic drugs, and particularly relates to uricase, a pharmaceutical composition thereof and application thereof.

Background

Gout and hyperuricemia are common diseases which are harmful to human health at present, and the incidence rate of the hyperuricemia reaches 10 percent of the population according to statistics. Gout is an acute arthritis caused by precipitation of urate crystals in the joints due to excessive blood uric acid levels. On one hand, with the change of diet and living habits of people, the intake of high-protein and high-purine foods is increased, and the number of gout patients is increased year by year; on the other hand, after chemotherapy and radiotherapy of some malignant tumors, especially leukemia and lymphoma patients, a large amount of uric acid is deposited in the kidney in a short period of time, so that the kidney is damaged. Over the last several decades, many scientists have devoted themselves to the use of uricase from other species for the treatment of human hyperuricemia-related diseases. However, one of the biggest problems in applying foreign proteins to the human body is an anti-protein reaction and an allergic reaction caused by the immunogenicity of the proteins. Later scientists used ethylene glycol (PEG) to covalently modify proteins, demonstrating that it was possible to reduce protein immunogenicity, increase protein solubility and extend the half-life of proteins.

There are two clinically approved uricase drugs currently available, one being krysteka and the other being Elitek. Krystexxa (polyethyleneglycol recombinant uricase), a pegylated uricase approved for the treatment of chronic gout in adult patients refractory to conventional therapy, is a chimeric protein of highly pegylated porcine and baboon uricase sequences that can be administered within 2 hours by intravenous injection. Krystexxa contains a black box warning against anaphylaxis and infusion reactions. While Elitek (labyrinase) is a modified recombinant aspergillus flavus uricase that was indicated for the initial management of plasma uric acid levels in children and adult patients with leukemias, lymphomas, and solid tumor malignancies who received anti-cancer therapy that is expected to result in tumor lysis and subsequent elevation of plasma uric acid. However, elitek has a half-life in vivo of 16-21 hours and must be administered daily via intravenous infusion. Likewise, elitek also has a black box warning against anaphylaxis and hemolysis.

In view of the above, there is an urgent need in the art to develop safer, more convenient, and less immunogenic drugs for treating hyperuricemia.

Disclosure of Invention

In one aspect, the invention provides a uricase comprising the amino acid sequence as set forth in SEQ ID NO:9 or a variant thereof which retains enzymatic activity, wherein the variant has an amino acid sequence which is substantially identical to the amino acid sequence set forth in SEQ ID NO: mutations of 9 include: less than 17 amino acid residues from the N-terminus and/or less than 10 amino acid residues from the C-terminus.

In some embodiments, the variant has a sequence relative to SEQ ID NO: mutations of 9 include: the N-terminal amino acid sequence MTATAETSTGTKVVLGGQ is replaced by MANIILGK, and/or the C-terminal amino acid sequence SRADHPIWSN is replaced by C. .

In some embodiments, the uricase comprises the amino acid sequences as set forth in SEQ ID NOs: 10 or SEQ ID NO:11, and (c) the sequence shown in fig. 11.

In some embodiments, the variant has a sequence relative to SEQ ID NO:9 further comprises introducing 1, 2, 3, 4 or 5 surface accessible cysteine residues in 1, 2, 3, 4 or 5 positions selected from 35, 82, 142, 194, 216, 287 and 288, wherein the position numbering is relative to SEQ ID NO:3, position number.

In some embodiments, the variant has a sequence relative to SEQ ID NO:9 further comprises the introduction of 2 surface accessible cysteine residues at 2 positions selected from positions 35, 82, 142, 194, 216, 287 and 288, wherein the position numbering is relative to SEQ ID NO: position number of 3.

In some embodiments, the variant has a sequence relative to SEQ ID NO: mutations of 9 also include the inclusion of cysteine residues in the following two positions: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, wherein the position numbering is relative to SEQ ID NO: position number of 3.

In some embodiments, the variant has a sequence relative to SEQ ID NO: the mutations of 9 further include: a substitution mutation at position 205 and/or 208, wherein position numbering is relative to SEQ ID NO:3, position number. Preferably, the substitution mutation is a substitution of S at position 205 with D or E, or a substitution of G at position 208 with R, K or S. In some embodiments, the substitution mutation is S205D or G208R, or a combination thereof.

In some embodiments, the uricase of the invention comprises the amino acid sequence as set forth in SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. the amino acid sequence of SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 18. SEQ ID NO:19 or SEQ ID NO:20, or a pharmaceutically acceptable salt thereof.

In yet another aspect, the invention provides, in one aspect, a uricase conjugate (i.e., a modified uricase) comprising a uricase described herein and a polymer covalently linked or expressed fused to the uricase. In some embodiments, the polymer is selected from the group consisting of polyethylene glycol (PEG), phosphorylcholine polymers, polymers of repeating peptides or "X-TEN" sequences, and carbohydrate-based polymers. In some embodiments, the polymer is expressed fused to the N-terminus and/or C-terminus of the uricase, or is covalently linked through a cysteine residue present on the uricase.

In some embodiments, the polymer is PEG, which is covalently linked through a cysteine residue present on the uricase. Preferably, the uricase has 1, 2, 3, 4, or 5 surface accessible cysteine residues in 1, 2, 3, 4, or 5 positions selected from the group consisting of positions 35, 82, 142, 194, 216, 287, and 288, wherein the position numbering is relative to the amino acid sequence of SEQ ID NO:3, the PEG being covalently linked through cysteine residues at these positions. More preferably, said uricase comprises cysteine residues in the following two positions, respectively: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, wherein the position numbering is relative to the position of SEQ ID NO:3, the PEG is covalently linked through a cysteine residue at these positions.

In some embodiments, the uricase comprises a cysteine residue at position 288, and the PEG is covalently linked through the cysteine residue at that position, wherein the position numbering is relative to the position of SEQ ID NO: position number of 10.

In some embodiments, the uricase has the amino acid sequence set forth in SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 18. the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:20, said PEG is covalently linked to said amino acid sequence at positions 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, 82 and 287, 35 and 194, and 142 and 216 of said amino acid sequence, respectively, wherein the position numbering is relative to SEQ ID NO:3, position number.

In some embodiments, the uricase has the amino acid sequence set forth in SEQ ID NO: 13. SEQ ID NO:15 and SEQ ID NO:16, said PEG is further covalently attached to said amino acid sequence at position 288 of said amino acid sequence, wherein the position numbering is relative to SEQ ID NO:10, position number.

In some embodiments, the uricase has the amino acid sequence set forth in SEQ ID NO:17, the PEG is covalently linked through cysteine residues of 35 and 194, wherein the position numbering is relative to SEQ ID NO:3, position number.

In yet another aspect, the present invention provides a pharmaceutical composition comprising: (1) a buffer solution; (2) A uricase or a uricase conjugate according to any one of the embodiments herein.

In some embodiments, the uricase conjugate is a PEG-modified uricase of the invention.

In some embodiments, the pH of the pharmaceutical composition is about 6.0 to about 8.5, preferably about 7.0 to about 8.5, and preferably about 7.5 to about 8.1.

In some embodiments, the uricase of the above pharmaceutical composition comprises the amino acid sequences shown as SEQ ID NO 12, SEQ ID NO 13, or SEQ ID NO 17, respectively.

In some embodiments, the concentration of uricase or uricase-conjugate in the pharmaceutical composition is about 1 to about 300mg/mL, preferably about 1 to about 250mg/mL, preferably about 1 to about 200mg/mL, preferably about 5 to about 80mg/mL, more preferably about 5 to about 40mg/mL; more preferably 5-20mg/mL; more preferably, the uricase concentration is about 5mg/mL,6mg/mL,7mg/mL,8mg/mL,9mg/mL,10mg/mL,12mg/mL,14mg/mL,16mg/mL,18mg/mL,20mg/mL,25mg/mL,30mg/mL,35mg/mL,40mg/mL,50mg/mL,60mg/mL,70mg/mL,80mg/mL,90mg/mL,100mg/mL,110mg/mL,120mg/mL,130mg/mL or 140mg/mL, preferably about 8mg/mL,10mg/mL or 20mg/mL.

In some embodiments, the buffer is selected from the group consisting of phosphate buffer, tris-hcl buffer, citrate buffer, and histidine buffer; preferably, the buffer is phosphate buffer or Tris-hydrochloric acid buffer; more preferably, the buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

In some embodiments, the buffer is a phosphate buffer, and preferably, the phosphate buffer is a sodium phosphate dibasic-sodium phosphate monobasic buffer. In some embodiments, the phosphate buffer is made up of 1-30mM disodium phosphate and 1-30mM sodium dihydrogen phosphate. In some embodiments, the phosphate buffer is made up of 17mM disodium phosphate and 3mM sodium dihydrogen phosphate monohydrate. In some embodiments, the phosphate buffer is made up of 18.5mM sodium phosphate dibasic and 1.5mM sodium phosphate monobasic monohydrate.

In some embodiments, the buffer is Tris-HCl (Tris-HCl) buffer. In some embodiments, the Tris-HCl buffer is made up of 1-30mM HCl and 1-30mM Tris (Tris hydroxymethyl aminomethane). In some embodiments, the Tris-HCl buffer is made up of 11.8mM Tris-HCl and 8.2mM Tris. In some embodiments, the Tris-HCl buffer is made up of 16.5mM Tris-HCl and 3.5mM Tris. In some embodiments, the Tris-HCl buffer is prepared from 20mM Tris, with concentrated HCl added to adjust the pH to 7.5 or 8.0.

In some embodiments, the buffer is a histidine buffer, preferably the histidine buffer is selected from a histidine-hydrochloride buffer or a histidine-acetate buffer.

In some embodiments, the buffer is a citrate buffer, and preferably, the citrate buffer is a citrate-sodium citrate buffer.

In some embodiments, the buffer is at a concentration of about 1mM to about 200mM, preferably about 5mM to about 200mM, preferably about 10mM to about 50mM, preferably about 10mM to about 30mM; preferably about 20 to 30mM, and the above-mentioned buffer concentration is, for non-limiting examples, about 5mM,10mM, 1mM, 10mM, 25mM,30mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 105mM, 110mM, 115mM, 120mM, 130mM, 140mM, 150mM, 160mM,170mM or 180mM, or a range wherein any two of these ranges are defined as endpoints, preferably 10mM,15mM,20mM or 30mM.

In some embodiments, the pH of the buffer is about 6.0 to about 8.5, preferably about 6.5 to about 8.5, preferably about 7.0 to about 8.5, preferably about 7.5 to about 8.1, and non-limiting examples of the pH of the buffer are about 6.0,6.1,6.2,6.3,6.4,6.5,6.6,6.7,6.8,6.9,7.0,7.1,7.2,7.3,7.4,7.5,7.6,7.7,7.8,7.9,8.0,8.1,8.2,8.3,8.4,8.5, preferably about 7.5,7.6,7.7,7.8,7.9,8.0, or 8.1, more preferably about 7.8.

In some embodiments, the pharmaceutical composition further comprises a stabilizer selected from one or more of arginine hydrochloride, sodium chloride, mannitol, sucrose and trehalose; preferably, the stabilizer is selected from mannitol, trehalose, a combination of mannitol and sodium chloride or a combination of trehalose and sodium chloride.

In some embodiments, the concentration of the stabilizer is about 10mM to 400mM, preferably 20mM to 300mM, and more preferably 50mM to 200mM.

In some embodiments, the stabilizing agent is sodium chloride at a concentration of about 30 to 200 mM; or the stabilizing agent is mannitol at a concentration of about 100-300 mM; or the stabilizer is sucrose at a concentration of about 100-300 mM; or the stabilizer is trehalose at a concentration of about 100-300 mM; or the stabilizer is arginine hydrochloride at a concentration of about 30-200 mM.

In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM trehalose; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose.

In some embodiments, the stabilizer is sodium chloride. In some embodiments, the stabilizing agent is sodium chloride at a concentration of about 30mM to about 200mM, preferably at a concentration of about 50mM to about 190mM, preferably about 100mM to about 180mM, preferably about 120mM to about 170mM, preferably about 130mM to about 150mM, non-limiting examples of such concentrations of sodium chloride are about 100mM,110mM,120mM,125mM,130mM,135mM,140mM,145mM,150mM,155mM,160mM,170mM,180mM,190mM,200mM, preferably 135mM or 140mM.

In some embodiments, the stabilizing agent is mannitol. In some embodiments, the stabilizing agent is mannitol in a concentration of about 100mM to about 300mM, preferably about 150mM to about 300mM, preferably about 200mM to about 300mM, and non-limiting examples of such mannitol concentrations are about 200mM,210mM,220mM,230mM,240mM,250mM,260mM,270mM,280mM, preferably 240mM.

In some embodiments, the stabilizer is sucrose. In some embodiments, the stabilizing agent is sucrose at a concentration of about 100mM to about 300mM, preferably about 150mM to about 300mM, and more preferably about 200mM to about 300mM, non-limiting examples of such sucrose concentrations are about 200mM,210mM,220mM,230mM,240mM,250mM,260mM,270mM,280mM, and preferably about 220mM.

In some embodiments, the stabilizing agent is trehalose. In some embodiments, the stabilizing agent is trehalose at a concentration of about 100mM to about 300mM, preferably about 150mM to about 300mM, more preferably about 200mM to about 300mM, non-limiting examples of such trehalose concentrations are about 180mM,200mM,210mM,220mM,230mM,240mM,250mM,260mM,270mM,280mM, more preferably about 220mM.

In some embodiments, the stabilizing agent is arginine hydrochloride. In some embodiments, the stabilizer is arginine hydrochloride at a concentration of about 30-200mM, preferably about 50-190mM, preferably about 100-180mM, preferably about 120-170mM, preferably about 130-150mM, non-limiting examples of such arginine hydrochloride concentrations are about 100mM,110mM,120mM,125mM,130mM,135mM,140mM,145mM,150mM,155mM,160mM,170mM,180mM,190mM,200mM, preferably 135mM or 140mM.

In some embodiments, the stabilizing agent is a combination of sodium chloride and mannitol. In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol, preferably a combination of about 30-150mM sodium chloride and about 40-200mM mannitol, preferably a combination of about 30-100mM sodium chloride and about 100-180mM mannitol, a non-limiting example of which is a combination of about 50mM sodium chloride and about 140mM mannitol.

In some embodiments, the stabilizer is a combination of sodium chloride and sucrose. In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose, preferably a combination of about 30-150mM sodium chloride and about 40-200mM sucrose, preferably a combination of about 30-100mM sodium chloride and about 100-180mM sucrose, a non-limiting example of which is a combination of about 50mM sodium chloride and about 140mM sucrose.

In some embodiments, the stabilizing agent is a combination of sodium chloride and trehalose. In some embodiments, the stabilizing agent is a combination of about 30-200mM sodium chloride and about 30-200mM trehalose, preferably a combination of about 30-150mM sodium chloride and about 40-200mM trehalose, preferably a combination of about 30-100mM sodium chloride and about 100-180mM trehalose, a non-limiting example of which is a combination of about 50mM sodium chloride and about 140mM trehalose.

In some embodiments, the above pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20, or poloxamer 188.

In some embodiments, the surfactant is selected from polysorbate 80.

In some embodiments, the surfactant is selected from polysorbate 20.

In some embodiments, the surfactant concentration is from about 0.001% to about 0.1%, preferably from about 0.01% to about 0.1%, preferably from about 0.02% to about 0.08%, calculated as w/v; by way of non-limiting example, the concentration of the above surfactant is about 0.02%,0.04% or 0.08%, preferably 0.02%.

In some embodiments, the pharmaceutical composition herein comprises, or consists of, the components set forth in any one of (1) to (5) below:

(1) (a) about 5mg/mL to 40mg/mL of uricase or uricase conjugate; (b) about 10 to about 30mM phosphate buffer, at a pH of about 7.5 to about 8.1; (c) about 100 to 300mM mannitol; and (d) about 0.01% to 0.1% polysorbate 80; or

(2) (a) about 5mg/mL to 40mg/mL of uricase or a uricase conjugate; (b) About 10 to 30mM Tris-HCl buffer, pH about 7.5 to 8.1; (c) about 100 to 300mM trehalose; and (d) about 0.01% to 0.1% polysorbate 80; or

(3) (a) about 5mg/mL to 40mg/mL of uricase or a uricase conjugate; (b) about 10 to 30mM phosphate buffer, pH about 7.5 to 8.1; (c) about 100 to 300mM trehalose; and (d) about 0.01% to 0.1% polysorbate 80; or

(4) (a) about 5mg/mL to 40mg/mL of uricase or a uricase conjugate; (b) about 10 to about 30mM phosphate buffer, at a pH of about 7.5 to about 8.1; (c) A combination of about 30-200mM sodium chloride and about 30-200mM mannitol; and (d) about 0.01% to 0.1% polysorbate 80; or

(5) (a) about 5mg/mL to 40mg/mL of uricase or a uricase conjugate; (b) about 10 to about 30mM phosphate buffer, at a pH of about 7.5 to about 8.1; (c) A combination of about 30-200mM sodium chloride and about 30-200mM trehalose; and (d) about 0.01% to 0.1% polysorbate 80.

Preferably, the uricase is as described in any one of the embodiments herein.

In some embodiments, the pharmaceutical composition comprises, or consists of, a component as set forth in any one of (1) to (5) below, or each of (1) to (5):

(1) (a) about 8mg/mL uricase or uricase conjugate; (b) about 20mM phosphate buffer, pH about 7.5-7.8; (c) about 240mM mannitol; and (d) about 0.02% polysorbate 80; or

(2) (a) about 8mg/mL uricase or uricase conjugate; (b) about 20mM Tris-HCl buffer, pH about 7.5-8.0; (c) trehalose at about 220 mM; and (d) about 0.02% polysorbate 80; or

(3) (a) about 8mg/mL of uricase or a uricase conjugate; (b) about 20mM phosphate buffer, pH about 7.5-7.8; (c) about 220mM trehalose; and (d) about 0.02% polysorbate 80; or

(4) (a) about 8mg/mL uricase or uricase conjugate; (b) about 20mM phosphate buffer at a pH of about 7.5 to about 7.8; (c) A combination of about 50mM sodium chloride and about 140mM mannitol; and (d) about 0.02% polysorbate 80; or

(5) (a) about 8mg/mL uricase or uricase conjugate; (b) about 20mM phosphate buffer at a pH of about 7.5 to about 7.8; (c) A combination of about 50mM sodium chloride and about 140mM trehalose; and (d) about 0.02% polysorbate 80.

Preferably, the uricase or uricase conjugate is as described in any embodiment herein.

In some embodiments, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.

In some embodiments, the pharmaceutical composition is a liquid formulation.

In some embodiments, the liquid formulation or lyophilized formulation described above is stable at 2-8 ℃ for at least 2 months, 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.

In some embodiments, the aqueous solution or lyophilized formulation is stable at 23-27 ℃ for at least 2 weeks, at least 4 weeks, or at least 8 weeks.

In some embodiments, the aqueous solution or lyophilized formulation is stable at 40 ℃ for at least 7 days, at least 14 days, or at least 28 days.

The present invention also provides an injection comprising the pharmaceutical composition according to any of the embodiments of the present invention and 0.9wt% sodium chloride solution or 5wt% aqueous glucose solution; preferably, the concentration of the uricase in the injection containing 0.9 weight percent of sodium chloride solution is 0.2 to 4mg/mL, and the concentration of the uricase in the injection containing 5 weight percent of glucose aqueous solution is 1 to 4mg/mL; preferably, the injection has a pH of 7.5 to 8.1.

The invention also provides application of the uricase, the uricase conjugate or the pharmaceutical composition in preparing medicines for reducing the uric acid level in body fluid and tissues of mammals or treating hyperuricemia.

Drawings

FIG. 1: enzyme specific activities of various uricases were compared.

FIG. 2 is a schematic diagram: thermal stability experiments for various uricases.

FIG. 3: thermal stability studies of various uricases. 3a: carrying out PR-HPLC analysis on protein molecules at 4 ℃;3b: protein molecules were analyzed by SEC-HPLC at 4 ℃.

FIG. 4: and (3) carrying out enzyme activity kinetic study evaluation on the uricase modified by PEGylation.

FIG. 5: evaluation of thermostability of uricase modified by PEGylation.

FIG. 6: and (3) evaluating the in vivo efficacy of the uricase modified by the PEGylation.

Detailed Description

Definitions and explanations

In order that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

The term "pharmaceutical composition" or "formulation" means a mixture containing one or more of the antibodies described herein and other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient, and exert biological activity.

The term "liquid formulation" refers to a formulation in the liquid state and is not intended to refer to a resuspended lyophilized formulation. The liquid formulations of the present invention are stable upon storage and their stability is independent of lyophilization (or other state change methods, such as spray drying).

The term "aqueous liquid formulation" refers to a liquid formulation that uses water as a solvent. In some embodiments, the aqueous liquid formulation is one that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).

The term "excipient" refers to an agent that may be added to a formulation to provide a desired characteristic (e.g., consistency, improved stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

As used herein, "about" when referring to a measurable value (e.g., amount, duration, etc.) is intended to encompass variations of ± 20% or ± 10% from the particular value, including ± 5%, ± 1% and ± 0.1%, as such variations are suitable for performing the disclosed methods.

The term "buffer pH of about 6.0-8.5" refers to an agent that, through the action of its acid/base conjugate components, renders a solution containing the agent resistant to pH changes. The buffer used in the formulation of the present invention may have a pH in the range of about 6.0 to about 8.5, or a pH in the range of about 7.0 to about 8.5, or a pH in the range of about 7.5 to about 8.1.

Examples of "buffers" that control the pH within this range herein include acetate (e.g., sodium acetate), tris hydrochloric acid, succinate (e.g., sodium succinate), gluconic acid, histidine hydrochloride, methionine, citrate, phosphate, citrate/phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof, and other organic acid buffers.

A "phosphate buffer" is a buffer that includes phosphate ions. Examples of the phosphate buffer include disodium hydrogenphosphate-sodium dihydrogenphosphate, dipotassium hydrogenphosphate-potassium dihydrogenphosphate, and the like. The preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

"Tris-HCl buffer" is Tris-HCl buffer, which is also Tris-HCl buffer. The Tris-HCl buffer comprises Tris and HCl.

"histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include salts of histidine and histidine, such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, and the like, such as histidine buffer containing histidine and histidine hydrochloride.

A "citrate buffer" is a buffer that includes citrate ions. Examples of citrate buffers include sodium citrate-citrate, potassium citrate-citrate, calcium citrate-citrate, magnesium citrate-citrate, and the like. The preferred citrate buffer is a citric acid-sodium citrate buffer.

The term "stabilizer" means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or salts thereof (e.g., arginine hydrochloride), glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), octopine (opines), alanine (alanine), octopine, glycinol (strombine)) and the N-oxide of Trimethylamine (TMAO), human serum albumin (hsa), albumin (bsa), α -bovine serum casein, globulin, α -lactalbumin, GABA, lysozyme, myoglobin, ovalbumin and rnasea a. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., may also serve to control osmotic pressure. The stabilizer used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars. The preferred salt is sodium chloride, the preferred sugars are sucrose and trehalose, and the preferred polyol is mannitol. Preferred amino acids are arginine or its salts (e.g. arginine hydrochloride), glycine, proline. Preferred stabilizers are sodium chloride, mannitol, sucrose, trehalose, arginine hydrochloride, sodium chloride-mannitol, sodium chloride-sucrose and sodium chloride-trehalose.

The term "surfactant" generally includes agents that protect proteins, such as antibodies, from air/solution interface-induced stress, solution/surface-induced stress to reduce aggregation of the antibodies or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenylpolyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronics), sodium Dodecyl Sulfate (SDS).

The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using an osmometer of the vapor pressure or freezing point depression type.

The term "stable" formulation is one in which the antibody substantially retains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. The pharmaceutical preparation may be stable even if the contained antibody fails to maintain 100% of its chemical structure or biological function after storage over a certain period of time. In certain instances, an antibody structure or function that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% after storage over a period of time may also be considered "stable". Various analytical techniques for measuring Protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-301, vincent Lee, marcel Dekker, inc., new York, n.y., pubs. (1991)), and Jones, a., 1993, adv. 29-90 (both incorporated by reference).

After storage of the formulation at a temperature and for a period of time, its stability can be measured by determining the percentage of native antibody remaining therein (among other methods). The percentage of native antibody may be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SEC-HPLC ]), among other methods, "native" referring to unaggregated and undegraded. In some embodiments, the stability of a protein is determined as a percentage of monomeric protein in a solution having a low percentage of degraded (e.g., fragmented) and/or aggregated protein. In some embodiments, the formulation is stable for storage at room temperature, about 25-30 ℃, or 40 ℃ for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, up to no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.

Stability can be measured by determining the percentage of antibodies ("acidic form") that migrate during ion exchange in the fraction of this main fraction of antibodies ("predominantly charged form") that is more acidic (among other methods), wherein stability is inversely proportional to the percentage of antibodies in acidic form. The percentage of "acidified" antibody can be measured by, among other methods, ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ]). In some embodiments, an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in acidic form can be detected in the formulation after storage of the formulation at a temperature and for a time. The certain time period of storage prior to measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, a temperature that allows for storage of the pharmaceutical formulation can be any temperature in the range of about-80 ℃ to about 45 ℃, e.g., storage at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 2-8 ℃, about 5 ℃, about 25 ℃, or about 40 ℃.

An antibody "retains its physical stability" in a pharmaceutical composition if it shows substantially no signs of, for example, aggregation, precipitation and/or denaturation when visually inspected for color and/or clarity or measured by UV light scattering or by pore size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate, covalently or non-covalently, to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.

Stability, e.g., physical stability, of a formulation can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements correlate with the turbidity of the formulation. Turbidity of a formulation is, in part, an inherent property of proteins dissolved in solution and is typically measured by nephelometry and is measured in Nephelometric Turbidity Units (NTU).

Turbidity levels that vary with, for example, the concentration of one or more components in a solution (e.g., protein and/or salt concentration) are also referred to as "opacification" or "opacified appearance" of a formulation. Turbidity levels can be calculated with reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining the turbidity level of a pharmaceutical composition may be based on the "European Pharmacopoeia" standards (European Pharmacopoeia), fourth edition, "European commission for Quality of Medicine instructions" (EDQM), strasbourg, france). According to the european pharmacopoeia standard, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension according to the european pharmacopoeia standard having a turbidity of about 3. Nephelometric turbidity measurements can detect rayleigh scattering in the absence of association or non-ideal effects, which typically varies linearly with concentration. Other methods for assessing physical stability are well known in the art.

An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given point in time is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be assessed, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical changes may include size changes (e.g., clipping), which may be assessed using, for example, pore size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS). Other types of chemical changes include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

An antibody in a pharmaceutical composition "retains its biological activity" if it is biologically active for its intended purpose. For example, a formulation of the invention may be considered stable if, after storage of the formulation at a temperature, e.g., 5 ℃,25 ℃, 45 ℃ or the like, for a period of time (e.g., 1 to 12 months), the formulation comprises uricase that binds to TIGIT with an affinity that is at least 90%, 95% or more of the binding affinity of the antibody prior to said storage. Binding affinity can also be measured using, for example, ELISA or plasmon resonance techniques.

In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody, in a pharmacological sense, refers to an amount effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody is effective to treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of the asymptomatic phase of a disease, or the prevention of damage or disability resulting from the affliction of the disease. The ability of a drug to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by assaying the activity of the agent in an in vitro assay. A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of a drug that inhibits the development or recurrence of a disease when administered to a subject at risk of developing the disease or a subject having a relapse of the disease, either alone or as combined with other therapeutic drugs.

The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects/patients include human and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a particular embodiment of the invention, the subject is a human.

The terms "administering," "administering," and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those of skill in the art. Routes of administration of anti-PD-1 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "parenteral administration" refers to modes of administration other than enteral or topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion, and via in vivo electroporation.

An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or sign of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending upon factors such as the condition to be treated, the general health of the patient, the method of administration and the dosage and severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects. The results may result in an improvement in the diagnostic measure or parameter of at least 5%, typically at least 10%, more typically at least 20%, most typically at least 30%, preferably at least 40%, more preferably at least 50%, most preferably at least 60%, ideally at least 70%, more ideally at least 80%, most ideally at least 90%, where 100% is defined as the diagnostic parameter exhibited by a normal subject (see, e.g., maynard et al (1996) A Handbook of SOPs for Good Clinical Practice, interpharm Press, boca Raton, FL; dent (2001) Good Laboratory and Good Clinical Practice, urch publication, london, UK).

Uricase

Urate oxidase (uricase)

Urate oxidase (uricase) is a class of enzymes that catalyzes the oxidation of uric acid, the oxidation product being the more soluble allantoin. Allantoin belongs to purine metabolites and is more easily excreted. It is a homotetrameric enzyme composed of four identical 34kDa subunits, which serves as the starting material for the start of a series of reactions to convert uric acid to a more soluble and more excretable product (allantoin), i.e., uricase catalyzes Uric Acid (UA) with O ₂ And H ₂ O reaction to form 5-hydroxy-isocyanurate (HIU) and H ₂ O ₂ Is released. HIU is an unstable product that undergoes non-enzymatic hydrolysis to form 2-oxo-4-hydroxy-5-ureidoimidazole (OHCU) and subsequent spontaneous decarboxylation to form racemic allantoin. Two additional enzymes (HIU hydrolase and OHCU decarboxylase) are expressed in species containing functional uricase and catalyze these reactions more rapidly to produce(s) -allantoin. Functional uricases are found in a wide variety of organisms, such as archaea, bacteria, and eukaryotes. However, uricase is not expressed in humans and some primates. This is due to the presence of a nonsense mutation at codon 33, codon 187, and a mutation at the splice acceptor site in intron 2. In summary, the lack of uricase expression in humans results in high systemic UA levels, and in some casesIn cases leading to hyperuricemia such as gout and tumor lysis syndrome. According to conservative estimation, the number of patients with hyperuricemia in China reaches 1.2 hundred million, and the hyperuricemia becomes the fourth highest after hypertension, hyperglycemia and hyperlipidemia. Gout is defined as an inflammatory arthritis in which serum UA levels exceed the solubility of UA in body fluids, and serum UA levels above 6.8mg/dL can lead to the formation of UA crystals in tissues, causing an acute inflammatory response. Gout causes chronic pain, dysfunction in work and home life, and impaired health-related quality of life, among others. Tumor Lysis Syndrome (TLS) refers to a group of clinical syndromes that a large amount of tumor cells are lysed in a short period to release intracellular metabolites, resulting in hyperuricemia, hyperkalemia, hyperphosphatemia, hypocalcemia, and acute renal failure as main manifestations.

The uricase of the invention comprises the amino acid sequence shown as SEQ ID NO:9 or a variant thereof which retains enzymatic activity, wherein the variant has an amino acid sequence which is substantially identical to the amino acid sequence set forth in SEQ ID NO: the mutations of 9 include: less than 17 amino acid residues from the N-terminus and/or less than 10 amino acid residues from the C-terminus. Variants of uricase of the invention have the following features relative to SEQ ID NO: the mutation of 9 may further comprise introducing 1, 2, 3, 4 or 5 surface accessible cysteine residues in 1, 2, 3, 4 or 5 positions selected from positions 35, 82, 142, 194, 216, 287 and 288, wherein the position numbering is relative to the position of SEQ ID NO: position number of 3. Variants of uricase of the invention have the following features relative to SEQ ID NO: the mutation of 9 may further comprise: a substitution mutation at position 205 and/or 208, wherein position numbering is relative to SEQ ID NO: position number of 3.

In a preferred embodiment, the variant has a sequence relative to SEQ ID NO: the mutations of 9 include: the N-terminal amino acid sequence MTATAETSTGTKVVLQis replaced by MANIILGK, and/or the C-terminal amino acid sequence SRADHPIWSN is replaced by C.

In a preferred embodiment, said uricase variant has a sequence relative to SEQ ID NO: the mutation of 9 may further comprise the introduction of 2 surface accessible cysteine residues at 2 positions selected from positions 35, 82, 142, 194, 216, 287 and 288, wherein the position numbering is relative to the position of SEQ ID NO: position number of 3. In a further preferred embodiment, the variant comprises a cysteine residue in two of the following positions: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, wherein the position numbering is relative to the position of SEQ ID NO: position number of 3.

In a preferred embodiment, said variant of uricase has a sequence relative to SEQ ID NO: the mutation of 9 further comprises the substitution of S at position 205 with D or E and the substitution of G at position 208 with R, K or S. In some embodiments, the substitution mutation is S205D or G208R, or a combination thereof.

In a preferred embodiment, the amino acid sequences of said variants of uricase comprise the amino acid sequences as set forth in SEQ ID NOs: 10-20, wherein the amino acid sequence of SEQ ID NO 12-20 is as follows:

AG-2C12/AG-2C12-PEG (SEQ ID NO:12, cys mutation based on SEQ ID NO: 9)

AG-2C13/AG-2C13-PEG (SEQ ID NO:13, cys mutation based on SEQ ID NO: 10)

AG-2C16 (SEQ ID NO:14, cys mutation based on SEQ ID NO: 9)

AG-2C17 (SEQ ID NO:15, cys mutation based on SEQ ID NO: 10)

AG-2C13-1 (SEQ ID NO:16, cys mutation based on SEQ ID NO: 10)

AG-2C01-2/AG-2C01-2-PEG (SEQ ID NO:17, cys mutation based on SEQ ID NO: 11)

AG-2C05-1 (SEQ ID NO:18, cys mutation based on SEQ ID NO: 9)

AG-2C01-1 (SEQ ID NO:19, cys mutation based on SEQ ID NO: 11)

AG-2C12-1 (SEQ ID NO:20, cys mutation based on SEQ ID NO: 9)

Uricase conjugate (modified uricase)

The uricase conjugates of the invention comprise a uricase as described herein and a polymer covalently linked or expressed in fusion with the uricase. In some embodiments, the polymer is selected from the group consisting of polyethylene glycol (PEG), phosphorylcholine polymers, polymers of repeating peptides or "X-TEN" sequences, and carbohydrate-based polymers. In some embodiments, the polymer is expressed fused to the N-terminus and/or C-terminus of the uricase, or is covalently linked through a cysteine residue present on the uricase.

In this contextIn (1), polyethylene glycol (PEG) is of the structure H- (O-CH) ₂ -CH ₂ ) _n -OH. The PEG reagent most commonly used for covalent attachment of proteins is of the structure CH ₃ -O-(CH ₂ -CH ₂ -O) _n Monomethoxy poly (ethylene glycol) derivatives of X, wherein X contains a linear linker and a reactive functional group (linear PEG), and n is an integer from 110 to 18200. In some cases, X may contain branching elements such that the PEG reagent contains one reactive functional group and more than one PEG polymer chain (branched PEG) or more than one reactive functional group and a PEG polymer chain (forked PEG). The PEG reagent may comprise about 5, 10, 20, 40, 60 and 80KDa of total PEG polymer.

In some embodiments, a thiol-reactive PEG may be used to react with a thiol group on at least one cysteine. For example, PEG-maleimide may be used, as well as PEG-orthopyridyl disulfide, PEG-vinyl sulfone, and PEG-iodoacetamide. In other embodiments, the thiol-reactive PEG may be a linear or branched structure with a single thiol-reactive moiety, or may be a forked structure with two or more reactive groups per PEG molecule.

Various methods are known in the art to PEGylate one or more cysteines in uricase. The present invention can be adopted.

In a preferred embodiment, the polymer is PEG, and the uricase conjugate of the invention is PEG-modified uricase, also denoted herein as PEG-uricase. PEG may be covalently linked through a cysteine residue present on said uricase. Preferably, the uricase has 1, 2, 3, 4, or 5 surface accessible cysteine residues in 1, 2, 3, 4, or 5 positions selected from the group consisting of positions 35, 82, 142, 194, 216, 287, and 288, wherein the position numbering is relative to the position of SEQ ID NO:3, the PEG being covalently linked through cysteine residues at these positions. More preferably, said uricase comprises cysteine residues in the following two positions, respectively: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, wherein the position numbering is relative to the position of SEQ ID NO:3, the PEG being covalently linked through cysteine residues at these positions.

In some embodiments, the uricase has the amino acid sequence set forth in SEQ ID NO: 12. SEQ ID NO: 13. the amino acid sequence of SEQ ID NO: 14. SEQ ID NO: 15. the amino acid sequence of SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 18. the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:20, said PEG is covalently attached to said amino acid sequence at positions 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, 82 and 287, 35 and 194, and 142 and 216, respectively, of said amino acid sequence, wherein the position numbering is relative to SEQ ID NO:3, position number.

Diseases and their treatment or prevention

The invention also provides methods of treating or preventing hyperuricemia in a bodily fluid or tissue of a mammal using a uricase or a uricase conjugate (e.g., PEG-uricase) of the invention. The methods generally comprise administering to a subject in need thereof a therapeutically or prophylactically effective amount of an improved uricase or uricase-conjugate (e.g., PEG-uricase) according to any one of the embodiments of the invention, or a pharmaceutical composition comprising the uricase or uricase-conjugate (e.g., PEG-uricase). Suitable modes of administration may be selected as appropriate, including but not limited to oral, intravenous, subcutaneous, intramuscular, intraarterial, by inhalation, aerosol delivery.

The subject typically suffers from a disease associated with elevated uric acid, i.e., a disease that benefits from a decrease in uric acid. Typically, diseases associated with elevated uric acid include hyperuricemia such as gout and tumor lysis syndrome.

Pharmaceutical preparation

The pharmaceutical composition is a high-stability pharmaceutical composition containing uricase or uricase conjugate. The invention discovers that the trehalose can obviously improve the stability of the pharmaceutical composition.

The pharmaceutical composition provided by the invention is a liquid preparation with high stability. In particular, the present inventors have found that formulations containing phosphate buffers are significantly more stable than other buffers.

The present invention provides a pharmaceutical composition comprising: (1) a buffer solution; (2) Uricase or a uricase conjugate according to any embodiment of the invention.

The uricase or uricase conjugate in the pharmaceutical composition of the invention may be uricase comprising the amino acid sequence set forth in any one of SEQ ID Nos. 3-20 or a conjugate thereof, particularly PEG-modified uricase.

The pharmaceutical compositions of the invention may have a uricase or uricase conjugate concentration of about 1 to about 300mg/mL, preferably about 1 to about 250mg/mL, preferably about 1 to about 200mg/mL, preferably about 5 to about 80mg/mL, more preferably about 5 to about 40mg/mL; more preferably 5-20mg/mL; more preferably, the uricase concentration is about 5mg/mL,6mg/mL,7mg/mL,8mg/mL,9mg/mL,10mg/mL,12mg/mL,14mg/mL,16mg/mL,18mg/mL,20mg/mL,25mg/mL,30mg/mL,35mg/mL,40mg/mL,50mg/mL,60mg/mL,70mg/mL,80mg/mL,90mg/mL,100mg/mL,110mg/mL,120mg/mL,130mg/mL or 140mg/mL, preferably about 8mg/mL,10mg/mL or 20mg/mL.

The buffer in the pharmaceutical composition of the present invention may be selected from the group consisting of phosphate buffer and Tris-hcl buffer to provide a pH of 6.0 to 8.5, preferably about 6.5 to 8.5, preferably about 7.0 to 8.5, preferably about 7.5 to 8.1, more preferably about 7.8, to the pharmaceutical composition of the present invention. In another aspect, the pH of the buffer used in the pharmaceutical compositions of the present invention may be from 6.0 to 8.5, preferably from about 6.5 to 8.5, preferably from about 7.0 to 8.5, preferably from about 7.5 to 8.1, more preferably about 7.8.

A particularly preferred buffer in the pharmaceutical composition of the invention is a phosphate buffer. Preferably, the phosphate buffer has a pH of about 7.0 to about 8.5, preferably about 7.5 to 8.5, more preferably about 7.5 to 8.1. Preferably, the phosphate buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer. In some embodiments, the phosphate buffer is made up of 1-30mM disodium phosphate and 1-30mM sodium dihydrogen phosphate. In some embodiments, the phosphate buffer is made up of 17mM disodium phosphate and 3mM sodium dihydrogen phosphate monohydrate. In some embodiments, the phosphate buffer is made up of 18.5mM disodium phosphate and 1.5mM monosodium phosphate monohydrate.

The buffer in the pharmaceutical composition of the present invention may be a Tris hydrochloride buffer. In some embodiments, the Tris-HCl buffer is made up of 1-30mM HCl and 1-30mM Tris (Tris hydroxymethyl aminomethane). In some embodiments, the Tris-HCl buffer is made up of 11.8mM Tris-HCl and 8.2mM Tris. In some embodiments, the Tris-HCl buffer is made up of 16.5mM Tris-HCl and 3.5mM Tris. In some embodiments, the Tris-HCl buffer is prepared from 20mM Tris, with concentrated HCl added to adjust the pH to 7.5 or 8.0.

Thus, in the pharmaceutical composition of the present invention, the buffer may be a phosphate buffer having a pH of 7.5 to 8.1, which is present in the pharmaceutical composition at a concentration of 10 to 30mM; and the pharmaceutical composition comprises 5-40mg/mL of uricase or modified uricase according to any one of the preceding embodiments, in particular, the PEG-modified uricases AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG described herein.

In some embodiments, the pharmaceutical compositions of the present invention further comprise a stabilizer. Preferably, the stabilizer is selected from one or more of arginine hydrochloride, sodium chloride, mannitol, sucrose and trehalose; preferably, the stabilizer is selected from mannitol, trehalose, a combination of mannitol and sodium chloride or a combination of trehalose and sodium chloride; more preferably trehalose or a combination of trehalose and sodium chloride. The concentration of the stabilizer in the pharmaceutical composition of the present invention is about 10mM-400mM, preferably 20mM-300mM, more preferably 50mM-250mM. In some embodiments, the stabilizing agent is sodium chloride at a concentration of about 30-200mM, preferably 100-180; or the stabilizing agent is mannitol in a concentration of about 100-300mM, preferably 200-300 mM; or the stabilizer is sucrose at a concentration of about 100-300mM, preferably 200-300 mM; or the stabilizer is trehalose at a concentration of about 100-300mM, preferably 200-300 mM; or the stabilizer is arginine hydrochloride at a concentration of about 30-200mM, preferably about 100-180 mM. In some embodiments, the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol, preferably about 30-100mM sodium chloride and about 100-180mM mannitol; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose, preferably about 30-100mM sodium chloride and about 100-180mM sucrose; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM trehalose, preferably about 30-100mM sodium chloride and about 100-180mM trehalose.

Thus, in some embodiments, the pharmaceutical compositions of the present invention comprise: (a) 10-30mM buffer, which is phosphate buffer with pH 7.5-8.1; (b) 5-40mg/mL of a uricase or a modified uricase according to any one of the preceding embodiments, in particular, the PEG-modified uricases AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG described herein; and (c) 20mM to 300mM of a stabilizer, preferably mannitol at a concentration of about 200mM to 300mM, or trehalose at a concentration of about 200mM to 300mM, or a combination of about 30mM to 100mM sodium chloride and about 100mM to 180mM trehalose, or a combination of about 30mM to 100mM sodium chloride and about 100mM to 180mM mannitol. In some embodiments, the stabilizing agent is trehalose at about 200-300 mM. In some embodiments, the stabilizing agent is a combination of about 30-100mM sodium chloride and about 100-180mM trehalose.

In some embodiments, the pharmaceutical compositions of the present invention further comprise a surfactant. Preferred surfactants are selected from polysorbate 80, polysorbate 20 and poloxamer 188. The most preferred surfactant is polysorbate 80. The concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001% to about 0.1%, preferably about 0.02% to about 0.08%, preferably about 0.02% to about 0.04%, in w/v. By way of non-limiting example, the concentration of surfactant in the pharmaceutical composition of the present invention is about 0.02%,0.04% or 0.08%, preferably 0.02%.

Thus, in some embodiments, the pharmaceutical compositions of the present invention comprise: a buffer which is a phosphate buffer having a pH of 7.5 to 8.1 and has a concentration of 10 to 30mM in the pharmaceutical composition; 5-40mg/mL of a uricase or a modified uricase according to any one of the preceding embodiments, in particular, the PEG-modified uricases AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG described herein; 100mM to 300mM of a stabilizer, preferably, the stabilizer is a combination of about 30 to 100mM of sodium chloride and about 100 to 180mM of trehalose, or about 200 to 300mM of trehalose; and 0.02% -0.04% by w/v of polysorbate 80.

The pharmaceutical composition of the present invention may be a liquid formulation, or a lyophilized formulation.

Medical uses and methods

The invention also provides uricase or uricase conjugate or pharmaceutical composition according to any embodiment of the invention for reducing uric acid levels in body fluids and tissues of mammals or treating or preventing hyperuricemia, the use of the uricase or uricase conjugate or pharmaceutical composition according to any embodiment of the invention in the preparation of a medicament for reducing uric acid levels in body fluids and tissues of mammals or treating hyperuricemia, and administering to an individual or patient in need thereof a therapeutically effective amount of the uricase or uricase conjugate or pharmaceutical composition according to any embodiment of the invention to reduce uric acid levels in body fluids and tissues of mammals or treat hyperuricemia.

The invention will be elucidated hereinafter by means of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. The following examples will apply to the following process.

Protein content detection method

Protein concentration was measured using an ultraviolet spectrophotometer. The percentage extinction coefficient (E1%) was set at 1.221 (g/ml) -1cm- ¹ . The BIO MATE 3S instrument was used. 2 solutions were run in parallel for each sample, and the run was repeated 3 times for each solution and averaged. And calculating the concentration of the corresponding detection product according to the extinction coefficient and the OD value.

Size exclusion chromatography (SEC-HPLC) purity detection method

SEC-HPLC purity was determined by HPLC (Waters e2695 instrument) fitted with a SEC column (TOSOH G4000sxl SEC 7.8mm ID x 30cm,8 μm). The mobile phase composition was 50mM Tris,150mM NaCl, pH 7.4. The relative percentages of the main peak, high polymer and low polymer were calculated using area normalization. Details are given in the following table:

chromatographic conditions	Chromatographic parameters
		Wavelength of detector	280nm
Auto sampler temperature	25±3℃
		Column temperature
	5±3℃
		Flow rate of flow	0.50ml/min
Sample introduction volume	25μl
		Mode of operation	Isocratic mode
Run time	30.00min

Purity detection method of reversed phase high performance liquid chromatography (RP-HPLC)

RP-HPLC purity was determined by HPLC (Agilent 1260) fitted with a reverse phase chromatography column (Advancobio RP-mAb C4.6 mm ID. Times.150mm, 3.5 μm). The mobile phase composition is A phase: 0.1% aqueous tfa solution; phase B: 0.1% TFA ACN. And calculating the relative percentage of the double-PEG modified protein peak, the single-PEG modified protein peak and the naked protein by using an area normalization method. Details are given in the table below:

method for detecting purity of non-reduced sodium dodecyl sulfate by capillary electrophoresis (NR-CE-SDS)

The purity of NR-CE-SDS was measured by a capillary electrophoresis apparatus (Maurice apparatus) equipped with a CE-SDS cartridge. mu.L of the sample solution (1 mg/mL) was added with 5. Mu.L of 0.8M iodoacetamide, incubated at 70 ℃ for denaturing heat treatment for 5min, and detected by capillary electrophoresis (Maurice). And respectively calculating the purities of the protein, the single-PEG modified protein peak and the double-PEG modified protein peak according to the percentage of the corrected peak areas of the protein, the single-PEG modified protein and the double-PEG modified protein to the sum of all the corrected peak areas.

Enzyme activity detection method

The uric acid solution was diluted to 300. Mu.M with buffer and 250. Mu.L/well was added

Putting the plate into a flat-bottom black Microplate, setting the plate into a Multi-Mode Microplate Reader with the preset temperature of 30 ℃, quickly transferring the sample diluted to 8.0 mu g/mL into the plate added with uric acid solution in an amount of 50 mu L/hole after balancing for 10min, shaking for 15s, reading once every 2min under the condition of 292nm, and reading for 20min.

Buffer solution: 35.3mM Tris base; 14.7mM Tris HCl;150mM NaCl,1.3mM EDTA (ethylenediaminetetraacetic acid); 0.02% polysorbate 80;220mM sucrose, final pH 8.4. + -. 0.2.

Other methods and materials used in the examples are, unless otherwise indicated, conventional in the art.

Examples

Example 1: construction of uricase expression vector

Uricase nucleotide sequence (Suzhou Jinwei Zhi Biotech science and technology Co., ltd.) was synthesized and constructed on PET28a expression vector. The 5 'cleavage site of the target gene is NcoI, and the 3' cleavage site is XhoI.

Taking AG-2C13 molecule construction as an example, the nucleotide sequence after codon optimization is as follows:

atgaccgcgaccgccgaaaccagcacgggtaccaaagttgttctgggccagaaccagtacggcaaagccgaagttcgtctggttaaggttacccgcaacacgtgccgccatgagattcaagatctgaatgtgaccagccagctccgcggcgatttcgaagccgcgcataccgccggtgataatgcccatgttgttgcgacggacacgcagaaaaacacggtttacgccttcgcccgcgatggtttcgcgaccaccgaagaatttctgctccgtctgggcaaacacttcaccgaaggttttgactgggttaccggtggtcgttgggccgcccagcagtttttctgggaccgcatcaacgaccacgaccacgcctttagccgcaataaaagcgaggtgcgtacggccgttctggaaatcagcggtagcgaacaagccatcgtggccggcattgaaggtctgaccgttctgaagagcaccggcagcgaatttcatggcttcccgcgcgacaagtacacgacgctgccagaaaccgaggatcgcattctggccaccgatgtgagtgcccgttggcgctataacacggtggaggtggatttcgatgccgtttacgcgagcgttcgcggtctgctgctgaaagcgtttgccgaaacccatagtctggcgctgcagcagaccatgtacgaaatgggtcgcgcggttatcgagacgcacccggaaatcgacgagatcaagatgagtctgccgaacaagcaccactttctggttgatctgagcccgttcggccaagataatccgaacgaggttttttacgcggccgatcgcccgtacggtctgatcgaggccaccatccagcgcgaaggctgctaa(SEQ ID NO:9)。

expression of uricase:

coli BL21 (DE 3) (Merck) transformation experiments were performed and transformed colonies were picked on LB/Kana plates. Escherichia coli containing a uricase expression vector was cultured overnight at 37 ℃ in 5ml of LB liquid medium (containing 50um/ml kanamycin), and the above seed solution was inoculated into a shake flask containing 200ml of LB medium at an inoculation ratio of 3% and cultured at 37 ℃. When the thallus grows to OD 600 of 0.6-0.8, the culture temperature is reduced to 25 ℃. IPTG was added to a final concentration of 0.3mM to induce expression of the target uricase protein.

And (3) purifying uricase:

crushing the bacteria by a high-pressure homogenizing method or an ultrasonic method, centrifuging, further purifying by an anion exchange method to obtain the protein with the purity of more than 95%, and performing subsequent characterization analysis. The amino acid sequence of the protease was obtained by DNA sequencing.

Taking AG-00 as an example, the sequence is verified to be consistent with the optimized DNA sequence, and the amino acid sequence is SEQ ID NO:1, as follows:

synthesis of control uricase

A control uricase MEDI4945 was synthesized using a method similar to that described above, having the amino acid sequence of SEQ ID NO:2.MEDI4945 is a uricase disclosed in International application No. PCT/US2016/032415, and its amino acid sequence is shown below:

MATAETSTGCKVVLGQNQYGKAEVRLVKVTRCTARHEIQDLNVTSQLSGDFEAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGLTVLKSTGSEFHGFPRDKYTTLQETTDRILATDVSARWRYNTVEVDFDAVYASVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLQPFGQDNPNEVFYAADRPYGLIEATIQREGSRAD

example 2: sequence evolution of uricase from Micrococcus

A. Optimizing C-terminal

Converting the amino acid sequence of SEQ ID NO:1 truncates the C-terminal 5 amino acids to eliminate the C-terminal Cys, thereby generating SEQ ID NO:3. namely, by a method similar to that of example 1, DNA specific primers were designed, and amino acids 298 to 302 at the C-terminal end thereof were deleted by PCR amplification using AG-00 sequence as a template, and the constructed product was subjected to DNA sequencing verification, and the resulting molecule was named: AG-00-1, the sequence is as follows:

n-terminal amino acid substitutions

Using more than 15 aligned uricase databases, SEQ ID NO:1 are not highly conserved with the same genus of uricases, and therefore, by a method similar to example 1, specific primers were designed to replace amino acids 1-17 of the AG-00 uricase molecule with an amino acid sequence (MTNIILGK) by PCR amplification using AG-00 as a template, and the constructed product was verified by DNA sequencing to yield SEQ ID NO:4, the molecule is named as: AG-00-2, sequence shown below:

C. site-directed mutagenesis

By the same method as in example 1, on the basis of AG-00 sequence, DNA specific primers were designed, glutamine (Q) at position 258 was mutated to serine (S) by the bridge PCR technique, and the constructed product was subjected to DNA sequencing verification to generate SEQ ID NO:5, named AG-01, with the sequence shown below:

in a similar way, glutamine (Q) 175 is mutated to proline (P), threonine (T) 178 is mutated to glutamic acid (E), and the constructed product is subjected to DNA sequencing verification to generate SEQ ID NO:6, named AG-02, with the sequence shown below:

in a similar way, the 175 th glutamine (Q) is mutated into proline (P), the 178 th threonine (T) is mutated into glutamic acid (E) and the 258 th glutamine (Q) is mutated into serine (S), and the constructed product is subjected to DNA sequencing verification to generate SEQ ID NO:7, named AG-05, with the sequence shown below:

D. optimizing N-terminal

It is known that when a protein is expressed in E.coli, the N-terminal methionine can be removed by Met aminopeptidase, depending on the size of the second amino acid residue after methionine, typical cleavage occurs if the second position is a smaller residue, cleavage does not occur if the second position is a bulky residue, or the Met aminopeptidase can cleave a moiety, resulting in heterogeneity, if the residue at the second position is neither bulky nor particularly small. Thus, to eliminate incomplete cleavage of the first amino acid by prokaryotic expression, SEQ ID NO:4 to alanine (a) to yield the amino acid sequence of SEQ ID NO:8, named AG-00-2A.

SEQ ID NO：8

E. Optimization of C-terminal, site-directed mutagenesis

By the same method as in example 1, on the basis of AG-00 sequence, DNA specific primers are designed, amino acids 298-302 at the C-terminal end are deleted by PCR amplification, glutamine (Q) 175 is mutated into proline (P), threonine (T) 178 is mutated into glutamic acid (E), and glutamine (Q) 258 is mutated into serine (S), and the constructed product is subjected to DNA sequencing verification to generate SEQ ID NO:9, named AG-1, with the sequence shown below:

AG-1(SEQ ID NO：9)

F. further optimization of C-terminal, site-directed mutagenesis

By a method similar to that in example 1, DNA specific primers are designed, an AG-00 sequence is used as a template, 288-301 th amino acids (14 amino acids in total) at the C terminal of the primer are deleted through PCR amplification, the protein structure is further simplified, the immunogenicity possibly brought by heterologous protein is reduced, and meanwhile, a terminal Cys is reserved and used as one of subsequent PEG modification sites; and (2) mutating the 175 th glutamine (Q) to proline (P), mutating the 178 th threonine (T) to glutamic acid (E) and mutating the 258 th glutamine (Q) to serine (S), and carrying out DNA sequencing verification on the constructed product to generate SEQ ID NO:10, designated as AG-2, having the sequence shown below:

AG-2(SEQ ID NO：10)

G. optimized C/N-terminal, site-directed mutagenesis

By the same method as example 1, on the basis of AG-00 sequence, DNA specific primers were designed, C-terminal 298-302 amino acids were deleted by PCR amplification, 1-17 amino acids of AG-00 uricase molecule were replaced with the amino acid sequence MANIILGK, 175-glutamine (Q) was mutated to proline (P), 178-threonine (T) was mutated to glutamic acid (E) and 258-glutamine (Q) was mutated to serine (S), the constructed product was subjected to DNA sequencing verification to generate SEQ ID NO:11, designated AG-3, having the sequence shown below:

SEQ ID NO：11

H. evaluation of enzymatic Activity of modified Micrococcus uricase

Using Tris-HCl buffer system (pH 8.5), 0.25mL of uric acid solution and 0.375mL of Tris-HCl buffer solution were added to a test tube, and the test tube was incubated at 37 ℃ for 10min in a water bath, 0.125mL of the above uricase solution (0.004 mg/mL) diluted appropriately was added to the test tube, and after 10min of accurate reaction, 0.05mL of 20 KOH was added to terminate the reaction. The absorbance of residual uric acid at 292nm was measured to evaluate the enzyme activity of the modified uricase of the genus Micrococcus, and as a result, as shown in FIG. 1, it was found that the introduction of the point mutation did not affect the enzyme activity of uricase.

Uricase activity is defined as: the amount of enzyme required to catalyze the decomposition of 1umol of uric acid per minute at 37 ℃ and pH8.5 was defined as 1 enzyme activity unit.

I. Evaluation of the thermostability of modified Micrococcus uricase

The modified uricase molecule is placed in a water bath at 50 ℃,60 ℃ and 70 ℃ for 30 minutes. After equilibration for 20 minutes at 37 ℃,20 ul of 0.004mg/ml uricase solution was added and immediately read by a microplate reader for 10 minutes. And measuring the uricase residual enzyme activity, and reacting according to the absorbance difference. The uricase enzyme activity was normalized by leaving it at 4 ℃ and the results are shown in FIG. 2. In vivo line assay: 50ul of uric acid at a concentration of 800uM, 30ul of buffer.

After AG-00 is subjected to heat treatment at 60 ℃ for half an hour, the enzyme activity is kept 47%, and the enzyme activity retention rates of AG-01, AG-02 and AG-05 are respectively 60%,75% and 78% when the AG-00 is treated under the same conditions. When the enzyme is treated at 70 ℃, the AG-00 enzyme activity is kept at 6 percent, and the enzyme activity retention rates of uricases AG-01, AG-02 and AG-05 are respectively 17 percent, 20 percent and 15 percent. Therefore, the introduction of point mutation improves the thermal stability of enzyme activity.

J. Evaluation of Tm value of modified Micrococcus uricase

The Tm value of the protein molecule was evaluated using the protein Thermo Shift assay kit. Using buffer (Na) ₂ HPO ₄ 2.34g/L；NaH ₂ PO ₄ ·H ₂ O is 0.48g/L; 75.31g/L of sucrose; tween 80.2 g/L; pH 7.6 + -2) diluting the uricase to 0.5715mg/mL; diluting the 1000X fluorescent dye in the kit into 8X fluorescent dye by using ultrapure water; 70 μ L of 0.5715mg/mL uricase and 10 μ L of 8 × fluorescent dye were mixed well in 1.5mL EP tube and dispensed into 8-plex PCR tube or 96-well PCR plate with a volume of 30 μ L for a total of 2 wells. The blank control sample is prepared by mixing 70 mu L of preparation buffer solution and 10 mu L of 8 Xfluorescent dye, and is subpackaged into 8-linked PCR tubes or 96-hole PCR plates with the volume of 30 mu L for 2 holes. The other system blank control is prepared by mixing 70 microliter of ultrapure water and 10 microliter of 8 multiplied by fluorescent dye, subpackaging into 8-linked PCR tubes or 96-well PCR plates with the volume of 30 microliter, and subpackaging 2 wells in total.

The results are shown in table 1 below.

As can be seen, the Tm D values (temperature of first protein denaturation) of AG-01, AG-02, AG-05 and AG-00-2 were all higher than those of the wild-type uricase molecule AG-00 and control MEDI4945. It can be seen that the introduction of point mutations improves the thermostability of uricase.

TABLE 1

Serial number	Sample name	Tm D(℃)
			1	MEDI4945	53.45
2	AG-00	58.12
			3	AG-00-1	56.59
4	AG-00-2	61.91
			5	AG-01	61.40
6	AG-02	58.92
			7	AG-05	60.38

Example 3: specific PEGylation

Therapeutic proteins modified with polyethylene glycol (PEG) can be randomly attached to selected protein residues (such as lysine side chains) or specific for a particular site. While pegylation specific for a particular site can be better controlled and result in a highly homogeneous pegylated product with defined biological activity. Among the methods for site-specific attachment, coupling to unpaired cysteine residues is most widely used, which requires the introduction of one or more free cysteine residues in the protein sequence. Whereas the C-terminally truncated AG-00-1 molecular sequence (SEQ ID NO: 3) does not contain cysteine, and thus cysteine can be introduced by amino acid mutation.

Cys mutation introduction

To select a potential site for Cys residue introduction in the AG-00-1 sequence, several factors are considered:

(1) The site must be far from the enzymatic active site to avoid the risk of affecting activity;

(2) Sites are not accessible to each other to avoid steric hindrance making adjacent Cys difficult to pegylate;

(3) The site must be on the protein surface to ensure efficient reaction with the thiol-reactive PEG reagent;

(4) Sites must not be in structural or conserved regions.

Through analysis, the sites which are found to meet the requirements comprise: a35, F82, S142, T194, E216, G287, and S288, therefore, the above several sites were selected as sites for Cys mutation (the above sites are relative to SEQ ID NO: 3).

Using a method similar to example 1, the following molecules were prepared by introducing Cys mutations based on AG-1, AG-2 and AG-3, respectively: AG-2C12, AG-2C13, AG-2C16, AG-2C17, AG-2C13-1, AG-2C01, AG-2C05-1, AG-2C01-1 and AG-2C12-1, the amino acid sequences of which are respectively shown in SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. the amino acid sequence of SEQ ID NO: 17. SEQ ID NO: 18. the amino acid sequence of SEQ ID NO:19 or SEQ ID NO: shown at 20.

Protein stability Studies

Each of the modified uricases was placed in phosphate buffer, pH7.0,1mM DTT, at 4 ℃ and the stability of each protein was compared using SEC-HPLC and RP-HPLC, and the results are shown in FIG. 3.

RP-HPLC (FIG. 3, 3a) results show: AG-2C13, AG-2C13-1, AG-2C12 and AG-2C01-2 have better purity. SEC-HPLC (FIG. 3, 3b) results show: AG-2C13, AG-2C17, AG-2C12, and AG-2C01-2 have good purity. The results of SEC-HPLC and RP-HPLC are combined, preferably with AG-2C13, AG-2C12, AG-2C01-2 molecules.

Uricase PEG modification

To 2mg of uricase (100 mM PBS buffer, pH =7.0, 150mM NaCl) were added TCEP.HCl (0.5 mg, tris (2-carboxyethyl) phosphine hydrochloride), M-MAL-PEG-10K (27.5 mg, maleimide polyethylene glycol, molecular weight 10000 Da) in this order, and reacted at room temperature for 2 hours. And purifying the reaction solution by using ion exchange chromatography to obtain the pegylated uricase.

The method for measuring the activity of the uricase comprises the following steps: a phosphate buffer system (pH 7.3) was used. Activity assay systems the absorbance of residual uric acid at 292nm was determined by reference to the above method (example 2).

Combining the selected Cys mutation sites in pairs, and performing PEG modification and uricase activity evaluation. The results are shown in Table 2.

TABLE 2

Name of molecule	Site mutation	Sequence numbering	Number of single subunits	Enzyme activity maintenance	Modified enzyme activity U/mg
						AG-2C12	S142C/E216C	12	2	98％	2.0
AG-2C13	A35C/S288C	13	2	90％	1.95
						AG-2C16	A35C/S142C	14	2	98％	1.69
AG-2C17	E216C/S288C	15	2	90％	1.85
						AG-2C13-1	A35C/S288C	16	2	113％	2.33
AG-2C01-2	A35C/T194C	17	2	99％	1.55
						AG-2C05-1	F82C/G287C	18	2	44％	0.67
AG-2C01-1	A35C/T194C	19	2	92％	1.43
						AG-2C12-1	S142C/E216C	20	2	102％	1.65

* Note: AG-2C13-1, AG-2C01-1 and AG-2C12-1 also included the following mutations compared to AG-2C13, AG-2C01-2 and AG-2C12, respectively: S205D and G208R.

Both cysteines introduced in the molecules listed in table 2 were well modified by PEG, demonstrating that the site of introduction was exposed on the surface of the molecule. And after a single subunit of uricase is doubly modified by mPEG, the enzyme activity retention rate is different from 44% to 113%. In addition, mutations at positions 205 and 208 can further enhance enzyme activity.

Example 4: PEGylation-modified uricase Activity assessment

Study of enzyme Activity of PEGylated urokinase

The mother liquor of the uricase working solution was 2mM uric acid using Tris-HCl buffer system (pH 8.5), and stored at 4 ℃. The enzyme activity determination process comprises the following steps:

set temperature 37 degrees, substrate (uric acid) concentration gradient: 600 micromolar, 500 micromolar, 400 micromolar, 200 micromolar, 100 micromolar, 50 micromolar, 25 micromolar, 12.5 micromolar; 50ul of the above uric acid solution and 30ul of the above Tris-HCl buffer were added to a 96-well plate, incubated at 37 ℃ for 20min, and then 20ul of an appropriately diluted uricase solution (0.004 mg/mL) was rapidly added to the 96-well plate. Setting a program: the reaction was started for 10min, with data read every 20 s. The results are shown in fig. 4 and table 3.

Control uricase MEDI4945: uricase disclosed in International application No. PCT/US 2016/032415.

The results show that the maximum reaction rates of uricase molecules are AG-2C01-2-PEG, AG-2C12-PEG, AG-2C13-PEG and MEDI4945 in sequence.

TABLE 3

Name of molecule	Km	Vmax	Kcat
				AG-2C01-2-PEG	451.4	13.84	5.4
AG-2C12-PEG	355.5	13.24	5.3
				AG-2C13-PEG	167.7	9.4	3.6
MEDI4945	101.5	6.8	2.6

Note: km: a Michaelis constant; vmax: a maximum reaction rate; kcat: the number of conversions, refers to the number of substrate conversions of a single enzyme molecule in one second.

Accelerated stability assessment of PEG-modified urokinase (37 deg.C)

The PEG-modified uricase is placed in an incubator at 37 ℃, and the buffer system is a phosphate buffer system, and the pH value is 7.0. The SEC-HPLC results are shown in FIG. 5. It can be seen that the stability of AG-2C01-2-PEG is better than that of AG-2C12-PEG and AG-2C13-PEG at 37 ℃.

Example 5: pharmaceutical composition buffer system, pH and stabilizer screening assay

In the liquid type pharmaceutical composition, the buffer system and pH closely affect the stability of the antibody, and each antibody having unique physicochemical properties has the optimum type and pH of the buffer. This example is directed to screening an optimal buffer system and pH for optimal stability of uricase disclosed herein for clinical use.

5.1 Experimental procedure

This example was performed with uricase (AG-2C 01-2-PEG) at concentrations of about 10mg/mL and 20mg/mL. The sample was Millipore Pellicon 3.11 m ² And (3) carrying out ultrafiltration concentration and liquid change on the membrane, placing the sample in a corresponding prescription after the liquid change, and placing the sample in a sealed centrifugal tube for buffer liquid screening. The buffer solution selects phosphate buffer solution and Tris hydrochloric acid buffer solution, and the pH value is from 7.5 and 8.0; stabilizers sodium chloride, arginine hydrochloride, mannitol, sucrose and trehalose were screened for comparative testing (as shown in table 4).

The samples were placed at 25. + -. 2 ℃ or 5. + -. 3 ℃ and taken out at week 0, week 2, week 4, week 8 (only 5. + -. 3 ℃) and month 3 (only 5. + -. 3 ℃) respectively for analytical testing. The evaluation indexes include: 1. visual appearance was observed; 2. protein concentration (uv spectrophotometry); 3. SEC-HPLC (size exclusion chromatography) to determine the percentage of protein monomers, aggregates and debris; 4. RP-HPLC (reversed phase high performance liquid chromatography) to determine protein purity; 5. NR-CE-SDS (non-reducing sodium dodecyl sulfate capillary electrophoresis) to detect the purity of the protein; 6. and (4) analyzing and detecting enzyme activity. The effect of different buffer systems and pH on uricase stability was examined and the results are shown in tables 5-1 to 5-6.

Table 4: prescription information in buffer system, pH and stabilizer screening experiments

5.2 results of the experiment

5.2.1 appearance and protein content

According to the results in table 5-1, no significant change in protein content occurred in all samples under accelerated or long term conditions.

According to the results in Table 5-2, no significant visible foreign matter was found at T0 and no significant opalescence was observed for all samples. After the mixture is placed for 4 weeks under an accelerated condition, protein precipitation occurs when the concentration of the FS2-8 and FS2-3 prescriptions is 20mg/ml (the subsequent detection is stopped by the FS 2-8), and the rest prescriptions have no obvious change; after being placed for 3 months under long-term conditions, protein precipitation appears in the prescription FS2-8 with the concentration of 20mg/ml and the prescription FS2-13, and the appearances of other samples do not change significantly.

Table 5-1: protein content assay data

Note: "/" indicates that: the detection is terminated.

Tables 5-2: appearance inspection data

Note: "/" indicates that: the detection is terminated.

5.2.2 SEC purity

According to the SEC purity results in tables 5-3, the FS2-8 (20 mg/ml) group terminated the assay; after being placed for 4 weeks under accelerated conditions, the formulas (FS 2-2 and FS 2-7) containing arginine hydrochloride have obvious increase of polymers; no significant reduction in SEC purity occurred for all prescriptions after 3 months of long term storage.

Tables 5 to 3: SEC-HPLC monomer, dimer and fragment content data

Note: "/" indicates that: the detection is terminated.

5.2.3 RP-HPLC purity

According to the RP-HPLC purity results in tables 5-4, the detection was terminated in the FS2-8 (20 mg/ml) group; after being placed for 4 weeks under the accelerated condition, the purity of four groups of FS2-2, FS2-7, FS2-12 and FS2-13 is reduced at a higher speed; after being placed under long-term conditions, 3M, FS2-3 (10/20 mg/ml) and four groups of FS2-4 and FS2-5 have slower purity reduction speed.

Tables 5 to 4: RP-HPLC protein purity screening data

Note: "/" indicates that: the detection is terminated.

5.2.4 Purity of NR-CE-SDS

FS2-8 (20 mg/ml) group terminated the detection according to the NR-CE-SDS purity results in tables 5-5; all samples did not show significant changes in purity after 4 weeks of accelerated or 3M after long term storage.

Tables 5 to 5: NR-CE-SDS purity screening data

Note: "/" indicates that: the detection is terminated.

5.2.5 enzymatic Activity

According to the results of enzyme activities in tables 5-6, no decrease in enzyme activity occurred in all formulations except FS2-8 (20 mg/ml) under accelerated or long-term conditions.

Tables 5 to 6: data on screening of enzyme activity

Note: "/" indicates that: the detection is terminated.

5.3 first round prescription screening conclusion

All formulations did not show significant differences as seen from the results of protein content, enzyme activity and NR-CE-SDS purity. From the appearance, FS2-8 (20 mg/ml) has poor appearance result; from SEC-HPLC results, FS2-2 and FS2-7 showed an increase in the number of the dimer peaks; from the RP-HPLC results, the results of the FS2-3 (10/20 mg/ml), FS2-4 and FS2-5 groups are better than those of the other groups.

In summary, FS2-3, FS2-4 and FS2-5 are preferred. Therefore, 20mM phosphate buffer system (pH7.5) is selected as the buffer system to enter the next round of screening experiment; and further evaluation of the three stabilizers sucrose, trehalose and mannitol was performed in the next round of experiment.

Example 6: stabilizer and surfactant screening

6.1 Experimental procedure

Uricase (AG-2C 01-2-PEG) was applied to Millipore Pellicon 3.11 m ² And (3) replacing the liquid of the membrane, placing the sample in a corresponding prescription after the liquid is replaced, filling the sample into a 2R penicillin bottle with the final protein concentration of about 8mg/ml (shown in table 6) in an aseptic manner, and performing stability lofting and detection.

1) Placing the sample in an environment with the temperature of 25 +/-2 ℃ or 5 +/-3 ℃, and taking out the sample at the 0 th week, the 1 st week, the 2 nd week, the 4 th week and the 8 th week respectively for analysis and detection; 2) Subjecting the sample to freeze-thaw cycling for 3 times; 3) The sample was shaken for 3 days. The evaluation indexes include: 1. visual inspection of the appearance; 2. protein concentration (uv spectrophotometry); 3. SEC-HPLC (size exclusion chromatography) to determine the percentage of protein monomers, aggregates and debris; 4. RP-HPLC (reversed phase high performance liquid chromatography) to determine protein purity; 5. NR-CE-SDS (sodium dodecyl sulfate capillary electrophoresis) to detect the purity of the protein; 6. and (4) analyzing and detecting enzyme activity. The results are shown in tables 7-1 to 7-6.

Table 6: stabilizer screening protocol

Note: "/" indicates that: was not added.

6.2 results of the experiment

6.2.1 appearance and protein concentration

According to the results in tables 7-1 and 7-2, no significant change in protein content occurred in all samples after 8 weeks of storage under accelerated and prolonged conditions; no visible foreign body was found at T0 for all samples, no visible opalescence was observed. After the mixture is placed for 8 weeks under accelerated conditions or long-term conditions, the four prescriptions FS3-1, FS3-3, FS3-4 and FS3-6 have no appearance change, and the opalescence of the other prescriptions is increased; after 3 times of freeze-thaw cycle, no obvious visible foreign matter and no obvious opalescence are found in the samples after shaking for 3 days.

Table 7-1: protein content screening data

Note: "/" indicates that: not detected.

Tables 7 to 2: appearance screening data

Note: "/" indicates that: and (4) not detecting.

6.2.2 SEC purity

According to the SEC purity results in tables 7-3, no significant change in SEC purity occurred for all samples placed for 8 weeks under accelerated or prolonged conditions; after 3 times of freeze-thaw cycles, no significant change in SEC purity occurred after shaking for 3 days.

Tables 7 to 3: SEC-HPLC MONOMER, MER AND FRAGMENT CONTENT DATA

Note: "/" indicates that: not detected.

6.2.3 RP-HPLC purity

According to the RP-HPLC purity results in tables 7-4, all samples showed purity reduction but showed no difference between groups upon standing under accelerated conditions for 8 weeks; after being placed for 8 weeks under long-term conditions, the purity of the product is not changed obviously; after 3 times of freeze-thaw cycles, no significant change in purity occurred after shaking for 3 days.

Tables 7 to 4: RP-HPLC purity screening data

Note: "/" indicates that: and (4) not detecting.

6.2.4 Purity of NR-CE-SDS

According to the purity results of NR-CE-SDS in tables 7 to 5, all samples showed purity decrease but showed no difference between groups upon standing under accelerated conditions for 8 weeks; after being placed for 8 weeks under long-term conditions, no significant change in purity occurred.

Tables 7 to 5: NR-CE-SDS purity screening data

6.2.5 results of enzyme Activity

According to the results of enzyme activity in tables 7-6, all samples did not show significant change in enzyme activity after being placed under accelerated conditions or long-term conditions for 8 weeks; after 3 times of freeze-thaw cycle, no significant change of enzyme activity appears after shaking for 3 days.

Tables 7 to 6: data on screening of enzyme activity

Note: "/" indicates that: not detected.

6.3 second round prescription screening conclusions

No significant differences were observed for all formulations as seen by protein content, enzyme activity and NR-CE-SDS purity, SEC-HPLC purity and RP-HPLC purity results. From the appearance results, four groups of FS3-1, FS3-3, FS3-4 and FS3-6 are superior.

In summary, three prescriptions, FS3-1, FS3-3, FS3-4 and FS3-6, are preferred. Considering that mannitol is easy to form crystals when frozen and has the risk of freeze-thaw, FS3-3 (20 mM phosphate, pH7.5, containing 220mM trehalose and 0.02% polysorbate 80) is selected as a preferred prescription.

Example 7: compatibility stability test

7.1 Experimental procedure

The six formulations FS 3-1-6 from example 2 were evaluated for stability after dilution with two media, 0.9% sodium chloride injection and 5% dextrose injection. Uricase (AG-2C 01-2-PEG) was diluted to different concentrations of the corresponding medium (Table 8) according to the following protocol, placed at 25 ℃ for 24h and then tested to investigate different medium pairs.

The evaluation indexes include: 1. visual inspection of the appearance; 2. protein concentration (uv spectrophotometry); 3. SEC-HPLC (size exclusion chromatography) determines the percentage of protein monomers, aggregates and debris. The results are shown in Table 9.

Table 8: compatibility scheme

7.2 results of the experiment

The uricase has good stability in the concentration range of 0.2 mg/ml-4 mg/ml of 0.9% sodium chloride injection. A fragment increase occurred at a low concentration of 0.2mg/ml SEC in 5% glucose injection.

Table 9: results of compatibility test

In conclusion, the stability of uricase is explored and researched by investigating different buffer systems, different pH conditions, different protein concentrations and different auxiliary material compositions, and the optimal liquid preparation formula is determined. According to the above experiment, the uricase of the invention selects phosphate buffer solution with pH7.5 to adjust pH, trehalose to adjust the osmotic pressure of the preparation, and polysorbate 80 to increase the solubility of the preparation.

Compatibility experiments show that the uricase is diluted by 0.9 percent sodium chloride injection and does not degrade obviously after being placed for 24 hours at room temperature within the range of 0.2 to 4mg/ml of protein concentration.

Considering that the present uricase has an isoelectric point in the range of 5.5 to 6.3, and that precipitation occurred at pH7.0 in early preliminary experiments, pH was finally increased to 7.8 to increase its solubility as far away from pI as possible, pH range 7.5 to 8.1.

Example 8: in vivo pharmacodynamic study

The effect of the test substance on serum uric acid levels was evaluated by means of a single intravenous (i.v.) administration, on a mouse model of exogenously uric acid-induced hyperuricemia injected intraperitoneally (i.p.). 120 normal mice (ICR-male) were randomly divided into 6 groups of 20 mice each. Group 1 and group 2 were administered with solvent as a blank control and a solvent control, group 3 was administered with MEDI 4945.1 mg/kg as a positive control, and

groups

4, 5, and 6 were administered with AG-2C13-PEG, AG-2C01-2-PEG, and AG-2C12-PEG (prescription FS 3-3) at a dose of 0.1mg/kg as a treatment group, respectively. On the next day after administration, except for the blank control group, all the experimental mice of the other groups were injected with uric acid for molding (250 mg/kg,10 ml/kg) in the abdominal cavity, and blood samples were collected for 30min and 3h after molding for animals 1 to 10 in each group, and for 1.5h and 6h for animals 11 to 20.

The results are shown in FIG. 6. The serum uric acid level of the animals in the AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG treatment groups at the dose of 0.1mg/kg is obviously lower than that of the solvent control group (p is less than 0.01) at each time point of 0.5h to 6h after the molding, the serum uric acid level of the animals in 3 treatment groups is lower than the lower detection limit (except the AG-2C13-PEG group, 8.35 mu mol/l at 1.5 h) at each time point of 0.5h to 1.5h after the molding. Under the measured dose (0.1 mg/kg), after single injection administration of AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG, the serum uric acid level is obviously reduced on an ICR mouse hyperuricemia model induced by exogenous uric acid in the abdominal cavity. Under the dosage, the urokinase AG-2C13-PEG, AG-2C01-2-PEG and AG-2C12-PEG have no obvious difference in effect at each time point after molding, but the drug effect is better than MEDI4945 after 3 h.

Sequence listing

<110> Shanghai Junshi biomedical science and technology Co., ltd

SUZHOU JUNMENG BIOSCIENCES Co.,Ltd.

<120> uricase, pharmaceutical composition thereof and use thereof

<130> 212407

<150> CN 202110381978.7

<151> 2021-04-09

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 302

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 2

<211> 290

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Ala Thr Ala Glu Thr Ser Thr Gly Cys Lys Val Val Leu Gly Gln

1 5 10 15

Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg Cys

20 25 30

Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu Ser

35 40 45

Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val Val

50 55 60

Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp Gly

65 70 75 80

Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe Thr

85 90 95

Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln Phe

100 105 110

Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn Lys

115 120 125

Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln Ala

130 135 140

Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly Ser

145 150 155 160

Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu Thr

165 170 175

Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr Asn

180 185 190

Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly Leu

195 200 205

Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln Thr

210 215 220

Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile Asp

225 230 235 240

Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp Leu

245 250 255

Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala Asp

260 265 270

Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser Arg

275 280 285

Ala Asp

290

<210> 3

<211> 297

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Gln Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn

290 295

<210> 4

<211> 288

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Thr Asn Ile Ile Leu Gly Lys Asn Gln Tyr Gly Lys Ala Glu Val

1 5 10 15

Arg Leu Val Lys Val Thr Arg Asn Thr Ala Arg His Glu Ile Gln Asp

20 25 30

Leu Asn Val Thr Ser Gln Leu Arg Gly Asp Phe Glu Ala Ala His Thr

35 40 45

Ala Gly Asp Asn Ala His Val Val Ala Thr Asp Thr Gln Lys Asn Thr

50 55 60

Val Tyr Ala Phe Ala Arg Asp Gly Phe Ala Thr Thr Glu Glu Phe Leu

65 70 75 80

Leu Arg Leu Gly Lys His Phe Thr Glu Gly Phe Asp Trp Val Thr Gly

85 90 95

Gly Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp Arg Ile Asn Asp His

100 105 110

Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val Arg Thr Ala Val Leu

115 120 125

Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala Gly Ile Glu Gly Leu

130 135 140

Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His Gly Phe Pro Arg Asp

145 150 155 160

Lys Tyr Thr Thr Leu Gln Glu Thr Thr Asp Arg Ile Leu Ala Thr Asp

165 170 175

Val Ser Ala Arg Trp Arg Tyr Asn Thr Val Glu Val Asp Phe Asp Ala

180 185 190

Val Tyr Ala Ser Val Arg Gly Leu Leu Leu Lys Ala Phe Ala Glu Thr

195 200 205

His Ser Leu Ala Leu Gln Gln Thr Met Tyr Glu Met Gly Arg Ala Val

210 215 220

Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys Met Ser Leu Pro Asn

225 230 235 240

Lys His His Phe Leu Val Asp Leu Gln Pro Phe Gly Gln Asp Asn Pro

245 250 255

Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr Gly Leu Ile Glu Ala

260 265 270

Thr Ile Gln Arg Glu Gly Ser Arg Ala Asp His Pro Ile Trp Ser Asn

275 280 285

<210> 5

<211> 302

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Gln Glu

165 170 175

Thr Thr Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 6

<211> 302

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 7

<211> 302

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn Ile Ala Gly Phe Cys

290 295 300

<210> 8

<211> 288

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Ala Asn Ile Ile Leu Gly Lys Asn Gln Tyr Gly Lys Ala Glu Val

1 5 10 15

Arg Leu Val Lys Val Thr Arg Asn Thr Ala Arg His Glu Ile Gln Asp

20 25 30

Leu Asn Val Thr Ser Gln Leu Arg Gly Asp Phe Glu Ala Ala His Thr

35 40 45

Ala Gly Asp Asn Ala His Val Val Ala Thr Asp Thr Gln Lys Asn Thr

50 55 60

Val Tyr Ala Phe Ala Arg Asp Gly Phe Ala Thr Thr Glu Glu Phe Leu

65 70 75 80

Leu Arg Leu Gly Lys His Phe Thr Glu Gly Phe Asp Trp Val Thr Gly

85 90 95

Gly Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp Arg Ile Asn Asp His

100 105 110

Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val Arg Thr Ala Val Leu

115 120 125

Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala Gly Ile Glu Gly Leu

130 135 140

Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His Gly Phe Pro Arg Asp

145 150 155 160

Lys Tyr Thr Thr Leu Gln Glu Thr Thr Asp Arg Ile Leu Ala Thr Asp

165 170 175

Val Ser Ala Arg Trp Arg Tyr Asn Thr Val Glu Val Asp Phe Asp Ala

180 185 190

Val Tyr Ala Ser Val Arg Gly Leu Leu Leu Lys Ala Phe Ala Glu Thr

195 200 205

His Ser Leu Ala Leu Gln Gln Thr Met Tyr Glu Met Gly Arg Ala Val

210 215 220

Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys Met Ser Leu Pro Asn

225 230 235 240

Lys His His Phe Leu Val Asp Leu Gln Pro Phe Gly Gln Asp Asn Pro

245 250 255

Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr Gly Leu Ile Glu Ala

260 265 270

Thr Ile Gln Arg Glu Gly Ser Arg Ala Asp His Pro Ile Trp Ser Asn

275 280 285

<210> 9

<211> 297

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn

290 295

<210> 10

<211> 288

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Cys

275 280 285

<210> 11

<211> 288

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Ala Asn Ile Ile Leu Gly Lys Asn Gln Tyr Gly Lys Ala Glu Val

1 5 10 15

Arg Leu Val Lys Val Thr Arg Asn Thr Ala Arg His Glu Ile Gln Asp

20 25 30

Leu Asn Val Thr Ser Gln Leu Arg Gly Asp Phe Glu Ala Ala His Thr

35 40 45

Ala Gly Asp Asn Ala His Val Val Ala Thr Asp Thr Gln Lys Asn Thr

50 55 60

Val Tyr Ala Phe Ala Arg Asp Gly Phe Ala Thr Thr Glu Glu Phe Leu

65 70 75 80

Leu Arg Leu Gly Lys His Phe Thr Glu Gly Phe Asp Trp Val Thr Gly

85 90 95

Gly Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp Arg Ile Asn Asp His

100 105 110

Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val Arg Thr Ala Val Leu

115 120 125

Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala Gly Ile Glu Gly Leu

130 135 140

Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His Gly Phe Pro Arg Asp

145 150 155 160

Lys Tyr Thr Thr Leu Pro Glu Thr Glu Asp Arg Ile Leu Ala Thr Asp

165 170 175

Val Ser Ala Arg Trp Arg Tyr Asn Thr Val Glu Val Asp Phe Asp Ala

180 185 190

Val Tyr Ala Ser Val Arg Gly Leu Leu Leu Lys Ala Phe Ala Glu Thr

195 200 205

His Ser Leu Ala Leu Gln Gln Thr Met Tyr Glu Met Gly Arg Ala Val

210 215 220

Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys Met Ser Leu Pro Asn

225 230 235 240

Lys His His Phe Leu Val Asp Leu Ser Pro Phe Gly Gln Asp Asn Pro

245 250 255

Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr Gly Leu Ile Glu Ala

260 265 270

Thr Ile Gln Arg Glu Gly Ser Arg Ala Asp His Pro Ile Trp Ser Asn

275 280 285

<210> 12

<211> 297

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Cys Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Cys Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn

290 295

<210> 13

<211> 288

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Cys Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Cys

275 280 285

<210> 14

<211> 297

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Cys Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Cys Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn

290 295

<210> 15

<211> 288

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Cys Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Cys

275 280 285

<210> 16

<211> 288

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Cys Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Asp Val Arg Arg

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Cys

275 280 285

<210> 17

<211> 288

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Met Ala Asn Ile Ile Leu Gly Lys Asn Gln Tyr Gly Lys Ala Glu Val

1 5 10 15

Arg Leu Val Lys Val Thr Arg Asn Thr Cys Arg His Glu Ile Gln Asp

20 25 30

Leu Asn Val Thr Ser Gln Leu Arg Gly Asp Phe Glu Ala Ala His Thr

35 40 45

Ala Gly Asp Asn Ala His Val Val Ala Thr Asp Thr Gln Lys Asn Thr

50 55 60

Val Tyr Ala Phe Ala Arg Asp Gly Phe Ala Thr Thr Glu Glu Phe Leu

65 70 75 80

Leu Arg Leu Gly Lys His Phe Thr Glu Gly Phe Asp Trp Val Thr Gly

85 90 95

Gly Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp Arg Ile Asn Asp His

100 105 110

Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val Arg Thr Ala Val Leu

115 120 125

Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala Gly Ile Glu Gly Leu

130 135 140

Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His Gly Phe Pro Arg Asp

145 150 155 160

Lys Tyr Thr Thr Leu Pro Glu Thr Glu Asp Arg Ile Leu Ala Thr Asp

165 170 175

Val Ser Ala Arg Trp Arg Tyr Asn Cys Val Glu Val Asp Phe Asp Ala

180 185 190

Val Tyr Ala Ser Val Arg Gly Leu Leu Leu Lys Ala Phe Ala Glu Thr

195 200 205

His Ser Leu Ala Leu Gln Gln Thr Met Tyr Glu Met Gly Arg Ala Val

210 215 220

Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys Met Ser Leu Pro Asn

225 230 235 240

Lys His His Phe Leu Val Asp Leu Ser Pro Phe Gly Gln Asp Asn Pro

245 250 255

Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr Gly Leu Ile Glu Ala

260 265 270

Thr Ile Gln Arg Glu Gly Ser Arg Ala Asp His Pro Ile Trp Ser Asn

275 280 285

<210> 18

<211> 297

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Cys Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Ser Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Ser Val Arg Gly

195 200 205

Leu Leu Leu Lys Ala Phe Ala Glu Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Cys Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn

290 295

<210> 19

<211> 288

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Met Ala Asn Ile Ile Leu Gly Lys Asn Gln Tyr Gly Lys Ala Glu Val

1 5 10 15

Arg Leu Val Lys Val Thr Arg Asn Thr Cys Arg His Glu Ile Gln Asp

20 25 30

Leu Asn Val Thr Ser Gln Leu Arg Gly Asp Phe Glu Ala Ala His Thr

35 40 45

Ala Gly Asp Asn Ala His Val Val Ala Thr Asp Thr Gln Lys Asn Thr

50 55 60

Val Tyr Ala Phe Ala Arg Asp Gly Phe Ala Thr Thr Glu Glu Phe Leu

65 70 75 80

Leu Arg Leu Gly Lys His Phe Thr Glu Gly Phe Asp Trp Val Thr Gly

85 90 95

Gly Arg Trp Ala Ala Gln Gln Phe Phe Trp Asp Arg Ile Asn Asp His

100 105 110

Asp His Ala Phe Ser Arg Asn Lys Ser Glu Val Arg Thr Ala Val Leu

115 120 125

Glu Ile Ser Gly Ser Glu Gln Ala Ile Val Ala Gly Ile Glu Gly Leu

130 135 140

Thr Val Leu Lys Ser Thr Gly Ser Glu Phe His Gly Phe Pro Arg Asp

145 150 155 160

Lys Tyr Thr Thr Leu Pro Glu Thr Glu Asp Arg Ile Leu Ala Thr Asp

165 170 175

Val Ser Ala Arg Trp Arg Tyr Asn Cys Val Glu Val Asp Phe Asp Ala

180 185 190

Val Tyr Ala Asp Val Arg Arg Leu Leu Leu Lys Ala Phe Ala Glu Thr

195 200 205

His Ser Leu Ala Leu Gln Gln Thr Met Tyr Glu Met Gly Arg Ala Val

210 215 220

Ile Glu Thr His Pro Glu Ile Asp Glu Ile Lys Met Ser Leu Pro Asn

225 230 235 240

Lys His His Phe Leu Val Asp Leu Ser Pro Phe Gly Gln Asp Asn Pro

245 250 255

Asn Glu Val Phe Tyr Ala Ala Asp Arg Pro Tyr Gly Leu Ile Glu Ala

260 265 270

Thr Ile Gln Arg Glu Gly Ser Arg Ala Asp His Pro Ile Trp Ser Asn

275 280 285

<210> 20

<211> 297

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Met Thr Ala Thr Ala Glu Thr Ser Thr Gly Thr Lys Val Val Leu Gly

1 5 10 15

Gln Asn Gln Tyr Gly Lys Ala Glu Val Arg Leu Val Lys Val Thr Arg

20 25 30

Asn Thr Ala Arg His Glu Ile Gln Asp Leu Asn Val Thr Ser Gln Leu

35 40 45

Arg Gly Asp Phe Glu Ala Ala His Thr Ala Gly Asp Asn Ala His Val

50 55 60

Val Ala Thr Asp Thr Gln Lys Asn Thr Val Tyr Ala Phe Ala Arg Asp

65 70 75 80

Gly Phe Ala Thr Thr Glu Glu Phe Leu Leu Arg Leu Gly Lys His Phe

85 90 95

Thr Glu Gly Phe Asp Trp Val Thr Gly Gly Arg Trp Ala Ala Gln Gln

100 105 110

Phe Phe Trp Asp Arg Ile Asn Asp His Asp His Ala Phe Ser Arg Asn

115 120 125

Lys Ser Glu Val Arg Thr Ala Val Leu Glu Ile Ser Gly Cys Glu Gln

130 135 140

Ala Ile Val Ala Gly Ile Glu Gly Leu Thr Val Leu Lys Ser Thr Gly

145 150 155 160

Ser Glu Phe His Gly Phe Pro Arg Asp Lys Tyr Thr Thr Leu Pro Glu

165 170 175

Thr Glu Asp Arg Ile Leu Ala Thr Asp Val Ser Ala Arg Trp Arg Tyr

180 185 190

Asn Thr Val Glu Val Asp Phe Asp Ala Val Tyr Ala Asp Val Arg Arg

195 200 205

Leu Leu Leu Lys Ala Phe Ala Cys Thr His Ser Leu Ala Leu Gln Gln

210 215 220

Thr Met Tyr Glu Met Gly Arg Ala Val Ile Glu Thr His Pro Glu Ile

225 230 235 240

Asp Glu Ile Lys Met Ser Leu Pro Asn Lys His His Phe Leu Val Asp

245 250 255

Leu Ser Pro Phe Gly Gln Asp Asn Pro Asn Glu Val Phe Tyr Ala Ala

260 265 270

Asp Arg Pro Tyr Gly Leu Ile Glu Ala Thr Ile Gln Arg Glu Gly Ser

275 280 285

Arg Ala Asp His Pro Ile Trp Ser Asn

290 295

Claims

1. A uricase comprising the amino acid sequence as set forth in SEQ ID NO:9 or a variant thereof which retains enzymatic activity, wherein the variant has an amino acid sequence which is substantially identical to the amino acid sequence set forth in SEQ ID NO: mutations of 9 include: less than 17 amino acid residues from the N-terminus and/or less than 10 amino acid residues from the C-terminus.

2. The uricase of claim 1, wherein the variant comprises any one of the following sequences relative to SEQ ID NO: the mutations of 9 include: the N-terminal amino acid sequence MTATAETSTGTKVVLGQ is replaced by MANIILGK, and/or the C-terminal amino acid sequence SRADHPIWSN is replaced by C; preferably, said uricase comprises the amino acid sequences as set forth in SEQ ID NOs: 10 or SEQ ID NO:11, and (c) the sequence shown in fig. 11.

3. The uricase of claim 1 or 2, wherein the uricase has a relative identity to SEQ ID NO: mutations of 9 also include: introducing 1, 2, 3, 4 or 5 surface accessible cysteine residues at positions selected from 35, 82, 142, 194, 216, 287 and 288, wherein the position numbering is relative to the position of SEQ ID NO:3, position number.

4. The uricase of claim 3, wherein the uricase comprises cysteine residues in the following two positions, respectively: 142 and 216, 35 and 288, 35 and 142, 216 and 288, 35 and 194, or 82 and 287, wherein the position numbering is relative to SEQ ID NO: position number of 3.

5. The uricase of any one of claims 1-4, wherein the uricase has a sequence selected from the group consisting of SEQ ID NO: mutations of 9 also include: a substitution mutation at position 205 and/or 208, preferably the substitution mutation is S205D or G208R or a combination thereof, wherein the position numbering is relative to SEQ ID NO: position number of 3.

6. The uricase of claims 1-5, wherein the uricase comprises the amino acid sequences set forth as SEQ ID NOs: 12. the amino acid sequence of SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16. SEQ ID NO: 17. the amino acid sequence of SEQ ID NO: 18. the amino acid sequence of SEQ ID NO:19 or SEQ ID NO:20, or a pharmaceutically acceptable salt thereof.

7. A modified uricase, wherein the modified uricase is the uricase of any one of claims 1-6 modified with polyethylene glycol (PEG); preferably, said PEG is covalently linked to the N-terminus and/or C-terminus of said uricase, and/or is covalently linked through a cysteine residue present on said uricase.

8. A pharmaceutical composition comprising:

(1) A buffer solution; and

(2) The uricase of any one of claims 1-6 or the modified uricase of claim 7.

9. The pharmaceutical composition of claim 8, wherein the buffer is selected from the group consisting of phosphate buffer, tris-hcl buffer, citrate buffer, and histidine buffer; preferably, the buffer is phosphate buffer or Tris-hydrochloric acid buffer; more preferably, the buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

10. The pharmaceutical composition of claim 9, wherein the buffer is at a concentration of about 10 to 50mM; preferably, the buffer has a concentration of about 10 to 30mM.

11. The pharmaceutical composition of claim 9, wherein the buffer has a pH of about 6.0 to about 8.5, preferably about 7.0 to about 8.5, preferably about 7.5 to about 8.1.

12. The pharmaceutical composition of claim 8, wherein the pharmaceutical composition further comprises a stabilizer selected from one or more of arginine hydrochloride, sodium chloride, mannitol, sucrose, and trehalose; preferably, the stabilizer is selected from mannitol, trehalose, a combination of mannitol and sodium chloride or a combination of trehalose and sodium chloride.

13. The pharmaceutical composition of claim 12, wherein the concentration of the stabilizing agent is about 20mM-300mM, preferably 50mM-250mM; preferably, the stabilizer is sodium chloride at a concentration of about 30-200 mM; or the stabilizer is arginine hydrochloride at a concentration of about 30-200 mM; or the stabilizer is mannitol in a concentration of about 100-300 mM; or the stabilizer is sucrose at a concentration of about 100-300 mM; or the stabilizer is trehalose at a concentration of about 100-300 mM; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM mannitol; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM trehalose; or the stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose; preferably, the stabilizer is about 100-300mM trehalose, about 100-300mM mannitol, about 30-200mM sodium chloride in combination with about 30-200mM mannitol, or about 30-200mM sodium chloride in combination with about 30-200mM trehalose; more preferably about 100-300mM trehalose or a combination of about 30-200mM sodium chloride and about 30-200mM trehalose.

14. The pharmaceutical composition according to claim 8, wherein the pharmaceutical composition further comprises a surfactant selected from polysorbate 80, polysorbate 20 or poloxamer 188, preferably the surfactant is polysorbate 80.

15. The pharmaceutical composition of claim 14, wherein the surfactant concentration is about 0.01% to about 0.1%, preferably the surfactant concentration is about 0.02% to about 0.08%, preferably about 0.02% to about 0.05%.

16. The pharmaceutical composition of claim 8, wherein the uricase or modified uricase is present at a concentration of about 1 to about 200mg/mL, preferably about 5 to about 80mg/mL, and more preferably about 5 to about 40mg/mL.

17. The pharmaceutical composition according to claim 8, which comprises a component represented by any one of the following (1) to (5):

(1) (a) from about 5mg/mL to 40mg/mL of uricase or modified uricase; (b) about 10 to 30mM phosphate buffer, pH about 7.5 to 8.1; (c) about 100 to 300mM mannitol; and (d) about 0.01% to 0.1% polysorbate 80; or

(2) (a) from about 5mg/mL to 40mg/mL of uricase or modified uricase; (b) About 10 to 30mM Tris-HCl buffer, pH about 7.5 to 8.1; (c) about 100 to 300mM trehalose; and (d) about 0.01% to 0.1% polysorbate 80; or

(3) (a) from about 5mg/mL to 40mg/mL of uricase or modified uricase; (b) about 10 to 30mM phosphate buffer, pH about 7.5 to 8.1; (c) about 100 to 300mM trehalose; and (d) about 0.01% to 0.1% polysorbate 80; or

(4) (a) from about 5mg/mL to 40mg/mL of uricase or modified uricase; (b) about 10 to 30mM phosphate buffer, pH about 7.5 to 8.1; (c) A combination of about 30-200mM sodium chloride and about 30-200mM mannitol; and (d) about 0.01% to 0.1% polysorbate 80; or

(5) (a) from about 5mg/mL to 40mg/mL of uricase or modified uricase; (b) about 10 to 30mM phosphate buffer, pH about 7.5 to 8.1; (c) A combination of about 30-200mM sodium chloride and about 30-200mM trehalose; and (d) about 0.01% to about 0.1% polysorbate 80.

18. The pharmaceutical composition according to claim 17, which comprises a component represented by any one of the following (1) to (5):

(1) (a) about 8mg/mL of uricase or modified uricase; (b) about 20mM phosphate buffer at a pH of about 7.5 to about 7.8; (c) about 240mM mannitol; and (d) about 0.02% polysorbate 80; or

(2) (a) about 8mg/mL of uricase or modified uricase; (b) about 20mM Tris-HCl buffer, pH about 7.5-8.0; (c) about 220mM trehalose; and (d) about 0.02% polysorbate 80; or

(3) (a) about 8mg/mL of uricase or modified uricase; (b) about 20mM phosphate buffer, pH about 7.5-7.8; (c) about 220mM trehalose; and (d) about 0.02% polysorbate 80; or

(4) (a) about 8mg/mL of uricase or modified uricase; (b) about 20mM phosphate buffer, pH about 7.5-7.8; (c) A combination of about 50mM sodium chloride and about 140mM mannitol; and (d) about 0.02% polysorbate 80; or

(5) (a) about 8mg/mL of uricase or modified uricase; (b) about 20mM phosphate buffer at a pH of about 7.5 to about 7.8; (c) A combination of about 50mM sodium chloride and about 140mM trehalose; and (d) about 0.02% polysorbate 80.

19. An injection comprising the pharmaceutical composition of any one of claims 8-18 and 0.9wt% sodium chloride solution or 5wt% dextrose in water; preferably, the concentration of the uricase in the injection containing 0.9 weight percent of sodium chloride solution is 0.2 to 4mg/mL, and the concentration of the uricase in the injection containing 5 weight percent of glucose aqueous solution is 1 to 4mg/mL; preferably, the injection has a pH of 7.5 to 8.1.

20. Use of the uricase of any one of claims 1-6 or the modified uricase of claim 7 or the pharmaceutical composition of any one of claims 8-18 for the preparation of a medicament for reducing uric acid levels in bodily fluids and tissues of a mammal or treating hyperuricemia.