Modified uricase and its use

Abstract

Described herein are modified uricase and methods for reducing uric acid levels by contacting a medium with the modified uricase. The modified uricase comprises a uricase polypeptide crosslinked by at least one bifunctional linking moiety comprising a poly(alkylene glycol) moiety. The bifunctional linking moiety has a molecular weight of about 1.5 kDa to about 4 kDa, and / or the modified uricase comprises a plurality of polypeptides having the amino acid sequence of SEQ ID NO: 2. Also described are polypeptides having the amino acid sequence of SEQ ID NO: 2. Also described are methods for preparing the modified uricase, comprising contacting a polypeptide with a crosslinking agent comprising a poly(alkylene glycol) moiety and at least two aldehyde groups to obtain a conjugate, and contacting the conjugate with a reducing agent.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 108,890, filed on 3 November 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] Description of sequence listings An ASCII file titled 89419.txt, containing 11,072 bytes, created on October 31, 2021, and filed concurrently with the filing of this application, is incorporated herein by reference.

[0003] In some embodiments, the present invention relates to treatment, and more specifically, to novel forms of uricase and their use, for example, in lowering uric acid levels. [Background technology]

[0004] Uric acid is a metabolic breakdown product of purine nucleotides. High blood uric acid levels (hyperuricemia) can cause gout and / or kidney stones, and high uric acid levels are associated with hemorrhagic shock [D'Alessandro et al., J Transl Med 2015, 13:253], malaria [Gallego-Delgado et al., Curr Rheumatol Rep 2014, 16:401], allergic asthma [Kool et al., Immunity 2011, 34:P527-P540], traumatic brain injury [Liu et al., Int J Med Sci 2018, 15:1072-1082], renal dysfunction and acute gastroenteritis [Matsuo et al., Sci Rep 2016, 6:31003], and multiple sclerosis [Piancone et al., Front Immunol 2018, It is associated with other medical conditions, including inflammatory bowel disease [Crane & Mongiardo, Immunol Invest 2014, 43:255-266], gastrointestinal infections [Crane et al., Infect Immun 2016, 84:976-988], and sterile inflammation and pregnancy complications [Nadeau-Vallee et al., Reproduction 2016, 152:R277-R292].

[0005] The usual primary treatment for gout is to manage the symptoms using, for example, steroidal or nonsteroidal anti-inflammatory drugs. Further medications include allopurinol and febuxostat, which are inhibitors of the enzyme xanthine oxidase (which produces uric acid), as well as probenecid, recinurate, and benzbromarone, which are thought to inhibit the reabsorption of uric acid in the kidneys.

[0006] Uricase, also known as uric acid oxidase in the art, is an enzyme that catalyzes the oxidation of uric acid to 5-hydroxyisouric acid (consuming O2 and producing H2O2). 5-hydroxyisouric acid is hydrolyzed to allantoin in most animals, plants, and bacteria. However, uricase is absent in humans (and some other great apes), and therefore humans are particularly prone to high blood uric acid levels.

[0007] Rasburicase (marketed as Elitek®) is a tetrameric uricase cloned from Aspergillus flavus and is approved in the United States and Europe for the prevention and treatment of tumor lysis syndrome in cancer patients undergoing chemotherapy. Off-label use of rasburicase for the treatment of gout has also been reported [J Rheumatol 2007, 34:2093-2098]. Rasburicase has a half-life of 6 to 21 hours and must be administered daily by intravenous infusion.

[0008] Pegroticase (marketed as Krystexxa®) is a PEGylated tetrameric porcine-hihikimelauricase approved for the treatment of refractory gout. In each of the four monomers, an average of 10 of the 30 lysine residues are conjugated by a 10 kDa PEG chain.

[0009] Because uricase is a protein that does not naturally exist in humans, it is highly immunogenic. Both rasburicase and pegroticase can cause anaphylaxis as a serious side effect. The PEG portion of pegroticase can reduce the immune response to the uricase backbone, but the PEG portion itself can function as an antibody target [Zhang et al., J Control Release 2016, 244:184-193, Hershfield et al., Arthritis Res Ther 2014, 16:R63, Ganson et al., Arthritis Res Ther 2006, 8:R12].

[0010] In a Phase 3 clinical trial of pegroticase, 26% of patients experienced an infusion reaction, and 6.5% experienced a reaction characterized as anaphylaxis [Baraf et al., Arthritis Res Ther 2013, 15:R137, Strand et al., J Rheumatol 2012, 39:1450-1457].

[0011] In Phase 2 and Phase 3 trials lasting up to 6 months, antibodies against pegroticase were detected (by various methods) in over 80% of patients at some point, and peak titers were associated with loss of efficacy and infusion reactions [Sundy et al., JAMA 2011, 306:711-720, Sundy et al., Arthritis Rheum 2008, 58:2882-2891].

[0012] International Publication No. 00 / 07629 describes PEG-covalent uricases having an average of 2 to 10 PEG chains per uricase subunit and an average PEG molecular weight between approximately 5 kDa and 100 kDa.

[0013] International Publication No. 2011 / 107992 describes multimeric protein structures including monomers of therapeutic proteins such as TNF-α, luteinizing hormone, immunoglobulins, TNF-α receptors, CTLA-4, uric acid oxidase, VEGF, PDGF, VEGF receptors, PDGF receptors, interleukin-17, or fragments thereof, in which such monomers are covalently linked to one another via linking regions.

[0014] Koyama et al. [J Biochem 1996, 120:969-973] described Candida utilis uricase and its mutant in which a cysteine ​​residue is replaced with a serine residue, and concluded that only Cys 168 of the four cysteine ​​residues is involved in the enzyme's activity.

[0015] Chua et al. [Ann Intern Med 1988, 109:114-117] described Arthrobacter protoformiae uricase modified with monofunctional (methoxy-capped) PEG and reported that it did not induce antibody production for 3 weeks after administration.

[0016] Further background technologies include: Hershfield et al. (2009) [“Development of PEGylated mammalian urate oxidase as a therapy for patients with refractory gout,” Birkhauser Basel, in Veronese FM (ed.) PEGylated Protein Drugs: Basic Science and Clinical Applications. Milestones in Drug Therapy], Nyborg et al. [PLoS ONE 2016,11:e0167935], and Veronese [Biomaterials]. [2001,22:405-417], U.S. Patent Nos. 4,179,337, 6,913,915, 8,188,224, and 9,885,024; U.S. Patent Application Publication Nos. 2007 / 0274977 and 2008 / 0159976; and International Publication Brochures Nos. 2011 / 107990, 2011 / 107991, 2016 / 187026, 2018 / 010369, and 2019 / 010369. [Overview of the project]

[0017] According to one aspect of several embodiments of the present invention, a modified uricase is provided, comprising a uricase polypeptide crosslinked by at least one difunctional linkage moiety containing a poly(alkylene glycol) moiety, wherein the molecular weight of the difunctional linkage moiety is in the range of about 1.5 kDa to about 4 kDa.

[0018] According to one aspect of some embodiments of the present invention, there is provided a modified uricase comprising a plurality of polypeptides having the amino acid sequence of SEQ ID NO: 2, wherein the polypeptides are crosslinked by at least one bifunctional linking moiety comprising a poly(alkylene glycol) moiety.

[0019] According to one aspect of some embodiments of the present invention, there is provided a polypeptide having the amino acid sequence of SEQ ID NO: 2.

[0020] According to one aspect of some embodiments of the present invention, there is provided a method for preparing a modified uricase according to any of the embodiments described herein for modified uricase, comprising: (a) contacting a polypeptide with a crosslinking agent comprising a poly(alkylene glycol) moiety and at least two aldehyde groups to obtain a conjugate of the polypeptide and the crosslinking agent, and (b) contacting the conjugate with a reducing agent.

[0021] According to one aspect of some embodiments of the present invention, there is provided a method for reducing the level of uric acid in a medium, comprising contacting the medium with a modified uricase according to any of the embodiments described herein for modified uricase.

[0022] In some of any embodiments of the present invention, the polypeptide is a recombinant polypeptide.

[0023] In some of any embodiments of the present invention, the polypeptide is a plant recombinant polypeptide.

[0024] In some of any embodiments of the present invention regarding the modified uricase, the molecular weight of the bifunctional linking moiety ranges from about 1.5 kDa to about 4 kDa.

[0025] In some of any embodiments of the present invention regarding the modified uricase, the molecular weight of the bifunctional linking moiety ranges from about 2 kDa to about 3.5 kDa.

[0026] In some embodiments of the present invention relating to modified uricases, the bifunctional linkage includes an alkylene group covalently bonded to the nitrogen atom of an amine group in the uricase polypeptide.

[0027] In some embodiments of the present invention relating to amine groups in polypeptides, the amine group is contained within the side chain of a lysine residue.

[0028] In some embodiments of the present invention relating to modified uricases, the uricase polypeptide is conjugated to at least eight bifunctional linkages on average.

[0029] In some of the embodiments of the present invention relating to multiple polypeptides, each polypeptide is linked to at least eight bifunctional linkages on average.

[0030] In some embodiments of the present invention relating to modified uricases, at least 30% of the lysine residue side chains in the modified uricase are covalently bonded to at least one bifunctional linkage moiety.

[0031] In some embodiments of the present invention relating to modified uricases, the bifunctional linkage is represented by the following formula I. -CH2-L1-[O-(CH2)m]nO-L2-CH2- Equation I (In the ceremony L1 and L2 are, independently, either hydrocarbon portions or absent. m is an integer in the range of 2 to 10. n is an integer in the range of 2 to 1000. It holds.

[0032] In some embodiments of the present invention relating to Formula I, n is in the range of 30 to 100.

[0033] In some embodiments of the present invention relating to Formula I, at least one or both of L1 and L2 are unsubstituted alkylenes.

[0034] In some embodiments of the present invention relating to modified uricases, the poly(alkylene glycol) portion is a polyethylene glycol portion.

[0035] In some embodiments of the present invention relating to modified uricases, the modified uricase is in the form of a tetramer.

[0036] In some embodiments of the present invention relating to modified uricases, the modified uricase is in the form of a cross-linked tetramer.

[0037] In some embodiments of the present invention relating to modified uricases, the modified uricase comprises at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4 and their homologs.

[0038] In some embodiments of the present invention relating to modified uricases, the modified uricase comprises at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 and their homologs.

[0039] In some embodiments of the present invention relating to modified uricases, the modified uricase is characterized by a plasma half-life of at least 50 hours in rats.

[0040] In some embodiments of the present invention relating to modified uricases, the modified uricases are intended for use in treating diseases or disorders in which uricase activity is beneficial.

[0041] In some embodiments of the present invention relating to modified uricases, the modified uricases are intended for use in treating diseases or disorders associated with excessive uric acid levels.

[0042] In some embodiments of the present invention relating to a disease or disorder, the disease or disorder is selected from the group consisting of gout, diabetes mellitus, kidney stones, tumor lysis syndrome, hemorrhagic shock, malaria, allergic inflammation, renal dysfunction, viral infection, acute gastroenteritis, placenta, aseptic inflammation, pregnancy complications, multiple sclerosis, inflammatory bowel disease, gastrointestinal infection, and Reschnaihan syndrome.

[0043] In some embodiments of the present invention relating to a treatment, the treatment includes administering a modified uricase at least once every week.

[0044] In some embodiments of the present invention relating to treatment, the treatment includes administering a modified uricase at least every two months.

[0045] In some embodiments of the present invention relating to treatment, the treatment includes administering a modified uricase at a dose of 8 mg / month or less.

[0046] In some embodiments of the present invention relating to the method, the reducing agent is selected from the group consisting of picoline borane complexes and cyanoboron hydride.

[0047] In some embodiments of the present invention concerning the method, the crosslinking agent is represented by the following formula II. HC(=O)-L1-[O-(CH2)m]nO-L2-C(=O)H Formula II (In the ceremony L1 and L2 are hydrocarbon portions, respectively. m is an integer in the range of 2 to 10. n is an integer in the range of 2 to 1000. It holds.

[0048] In some embodiments of the present invention relating to the method, the molecular weight of the crosslinking agent is in the range of about 1.5 kDa to about 4 kDa.

[0049] In some embodiments of the present invention relating to the method, the method includes contacting a polypeptide in tetrameric form with a crosslinking agent.

[0050] In some embodiments of the present invention relating to the method, the molar ratio of the crosslinking agent to the polypeptide is in the range of 100:1 to 10,000:1.

[0051] In some embodiments of the present invention relating to the method, the medium is a target tissue requiring administration, and the method comprises the step of administering a modified uricase to the target.

[0052] In some embodiments of the present invention relating to administration to a subject, the subject suffers from a disease or disorder selected from the group consisting of gout, diabetes mellitus, kidney stones, tumor lysis syndrome, hemorrhagic shock, malaria, allergic inflammation, renal dysfunction, viral infection, acute gastroenteritis, placenta, aseptic inflammation, pregnancy complications, multiple sclerosis, inflammatory bowel disease, gastrointestinal infection, and Reschneihan syndrome.

[0053] In some embodiments of the present invention relating to administration to a subject, administration is performed at least once every week.

[0054] In some of the embodiments of the present invention relating to administration to a subject, administration is performed at least every two months.

[0055] In some embodiments of the present invention relating to administration to a subject, the dose of uricase administered to the subject is 8 mg / month or less.

[0056] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Similar or equivalent methods and materials may be used in carrying out or testing embodiments of the present invention, but exemplary methods and / or materials are described below. In case of any conflict, including definitions, the present patent specification shall prevail. Furthermore, the materials, methods, and examples are merely illustrative and not necessarily intended to be limiting.

[0057] Several embodiments of the present invention are described herein with reference to the accompanying drawings for illustrative purposes only. The following details are particularly detailed with reference to the drawings, emphasizing that the details shown are for illustrative purposes only and serve to illustrate embodiments of the present invention. In this regard, the use of the drawings will make it clear to those skilled in the art how embodiments of the present invention can be put into practice. [Brief explanation of the drawing]

[0058] [Figure 1] Figure 1 illustrates modified uricase according to several embodiments of the present invention, and also shows a method for preparing modified uricase according to several embodiments of the present invention by contacting uricase with an aldehyde-containing crosslinking agent and a reducing agent. [Figure 2] Figure 2 shows gel images obtained by SDS-PAGE analysis (under denaturation conditions) of recombinant wild-type Candida uricase (prU-C) and C250K mutant (prU-C250K) before (-) and after (+) freeze / thaw (Fr / Th) cycles at a concentration of 0.3 mg / mL (measured by optical density) in 25 mM Tris (pH 8.4) (estimated molecular weight values ​​are shown in the center). High molecular weight structures related to uricase (structures formed from multiple subunits, such as dimers and tetramers) are shown in curly braces on the left. [Figure 3]Figure 3 is a graph showing the enzyme activity (normalized to the activity at t=0) of uricase variants (prU-A, prU-C, prU-C250K, and prU-G) as a function of incubation time in human plasma (2 μg / mL uricase at 37°C), normalized to the activity at t=0. [Figure 4] Figure 4 shows gel images obtained by SDS-PAGE analysis of unmodified prU-C and prU-C crosslinked with PEG-bisaldehyde (bisPEG ALD) containing PEG of 1000Da, 2000Da, 5000Da, or 10,000Da (molecular weight markers are shown on the right). [Figure 5] Figure 5 shows gel images obtained by SDS-PAGE analysis of unmodified prU-A (lanes marked "-") and prU-A crosslinked with PEG-bisaldehyde (bis-Ald-PEG) containing PEG in amounts of 600Da, 1000Da, 2000Da, 3400Da, 5000Da, or 10000Da (molecular weight markers are shown on the right). [Figure 6] Figure 6 shows gel images obtained by SDS-PAGE analysis of unmodified prU-G and prU-G crosslinked with 200 or 1000 equivalents of bis-Ald-PEG containing PEG of 2000 Da, 3400 Da, or 5000 Da (molecular weight markers are shown on the left). [Figure 7] Figure 7 shows gel images obtained by SDS-PAGE analysis of unmodified prU-A and prU-A that is bifunctional with an aldehyde (ALD) or N-hydroxysuccinimide (NHS) functional group and crosslinked with 2000 Da of PEG1000 equivalents (molecular weight markers are shown on the left). [Figure 8]Figures 8A and 8B show illustrations and bar graphs. Figure 8A is an illustration of rat immunization with prU-A crosslinked with 2000 Da bis-AldPEG or 2000 Da bis-NHSPEG and an alum adjuvant (the upper arrow indicates the day of immunization, and the lower arrow indicates the day of serum collection). Figure 8B is a bar graph showing the antibody titers against the test protein in rats immunized with prU-A crosslinked with bis-Ald-PEG (rats 7-12) or bis-NHS-PEG (rats 13-18), according to the schedule in Figure 8A. The three bar graphs for each rat show the antibody titers at day 30 (left bar), day 50 (middle bar), and day 72 (right bar). [Figure 9] Figures 9A and 9B show graphs. These graphs show the inhibition rate (%) of binding of antibodies against prU-A crosslinked with bis-Ald-PEG (Figure 9A) or bis-NHS-PEG (Figure 9B) to such crosslinked prU-A, compared to inhibition by unmodified prU-A, as determined by competitive ELISA. Serum samples from rats immunized with the test substance were pre-incubated with high concentrations of modified or unmodified prU-A (x-axis: "inhibitor"). The binding rate after inhibition by pre-incubation with either modified or unmodified prU-A is shown as a function of the concentration of the protein (inhibitor) used in pre-incubation. [Figure 10] Figure 10 is a bar graph showing the titer of antibodies (against the tested modified prU) in individual rats immunized with alum adjuvant, using either prU-A (rat 1-5) crosslinked with PEG-bisaldehyde having 2000 Da of PEG, or prU-A (rat 11-15) crosslinked with PEG-bisaldehyde having 3400 Da of PEG, or prU-C (rat 21-25) crosslinked with PEG-bisaldehyde having 2000 Da of PEG, or prU-C (rat 26-30) crosslinked with PEG-bisaldehyde having 3400 Da of PEG. Blood samples were collected on day 30 (blood collection 1), day 51 (blood collection 2), and day 72 (blood collection 3), and immunization was performed on day 1, day 21, day 42, and day 63. [Figure 11]Figure 11 is a table showing the recognition of unmodified prU-C250K, prU-C250K modified with monofunctional 10kDa PEG, and prU-C250K modified with bifunctional 3400Da PEG by pre-existing antibodies in healthy human donors using ELISA. The values ​​represent the OD ratio between the sample and negative control ELISA results, and only the results for donors showing a positive response with an OD ratio of at least 2 (34 out of 102 donors, each represented by a row) are highlighted. [Figure 12] Figure 12 is a graph showing the stability of two batches (346 and 347) of prU-C250K crosslinked with bis-Ald-PEG3400Da in human plasma at 37°C, evaluated by enzyme activity (normalized to activity at t=0). [Figure 13] Figures 13A and 13B are diagrams and bar graphs. Figure 13A is a diagram showing the pharmacokinetic (PK) and immunogenicity studies comparing unmodified prU-C250K with cross-linked prU-C250K-bis-Ald-PEG3400Da in female rats. The studies included six intravenous (IV) administrations (upper arrows), followed by evaluation of PK and antibody titers (lower arrows indicate the blood collection date for analysis, thick arrows indicate the antibody evaluation date, and thin arrows indicate the PK time point). Figure 13B is a bar graph showing the titers of anti-prU-C250K antibodies on day 0 (Pre1), day 14 (blood collection 1), day 31 (blood collection 2), day 44 (blood collection 3), day 59 (blood collection 4), and day 73 (blood collection 5) in six rats immunized with unmodified prU-C250K. [Figure 14-1]Figures 14A, 14B, 14C, and 14D are graphs showing the pharmacokinetic profile of prU-C250K-bis-Ald-PEG3400. They show the natural logarithm (LN) of plasma protein concentration over time in naive rats (Figures 14A and 14B) or rats administered six times (Figures 14C and 14D), as well as the linear fit to the data (dashed lines, equations, and R² values). Total prU-C250K-bis-Ald-PEG3400 concentrations were measured by complementary ELISA assays (Figures 14A and 14C). Active protein concentrations were assessed by measuring uricase activity (Figures 14B and 14D). The calculated half-lives and area under the curve (AUC) were 54.0 hours and 61.07 mg / min / mL (Figure 14A), 64.8 hours and 65.95 mg / min / mL (Figure 14B), 70.5 hours and 80.4 mg / min / mL (Figure 14C), and 68.4 hours and 58.5 mg / min / mL (Figure 14D). [Figure 14-2] Same as above [Figure 15] Figure 15 is a Michaelis-Menten plot showing the reaction rates of exemplary cross-linked uricase (square) and pegroticase (circle) as a function of substrate (UA) concentration. The reaction rates were measured by incubation at 37°C for 5 minutes and evaluation of the linear increase in fluorescence of the Ampliflu® probe oxidized with the generation of H2O2 (each data point represents the average of three consecutive experiments). [Figure 16] Figure 16 is a graph showing the plasma concentrations of exemplary cross-linked uricase and pegroticase in rats as a function of time after the first injection of the enzyme. [Figure 17] Figure 17 is a graph showing the plasma concentrations of exemplary cross-linked uricase and pegroticase in rats as a function of time after the fourth injection of the enzyme. [Figure 18] Figure 18 is a bar graph showing the plasma half-lives of exemplary cross-linked uricase and pegroticase in rats after the first (naive) and fourth (repeated) injections (based on the data shown in Figures 16 and 17). [Modes for carrying out the invention]

[0059] In some embodiments, the present invention relates to treatment, and more specifically, to novel forms of uricase and their use, for example, in lowering uric acid levels.

[0060] Before describing in detail at least one embodiment of the present invention, it should be understood that the uses of the present invention are not necessarily limited to the details shown in the following embodiments for carrying out the invention or the details illustrated by the examples. Other embodiments of the present invention are possible, or it can be carried out or realized in various ways.

[0061] As discussed above in this specification, uricase is a heterologous protein to humans and therefore highly immunogenic, which poses a significant obstacle to its therapeutic use. In addition, uricase is generally administered by injection or infusion. This administration method can be very inconvenient and may be unstable during handling or storage.

[0062] In the search for improved uricases, the inventors designed and successfully implemented modified uricases exhibiting reduced and nearly inactivated immunogenicity, as well as good stability during storage and in vivo. The inventors demonstrated that the properties of such cross-linked uricases are desirable compared to those of PEGylated uricases known in the art (for example, in terms of low immunogenicity in vivo and an enhanced half-life).

[0063] During the implementation of the present invention, the inventors discovered mutant uricase polypeptides associated with improved performance, including reduced immunogenicity and reduced aggregation.

[0064] Please refer to the various diagrams here. Figure 2 shows that an exemplary uricase mutant (SEQ ID NO: 2) is associated with a reduction in uricase aggregation.

[0065] Figure 3 shows an exemplary uricase variant that exhibits stability in human plasma. Figure 12 shows that the cross-linked uricase according to the exemplary embodiment exhibits high stability in human plasma. Figures 14A to 14D show that the in vivo half-life of the cross-linked uricase according to the exemplary embodiment exceeds 50 hours in rats even after repeated administration.

[0066] Figures 4, 5, and 6 show that the crosslinking efficiency of various uricase variants by polyethylene glycol linkers is maximized when the linkers have a molecular weight greater than 1000 Da and less than 5000 Da.

[0067] Figure 7 shows that both bis-NHS crosslinking agents and bis-aldehyde crosslinking agents efficiently crosslink uricase, and that crosslinking with bis-aldehyde agents resulted in more efficient modification than with bis-N-hydroxysuccinimide agents.

[0068] Figures 8A and 8B show that crosslinking using bis-aldehyde agents significantly reduces the in vivo immunogenicity of crosslinked uricases compared to crosslinking using bis-N-hydroxysuccinimide crosslinking agents.

[0069] Figure 10 shows that low levels of immunogenicity can be obtained in vivo by cross-linking various uricase variants. In contrast, Figures 13A-13B show that unmodified uricase is considerably immunogenic.

[0070] Figure 11 shows that crosslinking with an exemplary (short) PEG linkage results in lower antigenicity (expressed by the amount of pre-existing antibodies in a healthy human donor) compared to modification with 10 kDa PEG (the modification used in pegroticase).

[0071] Figure 15 shows that, in vitro, exemplary cross-linked uricases exhibit considerably stronger uricase activity than pegroticases.

[0072] Figures 16-18 show that, in vivo, exemplary cross-linked uricases have a longer plasma half-life than pegroticases.

[0073] Modified uricase: According to one aspect of several embodiments of the present invention, a modified uricase is provided. The modified uricase comprises at least one uricase polypeptide (as defined herein), the at least one uricase polypeptide being crosslinked by at least one linking moiety comprising a poly(alkylene glycol) moiety, preferably by at least one bifunctional linking moiety comprising a poly(alkylene glycol) moiety.

[0074] In this specification, the term “modified uricase” refers to any structure containing at least one uricase polypeptide (as defined herein) to which one or more additional parts (other than uricase) are covalently linked, and is not intended to be limited beyond what is expressly described herein.

[0075] In this specification, the terms “crosslinked,” “crosslinked,” and any variation thereof refer to individual parts that are covalently bonded to another molecule at two or more different sites on one molecule (e.g., covalently bonded to two or more different atoms). Crosslinking may be intramolecular, i.e., the aforementioned individual parts are covalently bonded to two or more different sites on a single molecule, e.g., a single polypeptide (e.g., the conjugates shown below): [ka] As shown below, part B is covalently bonded to part A at two different sites), or the bond may be intermolecular, that is, the aforementioned individual parts are covalently bonded to two or more different molecules, for example, two or more polypeptides (for example, as in the case of conjugate ABA, part B is covalently bonded to two different A parts), or the bond may be a combination of intramolecular and intermolecular crosslinking (for example, the conjugate shown below) [ka] As shown, two B portions are involved in intramolecular crosslinking, and one is involved in intermolecular crosslinking.

[0076] While not always the case, typically, the individual parts described above are described as "crosslinking" in this specification, and one or more other molecules are described as "being crosslinked" (for example, in conjugate ABA, part B is typically described as "crosslinking" two A parts, and the two A parts are "crosslinked" by part B).

[0077] In this specification, the term “linking portion” refers to any portion (a molecular component) that is covalently bonded to another portion and / or molecule at two or more different sites; that is, a portion that cross-links one or more other molecules (e.g., one or more polypeptides) as defined herein. A “bifunctional linking portion” refers to a linking portion that is covalently bonded to two (but not more) different sites.

[0078] It should be understood that the crosslinking and linking moieties described herein are different from various polypeptide modifications known in the art, including the attachment of moieties to single sites on polypeptides. For example, PEGylation typically involves the attachment of polyethylene glycol (PEG) to a single site. As shown in the Examples section herein, uricases modified with linking moieties may exhibit properties significantly different from corresponding uricases modified with monofunctional, chemically similar moieties, such as those resulting from typical PEGylation (as seen in pegroticase).

[0079] While not bound by any particular theory, it is thought that the linkage region is more sterically confined than the monofunctional region, which can increase the total number of sites bound to it and / or provide more efficient masking of polypeptides from its surroundings (for example, with respect to a given number and / or mass of the modified region).

[0080] In this specification, the term “uricase” encompasses any enzyme classified as EC 1.7.3.3 (catalyzing the oxidation of urate to 5-hydroxyurate, involving the conversion of O2 to H2O2) or any enzyme classified as EC 1.14.13.113 (catalyzing the oxidation of urate to 5-hydroxyurate, involving the oxidation of FADH and the conversion of O2 to H2O2), and includes both proteins having the amino acid sequence of a naturally occurring enzyme and proteins having a homologous amino acid sequence (for example, in accordance with any embodiment described herein in relation to homologs, this term is defined herein).

[0081] In some of the embodiments described herein, the uricase is EC 1.7.3.3 uricase.

[0082] In this specification, the term “uricase polypeptide” refers to a separate polypeptide chain contained in uricase. For example, a tetrameric uricase may contain four uricase polypeptides, or uricase may contain only one uricase polypeptide (e.g., consisting of a uricase polypeptide). A uricase polypeptide is optionally substituted with one or more substituents (e.g., other than the linking moieties described herein), for example, a sugar moiety and / or a lipid moiety, and / or any other substituents known in the art to be bound to naturally occurring polypeptides.

[0083] In some of the various embodiments described herein, the modified uricase is in the form of a multimeric structure; that is, the modified uricase contains multiple uricase polypeptide chains. Such a multimeric form may be, for example, a dimer, trimer, tetramer, hexamer, octamer, or a larger multimeric form. In some such embodiments, the multimeric form is structurally similar (e.g., in the number and / or orientation of the uricase polypeptide chains) to the unmodified form of uricase, for example, a tetramer of many uricase variants.

[0084] At least some of the uricase polypeptides in the polymer structure may optionally be covalently bonded to one another by intermolecular crosslinking, for example, by the linking moieties described herein. Alternatively or additionally, at least some of the uricase polypeptides in the polymer structure may optionally be related to other polypeptides in the polymer structure only by non-covalent interactions (for example, all crosslinking by the linking moieties described herein are intramolecular crosslinking).

[0085] As will be apparent to those skilled in the art, the modified uricases described herein have complex polymericity (for example, due to the presence of one or more polypeptides and / or one or more polymeric linkages), and are therefore typically produced in the form of a collection of similar but somewhat different molecular and / or polymeric structures. For example, the number of linkages and / or the positions in which one or more linkages attach to the uricase polypeptide can vary.

[0086] In some of the various embodiments, a uricase polypeptide (e.g., each of the multiple uricase polypeptides in the polymer structure described herein) is bound to, on average, at least two linkages (as in any of the embodiments described herein), and optionally at least three, at least four, at least five, at least six, at least seven, at least eight, at least ten, at least twelve, at least fourteen, and even at least sixteen linkages. The aforementioned linkages are optionally bifunctional linkages. In some such embodiments, each of the aforementioned linkages is bound to two lysine residues.

[0087] In some of the embodiments, at least 10% of the lysine residue side chains in the modified uricase are bound to a linking portion (for example, according to any embodiment described herein relating to a linking portion that binds to a lysine residue side chain), and optionally, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and even at least 90% of the lysine residue side chains may be bound to the linking portion. The aforementioned linking portion is optionally a bifunctional linking portion. In some embodiments, substantially all of the lysine residue side chains are bound to the linking portion.

[0088] Measuring the average number of binding sites attached to each polypeptide or the average number of lysine residue side chains attached to the binding sites determines the average value of a population of modified uricase molecules and / or polymer structures (e.g., the population discussed above herein). For example, each binding site attached to two polypeptides corresponds to 0.5 binding sites per polypeptide.

[0089] In addition to the linking moieties described herein, modified uricases may optionally be further modified by one or more additional moieties, for example, one or more moieties having a structure similar to the linking moieties described herein (e.g., comprising a poly(alkylene glycol) moiety described herein and / or being bound to a polypeptide lysine residue in the manner described herein for the linking moieties), but being bound to the uricase polypeptide at only one site. Such (monofunity) moieties may be generated, for example, by an incomplete crosslinking reaction in which a binding site (e.g., a lysine residue) that may be present on the polypeptide is attached to another site and / or is sterically blocked.

[0090] Furthermore, in any of the embodiments herein relating to a bifunctional linkage, the modified uricase may optionally be further modified by one or more non-bifunctional linkages, e.g., linkages (optionally branched linkages) bound to three, four or more polypeptide sites. In some such embodiments, the bifunctional linkage may be generated, for example, by an incomplete crosslinking reaction of a compound containing three or more functional groups that can bind to a polypeptide, such that a binding site that may be present on the polypeptide (e.g., a lysine residue) is bound to multiple sites and / or sterically blocked by multiple sites, thus inhibiting binding at a third site.

[0091] In some of the embodiments described herein, the modified uricase has a longer in vivo half-life than the unmodified uricase (i.e., without the linking portion described herein). In some such embodiments described herein, the half-life of the modified uricase is at least 20% longer than the half-life of the unmodified uricase. In some embodiments, the half-life of the modified uricase is at least 50% longer than the half-life of the unmodified uricase. In some embodiments, the half-life of the modified uricase is at least 100% longer, i.e., at least twice as long, than the half-life of the unmodified uricase. In some embodiments, the half-life of the modified uricase is at least three times longer than the half-life of the unmodified uricase. In some embodiments, the half-life of the modified uricase is at least five times longer than the half-life of the unmodified uricase. In some embodiments, the half-life of the modified uricase is at least 10 times that of the unmodified uricase used for comparison. In some embodiments, the half-life of the modified uricase is at least 20 times that of the unmodified uricase used for comparison. In some embodiments, the half-life of the modified uricase is at least 50 times that of the unmodified uricase used for comparison. In some embodiments, the half-life of the modified uricase is at least 100 times that of the unmodified uricase used for comparison.

[0092] The half-life of (modified and / or unmodified) uricase may be determined, for example, by measuring the amount of test uricase in the blood (e.g., in plasma) over time after injection of the test uricase into a subject (e.g., in humans and / or rats). As illustrated herein, the amount of uricase may be measured using an antibody against the test uricase (e.g., by ELISA) and / or by measuring the amount of enzyme activity characteristic of the uricase.

[0093] In some of the various embodiments described herein, the modified uricase has a plasma half-life of at least 40 hours in rats (measured, for example, by antibody recognition and / or enzyme activity). In some such embodiments, the half-life is at least 50 hours. In some such embodiments, the half-life is at least 60 hours. In some such embodiments, the half-life is at least 70 hours. In some such embodiments, the half-life is at least 80 hours. In some such embodiments, the half-life is at least 100 hours. In some embodiments, the half-life is at least 1 week, or at least 2 weeks, or at least 3 weeks, or at least 4 weeks.

[0094] The longer half-life of the modified uricase according to any of the embodiments described herein may optionally be related to a larger molecular weight of the modified uricase (which may reduce the rate at which it is removed from the bloodstream, for example, by filtration in the kidneys) and / or lower immunogenicity of the modified uricase (which may reduce the rate of inactivation and / or destruction by the immune system).

[0095] Connecting part: The linking portion according to any of the embodiments described herein may optionally be linked to a uricase polypeptide according to any of the embodiments described herein (e.g., those in each of the respective sections herein) in any manner described herein (e.g., according to any of the embodiments described herein with respect to the crosslinking properties and / or overall structure of the modified uricase).

[0096] As discussed herein, the linking portion includes a poly(alkylene glycol) portion.

[0097] As used herein, the term "poly(alkylene glycol)" encompasses a group of polyether polymers sharing the following general formula: -[O-(CH2)m]nO- (wherein m represents the number of methylene groups present in each alkylene glycol unit, and n represents the number of repeating units, thus representing the size or length of the polymer). For example, when m=2, the polymer is called polyethylene glycol, and when m=3, the polymer is called polypropylene glycol.

[0098] In some embodiments, m is an integer greater than 1 (for example, m = 2, 3, 4, etc.).

[0099] Optionally, m may differ between units of the poly(alkylene glycol) chain. For example, the poly(alkylene glycol) chain may contain both ethylene glycol (m=2) units and propylene glycol (m=3) units linked together.

[0100] The term "poly(alkylene glycol)" also encompasses its analogues in which the oxygen atom is substituted with another heteroatom, such as S and -NH-. The term further encompasses the above derivatives in which one or more of the methylene groups constituting the polymer are substituted. Examples of any substituents on the methylene group include, but are not limited to, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, hydroxy, oxo, thiol, and thioalkoxy. In some embodiments, the substituent on the methylene group (if present) is alkyl, optionally C 1-4 - Alkyl, and optionally methyl.

[0101] As used herein, the term “alkylene glycol unit” includes the -O-(CH2)m- group or its analogues described herein that form the main chain of poly(alkylene glycol), wherein (CH2)m (or its analogues) is bonded to an oxygen atom (or its heteroatom analogue) at the end of poly(alkylene glycol) (represented by the formula -[O-(CH2)m]nO-) or its heteroatom analogue, or at the end of a heteroatom belonging to another alkylene glycol unit or uricase polypeptide (in the case of a terminal unit), and O (or the aforementioned terminal oxygen atom) or its heteroatom analogue is bonded to (CH2)m (or its analogue) of another alkylene glycol unit, or to a functional group that forms a bond with a uricase polypeptide (as in any of the embodiments described herein).

[0102] An alkylene glycol unit may be branched to connect to three or more adjacent alkylene glycol units, each of which is part of a poly(alkylene glycol) chain. Such a branched alkylene glycol unit is bonded to one adjacent alkylene glycol unit via its heteroatom, and the heteroatoms of the remaining adjacent alkylene glycol units are each bonded to the carbon atoms of the branched alkylene glycol unit. Furthermore, a heteroatom (e.g., nitrogen) may bond to two or more carbon atoms of an alkylene glycol unit which is part of it, to form a branched alkylene glycol unit (e.g., [(-CH2)m]2N-, etc.).

[0103] In exemplary embodiments, at least 50% of the alkylene glycol units are identical, for example, they contain the same heteroatoms and the same m value. Optionally, at least 70%, optionally at least 90%, and optionally 100% of the alkylene glycol units are identical. In exemplary embodiments, the heteroatoms bonded to identical alkylene glycol units are oxygen atoms, and / or the alkylene glycol units are unsubstituted. In further exemplary embodiments, m is 2 for identical units.

[0104] In one embodiment, the poly(alkylene glycol) is a single linear chain (preferably polyethylene glycol (PEG)), and the two ends of the chain are independently bonded directly or indirectly (for example, via functional groups described herein) to the uricase polypeptide.

[0105] As used herein, the term “polyethylene glycol” refers to the poly(alkylene glycol) as defined herein above, wherein at least 50%, at least 70%, at least 90%, and preferably 100%, of the alkylene glycol units are -CH2CH2-O-. Similarly, the phrase “ethylene glycol units” is defined herein as units of -CH2CH2O-.

[0106] According to any embodiment, the connecting portion comprises polyethylene glycol or an analogue thereof, which is represented by the following general formula. -(Y1-CR1R2-CR3R4)n-Y2- (In the formula, Y1 and Y2 are each independently O, S, or NR5 (optionally O), n is an integer of any kind between 2 and 1000 (arbitrarily between 10 and 300, and arbitrarily between 30 and 100), but higher values ​​of n are also intended. Each of R1, R2, R3, R4, and R5 is independently hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, hydroxy, oxo, thiol, and / or thioalkoxy.

[0107] In some of the embodiments, R1, R2, R3, R4, and R5 are each independently hydrogen or alkyl, and optionally hydrogen or C 1-4- It is alkyl, and optionally hydrogen or methyl. In an exemplary embodiment, R1, R2, R3, R4, and R5 are each hydrogen.

[0108] Polyethylene glycol or its analogues may optionally contain copolymers, for example, the Y1-CR1R2-CR3R4 units in the above formula are not all identical to one another.

[0109] In some embodiments, at least 50% of the Y1-CR1R2-CR3R4 units are identical. Optionally, at least 70%, at least 90%, and at least 100% of the Y1-CR1R2-CR3R4 units are identical.

[0110] Optionally, the connecting portion branches, for example, for one or more Y1-CR1R2-CR3R4 units in the above formula, such that at least one of R1, R2, R3, R4, and R5 is -(Y1-CR1R2-CR3R4)p-Y2-, where R1-R5 and Y1 and Y2 are as defined herein, and p is an integer as defined herein for n (e.g., 2-1000) according to any of the embodiments.

[0111] The linking portion optionally comprises at least two functional groups, each functional group forming a covalent bond with the uricase polypeptide. Examples of functional groups include alkylene groups and carbonyl (-C(=O)-). The alkylene or carbonyl may optionally be bonded to a nitrogen atom of the polypeptide (e.g., an amine group) so as to form together an amine group or an amide group, respectively. The functional groups may optionally be terminal groups of the linking portion, and the entire length of the poly(alkylene glycol) is between the two functional groups. Each functional group may optionally be directly bonded to the poly(alkylene glycol) moiety (as in any of the embodiments described herein) or indirectly bonded via a linking group (as defined herein), and the linking group may optionally be a hydrocarbon moiety.

[0112] In any of the embodiments described herein, the linking portion (optionally, a bifunctional linking portion) includes an alkylene group (e.g., an unsubstituted alkylene group) covalently bonded to the nitrogen atom of an amine group in the polypeptide, e.g., a lysine residue side chain and / or an N-terminal amine group.

[0113] As illustrated herein, such alkylene groups covalently bonded to a nitrogen atom can optionally be obtained by reacting an aldehyde group with an amine group in the presence of a reducing agent (for example, by the method described herein).

[0114] While not bound by any particular theory, crosslinking via alkylene groups covalently bonded to polypeptide nitrogen atoms is advantageously considered to be less immunogenic than alternative crosslinking techniques, such as forming amide bonds between carbonyl (-C(=O)-) groups (optionally induced by carboxylate group condensation) and polypeptide amine groups.

[0115] Figure 1 schematically shows modified uricase (e.g., in tetramer form) according to several embodiments of the present invention, where some of the PEG moieties are bonded to multiple amine groups of the polypeptide (e.g., by reductive amination according to any of the embodiments described herein), some of the PEG moieties are bonded to a single amine group of the polypeptide, and unreacted functional groups (e.g., aldehydes) remain (optionally produced by incomplete crosslinking reactions according to any of the embodiments described herein). Furthermore, there may be amine groups that are not bonded to any moiety (i.e., -NH2 groups). As shown herein, the modified uricase may optionally be produced by the reaction of uricase (e.g., uricase tetramer) with a bisaldehyde reagent in the presence of a reducing agent.

[0116] In the case of uricases containing multiple polypeptide units (e.g., a uricase tetramer), the PEG moiety may optionally (but not necessarily) be attached to the polypeptides in such a way that some or all of the polypeptides (e.g., all four polypeptides of the tetramer) are crosslinked to each other (directly and / or indirectly via one or more intervening polypeptides). Optionally, the crosslinking is such that the polypeptides do not dissociate under denaturation conditions.

[0117] Modified uricases different from those illustrated in Figure 1 are also conceivable by having a different quaternary structure (e.g., a structure other than a tetramer), being formed by a different reaction (e.g., a reaction other than with a bisaldehyde agent in the presence of a reducing agent), containing a different functional group (e.g., something other than an aldehyde), containing a different group for attaching polypeptides to the linking portion (e.g., something other than an -NH- group), and / or containing a different linking portion (e.g., containing a polymer other than PEG).

[0118] In any of the embodiments described herein, the bifunctional linkage is represented by the following formula I. -CH2-L1-[O-(CH2)m]nO-L2-CH2- Equation I (In the ceremony L1 and L2 are either (as defined herein) linkages or absent (optionally, they may be the same or different linkage hydrocarbon portions), preferably the linkages are hydrocarbon portions. m is an integer in the range of 2 to 10. n is an integer in the range of 2 to 1000.

[0119] In some arbitrary embodiments of this specification relating to formulas including the variable m, m is 2, 3, or 4. In some embodiments, m is 2 or 3. In some embodiments, m is 2 such that the linking portion includes a polyethylene glycol portion (having n ethylene glycol subunits).

[0120] In some arbitrary embodiments of this specification relating to expressions containing the variable n, n is at least 10 (e.g., 10 to 300, or 10 to 100). In some such embodiments, n is at least 30 (e.g., 30 to 300, or 30 to 100, or 30 to 80, or 30 to 60). In some embodiments, n is at least 40 (e.g., 40 to 300, or 40 to 100, or 40 to 80, or 40 to 60). In some embodiments, n is at least 50 (e.g., 50 to 300, or 50 to 100, or 50 to 80, or 50 to 60). In some embodiments, n is at least 60 (e.g., 60 to 300, or 60 to 100, or 60 to 80). In some embodiments, n is at least 60 (e.g., 60 to 300, or 60 to 100, or 60 to 80). In some embodiments, n is at least 70 (e.g., 70 to 300, or 70 to 100, or 70 to 80).

[0121] In some arbitrary embodiments of this specification relating to formulas including variables m and n, n is at least 10 (e.g., 10 to 300, or 10 to 100), and m is 2, 3, or 4, preferably 2 or 3, more preferably 2. In some such embodiments, n is at least 30 (e.g., 30 to 300, or 30 to 100, or 30 to 80, or 30 to 60). In some embodiments, n is at least 40 (e.g., 40 to 300, or 40 to 100, or 40 to 80, or 40 to 60). In some embodiments, n is at least 50 (e.g., 50 to 300, or 50 to 100, or 50 to 80, or 50 to 60). In some embodiments, n is at least 60 (e.g., 60 to 300, or 60 to 100, or 60 to 80). In some embodiments, n is at least 60 (e.g., 60 to 300, or 60 to 100, or 60 to 80). In some embodiments, n is at least 70 (e.g., 70 to 300, or 70 to 100, or 70 to 80).

[0122] In some of the embodiments described herein, L1 and L2 are independently substituted or unsubstituted alkylenes, and may optionally have 1 to 4 carbon atoms, 1 to 3 carbon atoms, and 1 or 2 carbon atoms. In some such embodiments, the alkylene is unsubstituted, for example, CH2 or CH2CH2.

[0123] The linking portion of Formula I (as in any of the embodiments) may optionally be bonded to a nitrogen atom of the polypeptide at one or both of its ends. In such embodiments, the terminal -CH2- (optionally in combination with at least a portion of L1 and / or L2) forms an alkylene (optionally an unsubstituted alkylene) bonded to a nitrogen atom of the polypeptide (as in any of the embodiments described herein).

[0124] In any of the several embodiments described herein, the molecular weight of the linking portion (optionally, a bifunctional linking portion) is at least about 1.5 kDa. In some such embodiments, the molecular weight of the linking portion is in the range of about 1.5 kDa to about 4 kDa. In some embodiments, the molecular weight of the linking portion is in the range of about 1.5 kDa to about 3.5 kDa. In some embodiments, the molecular weight of the linking portion is in the range of about 1.5 kDa to about 3 kDa. In some embodiments, the molecular weight of the linking portion is in the range of about 1.5 kDa to about 2.5 kDa. In some exemplary embodiments, the molecular weight of the linking portion is about 2 kDa.

[0125] In any of the several embodiments described herein, the molecular weight of the linking portion (optionally, a bifunctional linking portion) is at least about 2 kDa. In some such embodiments, the molecular weight of the linking portion is in the range of about 2 kDa to about 4 kDa. In some embodiments, the molecular weight of the linking portion is in the range of about 2 kDa to about 3.5 kDa. In some embodiments, the molecular weight of the linking portion is in the range of about 2 kDa to about 3 kDa. In some embodiments, the molecular weight of the linking portion is in the range of about 2 kDa to about 2.5 kDa.

[0126] In any of the several embodiments described herein, the molecular weight of the linking portion (optionally, a bifunctional linking portion) is at least about 2.5 kDa. In some such embodiments, the molecular weight of the linking portion is in the range of about 2.5 kDa to about 4 kDa. In some embodiments, the molecular weight of the linking portion is in the range of about 2.5 kDa to about 3.5 kDa.

[0127] In any of the several embodiments described herein, the molecular weight of the linking portion (optionally, a bifunctional linking portion) is at least about 3 kDa. In some such embodiments, the molecular weight of the linking portion is in the range of about 3 kDa to about 4 kDa. In some embodiments, the molecular weight of the linking portion is in the range of about 3 kDa to about 3.5 kDa. In some exemplary embodiments, the molecular weight of the linking portion is about 3.4 kDa.

[0128] In some of the embodiments described herein, the molecular weight of the linking portion (optionally, a bifunctional linking portion) is about 4 kDa or less. In some such embodiments, the molecular weight of the linking portion is about 3.5 kDa or less. In some embodiments, the molecular weight of the linking portion is about 3 kDa or less. In some embodiments, the molecular weight of the linking portion is about 2.5 kDa or less.

[0129] As illustrated herein, linking portions of the sizes described herein (e.g., at least about 1.5 kDa and / or about 4 kDa or less, and / or values ​​of the variables m and / or n as described herein) may be associated with a favorable combination of crosslinking efficiency and low immunogenicity compared to smaller and / or larger linking portions.

[0130] While not bound by any particular theory, excessively small ligatures may lead to ineffective masking of polypeptides (related to higher immunogenicity), for example, because the mass per ligament is smaller and / or the amount of ligatures bound to each polypeptide is smaller (e.g., the ligament is not long enough to efficiently bind to two distinct binding sites, such as a pair of lysine residues). Furthermore, excessively large ligatures may also lead to ineffective masking of polypeptides; for example, the binding of a large ligament may sterically inhibit the binding of further ligatures, leaving a gap in polypeptide masking (e.g., allowing antibody penetration).

[0131] polypeptide: The uricase polypeptide portion according to any embodiment described herein may optionally be combined with a linking portion according to any embodiment described herein (e.g., in any of the embodiments described herein with respect to the crosslinking properties and / or overall structure of the modified uricase) in any manner described herein (e.g., in any of the embodiments described herein).

[0132] A uricase polypeptide used in any of the embodiments described herein may associate with any one or more uricases (as defined herein) known in the art. A modified uricase comprising multiple cross-linked uricase polypeptides (as in any of the embodiments described herein) may optionally comprise a single uricase variant or a uricase polypeptide associated with various uricase variants.

[0133] During the term of this application and the final patent, it is anticipated that many related variants of uricase will be characterized (e.g., naturally occurring uricase variants) and / or developed (e.g., uricase variants not naturally occurring), and the scope of the terms “uricase” and “uricase polypeptide” is intended to include all such new variants and technologies a priori.

[0134] Examples of uricase polypeptides that may be used in any of the embodiments described herein include, but are not limited to, those derived from: ancient humans, pigs, baboons, Agrobacterium tumefaciens, Alicyclobacillus mali, Arthrobacter gangotriensis, Arthrobacter globiformis, Aspergillus flavus, Aspergillus udagawae, Aureobasidium pullulans EXF-150, Bacillus fastidiosus, Bacillus halodurans C-125, Bacillus subtilis str.168, Bacillus sp. FJAT-21352, Bacillus sp. TB-90, Bacillus beveridgei, Bactrocera latifrons (fruit fly), Blastomyces dermatitidis, Camelus ferus (wild two-humped camel), and Candida. utillis, Candidatus Solibacter usitatus, Chlamydomonas reinhardtii, Cicer arietinum (chickpea), Deinococcus radiodurans, Deinococcus geothermalis, Drechmeria coniospora, Erinaceus europaeus (common hedgehog), Escherichia coli ISC56, Galdieria sulphuraria, Glycine max (soybean), Granulicella tundricola, Kyrpidia tusciae DSM 2912, Magnaporthiopsis poae, Microbacterium sp.zzj4-1, Neonectria ditissima, Nicotiana tabacum (tobacco), Paenibacillus darwinianus, Paenibacillus odorifer, Phaseolus vulgaris (bean), Philocephala scopiformis, Pseudomonas aeruginosa, Pygoscelis adeliae (Adélie penguin), Rousettus aegyptiacus (Egyptian flying fox), Stomoxys calcitrans (barn fly), Terriglobus saanensis, Tolypocladium ophioglossoides, and Tolypocladium ophioglossoides CBS 100239. Furthermore, chimeras of two or more uricase polypeptides (e.g., the porcine-baboon chimeric polypeptide contained in pegroticase), as well as any homolog thereof (as defined herein). Exemplary uricase polypeptide amino acid sequences include SEQ ID NOs: 1, 2, 3, and 4.

[0135] In some optional embodiments described herein, the modified uricase comprises at least one polypeptide having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 and / or SEQ ID NO: 3 and / or their homologs (as defined herein). As illustrated herein, such sequences may optionally be associated with relatively low polypeptide immunogenicity.

[0136] In some optional embodiments described herein, the modified uricase comprises at least one polypeptide having the amino acid sequences of SEQ ID NO: 1 and / or SEQ ID NO: 2 and / or their homologs (as defined herein). As illustrated herein, such sequences may optionally be associated with relatively low immunogenicity, relatively high crosslinking ability (e.g., due to a large number of lysine residues in the sequence), and / or relatively high stability under physiological conditions (e.g., thermal stability at a temperature of about 37°C).

[0137] Throughout this specification, a “homolog” of a given polypeptide (e.g., a uricase polypeptide as described herein) means a polypeptide that exhibits at least 80% homology, preferably at least 90% homology, more preferably at least 95% homology, and more preferably at least 98% homology to a given polypeptide. In some embodiments, a homolog of a given polypeptide further has common enzymatic activity and / or therapeutic activity (e.g., uric acid oxidation) with the given polypeptide. The homology percentage refers to the percentage of amino acid residues in the first polypeptide sequence that match the corresponding residues of the second polypeptide sequence being compared to the first polypeptide. Generally, polypeptides are aligned to obtain maximum homology. Various strategies for performing amino acid or nucleotide sequence comparisons to assess homology are known in the art, for example, manual alignment, computer-assisted sequence alignment, and combinations thereof. Numerous algorithms for performing sequence alignment (generally computer-implemented) are widely available or can be created by those skilled in the art. Representative algorithms include, for example, the local homology algorithm by Smith and Waterman (Adv. Appl. Math., 1981, 2:482), the homology alignment algorithm by Needleman and Wunsch (J. Mol. Biol., 1970, 48:443), the similarity search method by Pearson and Lipman (Proc. Natl. Acad. Sci. (USA), 1988, 85:2444), and / or computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, Science Drive 575, Madison, Wisconsin). Easily available computer programs incorporating such algorithms include, for example, BLASTN, BLASTP, Gapped BLAST, PILEUP, and CLUSTALW.When using the BLAST and Gapped BLAST programs, you may use the default parameters for each program. Alternatively, the implementer may use non-default parameters based on experience and / or other requirements (see, for example, the website whose URL is www(dot)ncbi(dot)nlm(dot)nih(dot)gov).

[0138] In some optional embodiments relating to a homolog of a given polypeptide, the homolog exhibits at least 80% sequence homology, optionally at least 90% sequence homology, optionally at least 95% sequence homology, optionally at least 98% sequence homology, and optionally at least 99% sequence homology to the given polypeptide.

[0139] In some optional embodiments described herein, the uricase polypeptide has the amino acid sequence of SEQ ID NO: 2. In some such embodiments, the modified uricase comprises a plurality of polypeptides having SEQ ID NO: 2, optionally four polypeptides having SEQ ID NO: 2.

[0140] As discussed in the Examples section below, Sequence ID No. 2 corresponds to a naturally occurring uricase polypeptide (Sequence ID No. 1) from Candida utilis having the point mutation C250K (i.e., Cys250 is substituted with lysine). The polypeptide of Sequence ID No. 2 readily forms tetramers, similar to the polypeptide of Sequence ID No. 1.

[0141] Alternatively, the uricase polypeptide may optionally be a homolog of SEQ ID NO: 2, which has any residue other than Cys at a position homologous to Lys250 of SEQ ID NO: 2 (or Cys250 of SEQ ID NO: 1). Optionally, the homolog contains Lys at a position homologous to Lys250 of SEQ ID NO: 2.

[0142] While not bound by any particular theory, Cys250 plays a crucial role in polypeptide aggregation by forming intermolecular disulfide bonds, suggesting that removing Cys250 would significantly reduce undesirable aggregation. Furthermore, the additional lysine residue (due to the C250K mutation) is thought to facilitate crosslinking, for example, with crosslinking moieties suitable for binding to amine groups.

[0143] According to one embodiment of several embodiments of the present invention, a polypeptide having the amino acid sequence of SEQ ID NO: 2 is provided.

[0144] The uricase polypeptides of any embodiment of the present invention may optionally be purified (for example, from plant or animal tissue) or produced by recombinant DNA technology. In any of the several embodiments, the uricase polypeptide is a plant recombinant polypeptide; that is, it is produced in a plant by recombinant technology. An example of a plant that produces recombinant polypeptides is Nicotiana tabacum (tobacco).

[0145] A wide variety of techniques for producing recombinant polypeptides in various cells and / or organisms (including plants and plant cells) are known in the art.

[0146] Recombinant proteins may optionally be characterized by post-translational modifications (e.g., glycosylation) that are characteristic of the type of cell and / or organism (e.g., plant) from which the recombinant protein is produced. These characteristics are in contrast to, for example, the types of cells and / or organisms that naturally express polypeptides (or their closest naturally occurring homologs).

[0147] As used herein, the term “plant” includes the whole plant, grafted plants, plant ancestors and offspring, as well as parts of a plant including seeds, buds, stems, roots (including tubers), rootstocks, scions, and plant cells, tissues, and organs. A plant may also be any form including suspension cultures, embryos, meristematic regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores. Plants particularly useful in the method of the present invention include all plants belonging to the superfamily Green Plantae, especially monocots and dicots including legumes used as fodder or forage, ornamental plants, edible crops, trees, or shrubs, such as: Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsoophila tricolor, Andropogon spp., Arachis spp., Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp., Camellia sinensis, Cannabaceae, Cannabis indica, Cannabis, Cannabis sativa, Hemp, Industrial hemp, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronallia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp., Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp., Freycinetia banksli, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia spp., Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp., Taxodium distichum, Themeda triandra、Trifolium spp.、Triticum spp.、Tsuga heterophylla、Vaccinium spp.、Vicia spp.Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, corn, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, rapeseed, carrot, cauliflower, celery, collard greens, flax, kale, lentil, rapeseed, okra, onion, potato, rice, soybean, wheat, sugar beet, sugarcane, sunflower, tomato, squash tea, and / or trees. Alternatively, algae and other non-green plant subkingdoms may be used in the methods of some embodiments of the present invention.

[0148] Alternatively, polypeptides of some embodiments of the present invention may be chemically synthesized by any technique known to those skilled in the art of peptide synthesis. Outlines of many techniques for solid-phase peptide synthesis can be found in J.M. Stewart and J.D. Young, Solid Phase Peptide Synthesis, WH Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides, vol.2, p.46, Academic Press (New York), 1973. For classical solution synthesis, see G. Schroder and K. Lupke, The Peptides, vol.1, Academic Press (New York), 1965.

[0149] Generally, these methods involve sequentially adding one or more amino acids or appropriately protected amino acids to an elongating polypeptide chain. Typically, either the amino group or carboxyl group of the first amino acid is protected by an appropriate protecting group. The protected or derivatized amino acid can then be utilized in solution by attaching it to an inert solid support or by adding the next amino acid in a sequence having an appropriately protected complementary (amino or carboxyl) group under conditions suitable for forming an amide bond. The protecting group is then removed from this newly added amino acid residue, and then the next amino acid (appropriately protected) is added, and so on. After all the desired amino acids have been linked in the appropriate sequence, any remaining protecting groups (and any solid support) are removed sequentially or simultaneously to obtain the final polypeptide compound. By making simple modifications to this general procedure, it is possible to add two or more amino acids to an elongating chain at once, for example, by coupling a protected tripeptide with an appropriately protected dipeptide (under conditions that do not racemize the chiral center) to form a pentapeptide after deprotection. Further descriptions of peptide synthesis are found in U.S. Patent No. 6,472,505.

[0150] Large-scale polypeptide synthesis has been described by Andersson et al. [Biopolymers 2000; 55:227-250].

[0151] In this specification, the term “polypeptide” refers to a polymer comprising at least 10 amino acid residues (preferably at least 50 amino acid residues) linked by peptide bonds or analogues thereof (as described below herein), or optionally by peptide bonds themselves. The term “polypeptide” encompasses naturally occurring polypeptides (e.g., degradation products, chemically synthesized peptides, and / or recombinant polypeptides), including but not limited to naturally occurring proteins, naturally occurring protein fragments, and naturally occurring protein homologs and / or fragments thereof; and also encompasses peptidoids and semipeptoids, which are peptidomimetics (typically chemically synthesized polypeptides) and polypeptide analogues, which may have modifications (e.g., modifications other than crosslinking modifications as expressly described herein) that make polypeptides placed in the body more stable or increase their permeability to cells. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications, skeletal modifications, and residue modifications. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, CA Ramsden Gd., Chapter 17.2, F. Choplin, Pergamon Press (1992), which is incorporated herein by reference as if it were fully described herein. Further details in this regard are provided below herein.

[0152] The peptide bond (-CO-NH-) within the peptide may be substituted with, for example, an N-methylated amide bond (-N(CH3-CO-), an ester bond (-C(=O)-O-), a ketomethylene bond (-CO-CH2-), a sulfinylmethylene bond (-S(=O)-CH2-), or an α-aza bond (-NH-N(R)-CO-), where R is any alkyl (e.g., methyl), an amine bond (-CH2-NH-), a sulfide bond (-CH2-S-), an ethylene bond (-CH2-CH2-), a hydroxyethylene bond (-CH(OH)-CH2-), a thioamide bond (-CS-NH-), an olefin double bond (-CH=CH-), a fluorinated olefin double bond (-CF=CH-), a retroamide bond (-NH-CO-), or a peptide derivative (-N(R)-CH2-CO-), where R is a naturally occurring "normal" side chain on a carbon atom.

[0153] These modifications can occur at any of the bonds along the peptide chain, and even simultaneously at several (e.g., 2-3) bonds.

[0154] The natural aromatic amino acids Trp, Tyr, and Phe may be substituted with unnatural aromatic amino acids such as 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), naphthylalanine, cyclic methylated derivatives of Phe, halogenated derivatives of Phe, or O-methyl-Tyr.

[0155] Peptides of some embodiments of the present invention may also contain one or more modified amino acids or one or more non-amino acid monomers (e.g., fatty acids, complex carbohydrates, etc.).

[0156] The term "amino acid" or "amino acids" is understood to include 20 naturally occurring amino acids, amino acids often formed in vivo through post-translational modification, such as hydroxyproline, phosphotyrosine, phosphothreonine, and other non-ordinary amino acids, such as, but not limited to, 2-aminodipic acid, hydroxylysine, isodesmosine, norvaline, norleucine, and ornithine. Furthermore, the term "amino acid" includes both D-amino acids and L-amino acids.

[0157] Tables 1 and 2 below list naturally occurring amino acids (Table 1) and unconventional or modified amino acids (e.g., synthetic, Table 2) that may be used in some embodiments of the present invention.

[0158] [Table 1]

[0159] [Table 2-1]

[0160] [Table 2-2]

[0161] [Table 2-3]

[0162] Preparation of modified uricase: According to one aspect of several embodiments of the present invention, a method is provided for preparing a modified uricase according to any embodiment relating to a modified uricase and / or its components (e.g., a uricase polypeptide and / or a linking portion). This method is (a) A step of contacting a uricase polypeptide (according to any embodiment described herein) with a crosslinking agent (according to any embodiment described herein) containing a poly(alkylene glycol) moiety to obtain a conjugate of the polypeptide and the crosslinking agent, wherein the crosslinking agent contains at least two aldehyde (-C(=O)H) groups, and (b) The step of bringing the conjugate into contact with a reducing agent.

[0163] In any of the embodiments described herein, the crosslinking agent comprises two or fewer aldehyde (-C(=O)H) groups.

[0164] In any of the embodiments described herein, the crosslinking agent is represented by the following formula II. HC(=O)-L1-[O-(CH2)m]nO-L2-C(=O)H Formula II (wherein L1 and L2 are hydrocarbon parts, m is an integer in the range of 2 to 10, and n is an integer in the range of 2 to 1000 (for example, in the formula L1, L2, m and / or n are as defined according to any of the embodiments described herein relating to Formula I)) The agent of Formula II may optionally be used to obtain a linked part according to Formula I (according to any of the embodiments described herein), for example, by reacting each aldehyde group with an amine group (for example, to form an imine or hemiaminal intermediate) and reducing it to form an amine group.

[0165] Alternatively, the crosslinking agent may optionally contain three or more (e.g., three, four, or more) aldehyde groups. Such a crosslinking agent may optionally generate a bifunctional linkage (as in any of the embodiments described herein) by the reaction of only two aldehyde groups with the polypeptide, for example, no unreacted amine group remains near the third aldehyde group after the reaction of the two aforementioned aldehyde groups.

[0166] Figure 1 schematically illustrates a method according to one of several embodiments described herein.

[0167] Examples of suitable reducing agents include, but are not limited to, boranes and their complexes (e.g., picoline borane complexes), boron hydride (including borohydride salts, e.g., sodium borohydride), triacetoxyboron hydride (including triacetoxyboron hydride salts, e.g., sodium triacetoxyboron hydride), cyanoboron hydride (including cyanoboron hydride salts, e.g., sodium cyanoboron hydride), and any other reducing agents known in the art to be suitable for reductive amination processes. Exemplary reducing agents include, but are not limited to, 2-picoline borane complexes and sodium cyanoboron hydride.

[0168] The uricase polypeptide, crosslinking agent, and reducing agent may be combined in any order. For example, the crosslinking agent may be optionally added to a mixture containing the polypeptide and the reducing agent, or the polypeptide may be optionally added to a mixture containing the crosslinking agent and the reducing agent (for example, such that the conjugate of the polypeptide and the crosslinking agent is already in contact with the reducing agent at the time of conjugate formation). In some embodiments, the uricase polypeptide, crosslinking agent, and reducing agent are combined essentially simultaneously (for example, as a "one-pot reaction").

[0169] In any of the several embodiments described herein, the uricase polypeptide is in a polymeric form (a form comprising two or more uricase polypeptide chains) when contacted with a crosslinking agent. Such a polymeric form may be, for example, a dimer, trimer, tetramer, hexamer, octamer, or a larger polymeric form. In some such embodiments, the polymeric form is a naturally occurring form of the uricase polypeptide, for example, a tetramer in many uricase polypeptides. In any of the several embodiments described herein, contacting the polymeric form of the uricase polypeptide with a crosslinking agent can serve as an efficient technique for generating intermolecular crosslinks, resulting in the creation of one or more crosslinked portions bonded to different polypeptide chains (for example, according to any of the embodiments described herein).

[0170] In any of the several embodiments described herein, the molar ratio of the crosslinking agent (according to any of the embodiments described herein) to the uricase polypeptide in contact with the crosslinking agent (according to any of the embodiments described herein) is at least 100:1. In some such embodiments, the molar ratio is 100:1 to 10,000:1. In some such embodiments, the molar ratio is 100:1 to 5,000:1. In some embodiments, the molar ratio is 100:1 to 2,000:1. In some embodiments, the molar ratio is 100:1 to 1,000:1.

[0171] In any of the several embodiments described herein, the molar ratio of the crosslinking agent (according to any of the embodiments described herein) to the uricase polypeptide in contact with the crosslinking agent (according to any of the embodiments described herein) is at least 200:1. In some such embodiments, the molar ratio is 200:1 to 10,000:1. In some such embodiments, the molar ratio is 200:1 to 5,000:1. In some embodiments, the molar ratio is 200:1 to 2,000:1. In some embodiments, the molar ratio is 200:1 to 1,000:1. Exemplary ratios include 200:1 and 1,000:1.

[0172] In any of the several embodiments described herein, the molar ratio of the crosslinking agent (according to any of the embodiments described herein) to the uricase polypeptide in contact with the crosslinking agent (according to any of the embodiments described herein) is at least 500:1. In some such embodiments, the molar ratio is 500:1 to 10,000:1. In some such embodiments, the molar ratio is 500:1 to 5,000:1. In some embodiments, the molar ratio is 500:1 to 2,000:1.

[0173] In any of the several embodiments described herein, the molar ratio of the crosslinking agent (according to any of the embodiments described herein) to the uricase polypeptide in contact with the crosslinking agent (according to any of the embodiments described herein) is at least 1,000:1. In some such embodiments, the molar ratio is 1,000:1 to 10,000:1. In some such embodiments, the molar ratio is 1,000:1 to 5,000:1.

[0174] The molecular weight of the crosslinking agent may be optionally selected to result in a crosslinked portion having a molecular weight according to any of the embodiments described herein with respect to the molecular weight of the crosslinked portion. The relationship between a given molecular weight of a crosslinking agent and the crosslinked portion produced from this crosslinking agent in the method described herein will be apparent to those skilled in the art. For example, a drug of formula II typically has a portion of formula I (variable L , L2, m, and n have molecular weights that are 30 Da greater than (for example, essentially rounding error for molecular weights of 1 kDa or more) than (as defined similarly).

[0175] According to one aspect of several embodiments of the present invention, a modified uricase that can be obtained by the method described herein, one of each embodiment, is provided.

[0176] Formulations and indications: The modified uricases according to any of the embodiments described herein may optionally be used for the treatment of diseases or disorders in which uricase activity is beneficial, and / or for the treatment of diseases or disorders associated with excessive uric acid levels.

[0177] According to one aspect of several embodiments of the present invention, the use of a modified uricase according to any of the embodiments described herein is provided in the manufacture of a pharmaceutical for treating a disease or disorder in which uricase activity is beneficial.

[0178] According to one aspect of several embodiments of the present invention, the use of a modified uricase according to any of the embodiments described herein is provided in the manufacture of a pharmaceutical for treating a disease or disorder associated with excessive uric acid levels.

[0179] According to one aspect of several embodiments of the present invention, a method is provided for treating a disease or disorder in which uricase activity is beneficial, comprising the step of administering a therapeutically effective amount of modified uricase according to any of the embodiments described herein to a subject requiring treatment.

[0180] According to one aspect of several embodiments of the present invention, a method is provided for treating a disease or disorder associated with excessive uric acid levels, comprising administering a therapeutically effective amount of modified uricase according to any of the embodiments described herein to a subject requiring treatment.

[0181] Examples of conditions that can be treated by some embodiments (as described herein) include, but are not limited to, gout, diabetes mellitus, kidney stones, tumor lysis syndrome, hemorrhagic shock, malaria, allergic inflammation, renal dysfunction, viral infections such as influenza and COVID-19 (e.g., excessive uric acid levels are associated with antiviral drugs such as favipiravir), acute gastroenteritis, placenta, sterile inflammation and other pregnancy complications associated with uric acid (e.g., miscarriage, pre-eclampsia and preterm birth), multiple sclerosis, inflammatory bowel disease, gastrointestinal infections, and Leschneihan syndrome.

[0182] In some optional embodiments described herein, the treatment promotes the dissolution of solid (e.g., crystalline) uric acid in the body, for example, in the treatment of gout, kidney stones, placenta, aseptic inflammation, pregnancy complications, Lesch-Nyhan syndrome and / or tumor lysis syndrome.

[0183] In some optional embodiments described herein, the treatment reduces the inflammatory effect of uric acid, which may optionally be beneficial in the treatment of inflammatory conditions, such as gout, malaria, allergic inflammation, viral infections (e.g., COVID-19), acute gastroenteritis, placenta, aseptic inflammation, pregnancy complications, multiple sclerosis, and inflammatory bowel disease.

[0184] According to one aspect of several embodiments of the present invention, a method is provided for reducing the uric acid level in a medium, comprising the step of contacting the medium with a modified uricase according to any of the embodiments described herein. The medium may optionally be an in vivo or ex vivo physiological medium (e.g., tissue) or a non-physiological medium.

[0185] In some embodiments, the medium is the tissue of a (human or non-human) subject requiring administration, and the method involves administering the modified uricase to the subject. The subject may optionally be suffering from or at risk of suffering from any of the diseases or disorders described herein.

[0186] The modified uricases according to any of the embodiments described herein may optionally be used, either by themselves or by substitute, as part of a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0187] As used herein, “pharmaceutical composition” refers to one or more modified uricase preparations described herein, comprising other chemical components such as suitable carriers and additives that are pharmaceutically acceptable. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to a living organism.

[0188] Hereinafter, the term “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not cause significant irritation to a living organism and does not impair the biological activity and properties of the administered compound. Examples of carriers include, but are not limited to, propylene glycol, physiological saline, emulsions and mixtures of organic solvents and water, as well as solid (e.g., powder) and gaseous carriers.

[0189] In this specification, the term “additive” refers to an inert substance added to a pharmaceutical composition to further facilitate the administration of a compound. Examples of additives include, but are not limited to, calcium carbonate, calcium phosphate, various types of sugars and starches, cellulose derivatives, gelatin, vegetable oils and polymers such as polyethylene glycol.

[0190] Techniques for the prescription and administration of drugs can be found in the latest edition of “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

[0191] The pharmaceutical compositions of the present invention may be manufactured by methods well known in the art, for example, by conventional mixing, dissolving, granulation, sugar-coated tablet manufacturing, levigating, emulsification, encapsulation, sealing, or freeze-drying methods.

[0192] Therefore, pharmaceutical compositions for use according to the present invention may be formulated in the conventional manner using one or more pharmaceutically acceptable carriers, including additives and adjuvants that facilitate the processing of modified uricase into pharmaceutically usable preparations. The appropriate formulation will vary depending on the selected route of administration.

[0193] The modified uricases described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Formulations for injection or infusion may be provided in unit dosage forms, for example in ampoules, or optionally in multi-dose containers with preservatives. The compositions may be suspensions, solutions, or emulsions of oily or aqueous excipients and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants.

[0194] The pharmaceutical composition for parenteral administration comprises an aqueous solution of a modified uricase preparation in a water-soluble form. For injection or infusion, the modified uricase may optionally be formulated in an aqueous solution, preferably a physiologically compatible buffer, such as Hanks' solution, Ringer's solution, or physiological saline buffer, with or without the addition of an organic solvent, such as propylene glycol or polyethylene glycol.

[0195] Furthermore, the suspension of modified uricase may be prepared as a suitable oily injection suspension and emulsion (e.g., water-in-oil, oil-in-water, or water-in-oil emulsion). Suitable lipophilic solvents or excipients include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. The aqueous injection suspension may contain a substance that increases the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain a suitable stabilizer or agent to increase the solubility of the modified uricase and enable the preparation of high-concentration solutions.

[0196] Direct injection and / or infusion into the bloodstream (e.g., intravenous administration) may be particularly suitable for treating hyperuricemia (including any associated conditions) characterized by elevated blood uric acid levels. Administration into the bloodstream may also optionally be used to deliver modified uricase to specific tissues.

[0197] Alternatively or additionally, modified uricase may be injected topically into tissues where uric acid levels are excessive, for example. The tissues may optionally be those associated with uric acid precipitation, such as joints (e.g., in the case of gout) or kidneys (e.g., in the case of kidney stones).

[0198] For transmucosal administration, penetrating agents are used in the formulation. Such penetrating agents are generally known in the art.

[0199] For oral administration, the modified uricase of the present invention may be readily formulated by combining the modified uricase with a pharmaceutically acceptable carrier well known in the art. Such carriers enable the formulation of the modified uricase described herein as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions for oral administration by patients. Pharmaceutical preparations for oral use can be prepared using solid additives, and optionally the resulting mixture may be pulverized, and after adding suitable excipients as needed, the granular mixture may be processed to obtain tablets or sugar-coated tablet cores. Suitable additives include, in particular, fillers such as sugars containing lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, and sodium carboxymethylcellulose; and / or physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or sodium alginate salts thereof may be added.

[0200] The core of the sugar-coated tablet is coated with a suitable coating. For this purpose, a concentrated sugar solution may be used which optionally contains gum arabic, talc, polyvinylpyrrolidone, carbopole gel, polyethylene glycol, titanium dioxide, lacquer solution, and a suitable organic solvent or solvent mixture. For identification or to characterize different combinations of active-modified uricase dosages, dyes or pigments may be added to the tablet or sugar-coated tablet coating.

[0201] Examples of pharmaceutical compositions for oral administration include push-fit capsules made of gelatin, and soft-seal capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain an active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the modified uricase may be dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Further stabilizers may be added. All formulations for oral administration should be administered in a dosage appropriate to the chosen route of administration.

[0202] The modified uricase of the embodiment of the present invention may also be formulated into suppositories or rectal compositions such as retained enemas using conventional suppository bases, such as cocoa butter or other glycerides.

[0203] Oral and / or rectal administration may be particularly suitable for treating diseases or disorders of the gastrointestinal tract, such as conditions associated with inflammation of the gastrointestinal tract (e.g., inflammatory bowel disease and / or gastroenteritis).

[0204] In the case of buccal administration, the composition may take the form of tablets or lozenges prescribed in the conventional manner.

[0205] For administration by inhalation, modified uricase is conveniently delivered from a pressurized pack or nebulizer in the form of an aerosol spray presentation (typically including a powdered, liquefied, and / or gaseous carrier) using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dose unit may be determined by providing a valve for delivering a quantifiable amount. For use in inhalers or insufflators, for example, gelatin capsules and cartridges may be formulated to contain a powder mixture of modified uricase and a suitable powder base, such as but not limited to lactose or starch.

[0206] Alternatively, the modified uricase may be in powder form, prepared before use with a suitable vehicle, such as sterile water free of pyrogens.

[0207] Suitable pharmaceutical compositions for use in the context of the present invention include compositions containing an active ingredient in an amount effective to achieve the intended purpose. More specifically, the therapeutically effective amount means an amount of modified uricase effective to prevent, alleviate or improve the symptoms of a disease, or to prolong the survival of the subject being treated.

[0208] For any modified uricase used in the method of the present invention, the therapeutically effective dose or amount can be initially estimated from an animal activity assay. For example, the IC measured by the activity assay 50 Doses can be formulated in animal models to achieve a circulating concentration range that includes (for example, the concentration of the test protein structure that achieves a half-maximal increase in the biological activity of modified uricase). Using such information, useful doses in humans can be determined more accurately.

[0209] As demonstrated in the Examples section below, the therapeutically effective dose of the modified uricase of the embodiments of the present invention may be in the range of about 1 μg / kg body weight to about 500 mg / kg body weight. In some optional embodiments described herein, the therapeutically effective dose of the modified uricase is about 10 μg / kg body weight to about 2000 μg / kg body weight, and optionally about 25 μg / kg body weight to about 800 μg / kg body weight.

[0210] The toxicity and therapeutic efficacy of the modified uricases described herein are determined by standard pharmaceutical procedures in experimental animals, for example, by EC of the target protein structure. 50 ,I C 50 and LD 50 This can be determined by measuring the lethal dose (which causes death in 50% of test animals). The data obtained from these activity assays and animal studies can be used to formulate a range of dosages for use in humans.

[0211] The dosage may vary depending on the dosage form used and the route of administration. The precise prescription, route of administration, and dosage can be selected by each physician, taking into account the patient's condition. (See, for example, Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1 p.1).

[0212] Dosage and administration intervals can be individually adjusted to provide a plasma level of active uricase sufficient to maintain the desired effect, known as the minimum effective concentration (MEC). While the MEC varies from preparation to preparation, it can be estimated from in vitro data, e.g., the concentration required to achieve the desired level of activity in vitro. The dose required to achieve the MEC varies depending on the individual characteristics and administration route. Plasma concentrations can be measured using HPLC assays or bioassays.

[0213] The dosing interval can also be determined using the MEC value. The formulation should be administered using a regimen that maintains plasma levels above the MEC for 10-90%, preferably 30-90%, and most preferably 50-90%.

[0214] As discussed herein, the modified uricases described herein may exhibit long half-lives in the body. Such properties allow for the use of relatively infrequent administration (which may be particularly advantageous when administration is via inconvenient routes such as injection) and / or relatively low-dose administration (which may be particularly advantageous in reducing toxicity and / or the immune response that may arise against the modified uricase).

[0215] Some conditions treatable by uricase activity may not require a continuous and long-term minimum effective concentration of modified uricase. Therefore, modified uricase may be administered at a frequency that is not sufficient to continuously provide the minimum effective concentration. For example, a condition characterized by uric acid precipitation may optionally be treated by administering a sufficient amount of modified uricase to promote the partial or complete dissolution of the precipitated uric acid in the body, followed by an interval during which uricase activity is not required, for example, until sufficient time has elapsed for clinically significant levels of uric acid to precipitate again.

[0216] In some of the embodiments described herein, administration (e.g., by injection) is performed at least once a week (i.e., the treatment comprises multiple administrations separated by at least one week intervals), and optionally at intervals of up to six or twelve months (one year). In some such embodiments, the interval is at least two weeks. In some embodiments, the interval is at least one month (e.g., one to twelve months, or one to six months, or one to two months, optionally within the range of one or two months). In some embodiments, the interval is at least two months (e.g., within the range of two to twelve months, or two to six months). In some embodiments, the interval is at least three months (e.g., within the range of three to twelve months, or three to six months).

[0217] In some of the embodiments described herein, the frequency of administration and the dose per administration are selected such that the dose of modified uricase (e.g., by injection to an adult subject) is 60 mg / month or less (e.g., a 120 mg dose every three months is considered a 40 mg / month dose). In some such embodiments, the dose is 40 mg / month or less (e.g., 80 mg or less administered approximately every two months). In some such embodiments, the dose is 24 mg / month or less (e.g., 48 mg or less administered approximately every two months). In some such embodiments, the dose is 16 mg / month or less (e.g., 32 mg or less administered approximately every two months). In some such embodiments, the dose is 12 mg / month or less (e.g., 24 mg or less administered approximately every two months). In some such embodiments, the dose is 10 mg / month or less (e.g., 20 mg or less administered approximately every two months). In some embodiments, the dosage is 8 mg / month or less (for example, 16 mg or less administered approximately every two months). In some embodiments, the dosage is 6 mg / month or less (for example, 12 mg or less administered approximately every two months). In some embodiments, the dosage is 4 mg / month or less (for example, 8 mg or less administered approximately every two months). In some embodiments, the dosage is 2 mg / month or less. In some embodiments, the dosage is 1 mg / month or less.

[0218] In some of the embodiments described herein, the frequency and dose per administration are selected such that the dose of modified uricase is 2 mg / kg body weight / month or less. In some such embodiments, the dose is 0.8 mg / kg body weight / month or less (e.g., 1.6 mg / kg body weight or less administered approximately every two months). In some such embodiments, the dose is 0.4 mg / kg body weight / month or less (e.g., 0.8 mg / kg body weight or less administered approximately every two months). In some such embodiments, the dose is 0.2 mg / kg body weight / month or less (e.g., 0.4 mg / kg body weight or less administered approximately every two months). In some such embodiments, the dose is 0.1 mg / kg body weight / month or less (e.g., 0.2 mg or less administered approximately every two months). In some embodiments, the dose is 0.5 mg / kg body weight / month or less. In some embodiments, the dose is 0.25 mg / kg body weight / month or less.

[0219] Depending on the severity and responsiveness of the condition being treated, administration may also optionally be a single dose of the sustained-release composition described herein, and the course of treatment may last for several days to several weeks, or until a cure is achieved or a reduction in the disease state is achieved.

[0220] The amount of the composition administered will naturally vary depending on the patient receiving treatment, the degree of pain, the method of administration, and the judgment of the prescribing physician.

[0221] The compositions of the present invention may, if necessary, be provided in packs or dispenser devices such as FDA-approved kits, which may contain one or more unit dosage forms containing the active ingredient. The packs may include, for example, metal or plastic foil such as blister packs or pressurized containers (for inhalation). The packs or dispenser devices may be accompanied by instructions for administration. The packs or dispensers may also be accompanied by notices relating to the form of container prescribed by government agencies that regulate the manufacture, use or sale of pharmaceuticals, which reflect the agency's approval of the form of the composition for human or veterinary administration. Such notices may be, for example, labels approved by the FDA for prescription drugs or product inserts. Compositions comprising any modified uricase of any embodiment of the present invention, formulated on a suitable pharmaceutical carrier, may also be prepared, placed in appropriate containers, and labeled for the treatment or diagnosis of the indicated conditions, as detailed herein.

[0222] Accordingly, according to one embodiment of the present invention, the pharmaceutical composition described herein is packaged in packaging material for use in the treatment of a condition in which the activity of the modified uricase is beneficial, as described herein, and is identified by printing in or on the packaging material.

[0223] Additional definitions: In this specification, the terms “hydrocarbon” and “hydrocarbon moiety” refer to organic moieties that include a carbon chain primarily substituted with hydrogen atoms as their basic structure. Hydrocarbons may be saturated or unsaturated and may consist of aliphatic, alicyclic, or aromatic moieties, which may optionally be substituted with one or more substituents (other than hydrogen). Substitutive hydrocarbons may have one or more substituents, each substituent independently of, for example, cycloalkyl, alkenyl, alkynyl, aryl, heteroaryl, heteroalicyclic, amine, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino. Hydrocarbons may be terminal groups or linking groups, and these terms are as defined herein. Preferably, the hydrocarbon moiety has 1 to 20 carbon atoms. In this specification, where a numerical range, for example "1 to 20," is described, this means that the group may contain 20 or fewer carbon atoms, in this case the hydrocarbon may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, and so on. Optionally, the hydrocarbon may be a medium-sized hydrocarbon having 1 to 10 carbon atoms. Optionally, the hydrocarbon may have 1 to 4 carbon atoms.

[0224] In this specification, the term "linking group" refers to a group (e.g., a substituent) that is bonded to two or more parts of a compound, while the term "terminal group" refers to a group (e.g., a substituent) that is bonded to a single part of a compound via one atom.

[0225] As used throughout this specification, the term “alkyl” refers to any saturated aliphatic hydrocarbon, including linear and branched groups. Preferably, alkyl groups have 1 to 20 carbon atoms. More preferably, alkyl groups are of medium size, having 1 to 10 carbon atoms. Most preferably, unless otherwise indicated, alkyl groups are lower alkyl groups, having 1 to 4 carbon atoms. Alkyl groups may or may not be substituted.

[0226] If substituted, substituents may be, for example, cycloalkyl, aryl, heteroaryl, heteroalicyclic groups, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, which are defined herein.

[0227] In this specification, the term "alkenyl" refers to an unsaturated aliphatic hydrocarbon containing at least one carbon-carbon double bond, including linear and branched groups. Preferably, the alkenyl group has 2 to 20 carbon atoms. More preferably, the alkenyl is a medium-sized alkenyl having 2 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkenyl is a lower alkenyl having 2 to 4 carbon atoms. The alkenyl group may or may not be substituted.

[0228] A substituted alkenyl may have one or more substituents, each substituent independently of, for example, an alkynyl, cycloalkyl, alkynyl, aryl, heteroaryl, heteroalicyclic group, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino.

[0229] In this specification, the term "alkynyl" refers to an unsaturated aliphatic hydrocarbon containing at least one carbon-carbon triple bond, including linear and branched groups. Preferably, the alkynyl group has 2 to 20 carbon atoms. More preferably, the alkynyl is a medium-sized alkynyl having 2 to 10 carbon atoms. Most preferably, unless otherwise indicated, the alkynyl is a lower alkynyl having 2 to 4 carbon atoms. The alkynyl group may or may not be substituted.

[0230] A substituted alkynyl may have one or more substituents, each substituent independently of, for example, a cycloalkyl, alkenyl, aryl, heteroaryl, heteroalicyclic group, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, or amino.

[0231] The term "alkylene" refers to a saturated or unsaturated aliphatic hydrocarbon linking group as defined herein, and differs from alkyl groups (in the case of saturation) or alkenyl or alkynyl groups (in the case of unsaturation) as defined herein, except that alkylene is a linking group and not a terminal group.

[0232] A "cycloalkyl" group refers to a saturated group on an unsaturated all-carbon monocyclic or fused ring (i.e., a ring sharing adjacent pairs of carbon atoms) in which one or more rings do not have a fully conjugated pi-electron system. Examples of cycloalkyl groups, but not limited to, include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexadiene, cycloheptane, cycloheptatriene, and adamantane. Cycloalkyl groups may or may not be substituted. If substituted, substituents may be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as defined herein. If the cycloalkyl group is unsaturated, the cycloalkyl group may contain at least one carbon-carbon double bond and / or at least one carbon-carbon triple bond. A cycloalkyl group may be a terminal group bonded to a single adjacent atom, as defined herein, or a linking group connecting two or more parts, as defined herein.

[0233] The "aryl" group refers to an all-carbon monocyclic or fused-ring polycyclic (i.e., a ring sharing adjacent carbon atom pairs) terminal group having a fully conjugated pi-electron system. Examples of aryl groups include, but are not limited to, phenyl, naphthalenyl, and anthracenyl. The aryl group may or may not be substituted. If substituted, substituents may be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as defined herein.

[0234] A "heteroaryl" group refers to a monocyclic or fused ring (i.e., a ring sharing adjacent pairs of atoms) terminal group having one or more atoms in the ring, such as nitrogen, oxygen, and sulfur, and further having a fully conjugated pi-electron system. Examples of heteroaryl groups, but not limited to, include pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine, quinoline, isoquinoline, and purine. Heteroaryl groups may or may not be substituted. If substituted, substituents may be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as defined herein.

[0235] The term "arirene" refers to a monocyclic or fused-polycyclic linking group as defined herein, and includes linking groups that differ from aryl or heteroaryl groups only in that arirenes are linking groups and not terminal groups, as defined herein.

[0236] A "heteroalicyclic" group refers to a monocyclic or fused cyclic group having one or more atoms, such as nitrogen, oxygen, and sulfur, within its ring. The ring may also have one or more double bonds. However, the ring does not have a fully conjugated π-electron system. Heteroalicyclic groups may or may not be substituted. If substituted, substituents may be, for example, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, halo, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, sulfinyl, sulfonyl, sulfonate, sulfate, cyano, nitro, azide, phosphonyl, phosphinyl, oxo, carbonyl, thiocarbonyl, urea group, thiourea group, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, S-thiocarbamyl, C-amide, N-amide, C-carboxy, O-carboxy, sulfonamide, guanyl, guanidinyl, hydrazine, hydrazide, thiohydrazide, and amino, as defined herein. Typical examples include piperidine, piperazine, tetrahydrofuran, tetrahydropyran, and morpholine. A heteroalicyclic group may be a terminal group bonded to a single adjacent atom as defined herein, or a linking group connecting two or more parts as defined herein.

[0237] In this specification, the terms "amine" and "amino" refer to the -NR'R'' group or -N, respectively. +Refers to any of the R’R’’R’’’ groups, where R’, R’’, and R’’’ are each, as defined herein, hydrogen or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heteroalicyclic (bonded to the amine nitrogen through a ring carbon), aryl or heteroaryl (bonded to the amine nitrogen through a ring carbon). Optionally, R’, R’’, and R’’’ are hydrogen or alkyl containing 1 to 4 carbon atoms. Optionally, R’ and R’’ (and R’’’ if present) are hydrogen. When substituted, the carbon atoms of the R’, R’’ or R’’’ hydrocarbon moiety bonded to the nitrogen atom of the amine are not substituted with oxo (unless otherwise specified), and R’, R’’, and R’’’ are not, for example, carbonyl, C-carboxy or amide as these groups are defined herein.

[0238] The “azide” group is -N=N + =N - group.

[0239] The “alkoxy” group refers to both -O-alkyl groups and -O-cycloalkyl groups as defined herein.

[0240] The “aryloxy” group refers to both -O-aryl groups and -O-heteroaryl groups as defined herein.

[0241] The “hydroxy” group refers to the -OH group.

[0242] The “thiohydroxy” or “thiol” group refers to the -SH group.

[0243] The “thioalkoxy” group refers to both -S-alkyl groups and -S-cycloalkyl groups as defined herein.

[0244] The “thioaryloxy” group refers to both -S-aryl groups and -S-heteroaryl groups as defined herein.

[0245] The "carbonyl" group refers to a -C(=O)-R' terminal group (where R' is defined as above), or a -C(=O)- linking group.

[0246] The "aldehyde" group refers to a -C(=O)H group.

[0247] The "thiocarbonyl" group refers to a -C(=S)-R' group, where R' is as defined herein.

[0248] "Carboxyl", "carboxy" or "carboxylate" refers to both the "C-carboxy" group and the "O-carboxy" group as defined herein.

[0249] The "C-carboxy" group refers to a -C(=O)-O-R' group, where R' is as defined herein.

[0250] The "O-carboxy" group refers to a R'C(=O)-O group, where R' is as defined herein.

[0251] The "oxo" group refers to a =O group.

[0252] The "halo" group refers to fluorine, chlorine, bromine or iodine.

[0253] The "sulfinyl" group refers to a -S(=O)-R' group, where R' is as defined herein.

[0254] The "sulfonyl" group refers to a -S(=O)2-R' group, where R' is as defined herein.

[0255] The "sulfonate" group refers to a -S(=O)2-O-R' group, where R' is as defined herein.

[0256] The "sulfate" group refers to a -O-S(=O)2-O-R' group, where R' is as defined herein.

[0257] The "sulfonamide" or "sulfonamido" group encompasses both the S-sulfonamide group and the N-sulfonamide group as defined herein.

[0258] The "S-sulfonamide" group refers to the -S(=O)2-NR'R'' group, where R' and R'' are defined herein.

[0259] The "N-sulfonamide" group refers to the R'S(=O)2-NR'' group, where R' and R'' are defined herein.

[0260] The "O-carbamyl" group refers to the -OC(=O)-NR'R'' group, where R' and R'' are defined herein.

[0261] The "N-carbamyl" group refers to the R'OC(=O)-NR''- group, where R' and R'' are defined as herein.

[0262] The "O-thiocarbamyl" group refers to the -OC(=S)-NR'R'' group, where R' and R'' are defined as herein.

[0263] The "N-thiocarbamyl" group refers to the R'OC(=S)NR''- group, where R' and R'' are defined as herein.

[0264] The "S-thiocarbamyl" group refers to the -SC(=O)-NR'R'' group, where R' and R'' are defined as herein.

[0265] The "amide" or "amido" group includes the C-amide and N-amide groups as defined herein.

[0266] The "C-amide" group refers to the -C(=O)-NR'R'' group, where R' and R'' are defined as herein.

[0267] The "N-amide" group refers to the R'C(=O)-NR''- group, where each of R' and R'' is as defined herein.

[0268] The "urea group" refers to the -N(R')-C(=O)-NR''R''' group, where each of R', R'', and R''' is as defined herein.

[0269] The "thiourea group" refers to the -N(R')-C(=S)-NR''R''' group, where each of R', R'', and R''' is as defined herein.

[0270] The "nitro" group refers to the -NO2 group.

[0271] The "cyano" group refers to the -C≡N group.

[0272] The term "phosphonyl" or "phosphonate" represents the -P(=O)(OR')(OR'') group, where R' and R'' are as defined above herein.

[0273] The term "phosphate" represents the -O-P(=O)(OR')(OR'') group, where each of R' and R'' is as defined above herein.

[0274] The term "phosphinyl" represents the -PR'R'' group, where each of R' and R'' is as defined above herein.

[0275] The term "hydrazine" represents the -NR'-NR''R''' group, where R', R'', and R''' are as defined herein.

[0276] As used herein, the term "hydrazide" represents the -C(=O)-NR'-NR''R''' group, where R', R'', and R''' are as defined herein.

[0277] As used herein, the term "thiohydrazide" refers to the -C(=S)-NR'-NR''R'' group, where R', R'' and R''' are as defined herein.

[0278] The "guanidinyl" group refers to the -RaNC(=NRd)-NRbRc group, where Ra, Rb, Rc, and Rd may be as defined herein for R' and R''.

[0279] The "guanyle" or "guanine" group refers to the RaRbNC(=NRd)- group, where Ra, Rb, and Rd are as defined herein.

[0280] "Imine" refers to the -C(=NR'')-R' group, where R' and R'' are as defined above in this specification.

[0281] "Hemiaminal" is a -C(R')(OH)-NR''R''' group, where R', R'' and R''' are as defined herein.

[0282] As used herein, the term "about" refers to ±10% or ±5%.

[0283] The terms "comprises," "comprising," "includes," "including," and "having," as well as their conjugations, all mean "including but not limited to."

[0284] The term "consisting of" means "to include or be limited to."

[0285] The term "consisting essentially of" means that the composition, method, or structure may include additional components, steps, and / or parts, provided that such additional components, steps, and / or parts do not substantially alter the fundamental and novel characteristics of the composition, method, or structure described in the claims.

[0286] As used herein, the singular pronouns "a," "an," and "the" also refer to the plural unless the context clearly indicates otherwise. For example, "compound" or "at least one compound" may include multiple compounds, and may also include mixtures thereof.

[0287] Throughout this application, various embodiments of the invention may be described in range form. It should be understood that the use of range form is solely for convenience and brevity and should not be interpreted as a limitation that restricts the flexibility of the scope of the invention. Therefore, range descriptions should be considered to specifically disclose all possible subranges and the individual numerical values ​​within those ranges. For example, a range description such as 1-6 should be considered to specifically disclose not only subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, and 3-6, but also the individual numerical values ​​within those ranges, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the size of the range.

[0288] Wherever a numerical range is indicated in this specification, it is intended to include any number (fraction or integer) within the indicated range. The phrases "range between" the first indicator and the second indicator, and "range from" the first indicator to the second indicator, are used interchangeably in this specification and are intended to include the first indicator and the second indicator, and all fractions and integers between the first indicator and the second indicator.

[0289] As used herein, the term “method” means a form, means, technique and procedure for achieving a given task, including, but not limited to, those known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine, or those readily available to practitioners from known forms, means, techniques and procedures.

[0290] It should be understood that certain features of the present invention described in relation to separate embodiments for clarity may also be provided in combination in a single embodiment. Conversely, several features of the present invention described in relation to a single embodiment for brevity may also be provided separately, in any preferred partial combination, or as appropriate, for any other described embodiment of the present invention. Certain features described in relation to various embodiments should not be considered essential features of the embodiment unless the embodiment is inoperable without that element.

[0291] The various embodiments and aspects of the present invention described herein and claimed in the claims are experimentally supported in the following examples. [Examples]

[0292] Examples are provided below. These examples illustrate, in a non-limiting manner, several embodiments of the present invention in conjunction with the above description.

[0293] Materials and methods material: The chimeric cHu3.3 human anti-PEGIgG1 antibody was obtained from Academia Sinica (Taiwan).

[0294] 2-Picolinborane and sodium borocyanohydride (NaBH3CN) were obtained from Sigma Aldrich.

[0295] Monofunctional polyethylene glycol nitrophenyl carbonate (mPEG(10K)-NPC) was obtained from Creative PEGWorks.

[0296] The polyethylene glycol bisaldehyde (bis-Ald-PEG) reagent was obtained from Creative PEGWorks.

[0297] Polyethylene glycol bis-N-hydroxysuccinimide (2kDa) (bis-NHS-PEG2000) was obtained from Iris Biotech GmbH.

[0298] Vector development for expressing uricase variants in BY2 cells: For the transformation and expression of different uricase sequences in BY2 cells, an expression system based on the geminivirus bean yellow dwarf virus (BeYDV) replicon [Chen et al., Hum Vaccin 2011, 7:331-338, Mor et al., Biotechnol Bioeng 2003, 81:430-437] was used.

[0299] BY2 cell transformation, uricase expression, and isolation of selected strains: Genetic transformation of BY2 cells using the molecular constructs described above was performed using an Agrobacterium-mediated transformation procedure as described in An et al. [EMBO J 1985,4:277-284]. Transformed cells were selected using kanamycin as a selector. Once a viable kanamycin-resistant cell suspension was prepared, individual cell lines (clones) were isolated and screened using this suspension. Individual cell lines were established by spreading a highly diluted, fixed volume of the transgenic cell suspension onto solid medium. Cells were grown until small calluses (plant cell aggregates) developed. Each callus, representing a single clone, was then resuspended in liquid medium and sampled. Individual transformed cell lines were isolated and screened for uricase expression levels. The line showing the highest expression level was selected for further process development.

[0300] Plant cell suspension: N. tabacum cv. BY2 cells were cultured at 25°C in liquid MS-BY2 medium [Nagata & Kumagai, Methods Cell Sci 1999, 21:123-127] while being constantly stirred (85 rpm) on an orbital shaker. This suspension was grown in 50 mL volumes in a 250 mL Erlenmeyer flask and subcultured weekly at a concentration of 2.5% (v / v).

[0301] Preparation of "pegroticase-like" PEGylated uricase: To use as a "pegroticase-like" control, prU-C250K uricase (SEQ ID NO: 2) was diluted to 1 mg / mL in 100 mM phosphate buffer (pH 8), and 1000 molar equivalents of monofunctional polyethylene glycol nitrophenyl carbonate were added to prepare monofunctional PEG-modified uricase. The reaction was allowed to proceed at room temperature for 2 hours. Purification was performed by 3 cycles of dialysis to 100 mM phosphate buffer (pH 8) using an Amicon® system with a 100 K cutoff (14000 G, 4 min).

[0302] Evaluation of antibody levels by ELISA: A MaxiSorp® 96-well microtiter plate was coated with a 5 μg / mL uricase sample in phosphate-buffered saline, incubated overnight at 4°C, washed, and blocked with 2% bovine serum albumin at room temperature for 2 hours. The plate was then washed to remove unbound proteins, and 100 μL of serum was added. After further incubation at room temperature for 2 hours with shaking at 600 rpm, unbound compounds were washed away, and mouse anti-human IgG alkaline phosphatase was added to each well at a 1:5000 dilution. The mixture was incubated at room temperature for 1.5 hours with shaking at 600 rpm. After the final washing step, BluePhos® phosphatase substrate was added, and the reaction was stopped using alkaline phosphatase stop solution. Final absorbance was measured at 630 nm using a microplate reader (Tecan).

[0303] MALDI-TOF mass spectrometry: Sample preparation: A matrix solution was prepared by mixing 375 μL of ethanol solution of 20 mg / mL 2,5-DHAP (2,5-dihydroxyacetophenone) with 125 μL of aqueous solution of 18 mg / mL DAC (diammonium hydrogen citrate). 2 μL of the sample solution was mixed with 2 μL of 2% trifluoroacetic acid solution, and then 2 μL of the matrix solution was added. When the resulting three-component mixture was pipetted up and down, the mixture, which was initially transparent, became opaque as crystallization began. 0.5 μL of this mixture was spread onto a MALDI steel target plate. After the solvent evaporated, the target plate was inserted into a mass spectrometer.

[0304] Mass Spectrometry: MALDI-TOF mass spectra were acquired using a MALDI-TOF / TOF Autolex® kinetic mass spectrometer (Bruker Daltonik GmbH). The mass spectrometer was equipped with a SmartBeam® II solid-state laser (modified Nd:YAG laser; λ=355nm), and cations were detected (operated) in linear mode within the range of 20000–200000 m / z or 60000–200000 m / z. Laser fluence was optimized for each sample. The laser was operated at a frequency of 2 kHz, and spectra were accumulated in multiples of 1000 laser shots, totaling 2000 shots.

[0305] Size exclusion chromatography (SEC): Size exclusion chromatography was performed using the Dionex® UltiMate® 3000 HPLC system.

[0306] The unmodified uricase was analyzed by measuring absorbance at 214 nm using a Superose® 12 10 / 300 GL column with a 50 mM borate buffer (pH 8) containing 100 mM NaCl at a flow rate of 0.4 mL / min.

[0307] Cross-linked uricase was analyzed by connecting two TSKgel® G5000PWXL, 7.8 × 300 mm columns in series, using a column temperature of 50°C, and measuring absorbance at 214 nm with a flow rate of 0.3 mL / min of 50 mM Tris buffer (pH 8.0) containing 100 mM NaCl.

[0308] Uricase activity assay: The specific activity of uricase was measured by an indirect fluorescence quantitative assay that detects H2O2 byproducts released after uric acid oxidation by uricase. Specifically, 400 μM uric acid was dissolved and added to 0.1 M sodium phosphate buffer (pH 7.4) with 0.1% BSA added, containing a sample with an unknown uricase concentration. At 37°C in the presence of horseradish peroxidase, a fluorescent probe (Ampliflu®) was reacted with H2O2 in a 1:1 stoichiometric ratio to obtain a highly fluorescent product with excitation wavelengths of 530–560 nm and emission wavelengths of 590 nm. The increase in fluorescence was recorded for 10 minutes using a microplate reader, and the sample was quantified according to the standard curve for uricase.

[0309] To perform the analysis using the Michaelis-Menten equation, the catalytic rate was measured as described above for increasing enzyme and uric acid concentrations (uric acid concentrations ranging from 1.56 μM to 200 μM) at a rate of 60 ng / mL. Kinetic parameters were calculated from substrate (UA) concentration versus reaction rate (V) plots using GraFit software (Erithacus Software Limited, 2010). One unit (U) of uricase activity was defined as the amount of enzyme required to convert 1 μmol of uric acid per minute to allantoin at 37°C and pH 8.0.

[0310] Optical density (OD): The purified proteins were quantified using a NanoDrop® 2000 spectrophotometer (Thermo Fisher Scientific Inc.) to determine the absorbance at 280 nm and the respective extinction coefficients (cm²). -1 (grams / liter) -1 ) was done based on this.

[0311] Example 1 In silico comparison of immunogenicity of uricase amino acid sequences To develop low-immunogenic uricases, the immunogenicity of 46 uricase sequences was estimated by in silico analysis.

[0312] Using the ProPred MHC class II binding peptide prediction server, we predicted MHC class II binding regions in sequences using a quantitative matrix, following the procedure described in [Singh & Raghava, Bioinformatics 2010, 17:1236-1237]. We identified the 9 most abundant human alleles (DRB1) that cover over 90% of the population, specifically focusing on MHC class II 9-mer peptide epitopes. * The following were determined: 0101, 0103, 0401, 0701, 0801, 1101, 1301, and 1015. Various 9-mer peptides were identified and scored based on deviations from the consensus binding sequence at a 5% threshold.

[0313] Peptides showing more than 15% similarity to the consensus sequence and predicted to bind to more than three MHC class II alleles were considered immunogenic.

[0314] The main strains are Agrobacterium tumefaciens, Alicyclobacillus mali, Arthrobacter gangotriensis, Arthrobacter globiformis, and Aspergillus flavus, Aspergillus udagawae, Aureobasidium pullulans EXF-150, Bacillus fastidiosus, Bacillus halodurans C-125, Bacillus subtilis str beveridgei, Bactrocera latifrons(Bacchus), Blastomyces dermatitidis, Camelus ferus (Camelus ferus), Candida utillis, Candidatus Solibacter usitatus, Chlamydomonas reinhardtii, Cicer arietinum (Cicer arietinum), Deinococcus radiodurans, Deinococcus geothermalis, Drechmeria coniospora, Erinaceus europaeus (Escherichia coli ISC56), Galdieria sulphuraria, Glycine max (Shadow), Granulicella tundricola, Kyrpidia tusciae DSM 2912, Magnaporthiopsis poae、Microbacterium sp.The specimens were from zzj4-1, Neonectria ditissima, Nicotiana tabacum (tobacco), Paenibacillus darwinianus, Paenibacillus odorifer, Phaseolus vulgaris (bean), Philocephala scopiformis, Pseudomonas aeruginosa, Pygoscelis adeliae (Adélie penguin), Rousettus aegyptiacus (Egyptian flying fox), Stomoxys calcitrans (barn fly), Terriglobus saanensis, Tolypocladium ophioglossoides, and Tolypocladium ophioglossoides CBS 100239.

[0315] The final candidates were selected based on (i) the number of lysine residues, (ii) the number of predicted immunogenic epitopes, and (iii) the score of the predicted immunogenic epitopes. The following uricases were selected for expression as recombinant uricases (PRu) in Nicotiana tabacum BY2 cells. Candida utilis uricase (prU-C) (SEQ ID NO: 1, Accession No. P78609): (Eight T cell epitopes were predicted with a maximum similarity score of 54% to the consensus binding sequence. The amino acid sequence contains 32 Lys residues available for protein modification), and Arthrobacter gangotriensis uricase (prU-G) (SEQ ID NO: 3, Accession No. EMR00187.1): (Five T cell epitopes are predicted, with a maximum similarity score of 42% to the consensus binding sequence. The amino acid sequence contains 12 Lys residues available for protein modification).

[0316] Uricases prU-G and prU-C were calculated to have significantly lower immunogenicity than the sequences of two clinically approved recombinant uricases, rasburicase and pegroticase. In particular, the uricase from Aspergillus flavus (prU-A) (SEQ ID NO: 4, accession number DB00049) had a maximum similarity score of 68% to the consensus binding sequence and was predicted to have 11 T cell epitopes (and 25 Lys residues available for protein modification), while the pig-baboon chimeric uricase used for pegroticase also had a maximum similarity score of 68% to the consensus binding sequence and was predicted to have 19 T cell epitopes (and 30 Lys residues available for protein modification).

[0317] For reference, Aspergillus flavus uricase (prU-A) (SEQ ID NO: 4) was also expressed in Nicotiana tabacum BY2 cells.

[0318] prU-C (SEQ ID NO: 1) contains peroxisomal targeting signal 1 (PTS1) as a tripeptide (TKL) at its C-terminus [Brocard & Hartig, Biochim Biophys Acta 2006, 1763:1565-1573] and was expressed in peroxisomes. prU-A was also expressed in peroxisomes, but prU-G was expressed in the cytoplasm.

[0319] Example 2 Effect of the C250K mutation on uricase Recombinant Candida utilis uricase (prU-C, SEQ ID NO: 1), prepared as described in the Materials and Methods section of this specification, was observed to undergo polymerization under conventional conditions. Further observation showed that dithiothreitol (DTT) inhibited the polymerization of prU-C, suggesting that polymerization is involved in the formation of disulfide bonds between cysteine ​​residues in multiple uricase molecules.

[0320] Cys250 of prU-C is one of the four Cys residues contained in prU-C and has been reported not to be essential for prU-C activity. Furthermore, comparison with the publicly available structures of other uricases (not listed) suggests that Cys250 of prU-C faces outward, and that such orientation may promote the formation of intermolecular disulfide bonds.

[0321] Considering the above, the immunogenicity of the prU-C C250K mutant (prU-C250K; SEQ ID NO: 2) was analyzed and expressed in Nicotiana tabacum BY2 cells as described above. With a maximum similarity score of 52% to the consensus binding sequence, 10 T cell epitopes were predicted. Similar to prU-C (SEQ ID NO: 1), prU-C250K (SEQ ID NO: 2) contains peroxisome target signaling molecule 1 (PTS1) as a tripeptide (TKL) at its C-terminus [Brocard & Hartig, Biochim Biophys Acta 2006, 1763:1565-1573] and was expressed in peroxisomes.

[0322] Next, the effect of the C250K point mutation on the storage stability of prU-C was evaluated before or after subjecting the protein to a freeze / thaw cycle in which it was stored overnight at -20°C at a concentration of 0.3 mg / mL in 25 mM Tris (pH 8.4) (determined by measuring optical density).

[0323] prU-C250K was analyzed before and after freezing by measuring its specific activity (quantified by fluorescence activity assay), the formation of high molecular weight (HMW) molecules (measured by size exclusion chromatography under native conditions), and SDS-PAGE under denaturation conditions.

[0324] As shown in Table 3 below, samples of the prU-C250K mutant contained only tetramers under native conditions before or after freezing, while wild-type prU-C contained 8.4% octamers before freezing and 20.2% octamers and 48.4% high molecular weight (HMW) isoforms after freezing (determined by SEC). Furthermore, as shown, the C250K mutation did not significantly affect specific activity.

[0325] Similarly, as shown in Figure 2, under denaturation conditions (in the absence of DTT), only the monomeric form of prU-C250K was observed before and after freezing, while some wild-type prU-C showed variability as molecular species containing multiple subunits such as dimers and tetramers (as determined by SDS-PAGE).

[0326] [Table 3]

[0327] These results indicate that the C250K mutation enhances the structural characteristics of uricase compared to the wild-type sequence, while preserving both activity and tetrameric structure (e.g., not the HMW species).

[0328] Furthermore, although not bound by any particular theory, the additional (33rd) Lys residue in the C250K mutant may further promote protein modification.

[0329] Example 3 Effect of uricase sequence on stability The stability of various uricase variants (prU-A, prU-C, prU-C250K, and prU-G) was compared under physiologically relevant conditions.

[0330] To evaluate thermal stability, uricase was analyzed by nano-differential scanning fluorescence (NFS). Specifically, purified protein samples were diluted with PBS to a final concentration of 0.5 mg / mL, and 10 μL of each sample was loaded into a capillary. After placing the plates in a capillary array, the plates were placed in a NFS differential scanning fluorescence analyzer and gradually heated from 15°C to 95°C at a rate of 1°C / min (excitation power 28%). The onset of the melting point (Tm) (the temperature at which the protein begins to denature) and the Tm (the temperature at which 50% of the protein is denatured) were measured by software as changes in fluorescence induced by changes in the protein's three-dimensional structure.

[0331] As shown in Table 4, prU-C and prU-C250K exhibited similar melting point parameters, but prU-A melted at a significantly lower temperature.

[0332] These results indicate that prU-C and prU-C250K are more stable than prU-A at physiological temperatures, and that the C250K mutation has little to no effect on thermal stability.

[0333] [Table 4]

[0334] To evaluate stability under human plasma conditions, uricase was diluted in human plasma (ex vivo) to a final concentration of 2 μg / mL and incubated at 37°C for 4 weeks. Protein stability was quantified by specific activity assay at the time indicated.

[0335] As shown in Figure 3, the unmodified prU-A, prU-C, prU-C250K, and prU-G proteins each showed a gradual decrease in activity after incubation in human plasma for 4 weeks. prU-A exhibited the greatest stability, while prU-G showed the lowest stability in human plasma. The similarly significant stability of prU-C and prU-C250K in the physiological matrix suggests that the C250K mutation has little to no effect on plasma stability.

[0336] Example 4 Effect of PEG crosslinking on uricase To crosslink uricase using polyethylene glycol bisaldehyde (bis-Ald-PEG), bisaldehyde PEG of various sizes (1 kDa to 10 kDa), up to 1000 moles per mole of uricase, was added to a solution of each mole of uricase in phosphate buffer (pH 8). A reducing agent was added to the resulting solution at a final concentration of 25 to 100 mM. The coupling reaction was allowed to proceed overnight at room temperature (approximately 23°C), for example, for at least 10 hours. Free PEG was then removed from the reaction mixture by chromatography and / or ultrafiltration. The effectiveness of the crosslinking was evaluated by SDS-PAGE, and enzyme activity was measured according to the standard curve for unmodified uricase. The ratio of active protein to total protein (measured by optical density (OD) at 280 nm) is expressed as the percentage of activity retained after the reaction.

[0337] Using the general procedure described above, the following experiments were conducted with various uricase variants crosslinked with bis-Ald-PEG of various sizes.

[0338] prU-C was crosslinked with 1000 molar equivalents (relative to total protein tetramer) of bis-Ald-PEG in the presence of 25 mM 2-picolinborane as a reducing agent. The PEG molecular weights were 1000Da, 2000Da, 5000Da, and 10,000Da.

[0339] As shown in Table 5 below, cross-linked prU-C retained much of the enzymatic activity of natural prU-C.

[0340] [Table 5]

[0341] As shown in Figure 4, natural prU-C showed a molecular weight of approximately 34 kDa in SDS-PAGE, corresponding to the molecular weight of a protein monomer (i.e., one subunit or a tetramer of approximately 136 kDa). Smaller bands correspond to dimers. In contrast, prU-C crosslinked with 2 kDa PEG showed a main band corresponding to approximately 315 kDa, with no bands corresponding to significantly lower molecular weights.

[0342] Since PEG molecules move with the mobility of a protein twice their molecular weight, the addition of 90 kDa PEG appears as a 180 kDa protein. Therefore, the main band of approximately 315 kDa corresponds to a fully crosslinked prU-C tetramer (approximately 136 kDa) modified with approximately 45 molecules of 2 kDa PEG. Furthermore, the absence of bands corresponding to molecular weights lower than the tetramer indicates efficient covalent crosslinking with no uncrosslinked monomers remaining.

[0343] As further shown in Figure 4, prU-C crosslinked with 1 kDa PEG was associated not only with a strong band corresponding to approximately 42 kDa, which represents the PEGylated monomer, but also with bands of approximately 80 kDa, 120 kDa, and 160 kDa, respectively, which represent the PEGylated dimer, trimer, and tetramer.

[0344] As further shown in Figure 4, (since PEG molecules move with the mobility of proteins twice their molecular weight,) prU-C crosslinked with 5 kDa PEG was associated with multiple bands having increments corresponding to 10 kDa, which match the increment of PEG1 molecule (5 kDa). This also included a faded band at a position slightly less than 55 kDa, which matched a single prU-C monomer modified with a single PEG molecule.

[0345] These results indicate that 1 kDa PEG and 5 kDa (or higher) PEG are considerably less efficient at crosslinking prU-C monomers into tetramers than 2 kDa PEG.

[0346] In another experiment, commercially available uricase A (rasburicase) was crosslinked with 500 molar equivalents of bis-Ald-PEG using 100 mM NaBH3CN as a reducing agent. The molecular weights of the PEG were 600 Da, 1000 Da, 2000 Da, 3400 Da, 5000 Da, and 10,000 Da.

[0347] As shown in Figure 5, rasburicase exhibited a molecular weight of approximately 34 kDa in SDS-PAGE, corresponding to the molecular weight of the protein monomer. Rasburicase reacted with 600 Da PEG showed major bands corresponding to the monomer and dimer (approximately 40 kDa and 72 kDa, respectively), as well as weaker bands corresponding to the trimer and tetramer (approximately 130 kDa and 160 kDa, respectively). Rasburicase crosslinked with 1000 Da PEG showed a major band corresponding to the tetramer, as well as weaker bands corresponding to the monomer, dimer, and trimer. Rasburicase crosslinked with 5000 Da or 10,000 PEG showed smears corresponding to the monomer and various numbers of PEG molecules (as described above with respect to Figure 4). Furthermore, as shown in Figure 5, rasburicase crosslinked with 2000 Da or 3400 Da PEG did not show bands corresponding to smaller molecular species, but rather bands corresponding to PEGylated tetramers.

[0348] These results indicate that crosslinking of rasburicase monomers to tetramers with 2 kDa and 3.4 kDa PEG was efficient, but crosslinking with 1 kDa (or less) or 5 kDa (or more) PEG was not efficient. These results are similar to those obtained with prU-C.

[0349] In another experiment, NaBH3CN was used as a reducing agent to crosslink prU-G with 200 molar equivalents or 1000 molar equivalents of bis-Ald-PEG. The PEG molecular weights were 2000 Da, 3400 Da, and 5000 Da.

[0350] As shown in Table 6 below, prU-G crosslinked with 2000 Da PEG maintained significantly higher activity than prU-G crosslinked with 5000 Da or 10,000 Da PEG, and the decrease in activity correlated with the amount of crosslinking agent used.

[0351] As shown in Figure 6, crosslinking with prU-G yielded relatively high levels of monomeric molecules (bands corresponding to less than 70 kDa on SDS-PAGE) for all tested bis-Ald-PEGs. Furthermore, the reaction with 1000 equivalents of PEG resulted in a higher molecular weight than the reaction with 200 equivalents of PEG. Under all tested conditions, prU-G enzyme activity was significantly reduced.

[0352] These results indicate that partial crosslinking of prU-G is dependent on PEG concentration and is less efficient than crosslinking of prU-A and prU-C.

[0353] As shown in the figure, crosslinking of prU-G with 5000Da PEG was less efficient than crosslinking with 2000Da or 3400Da PEG. This result is consistent with the results obtained for prU-C and prU-A.

[0354] [Table 6]

[0355] In summary, the above results indicate that uricase crosslinking is most efficient with PEG between 1 kDa and 5 kDa, and that crosslinking of several uricase variants (e.g., other than prU-G) under these conditions can produce crosslinked uricase with at least approximately 50% of the enzymatic activity of unmodified uricase.

[0356] Example 5 Effect of crosslinking agent type on modified uricase We compared the crosslinking of uricase using various crosslinking agents.

[0357] prU-A was crosslinked by reacting 1000 equivalents of bis-NHS-PEG (2000Da) in 100 mM phosphate buffer (pH 8) at room temperature for 2 hours, and then dialyzed against 100 mM phosphate buffer (pH 7.4). Furthermore, prU-A was crosslinked again by reacting 1000 equivalents of bis-Ald-PEG (2000Da) as described above. 100 mM NaBH3CN was used as a reducing agent. Protein concentration and enzyme activity were measured according to the procedure described above, and crosslinking efficiency and degree of modification were evaluated using SDS-PAGE according to the procedure described above.

[0358] As shown in Figure 7, both bis-NHS-PEG (2000 kDa) and bis-Ald-PEG (2000 kDa) resulted in efficient crosslinking of prU-A, as measured by SDS-PAGE. Crosslinked prU-A was mainly in the form of tetramers, and no bands corresponding to non-crosslinked molecular species (less than 140 kDa) were observed. As further shown in Figure 7, modifications using bis-NHS-PEG resulted in slightly lower molecular weights compared to modifications using bis-Ald-PEG, and bis-NHS-PEG resulted in fewer PEG molecules bound to prU-A.

[0359] Modified prU-A obtained using both bis-NHS-PEG(2000Da) and bis-Ald-PEG(2000Da) retained 59% and 78% of the initial enzyme activity, respectively, when the ratio of protein activity to total protein content (measured by OD at 280 nm) was measured.

[0360] These results demonstrate that the use of aldehyde functional groups and reducing agents is particularly effective in crosslinking uricase in terms of the number of PEG moieties introduced.

[0361] Example 6 Effect of uricase modification on immunogenicity The relative immunogenicity of prU-A crosslinked with either bis-NHS-PEG or bis-Ald-PEG was tested in animal studies according to the procedure described in Example 5. Samples of modified prU-A were mixed with Imject® alum adjuvant in a 1:1 ratio and subcutaneously injected at a dose of 1 mg (measured by OD) / kg every 3 weeks into 6-8 week old female Sprague Dawley rats (6 rats per group), as shown in Figure 8A. Serum was collected from each rat at the indicated time points, and the titer against the test substance was measured by ELISA (separately for each rat).

[0362] As shown in Figure 8B, immunization with prU-A crosslinked with bis-Ald-PEG(2000Da) resulted in significantly lower antibody titers compared to prU-A crosslinked with bis-NHS-PEG(2000Da). As further shown in Figure 8B, repeated injections generally led to an increase in antibody titers.

[0363] The properties of antibodies formed against cross-linked uricases were investigated using competitive ELISA. Samples were pre-incubated with unmodified uricase or uricase cross-linked with bis-NHS-PEG or bis-Ald-PEG(2000Da), and the ability of competing substances to inhibit the binding of the resulting antibodies was evaluated by ELISA.

[0364] As shown in Figures 9A and 9B, antibodies produced by immunization with bis-NHS-PEG-crosslinked prU-A were not inhibited by unmodified uricase, indicating that the antibodies recognized the PEG portion (Figure 9B). In contrast, antibodies produced by immunization with bis-Ald-PEG-crosslinked prU-A tended to recognize the core protein (Figure 9A).

[0365] In summary, the above results indicate that crosslinking using aldehyde groups is more effective in reducing the immunogenicity of crosslinked uricases than crosslinking using N-hydroxysuccinimide (NHS) groups, and that using NHS groups results in the production of large amounts of antibodies against the linked portion.

[0366] To further evaluate the effects of PEG molecular weight and uricase variants on immunogenicity, prU-A and prU-C were crosslinked with 2000 Da or 3400 Da of bis-Ald-PEG 1000 equivalents, respectively, followed by dialysing with 100 mM phosphate buffer (pH 8), and size exclusion chromatography (according to the procedure described in the Materials and Methods section above) to separate the high molecular weight morphology. The amount of bound PEG moiety was evaluated by MALDI mass spectrometry (according to the procedure described in the Materials and Methods section above) above.

[0367] As shown in Table 7, a slightly larger amount of PEG was incorporated into cross-linked prU-C than into cross-linked prU-A.

[0368] These results are consistent with the higher amount of lysine residues in the prU-C sequence compared to prU-A.

[0369] [Table 7]

[0370] Cross-linked prU-A and prU-C were mixed with Imject® alum adjuvant in a 1:1 ratio and subcutaneously injected at a dose of 1 mg (measured by OD) / kg every 3-4 weeks into female Sprague Dawley rats aged 6-8 weeks (5 rats per group) (using the same timeline as shown in Figure 8A). Serum was collected from each rat at the indicated time points, and the titer was measured by ELISA.

[0371] As shown in Figure 10, prU-C crosslinked with bis-Ald-PEG (3400 Da) resulted in the lowest antibody titer during immunization compared to crosslinked prU-A or prU-C crosslinked with 2000 Da PEG.

[0372] These results indicate that prU-C is somewhat less immunogenic than prU-A, that the methods described herein for reducing immunogenicity can be applied to different types of uricases, and that crosslinking with PEG at approximately 3400 Da is particularly effective in reducing immunogenicity.

[0373] The antigenicity of modified uricases was evaluated in human plasma based on the recognition level by existing antibodies.

[0374] Human serum samples from various naive patients (n=102) were tested for the presence of anti-PEG antibodies using ELISA (as described in the Materials and Methods section above). The assay was performed using two PEGylated variants of prU-C250K (prepared according to the above procedure): prU-C250K crosslinked with bis-Ald-PEG (3400Da) and prU-C250K PEGylated with monofunctional 10kDaPEG (similar to pegroticase), with unmodified prU-C250K used for comparison. A standard curve was constructed using a chimeric cHu3.3 human anti-PEG IgG1 antibody and used as a positive control. The reaction of a positive antibody was defined as an OD ratio of at least 2 relative to the blank, and the results for patients showing a positive reaction are shown in Figure 11.

[0375] As shown in Figure 11, screening of naive human blood samples revealed that 15 out of 102 donors (15%) had pre-existing antibodies that recognized monofunctional 10kDa PEG-modified uricase, while only 3 out of 102 donors (3%) (each included in the aforementioned group of 15 donors) had antibodies that recognized prU-C250K crosslinked with 3400Da PEG, and these antibodies had remarkably low titers. As further shown in Figure 11, 20 out of 102 donors (20%) had antibodies that recognized unmodified prU-C250K, but none of these donors had antibodies against prU-C250K crosslinked with 3400Da PEG.

[0376] These results indicate that PEG crosslinkers efficiently mask uricase proteins, and that when the PEG moiety (a key source of immunogenicity in PEGylated uricases) is crosslinked with bis-Ald-PEG (approximately 3400 Da), the immunogenicity is significantly lower than when modified with 10 kDa monofunctional PEG (similar to pegroticase).

[0377] To evaluate plasma stability, prU-C250K crosslinked with 3400 Da PEG was incubated in human plasma at a concentration of 2 μg / mL at 37°C ex vivo for 4 weeks. Uricase activity was assayed at the time indicated above according to the procedure described.

[0378] As shown in Figure 12, all test batches of prU-C250K crosslinked with 3400 Da PEG maintained full activity in human plasma for 28 days.

[0379] These results demonstrate that the exemplary cross-linked prU-C250K is highly stable in human plasma.

[0380] An example uricase (prU-C250K) was concentrated to 4.4 mg / mL using an Amicon® system (15 mL) with a cutoff of 30 kDa.

[0381] 40 mg of prU-C250K (10 mL) was diluted with 9.86 mL of 100 mM phosphate buffer (pH 8) to form a reaction mixture containing 50 μL of 200 mM DTT water, 1006 mg (1000 molar equivalents) of bis-Ald-PEG (3400Da), and 1 mL of 500 mM 2-picoline borane complex in ethanol. To this mixture, the protein and 25 mM 2-picoline borane complex were added to reach final concentrations of 2 mg / mL and 25 mM respectively. The reaction products were mixed by gentle shaking at room temperature for 17 hours. After the reaction, the samples were injected into a size exclusion chromatography column to remove high molecular weight (HMW) species. The fractions containing less than 5% HMW species were combined, dialyzed with 100 mM phosphate buffer (pH 8), and concentrated to 1.5 mg / mL (measured by OD).

[0382] Both cross-linked prU-C250K and unmodified prU-C250K were sterilized using a 0.22 μm filter, divided into fixed amounts, and stored at -20°C. Concentration and activity were measured as described above. When measured by MALDI mass spectrometry, the number of PEG moieties per uricase tetramer was approximately 37. The proportion of high molecular weight species (modified octamers), as measured by size exclusion chromatography, was approximately 2%.

[0383] The absence of endotoxins (<5 EU / mL) was confirmed using standard procedures, and the degree of masking of protein immunogenicity was measured by competitive ELISA.

[0384] The tested uricase was intravenously injected into 12 female Sprague Dawley rats aged 6-8 weeks at a dose of 10 U / kg (1.35 mg (measured by OD) / kg for cross-linked prU-C250K and 1.09 mg (measured by OD) / kg for unmodified prU-C250K) every two weeks (injections #1-5). Subsequently, as shown in Figure 13A, cross-linked prU-C250K was injected into both groups every four weeks (injection #6). Serum was collected from each group at the indicated time and its titer was measured.

[0385] As shown in Figure 13B, after five injections of unmodified prU-C250K, an increase in the anti-prU-C250K titer was observed in 4 out of 6 test animals.

[0386] In contrast, after six intravenous injections of prU-C250K crosslinked with 3400 Da PEG, the anti-prU-C250K-3400 titer was less than 1:50 in all rats tested (data not shown).

[0387] These results further confirm that cross-linking at the exemplary PEG portion significantly reduces the immunogenicity of uricase.

[0388] Example 7 Effect of uricase crosslinking on pharmacokinetics in repeated administration To evaluate the effect of repeated injections on the pharmacokinetics of cross-linked uricase, prU-C250K cross-linked with bis-Ald-PEG (3400Da) was intravenously injected into rats following the same procedure as described in Example 6 above. The half-life (T1 / 2) and area under the curve (AUC) of cross-linked prU-C250K were determined by either ELISA or an activity assay after the first and sixth injections.

[0389] As shown in Figures 14A to 14D, T after the first injection of cross-linked prU-C250K 1 / 2 The time interval was 54 hours when measured by ELISA and 64.8 hours when measured by activity assay, while the time interval after the 6th injection was 64.8 hours. 1 / 2 The time interval was 70.5 hours when measured by ELISA and 68.4 hours when measured by activity assay.

[0390] As further shown in Figures 14A to 14D, the AUC of cross-linked prU-C250K after the first injection was 61.07 mg when measured by ELISA. * The concentration was min / mL, and when measured by activity assay, it was 65.95 mg. * The concentration was min / mL, while after the 6th injection, the AUC was 70.5 mg when measured by ELISA. * The concentration was min / mL, and when measured by an activity assay, it was 58.5 mg. * The rate was minutes / mL.

[0391] In contrast, the unmodified prU-C250K T 1 / 2 It was less than one hour (data not included).

[0392] These results suggest that cross-linking uricase significantly extends its half-life in plasma, potentially enabling sustained therapeutic effects and administration at relatively infrequent intervals.

[0393] These results further demonstrate that repeated exposure to cross-linked prU-C250K did not shorten the relatively long in vivo half-life of the modified protein, further indicating that the low immunogenicity and sustained therapeutic effect of the modified protein can be maintained even after long-term treatment.

[0394] Example 8 Comparison of exemplary cross-linked uricases and pegroticases As described in the Materials and Methods section above, the enzymatic activity of exemplary cross-linked uricase (prU-C250K cross-linked with bis-Ald-PEG(3400Da)) and pegroticase was compared using uricase assays and Michaelis-Menten analysis.

[0395] As shown in Figure 15 and Table 8, cross-linked uricase has a considerably higher specific activity than pegroticase. cat and V max This was shown.

[0396] [Table 8]

[0397] These results suggest that at high uric acid concentrations (e.g., 200-400 μM, which is expected to be within the clinically appropriate uric acid concentration range), the enzyme reaction rate is k cat (and V max When it is roughly proportional to the K of pegroticase, cross-linked uricase is about 5 times more effective than pegroticase, and at low concentrations close to 30 μM (K of pegroticase) M In this study, cross-linked uricase was more than twice as effective as pegroticase, and the enzyme reaction rate was k cat / K M Even at very low uric acid concentrations, which are almost proportional to the ratio, cross-linked uricases are shown to be slightly more effective than pegroticases.

[0398] The in vivo efficacy of cross-linked uricase and pegroticase was also compared by intravenous injection at a dose of 1 mg / kg into female Sprague Dawley rats. Plasma samples were collected after the first injection (naive state pharmacokinetics) and after four injections repeated every three weeks (repeated injection pharmacokinetics), and the plasma half-life was calculated by measuring the active enzyme concentration at each time point.

[0399] As shown in Figures 16-18, the exemplary cross-linked uricases exhibited a longer plasma half-life than pegroticase both after a single administration (Figures 16 and 18) and after repeated (four) administrations (Figures 17 and 18).

[0400] In summary, the results above indicate that the enzymatic activity of the cross-linked uricase described herein is advantageous compared to the enzymatic activity of pegroticase, both in vitro and in vivo.

[0401] While the present invention has been described in conjunction with its specific embodiments, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, all such alternatives, modifications, and variations are included in the spirit and scope of the appended claims.

[0402] It is the applicant's intention that all publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application were specifically and individually referenced when it is mentioned that they are incorporated herein by reference. In addition, any citation or specification of any reference in this application should not be construed as an admission that such reference is available as prior art of the present invention. To the extent that chapter headings are used, such headings should not necessarily be construed as limiting. Furthermore, any priority documents of this application are incorporated herein by reference in their entirety. [Sequence Listing Free Text]

[0403] Sequence ID 2: Point mutation C250K of Sequence ID 1

Claims

1. A modified uricase comprising a uricase polypeptide crosslinked by at least one difunctional linkage moiety containing a poly(alkylene glycol) moiety, wherein the molecular weight of the difunctional linkage moiety is in the range of 1.5 kDa ± 10% to 4 kDa ± 10%.

2. The modified uricase according to claim 1, wherein the molecular weight of the bifunctional linkage portion is in the range of 2 kDa ± 10% to 3.5 kDa ± 10%.

3. The modified uricase according to claim 1, wherein the uricase polypeptide is bound to at least eight of the bifunctional linkage portions on average.

4. The modified uricase according to claim 1, wherein at least 30% of the lysine residue side chains in the modified uricase are covalently bonded to at least one bifunctional linkage portion.

5. The modified uricase according to claim 1, wherein the bifunctional linkage portion is represented by the following formula I. -CH ２ -L １ -[O-(CH ２ )m]n-O-L ２ -CH ２ - Equation I (In the formula, L １ and L ２ Each of these is either an independent hydrocarbon portion or absent. m is an integer in the range of 2 to 10. n is an integer in the range of 10 to 100.

6. The modified uricase according to claim 5, wherein n is in the range of 30 to 100.

7. The modified uricase according to claim 1, which is in the form of a crosslinked tetramer.

8. The modified uricase according to claim 1, comprising at least one polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and their homologs.

9. The modified uricase according to claim 1, wherein the uricase polypeptide is a recombinant plant polypeptide.

10. A modified uricase comprising a plurality of polypeptides having the amino acid sequence of SEQ ID NO: 2, wherein the polypeptides are crosslinked by at least one bifunctional linkage moiety containing a poly(alkylene glycol) moiety.

11. The modified uricase according to claim 10, wherein each of the polypeptides is bonded to at least eight of the bifunctional linkage portions on average.

12. The modified uricase according to claim 10, wherein at least 30% of the lysine residue side chains in the modified uricase are covalently bonded to at least one bifunctional linkage portion.

13. The modified uricase according to claim 10, wherein the bifunctional linkage portion is represented by formula I. -CH ２ -L １ -[O-(CH ２ )m]n-O-L ２ -CH ２ - Equation I (In the ceremony L １ and L ２ Each of these is either a hydrocarbon portion or absent. m is an integer in the range of 2 to 10. n is an integer in the range of 2 to 1000.

14. The modified uricase according to claim 13, wherein n is in the range of 30 to 100.

15. The modified uricase according to claim 10, wherein the molecular weight of the bifunctional linkage portion is in the range of 1.5 kDa ± 10% to 4 kDa ± 10%.

16. The modified uricase according to claim 10, which is in the form of a tetramer.

17. The modified uricase according to claim 10, wherein the polypeptide is a recombinant plant polypeptide.

18. A modified uricase according to any one of claims 1 to 17, for use in treating diseases or disorders in which uricase activity is beneficial, and / or diseases or disorders associated with excessive uric acid levels.

19. The modified uricase according to claim 18, for treatment of a disease or disorder selected from the group consisting of gout, diabetes mellitus, kidney stones, tumor lysis syndrome, hemorrhagic shock, malaria, allergic inflammation, renal dysfunction, viral infection, acute gastroenteritis, placenta, aseptic inflammation, pregnancy complications, multiple sclerosis, inflammatory bowel disease, gastrointestinal infection, and Lesch-Nyhan syndrome.

20. The modified uricase according to claim 18, wherein the treatment comprises administering the modified uricase at least every one week or at least every two weeks.

21. The modified uricase according to claim 18, wherein the treatment comprises administering the modified uricase at a dose of 8 mg / month or less.

22. A polypeptide having the amino acid sequence of SEQ ID NO:

2.

23. A method for preparing a modified uricase according to any one of claims 1 to 17, (a) A step of contacting a polypeptide with a crosslinking agent containing a poly(alkylene glycol) moiety and at least two aldehyde groups to obtain a conjugate of the polypeptide and the crosslinking agent, and (b) A method comprising the step of bringing the conjugate into contact with a reducing agent.

24. The method according to claim 23, wherein the reducing agent is selected from the group consisting of picoline borane complexes and cyanoboron hydride.

25. A method for reducing the level of uric acid in a medium, comprising the step of contacting the medium with a modified uricase described in any one of claims 1 to 17.

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