Uricase conjugate and its usage

Abstract

The present invention provides a novel uricase conjugate comprising a first domain and a second domain. The first domain comprises a uricase polypeptide, and the second domain is an unstructured random coil polypeptide domain comprising at least about 100 amino acids. Methods for using the uricase conjugate of the present invention in, for example, the treatment of tumor lysis syndrome and / or gout are also provided.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to U.S. Provisional Application No. 63 / 386,660, filed on 8 December 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] (Refer to the electronic sequence listing) The contents of the electronic sequence listing (INMD_186_01WO_SeqList_ST26.xml, size: 184,960 bytes, created: December 7, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0003] In humans, uric acid is produced after the breakdown of purines. Accumulation of uric acid in the blood (hyperuricemia) appears in diseases such as gout and tumor lysis syndrome.

[0004] Gout is a common and complex form of arthritis characterized by sudden, severe attacks of pain, swelling, redness, and tenderness in one or more joints. Gout is caused by the accumulation of uric acid crystals in the joints, leading to excruciating inflammation. Uric acid crystals form when high levels of uric acid are present in the blood.

[0005] Tumor lysis syndrome is a complication from the treatment of cancer, such as lymphoma and leukemia (including non-Hodgkin lymphoma, acute myeloid leukemia, and acute lymphoblastic leukemia). Tumor lysis syndrome occurs when numerous tumor cells lyse and release their contents into the bloodstream. Tumor lysis syndrome is characterized by high blood uric acid levels (hyperuricemia), as well as high blood potassium levels (hyperkalemia), high blood phosphate levels (hyperphosphatemia), low blood calcium levels (hypocalcemia), and blood urea nitrogen (BUN) levels higher than normal. The metabolic abnormalities seen in tumor lysis syndrome can ultimately lead to serious complications such as acute uric nephropathy, acute renal failure, seizures, cardiac arrhythmias, and death.

[0006] Uricase, sometimes called uric acid oxidase, is an enzyme that catalyzes the oxidation of uric acid to allantoin, a more soluble purine metabolite that is more easily excreted. Humans do not produce enzymatically active uricase due to several mutations in the uricase gene acquired during the evolution of higher primates; therefore, exogenously administered uricase provides a therapy for diseases exhibiting hyperuricemia (e.g., gout and tumor lysis syndrome).

[0007] The present invention addresses the need for effective treatment of gout, tumor lysis syndrome, and other diseases associated with hyperuricemia by providing novel uricase conjugates and methods for using them in the treatment of the aforementioned diseases. [Overview of the Initiative]

[0008] In one aspect of the present invention, a uricase conjugate comprising a first domain and a second domain is provided. The first domain comprises a uricase polypeptide or an amino acid variant thereof, and the second domain is a first random coil polypeptide domain comprising at least about 100 amino acids.

[0009] In one embodiment of the uricase conjugate provided herein, the uricase conjugate is a fusion protein of a first domain and a second domain. In a further embodiment, the first domain (uricase polypeptide) is located at the C-terminus of the second domain (first random coil polypeptide).

[0010] In one embodiment of the uricase fusion protein, an amino acid linker, for example, an amino acid linker containing about 2 to about 5 amino acids, is present between the first domain and the second domain. In a further embodiment, the linker is a glycerin-ser.

[0011] In another embodiment, the uricase conjugate is a uricase fusion protein comprising three domains: a uricase peptide domain (first domain), a first random coil polypeptide domain (second domain), and a second random coil polypeptide domain (third domain). The two random coil polypeptide domains may be the same or different. For each of the (first and second) random coil polypeptide domains, at least about 100 amino acids are present within the respective domain.

[0012] In the embodiments described herein, the random coil polypeptide domain comprises about 100 to about 600 amino acids, for example, about 11 to about 300 amino acids.

[0013] In one embodiment, the random coil polypeptide domain comprises a Pro-Ala-Ser(PAS) polypeptide. In a further embodiment, the PAS polypeptide has the amino acid sequence described in SEQ ID NO: 60. In another embodiment, the PAS polypeptide has the amino acid sequence described in SEQ ID NO: 61. In yet another embodiment, the PAS polypeptide has the amino acid sequence described in SEQ ID NO: 62.

[0014] In yet another embodiment, the PAS polypeptide has the amino acid sequence described in SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO: 58. In yet another embodiment, the PAS polypeptide has the amino acid sequence described in SEQ ID NO: 48. In one embodiment, the amino acid sequence of SEQ ID NO: 48 is encoded by a nucleotide sequence selected from SEQ ID NOs: 81 to 111.

[0015] In one embodiment, the random coil polypeptide domain includes an extended recombinant (XTEN) polypeptide. In a further embodiment, the XTEN polypeptide has the amino acid sequence described in SEQ ID NO: 74.

[0016] In one embodiment, the urease domain of the urease conjugate described herein comprises the amino acid sequence set forth in SEQ ID NO: 1.

[0017] In another embodiment, the urease domain of the urease conjugate comprises the amino acid sequence set forth in SEQ ID NO: 2.

[0018] In another embodiment, the urease domain of the urease conjugate comprises the amino acid sequence set forth in SEQ ID NO: 3.

[0019] In yet another embodiment, the urease domain of the urease conjugate comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0020] In still yet another embodiment, the urease domain of the urease conjugate comprises the amino acid sequence set forth in SEQ ID NO: 5.

[0021] In another embodiment, the urease domain of the urease conjugate described herein comprises an amino acid sequence selected from SEQ ID NOs: 6 to 39.

[0022] In another embodiment, the urease domain of the urease conjugate described herein comprises an amino acid sequence selected from SEQ ID NOs: 40 to 44.

[0023] In yet another embodiment, the urease domain of the urease conjugate described herein comprises the amino acid sequence set forth in SEQ ID NO: 40.

[0024] In yet another embodiment, the urease domain of the urease conjugate described herein comprises the amino acid sequence set forth in SEQ ID NO: 41.

[0025] In still yet another embodiment, the urease domain of the urease conjugate described herein comprises the amino acid sequence set forth in SEQ ID NO: 45.

[0026] In yet another embodiment, the urease domain of the urease conjugate described herein comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0027] In another embodiment, the urease domain of the urease conjugate comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0028] In yet another further embodiment, the urease domain is an amino acid variant of SEQ ID NO: 40. In a further embodiment, the amino acid variant comprises from about 10 to about 20 amino acid substitutions. In yet a further embodiment, the amino acid variant comprises from about 10 to about 16 or from about 10 to about 15 amino acid substitutions.

[0029] In yet another further embodiment, the urease domain is an amino acid variant of SEQ ID NO: 41. In a further embodiment, the amino acid variant comprises from about 10 to about 20 amino acid substitutions. In yet a further embodiment, the amino acid variant comprises from about 10 to about 16 or from about 10 to about 15 amino acid substitutions.

[0030] In one embodiment of the urease conjugate described herein, the urease conjugate is a fusion protein. In a further embodiment, the fusion protein forms a homotetramer when expressed.

[0031] In another embodiment of the urease fusion protein described herein, the urease fusion protein has an amino acid sequence selected from SEQ ID NOs: 63, 64, 65, 66, 67, 68, 69, 70, 75, 76, 77, 78, 79, 80, 112, and 113.

[0032] In another aspect of the invention, there is provided a nucleic acid molecule encoding the urease conjugate or a domain thereof described herein. The nucleic acid molecule is present in a vector in one embodiment to enable in vitro expression of the urease conjugate.

[0033] In yet another aspect of the present invention, a therapeutic method is provided, comprising administering to a subject in need of treatment an effective amount of one of the uricase conjugates described herein or a pharmaceutical composition containing the same. The therapeutic method in one embodiment is a method for treating hyperuricemia. Thus, in one embodiment provided herein, when an effective amount of the uricase conjugate of the present invention or a pharmaceutical composition containing the same is administered, the uric acid level in the subject, for example, in the subject's blood or plasma, decreases.

[0034] In another embodiment, the treatment method is a method for treating gout. In a further embodiment, the gout is refractory gout. In a further embodiment, when an effective amount of the uricase conjugate of the present invention or a pharmaceutical composition containing the same is administered, the uric acid level in the subject, for example, in the subject's blood or plasma, decreases.

[0035] In yet another embodiment, the treatment method is a method for treating tumor lysis syndrome. In yet another embodiment, when an effective amount of the uricase conjugate of the present invention or a pharmaceutical composition containing the same is administered, the uric acid level in a subject, for example, in the subject's blood or plasma, decreases.

[0036] In one embodiment of a method for treating a subject provided herein, the subject is an adult human subject.

[0037] In one embodiment of the method provided herein, the method includes parenteral administration to a subject. In a further embodiment, the parenteral administration is intravenous administration. In yet another embodiment, the method includes subcutaneous administration to a subject.

[0038] In another aspect, the Disclosure provides a method for recombinantly producing a uricase conjugate (e.g., recombinant uricase fusion protein) as disclosed herein. This method comprises (i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding a uricase conjugate (e.g., recombinant uricase fusion protein) as disclosed herein, such that the nucleic acid sequence is operably linked to a heterologous promoter under conditions that enable expression of the nucleic acid sequence encoding the uricase conjugate (e.g., recombinant uricase fusion protein) and recombinant production of the uricase conjugate (e.g., uricase fusion protein) by the host cell; and (ii) isolating the recombinantly produced uricase conjugate (e.g., recombinant uricase fusion protein). [Brief explanation of the drawing]

[0039] [Figure 1] This is a schematic diagram showing various stereochemical configurations of the uricase fusion protein of the present invention. [Figure 2A] These are images of an SDS-PAGE gel showing bands in lanes 1, 2, and 3, respectively, representing monomer NPAS20h-CPB41 uricase-CPAS20h, NPAS20h-CPB41 uricase-CPAS30h, and pegroticase, respectively, as detected by Coomassie brilliant blue staining. [Figure 2B] This is an image of an SDS-PAGE gel showing bands in lanes 1 and 2, respectively, that represent monomer CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h, as detected by Coomassie brilliant blue staining. [Figure 3A] This graph shows the plasma concentrations of endogenous uricase and uric acid in untreated control Wistar rats (n=3) at the corresponding pre-administration time point (time 0) and various corresponding post-administration time points up to 96 hours in pharmacokinetic (PK) study 1. Each data point is displayed as mean ± SD. [Figure 3B]This graph shows the plasma concentrations of pegroticase and uric acid at various time points before administration (time 0) and up to 96 hours after administration in Wistar rats (n=3) that received a single intravenous dose of 1 mg / kg body weight of pegroticase in PK Study 1. Each data point is displayed as mean ± SD. [Figure 3C] This graph shows the plasma concentrations of CPB40 uricase-CPAS10h and uric acid at various time points from before administration (time 0) to 96 hours after administration in Wistar rats (n=3) that received a single dose of 1.51 mg / kg body weight of CPB40 uricase-CPAS10h intravenously in PK Study 1. Each data point is displayed as mean ± SD. [Figure 3D] This graph shows the plasma concentrations of CPB40 uricase-CPAS20h and uric acid at various time points from before administration (time 0) to 96 hours after administration in Wistar rats (n=3) that received a single dose of 1.99 mg / kg body weight of CPB40 uricase-CPAS20h intravenously in PK Study 1. Each data point is displayed as mean ± SD. [Figure 3E] This graph shows the plasma concentrations of CPB40 uricase-CPAS30h and uric acid at various time points from before administration (time 0) to 96 hours after administration in Wistar rats (n=3) that received a single dose of 2.47 mg / kg body weight of CPB40 uricase-CPAS30h intravenously in PK Study 1. Each data point is displayed as mean ± SD. [Figure 3F] This graph shows the plasma concentrations of CPB40 uricase-CXTENh and uric acid at various time points before administration (time 0) and up to 96 hours after administration in Wistar rats (n=3) that received a single dose of 2.16 mg / kg body weight of CPB40 uricase-CXTENh in PK Study 1. Each data point is displayed as mean ± SD. [Figure 4A]This graph shows the plasma concentrations of endogenous uricase and uric acid in untreated control Wistar rats (n=3) at the corresponding pre-administration time point (time 0) and various corresponding post-administration time points up to 168 hours in PK Study 2. Each data point is displayed as mean ± SD. [Figure 4B] This graph shows the plasma concentrations of pegroticase and uric acid at various time points before administration (time 0) and up to 168 hours after administration in Wistar rats (n=3) that received a single intravenous dose of 1 mg / kg body weight of pegroticase in PK Study 2. Each data point is displayed as mean ± SD. [Figure 4C] This graph shows the plasma concentrations of NPAS20h-CPB41 uricase-CPAS20h and uric acid at various time points from before administration (time 0) to 168 hours after administration in Wistar rats (n=3) that received a single dose of NPAS20h-CPB41 uricase-CPAS20h at a dose of 2.99 mg / kg body weight in PK Study 2. Each data point is displayed as mean ± SD. [Figure 4D] This graph shows the plasma concentrations of NPAS20h-CPB41 uricase-CPAS30h and uric acid at various time points from before administration (time 0) to 168 hours after administration in Wistar rats (n=3) that received a single dose of NPAS20h-CPB41 uricase-CPAS30h at a dose of 3.47 mg / kg body weight in PK Study 2. Each data point is displayed as mean ± SD. [Figure 5A] This graph shows the plasma concentrations of endogenous uricase and uric acid in untreated control Wistar rats (n=3) at the corresponding pre-administration time point (time 0) and various corresponding post-administration time points up to 240 hours in PK Study 3. Each data point is displayed as mean ± SD. [Figure 5B]This graph shows the plasma concentrations of CPB40 uricase-CPAS20h and uric acid at various time points before administration (time 0) and up to 240 hours after administration in Wistar rats (n=3) that received a single dose of 1.99 mg / kg body weight of CPB40 uricase-CPAS20h intravenously in PK Study 3. Each data point is displayed as mean ± SD. [Figure 5C] This graph shows the plasma concentrations of NPAS20h-CPB41 uricase-CPAS 20h and uric acid at various time points before administration (time 0) and up to 240 hours after administration in Wistar rats (n=3) that received a single dose of NPAS20h-CPB41 uricase-CPAS20h at a dose of 2.99 mg / kg body weight in PK Study 3. Each data point is displayed as mean ± SD. [Modes for carrying out the invention]

[0040] In one embodiment, the present invention relates to a novel conjugate molecule comprising two domains: (i) a uricase enzyme and (ii) a random coil polypeptide to which it is conjugated. As described in a preferred embodiment, conjugation is achieved at the DNA level by operably linking a uricase DNA sequence to a DNA sequence encoding a random coil polypeptide, followed by recombinant expression of the sequential DNA sequences. Linking the two domains at the DNA level eliminates the need for in vitro coupling or modification steps to achieve conjugate synthesis, for example, required for the coupling of polyethylene glycol (PEG) to uricase in the case of the approved uricase molecule, KRYSTEXXA® (pegroticase).

[0041] In preferred embodiments described herein, the random coil polypeptide comprises three amino acids: proline (Pro), alanine (Ala), and serine (Ser). In further embodiments, the random coil polypeptide is composed of three amino acids: proline (Pro), alanine (Ala), and serine (Ser). When all amino acid residues in the polypeptide are Pro, Ala, and Ser, or substantially all amino acids in the polypeptide are Pro, Ala, and Ser, such polypeptide is referred to herein as a “PAS polypeptide”.

[0042] In another embodiment, the random coil polypeptide is an elongation recombinant (XTEN) polypeptide. In one embodiment, the XTEN polypeptide is one of the polypeptides disclosed in U.S. Patent Application Publication No. 2015 / 0037359, the contents of which are incorporated in whole by reference for any purpose. In one embodiment, the XTEN polypeptide is at least about 800 amino acids long and consists of six hydrophilic, chemically stable amino acids Ala, Asp, Gly, Pro, Ser, and Thr in a non-repeating manner. In one embodiment, the XTEN polypeptide is 864 residues long. In another embodiment, the XTEN polypeptide is an 864aa fragment of the XTEN polypeptide. In one embodiment, the XTEN polypeptide comprises the amino acid sequence described in SEQ ID NO: 74. The XTEN polypeptide may be conjugated to a uricase enzyme described herein via chemical conjugation, or may be produced as a fusion protein with a uricase enzyme.

[0043] In embodiments in which the uricase conjugate is produced via the recombinant expression of a single DNA sequence, the uricase conjugate is referred to herein as a uricase "fusion protein." Specifically, a "fusion protein" refers to a protein composed of multiple polypeptide components, which are typically not linked in their native state but whose respective N-terminuses and C-terminuses are linked via peptide bonds to form a single continuous polypeptide. A uricase fusion protein may be a combination of two, three, four, or more different proteins. A uricase fusion protein may also include fusions with heterologous and homologous leader sequences, with or without an N-terminal methionine residue, as well as fusion proteins containing additional sequences (e.g., polyhistidine tags) for the purification of the fusion protein.

[0044] In one embodiment, the uricase conjugate comprises at least two domains. The first of the at least two domains comprises the uricase enzyme, and the second of the at least two domains comprises a random coil polypeptide, e.g., a PAS polypeptide or an XTEN polypeptide, comprising at least about 100 amino acid residues. While we do not wish to be bound by theory, the random coil conformation mediates an increase in the in vivo and / or in vitro stability of the uricase enzyme. Furthermore, while we do not wish to be bound by theory, since the random coil polypeptide domain is not thought to adopt a stable structure or function on its own, the biological activity mediated by the uricase it conjugates is essentially conserved.

[0045] In another aspect of the present invention, a nucleic acid molecule is provided. In one embodiment, the nucleic acid molecule encodes a uricase conjugate (i.e., a uricase fusion protein). In some embodiments, nucleic acid vectors and cells containing a nucleic acid molecule encoding a uricase fusion protein are also provided. Another aspect of the present invention relates to compositions comprising the conjugate of the present invention, and to specific uses of the compositions, for example, for treating gout and tumor lysis syndrome.

[0046] As used herein, the term “domain” refers to any region / part of an amino acid sequence that can autonomously adopt a particular structure and / or function. Therefore, in the context of the present invention, “domain” may represent a functional domain or a structural domain. As described herein, the proteins of the present invention comprise at least one uricase domain and at least one domain / part that forms a random coil conformation (e.g., a PAS polypeptide domain). The uricase conjugate of the present invention may also comprise more than two domains. For example, as provided herein, since uricase exists as a homotetramer, in one embodiment, the polypeptide conjugate of the present invention comprises four uricase domains and four random coil domains. Furthermore, the fusion protein of the present invention may include, for example, additional linker structures between two domains / parts as defined herein or between another domain / part, such as protease-sensitive cleavage sites, affinity tags such as polyhistidine tags or Strep tags, targeting peptides such as signal peptides, retention peptides, membrane-transfer peptides, or additional effector domains such as antigen fragments for tumor targeting associated with enzymes for antitumor toxin or prodrug activation. In another embodiment of the uricase fusion protein, the uricase domain is a uricase monomer, and the fusion protein further includes a random coil domain on the C-terminal side of the uricase domain and a random coil domain on the N-terminal side of the uricase domain. In a further embodiment, the random coil domain comprises a PAS polypeptide.

[0047] Uricase (EC 1.7.3.3) is present in microorganisms (e.g., Bacillus fastidiosus, Candida mycoderma, and Aspergillus flavus), plants (e.g., beans and chickpeas), and animals (e.g., pigs, cattle, dogs, and baboons) (Suzuki Ket al., J. Biosci. Bioeng., 2004, 98:153-158). This enzyme initiates a series of reactions that convert uric acid (UA) into allantoin, a more soluble and readily excreted product. Briefly, uricase catalyzes the reaction of UA with O2 and water (H2O) to form 5-hydroxyisouric acid (HIU) and release hydrogen peroxide (H2O2). Subsequently, HIU undergoes non-enzymatic hydrolysis to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU), which then spontaneously decarboxylates to form allantoin. Nyborg et al. (2016). PLoS One 11(12):e0167935. Doi:10.1371 / journal.pone.0167935, Ramazzina et al. (2006). Nature Chemical Biology 2(2), pp.144-148.

[0048] Humans lack functional uricase due to three mutations that result in complete silencing of the human uricase gene. While it has been hypothesized that the lack of uricase is beneficial from an evolutionary standpoint, in modern humans, high uric acid levels can have negative consequences due to increased urate deposition and gout. Therefore, the uricase conjugate described herein is provided in one aspect of the present invention as a therapeutic agent for treating patients with elevated UA levels.

[0049] Active uricase is a tetrameric protein with four identical subunits (i.e., uricase is a homotetramer), each subunit having a molecular weight of approximately 34 kD and consisting of 301-304 amino acids. Uricase exhibits maximum enzymatic activity at pH 8.0 (Bayol A et al., Biophys. Chem. 1995, 54:229-235). Of all origins, uricase from Aspergillus flavus has the highest activity, reaching a maximum of 27 IU / mg, while uricase from Bacillus fastidiosus has the second highest activity at 13 IU / mg (Huang SH et al., Eur. J. Biochem., 2004, 271:517-523). Furthermore, uricase from beans has only 2-6 IU / mg of activity. Regarding recombinant mammalian uricases, the activity of porcine uricase can reach 5 IU / mg, while that of baboon uricase is only 1 IU / mg (Michael Het al., 2006, U.S. Patent No. 7,056,713, the whole of which is incorporated herein by reference), while human uricase has no activity.

[0050] The present invention is not limited by the source or amino acid sequence of the uricase enzyme domain of the conjugate molecule. For example, the uricase provided in the conjugate of the present invention is a wild-type uricase enzyme or an amino acid variant thereof. For example, in one embodiment, the uricase is canine, porcine, bovine, goat, or baboon uricase, or derived from canine, porcine, bovine, goat, or baboon uricase. In one embodiment, the uricase provided in the conjugate of the present invention is an amino acid variant of canine, porcine, bovine, goat, or baboon uricase. When used herein, the uricase amino acid variant has an amino acid sequence that differs from the corresponding wild-type uricase enzyme, but still retains uricase activity.

[0051] In one embodiment, uricase is an amino acid variant of a known uricase enzyme. In a further embodiment, the uricase amino acid variant has an amino acid sequence that is at least about 75%, for example, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% (including all values ​​and subranges in between) identical to the amino acid sequence of a known uricase enzyme. In a further embodiment, the known uricase enzyme has the amino acid sequence described in SEQ ID NO: 40 or SEQ ID NO: 41. Each of the uricase enzymes of SEQ ID NO: 40 and SEQ ID NO: 41 is a chimeric pig-baboon uricase containing amino acids (aa) 8-266 of porcine uricase (SEQ ID NO: 2) and aa 267-304 of baboon uricase (SEQ ID NO: 3). Therefore, the uricase enzymes of SEQ ID NO: 40 and SEQ ID NO: 41 are also referred to in this application as "CPB40 uricase" and "CPB41 uricase," respectively. The amino acid sequence of SEQ ID NO: 41 is otherwise identical to that of SEQ ID NO: 40, except for the absence of an N-terminal methionine. KRYSTEXXA® (pegroticase) is a hyper-PEGylated homotetrameric protein, in which each monomer is a single polypeptide chain composed of chimeric pig-baboon uricase having the amino acid sequence of SEQ ID NO: 40 or 41.

[0052] In one embodiment, uricase is a uricase amino acid variant. The uricase amino acid variant contains about 10 to about 20 amino acid substitutions, for example, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid substitutions. In another embodiment, the uricase amino acid variant contains about 10 to about 20 amino acid substitutions, for example, about 10 to about 18, about 10 to about 16, about 10 to about 14, about 10 to about 13, or about 10 to about 12 amino acid substitutions.

[0053] In one embodiment, the uricase amino acid variant is an amino acid variant of porcine-hihikimerauricase. In a further embodiment, the uricase amino acid variant is a variant of the uricase polypeptide sequence described in SEQ ID NO: 40. In a further embodiment, the uricase amino acid variant of SEQ ID NO: 40 contains about 10 to about 20 amino acid substitutions, for example, about 10 to about 18, about 10 to about 16, about 10 to about 14, about 10 to about 13, or about 10 to about 12 amino acid substitutions. In another embodiment, the uricase amino acid variant is a variant of the uricase polypeptide sequence described in SEQ ID NO: 41. In a further embodiment, the uricase amino acid variant of SEQ ID NO: 41 contains about 10 to about 20 amino acid substitutions, for example, about 10 to about 18, about 10 to about 16, about 10 to about 14, about 10 to about 13, or about 10 to about 12 amino acid substitutions.

[0054] The uricase conjugates provided herein are, in preferred embodiments, recombinant uricase fusion proteins. As used herein, “recombinant protein” refers to any artificially produced protein and is distinguished from naturally produced proteins (i.e., proteins produced in the tissues of animals that possess only the natural genes for the particular protein of interest). As described herein, a recombinant uricase fusion protein comprises a uricase domain conjugated via peptide bonds (directly or via amino acid linkers) to one or more random coil polypeptide domains.

[0055] In one embodiment, the uricase domain comprises a recombinant molecule containing segments of porcine and baboon liver uricase protein. In another embodiment, the uricase domain comprises recombinant modified baboon uricase. In one embodiment, the uricase domain comprises a chimeric porcine-baboon uricase (PBC uricase, SEQ ID NO: 1) containing amino acids (aa) 1-225 of porcine uricase (SEQ ID NO: 2) and aa 226-304 of baboon uricase (SEQ ID NO: 3). In another embodiment, the uricase is a chimeric porcine-baboon uricase containing aa 1-288 of porcine uricase and aa 289-304 of baboon uricase (PKS uricase, SEQ ID NO: 4), or a variant of one of the aforementioned, or a truncated version of one of the aforementioned.

[0056] In one embodiment, the uricase is a chimeric pig-baboon uricase.

[0057] In one embodiment, the uricase is porcine uricase having the amino acid sequence described in Sequence ID No. 5.

[0058] In one embodiment, the uricase is a humanized uricase, which may contain amino acids derived from inactive human uricase, substituted with the amino acid sequence of a non-human mammalian uricase, in order to retain its original activity and to improve homology with human uricase, thereby reducing immunogenicity in the human body. Such a humanized uricase enzyme is disclosed in U.S. Patent No. 8,586,535, which is incorporated in whole by reference.

[0059] In one embodiment, the uricase is either Aspergillus flavus uricase or derived from Aspergillus flavus uricase.

[0060] In one embodiment, the uricase is Arthrobacter globiformis uricase (NCBI accession number D0VWQ1), Deinococcus geothermalis uricase (NCBI accession number WP 011525965), Deinococcus radiodurans uricase (NCBI accession number WP_010887803), Granulicella tundricola uricase (NCBI reference sequence: WP 013581210.1), Solibacter usitatus uricase (NCBI accession number WP 011682147), Terriglobus saanensis uricase (NCBI accession number WP_013569963), Kyrpidia tusciae uricase (NCBI accession number ADG06709), or an amino acid variant of one of the above.

[0061] Uricase genes and proteins have been identified in several mammalian species, such as pigs, baboons, rats, rabbits, mice, and rhesus monkeys. The sequences of various uricase proteins are described herein by reference to their public database accession numbers, as follows: gi|50403728|sp|P25689, gi|20513634|dbj|BAB91555.1, gi|176610|AAA35395.1, gi|20513654|dbj|BAB91557.1, gi|47523606|ref|NP_999435.1, gi|66 78509|ref|NP_033500.1, gi|57463|emb|CAA31490.1, gi|20127395|ref|NP_446220.1, gi|137107|sp|P11645, gi|51458661|ref|XP_497688.1, gi|207619|gb|AAA42318.1, gi|26340770|dbj|BAC34047.1, and gi|57459|emb|CAA30378.1. Each of these sequences and their annotations in public databases accessible through the National Center for Biotechnology Information (NCBI) is incorporated in whole by reference for all purposes.

[0062] In one embodiment of the present invention, the uricase domain of the uricase conjugate comprises a mammalian uricase or an amino acid variant thereof. In a further embodiment, the mammalian uricase comprises an amino acid sequence of liver uricase from a pig, cattle, sheep, or baboon. In one embodiment of the present invention, the uricase is a chimeric uricase of two or more mammalian uricases. In a further embodiment, the mammalian uricase of the chimeric uricase is selected from liver uricase from a pig, cattle, sheep, or baboon.

[0063] In one embodiment, the uricase domain of the uricase conjugate includes a fungal or microbial uricase. In a further embodiment, the fungal or microbial uricase is Aspergillus flavus, Arthrobacter globiformis, or Candida utilis uricase. In yet another embodiment, the uricase domain of the uricase conjugate includes an invertebrate uricase. In a further embodiment, the invertebrate uricase is Drosophila melanogaster or Drosophila pseudoobscura uricase.

[0064] In another embodiment, the uricase domain of the uricase conjugate contains Candida utilis uricase.

[0065] In yet another embodiment, the uricase domain of the uricase conjugate contains plant uricase. In yet another embodiment, the plant uricase is nodule glycoside uricase.

[0066] In yet another embodiment, the uricase portion of the conjugate has an amino acid sequence selected from one of sequence numbers 6-39, or an amino acid variant thereof (Table 1).

[0067] [Table 1]

[0068] In yet another embodiment, the uricase is a synthetic uricase generated from a consensus uricase amino acid sequence derived from an alignment of 50 uricase sequences that share maximum identity with Arthrobacter globiformis uricase (SEQ ID NO: 39).

[0069] In one embodiment, the uricase enzyme may be a wild-type uricase enzyme or an engineered variant thereof. For example, in one embodiment, the uricase enzyme is recombinant mammalian uricase.

[0070] In one embodiment, uricase has the amino acid sequence disclosed in U.S. Patent No. 10,731,139, the contents of which are incorporated in whole by reference for any purpose.

[0071] Methods for mutating amino acids are well known in the art, and using such methods, one or more amino acids in the wild-type uricase enzyme can be mutated to produce uricase amino acid mutants. For example, the tripeptide Arg-Gly-Asp (RGD), which has been reported to mediate cell adhesion via integrin binding, can be mutated into the Ser-Gly-Asp (SGD) sequence, thereby causing the uricase domain to include the RGD sequence.

[0072] In one embodiment, the uricase portion of the conjugate is a truncated uricase. As used herein, “truncated uricase” refers to a uricase molecule having a shortened primary amino acid sequence compared to the amino acid sequence of a known uricase enzyme (e.g., a wild-type enzyme). In one embodiment, the truncation is at or around the N-terminus and / or C-terminus of the uricase. In one embodiment, the uricase is truncated at the N-terminus. In further embodiments, the N-terminal truncation begins at positions 1, 2, 3, 4, 5, or 6. In one embodiment, the amino-terminal truncation begins at position 2, thereby excluding the amino-terminal methionine (Met). In one embodiment, the amino-terminal Met may be removed by post-translational modification. In another embodiment, the amino-terminal Met is removed after the uricase has been produced as one of the fusion proteins described herein. In further embodiments, Met is removed by endogenous bacterial aminopeptidase.

[0073] In one embodiment of truncated uricase, 4 to 13 amino acids are truncated at its N-terminus. In another embodiment, 4 to 13 amino acids are truncated at its C-terminus. In one embodiment of truncated uricase, 4 to 13 amino acids are truncated at both its C-terminus and N-terminus. In yet another embodiment, 6 amino acids are truncated at its N-terminus. In yet another embodiment, uricase for use in a conjugate, e.g., one of the fusion proteins described herein, is truncated at its C-terminus by only 6 amino acids. In one embodiment of the present invention, uricase for use in a conjugate, e.g., one of the fusion proteins described herein, is truncated at both its carboxyl and amino termini by 6 amino acids.

[0074] In one embodiment of the uricase conjugate described herein, the uricase domain of the conjugate contains the amino acid sequence described in sequences selected from SEQ ID NOs: 40-44. Similar to the uricase enzymes of SEQ ID NOs: 40 and 41, the uricase enzymes of SEQ ID NOs: 42 and 43 are chimeric pig-baboon uricases containing aa8-220 of porcine uricase (SEQ ID NOs: 2) and aa221-301 of baboon uricase (SEQ ID NOs: 3), respectively. Except for the absence of an N-terminal methionine, the amino acid sequence of SEQ ID NOs: 43 is otherwise identical to the amino acid sequence of SEQ ID NOs: 42.

[0075] In a further embodiment, the uricase protein comprises the amino acid sequence described in SEQ ID NO: 1. In another embodiment, the uricase protein comprises the amino acid sequence described in SEQ ID NO: 40 or 41.

[0076] In one embodiment, the uricase protein comprises the amino acid sequence described in SEQ ID NO: 7. In yet another embodiment, the uricase protein comprises the amino acid sequence described in SEQ ID NO: 40, 41, 42, 43, or 44. In one embodiment of the present invention, uricase comprises the amino acid sequence of SEQ ID NO: 41. In another embodiment of the present invention, uricase comprises the amino acid sequence of SEQ ID NO: 43.

[0077] In yet another embodiment, the uricase domain includes one of the uricase polypeptides disclosed in International Publication No. 2016 / 187026, the contents of which are incorporated in their entirety by reference.

[0078] In one embodiment, the uricase domain includes rasburicase, which is commercially available under the trade name Elitek® and has the amino acid sequence described in SEQ ID NO: 45.

[0079] In another embodiment, the uricase domain includes a uricase having the amino acid sequence described in SEQ ID NO: 46, and also referred to as HZN-003.

[0080] In yet another embodiment, the uricase domain includes a uricase having the amino acid sequence described in Sequence ID No. 47, also known as SEL-212.

[0081] The drawbacks of prior art uricase therapies include (i) lack of solubility, (ii) immunogenicity, and (iii) rapid clearance from circulation via renal filtration, the last of which strongly hinders efficacy in both animal studies and human therapy. For this purpose, the present invention provides a uricase conjugate comprising a conformationally disordered (random coil) polypeptide domain containing the amino acid residues Pro, Ala, and Ser (PAS). The PAS sequence is a hydrophilic, uncharged biopolymer with biophysical properties similar to polyethylene glycol (PEG), and its chemical conjugation to drugs is an established method for extending plasma half-life and reducing immunogenicity (e.g., KRYSTEXXA® (pegroticase)). In contrast to PEG modification, PAS polypeptides provide fusion to uricase at the genetic level; that is, they are genetically encoding polypeptides, enabling the host (e.g., Escherichia coli, etc.) to produce a fully active uricase fusion protein, thus eliminating the need for in vitro coupling or modification steps.

[0082] As described above, the uricase conjugates provided herein comprise at least two domains. The uricase domain is described above. The second domain, referred to herein in some embodiments as the PAS domain, comprises an amino acid sequence containing at least about 100 amino acid residues that form a random coil conformation. The at least about 100 amino acid residues forming the random coil comprise the amino acids proline (Pro), alanine (Ala), and serine (Ser). In the PAS domain, all or substantially all of the amino acids are Pro, Ala, and Ser. While we do not wish to be bound by theory, the random coil conformation mediates an increase in the in vivo and / or in vitro stability of the uricase enzyme. Details relating to various types of PAS polypeptides and the nucleic acids encoding them for use in the present invention can be found in International Publication No. 2008 / 155134, the contents of which are incorporated in whole by reference for all purposes.

[0083] As used herein, the terms “random coil” or “random coil polypeptide domain” refer to a conformation of a polymer molecule, including an amino acid polymer, in which the individual monomer elements forming the polymer structure are oriented essentially randomly relative to adjacent monomer elements, but are still chemically bonded to those adjacent monomer elements. In particular, polypeptides or amino acid polymers employing / having / forming a “random coil” conformation substantially lack the defined secondary and tertiary structures. The properties of polypeptide random coils and methods for their experimental identification are known to those skilled in the art.

[0084] Random coils are formed under physiological conditions. For example, in one embodiment, the physiological conditions are parameters that are typically effective for higher organisms, particularly mammals, most preferably humans. Thus, the physiological conditions may be those commonly found in the body fluids of mammals. The physiological conditions may relate to corresponding parameters found in a healthy body, as well as parameters found in a diseased mammal or human patient. For example, a diseased mammal or human patient may have higher but physiological temperature conditions if the mammal or human is suffering from a fever.

[0085] Several buffers in the experimental setup (e.g., for use in determining protein structure, particularly circular dichroism (CD) measurement, and other methods for determining the structural properties of protein / amino acid stretches), solvents and / or excipients for pharmaceutical compositions are considered to represent physiological solutions and / or physiological conditions in vitro. Examples of such buffers include, for example, phosphate-buffered saline, Tris buffer, acetate buffer, citrate buffer, or similar buffers. Generally, the pH of buffers representing physiological solution conditions is in the range of 6.5 to 8.5, e.g., 7.0 to 8.0, e.g., 7.2 to 7.7, and the osmotic pressure may be in the range of 10 to 1000 mmol / kg H2O, more specifically in the range of 50 to 500 mmol / kg H2O, e.g., 200 to 350 mmol / kg H2O.

[0086] Methods for determining whether an amino acid polymer forms / adopts a random coil conformation are known in the art. Such methods include CD spectroscopy, which represents optical absorption spectroscopy that measures the difference in absorbance of right-circularly polarized and left-circularly polarized light by a substance. The secondary structure of a protein can be determined by CD spectroscopy using far-ultraviolet spectra with wavelengths of approximately 190–250 nm. At these wavelengths, α-helices, parallel and antiparallel β-sheets, and random coil conformations each produce characteristic shapes and sizes of CD spectra, so that different secondary structures commonly found in polypeptides can be analyzed. Therefore, by using CD spectroscopy, those skilled in the art can easily determine whether an amino acid polymer forms / adopts a random coil conformation under physiological conditions. Other established biophysical methods include nuclear magnetic resonance (NMR) spectroscopy, absorption spectroscopy, infrared and Raman spectroscopy, hydrodynamic volume measurement by size exclusion chromatography, analytical ultracentrifugation or dynamic / static light scattering, and measurement of friction coefficient or intrinsic viscosity.

[0087] In one embodiment, the random coil domain contains at least about 100 amino acid residues, at least about 150 amino acid residues, at least about 200 amino acid residues, at least about 250 amino acid residues, at least about 300 amino acid residues, at least about 350 amino acid residues, or at least about 400 amino acid residues. In another embodiment, the random coil domain contains up to about 1000 amino acid residues, up to about 900 amino acid residues, up to about 800 amino acid residues, up to about 700 amino acid residues, or up to about 600 amino acid residues. In one embodiment, the random coil domain contains up to about 500 amino acid residues or up to about 450 amino acid residues.

[0088] In one embodiment, the random coil polypeptide domain contains about 100 to about 3000 amino acid residues. In a further embodiment, the random coil domain contains about 100 to about 1000 amino acid residues. In one embodiment, the random coil polypeptide domain contains an amino acid sequence in which Pro residues account for about 4% to about 40% of the random coil polypeptide domain. In a further embodiment, alanine and serine residues constitute the remaining about 60% to about 96% of the random coil polypeptide. In some embodiments, the random coil polypeptide domain contains additional amino acids other than Ala, Ser, and Pro, i.e., trace components. As used in this context, the term “trace components” means that up to 10% of the amino acids in the random coil polypeptide domain are different from alanine, serine, and proline; for example, up to 8% of the amino acids in the random coil polypeptide domain, for example, up to 6% of the amino acids in the random coil polypeptide domain, for example, up to 5% of the amino acids in the random coil polypeptide domain, for example, up to 4% of the amino acids in the random coil polypeptide domain, for example, up to 3% of the amino acids in the random coil polypeptide domain, for example, up to 2% of the amino acids in the random coil polypeptide domain, and up to 1% of the amino acids in the random coil polypeptide domain are different from Ala, Ser, and Pro. In one embodiment of the random coil polypeptide domain, the polypeptide comprises amino acids other than Ala, Ser, and Pro, and the other amino acids are selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. In another embodiment, the other amino acids comprise one or more non-natural amino acids.

[0089] In another embodiment, the random coil polypeptide is an elongation recombinant (XTEN) polypeptide. In one embodiment, the XTEN polypeptide is one of the polypeptides disclosed in U.S. Patent Application Publication 2015 / 0037359, the contents of which are incorporated by reference in whole for any purpose. In one embodiment, the XTEN polypeptide is at least about 800 amino acids long and composed of six hydrophilic, chemically stable amino acids Ala, Asp, Gly, Pro, Ser, and Thr in a non-repeating manner. In one embodiment, the XTEN polypeptide is 864 residues long. In another embodiment, the XTEN polypeptide is an 864aa fragment of the XTEN polypeptide. The XTEN polypeptide may be conjugated to the uricase enzyme described herein via chemical conjugation, or may be produced as a fusion protein with the uricase enzyme.

[0090] In another embodiment, the random coil polypeptide domain includes multiple “amino acid repeats,” i.e., the same amino acid sequence appearing two or more times within the domain, where each “amino acid repeat” consists of Ala, Ser, and Pro residues (referred to herein as “PAS” or “APS”). In a further embodiment, six or fewer consecutive amino acid residues are identical in the random coil polypeptide domain, and the Pro residues constitute more than approximately 4% and less than approximately 40% of the amino acids in the random coil polypeptide domain. Non-limiting examples of “amino acid repeats” consisting of Ala, Ser, and Pro residues are provided herein; see, for example, SEQ ID NOs. 48, 50, 52, 54, 56, and 58 (Table 2A). Fragments and / or polymers of these sequences are used in several embodiments. A “fragment” contains at least three amino acids, including at least one Ala, one Ser, and / or one Pro.

[0091] [Table 2]

[0092] The aforementioned repeating sequences can be encoded by nucleic acid molecules having the sequences described in SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57 and / or SEQ ID NO: 59 (Table 2A).

[0093] In one embodiment, a nucleotide sequence encoding the amino acid repeat of SEQ ID NO: 48 is provided. In a further embodiment, the nucleotide sequence is selected from one of the nucleotide sequences listed in Table 2B, i.e., one of SEQ ID NOs: 81 to 111.

[0094] [Table 3-1]

[0095] [Table 3-2]

[0096] In one embodiment, the amino acid repeats used in the random coil PAS polypeptide domain comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, and the amino acid repeat comprises at least one Ala, Ser, and Pro residue. In one embodiment, the amino acid repeat does not comprise more than 100 amino acid residues. In one embodiment, the amino acid repeat comprises at least about 4%, at least about 5%, at least about 6%, at least about 10%, at least about 15%, or at least about 20% Pro residues. In further embodiments, the amino acid repeat comprises less than about 40%, for example, less than about 35% Pro residues.

[0097] In one embodiment, the random coil polypeptide domain includes five or fewer identical consecutive amino acid residues, for example, four or fewer identical consecutive amino acid residues, for example, three or fewer identical consecutive amino acid residues.

[0098] In one embodiment, the random coil polypeptide domain contains more than 4% Ala residues but less than 50% Ala residues, for example, more than 10% Ala residues but less than 50% Ala residues, for example, more than 20% Ala residues but less than 50% Ala residues.

[0099] In another embodiment, the random coil polypeptide domain contains more than 4% but less than 50% of Ser residues, for example, more than 10% but less than 50% of Ser residues, for example, more than 20% but less than 50% of Ser residues.

[0100] In one embodiment, the random coil polypeptide domain comprises approximately 35% Pro residues, approximately 50% Ala residues, and approximately 15% Ser residues. Alternatively, the random coil polypeptide domain comprises approximately 35% Pro residues, approximately 15% Ala residues, and approximately 50% Ser residues.

[0101] In one embodiment of the present invention, the uricase conjugate includes a random coil domain comprising the amino acid sequence described in SEQ ID NO: 60.

[0102] In one embodiment of the present invention, the uricase conjugate includes a random coil domain comprising the amino acid sequence described in SEQ ID NO: 61.

[0103] In one embodiment of the present invention, the uricase conjugate includes a random coil domain comprising the amino acid sequence described in Sequence ID No. 62.

[0104] In one embodiment of the random coil domain provided herein, the random coil domain includes an N-terminal Met residue when it is located at the N-terminus of the fusion protein. In another embodiment, the fusion protein does not include an N-terminal Met residue, for example, because it has been removed post-translation.

[0105] In one embodiment, the uricase conjugate of the present invention is a fusion protein. The fusion protein described herein comprises at least one uricase domain and at least one random coil polypeptide domain in a multidomain polypeptide. In an alternative embodiment, the uricase is bound to the random coil polypeptide domain via a non-peptide bond. Non-peptide bonds useful for crosslinking proteins are known in the art and may include disulfide bonds (e.g., between Cys side chains), thioether bonds, or non-peptide covalent bonds induced by chemical crosslinking agents (e.g., disuccinimidyl sberate (DSS) or sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (Sulfo-SMPB)), as well as non-covalent protein-protein interactions.

[0106] With respect to embodiments of fusion proteins, the two domains may be arranged in an order selected by those skilled in the art. For example, in one embodiment of a uricase conjugate fusion protein, the uricase domain is located at the amino(N-) terminus of the fusion protein, and the random coil polypeptide domain is located at the carboxy(C-) terminus of the fusion protein. However, this order may be reversed, for example, in one embodiment, the uricase domain is located in / at the carboxy(C-) terminus of the fusion protein, and the random coil polypeptide domain is located in / at the amino(N-) terminus. In yet another embodiment, the random coil polypeptide domain is located at both the C-terminus and N-terminus of the fusion protein, and the uricase domain is located between both random coil domains.

[0107] In one embodiment, the uricase fusion protein includes an N-terminal Met residue. In another embodiment, the uricase fusion protein does not include an N-terminal Met residue, for example, because it has been removed post-translation. Therefore, if an N-terminal Met is present in the sequence provided herein, an alternative embodiment includes a fusion protein of the same sequence but without the N-terminal Met. Similarly, if an N-terminal Met is not present in the fusion protein provided herein, an alternative embodiment includes a fusion protein of the same sequence but with the N-terminal Met present.

[0108] In one embodiment of the uricase fusion protein, an amino acid spacer sequence is present between the uricase domain and the random coil (e.g., PAS) domain. In one embodiment, the amino acid spacer sequence is 1 amino acid long, 2 amino acid long, 3 amino acid long, or 4 amino acid long. In a further embodiment, the amino acid spacer sequence is 2 amino acid long. In a further embodiment, the spacer sequence is Gly-Ser.

[0109] In one embodiment of the uricase conjugate described herein, the conjugate includes a purification tag at the C-terminus, N-terminus, or both the N-terminus and C-terminus. The purification tag is used to facilitate the purification of the uricase conjugate (i.e., uricase fusion protein) from an in vitro expression system, for example, by the use of immobilized metal affinity chromatography (IMAC). In one embodiment, the purification tag is located at the C-terminus of the uricase fusion protein. In a further embodiment, the purification tag is a polyhistidine tag (also called a "his tag"). In one embodiment, the his tag includes six histidine residues.

[0110] Alternative purification tags may also be used herein. For example, in one embodiment, a his-glu tag (HQ tag) is present at the C-terminus of one of the uricase conjugates described herein. In a further embodiment, the HQ tag has the amino acid sequence HQHQHQ (SEQ ID NO: 71). In another embodiment, the uricase conjugate includes a his-asp tag (HN tag) at its C-terminus to enable the purification of the conjugate. In one embodiment, the HN tag has the amino acid sequence HNHNHNHNHNHN (SEQ ID NO: 72). In yet another embodiment, the uricase conjugate includes a HAT peptide tag at its C-terminus to enable the purification of the conjugate. In one embodiment, the HAT peptide tag has the amino acid sequence KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 73).

[0111] Regarding embodiments of the uricase conjugate fusion proteins of the present disclosure, Figure 1 provides exemplary configurations of recombinant uricase fusion proteins comprising a uricase domain and one or two random coil PAS polypeptide or XTEN polypeptide domains. In one embodiment, the uricase fusion protein comprises a uricase domain and one random coil PAS polypeptide or XTEN polypeptide domain. In the uricase fusion protein, the uricase domain may be located at the C-terminus of one random coil PAS polypeptide or XTEN polypeptide domain, or at the N-terminus of one random coil PAS polypeptide or XTEN polypeptide domain. In another embodiment, the uricase fusion protein comprises a uricase domain and two random coil PAS polypeptide domains or two XTEN polypeptide domains. In the uricase fusion protein, one of the two random coil PAS polypeptide or XTEN polypeptide domains is at the N-terminus and the other is at the C-terminus, and the uricase domain is located between the two random coil PAS polypeptide or XTEN polypeptide domains. In some embodiments, the spacer sequence Gly-Ser(GS) between the uricase domain and the C-terminal random coil PAS polypeptide (e.g., PAS10, PAS20, or PAS30) or XTEN polypeptide domain, as shown in Figure 1, is optional and may not be present. In one embodiment, the uricase fusion protein has a polyhistidine tag (also called a "his tag") containing, for example, six histidine residues at the C-terminus, N-terminus, or both the C-terminus and N-terminus, for the purpose of purifying the uricase fusion protein. In some embodiments, the his tag is separated from its adjacent domain by the spacer sequence Gly-Ser(GS).

[0112] PAS10, PAS20, and PAS30 represent random coil PAS polypeptide domains containing 10, 20, and 30 tandem copies of a PAS sequence containing Pro, Ala, and Ser, such as the PAS sequence described in SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO: 58, respectively.

[0113] In one embodiment, PAS10, PAS20, and PAS30 each comprise 10, 20, and 30 tandem copies of the PAS sequence of SEQ ID NO: 48, respectively, thereby representing a random coil PAS polypeptide domain having the amino acid sequences described in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively.

[0114] In one embodiment, the DNA sequences encoding PAS10 of SEQ ID NO: 60, PAS20 of SEQ ID NO: 61, and PAS30 of SEQ ID NO: 62 each contain a total of 10, 20, and 30 tandem copies of one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 49 and SEQ ID NOs: 81 to 111, respectively.

[0115] In one embodiment, the DNA sequences encoding PAS10 of SEQ ID NO: 60, PAS20 of SEQ ID NO: 61, and PAS30 of SEQ ID NO: 62 each contain a total of 10, 20, and 30 tandem copies of the same (single) nucleotide sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NOs: 81-111, respectively.

[0116] In one embodiment, the DNA sequences encoding PAS10 of SEQ ID NO: 60, PAS20 of SEQ ID NO: 61, and PAS30 of SEQ ID NO: 62 each contain a total of 10, 20, and 30 tandem copies of two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 49 and SEQ ID NOs: 81 to 111, respectively.

[0117] In one embodiment, the DNA sequences encoding PAS10 of SEQ ID NO: 60, PAS20 of SEQ ID NO: 61, and PAS30 of SEQ ID NO: 62 each contain 10, 20, and 30 unique (i.e., different) nucleotide sequences selected from the group consisting of SEQ ID NOs: 49 and SEQ ID NOs: 81-111, linked together in tandem via 3',5'-phosphodiester bonds.

[0118] In an exemplary embodiment, the DNA sequence encoding PAS10 of SEQ ID NO: 60 includes each of SEQ ID NOs: 102-111, tandemly linked together in ascending order of ID numbers via a 3',5'-phosphodiester bond in the 5'-3' direction.

[0119] In an exemplary embodiment, the DNA sequence encoding PAS20 of SEQ ID NO: 61 includes each of SEQ ID NOs: 92-111, tandem-linked together in ascending order of ID numbers via a 3',5'-phosphodiester bond in the 5'-3' direction.

[0120] In an exemplary embodiment, the DNA sequence encoding PAS30 of SEQ ID NO: 62 includes each of SEQ ID NOs: 82-111, tandemly linked together in ascending order of ID numbers via a 3',5'-phosphodiester bond in the 5'-3' direction.

[0121] In another exemplary embodiment, the DNA sequence encoding PAS30 of SEQ ID NO: 62 includes SEQ ID NOs: 81 and 83-111, each tandem linked together in ascending order of ID numbers via a 3',5'-phosphodiester bond in the 5'-3' direction.

[0122] In one embodiment, the XTEN polypeptide domain of the uricase fusion protein contains the amino acid sequence of SEQ ID NO: 74.

[0123] In one embodiment, the uricase domain of the uricase fusion protein contains the chimeric pig-baboon uricase of SEQ ID NO: 1. In one embodiment, the uricase domain of the uricase fusion protein contains the chimeric pig-baboon uricase of SEQ ID NO: 4. In one embodiment, the uricase domain of the uricase fusion protein contains the chimeric pig-baboon uricase of SEQ ID NO: 40 (also referred to herein as "CPB40 uricase"). In one embodiment, the uricase domain of the uricase fusion protein contains the chimeric pig-baboon uricase of SEQ ID NO: 41 (also referred to herein as "CPB41 uricase"). In one embodiment, the uricase domain of the uricase fusion protein contains the chimeric pig-baboon uricase of SEQ ID NO: 42. In one embodiment, the uricase domain of the uricase fusion protein contains the chimeric pig-baboon uricase of SEQ ID NO: 43.

[0124] Table 3 shows the code names, sequence numbers, and domain compositions of exemplary uricase fusion proteins prepared and produced by the inventors of this application in E. coli. Table 3 also lists the DNA sequences encoding PAS10 in SEQ ID NO: 60, PAS20 in SEQ ID NO: 61, and PAS30 in SEQ ID NO: 62. The exemplary uricase fusion proteins include the amino acid sequence of either CPB40 uricase or CPB41 uricase in combination with one or two random coil PAS polypeptide domains, or one random coil XTEN polypeptide domain, according to the configuration in Figure 1 or variations thereof disclosed above. Details of the preparation and characterization of several exemplary uricase fusion proteins are described in "Examples".

[0125] [Table 4-1]

[0126] [Table 4-2]

[0127] In one embodiment, the present invention relates to a nucleic acid construct encoding a uricase conjugate (fusion protein) of the present invention. The nucleic acid molecule can be operably ligated to a suitable expression regulatory sequence known in the art to ensure proper transcription and translation of the polypeptide, and to a signal sequence to ensure targeting to cellular secretion or organelles. Such a vector may include further genes, such as marker genes, that enable selection of the vector under suitable conditions in a suitable host cell.

[0128] A "nucleic acid construct" refers to a nucleic acid sequence constructed to contain one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences derived from lambda phages), viral genomes containing non-natural nucleic acid sequences, and two or more operablely linked proteins (for example, to produce fusion proteins).

[0129] "Operatively linked" means an arrangement of elements configured so that the components described herein perform their normal functions. In the case of a promoter, a promoter operably linked to a coding sequence influences the expression of the coding sequence. Promoters or other regulatory elements do not need to be contiguous with the coding sequence insofar as they function to guide its expression. For example, an intervening untranslated but transcribed sequence may exist between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence. In the case of the different domains of the uricase conjugate described herein, the domains may be operably linked within a single DNA sequence to enable the expression of the uricase fusion protein.

[0130] A "vector" is a nucleic acid construct capable of introducing a gene sequence into a host cell. A "vector" is a nucleic acid construct capable of inducing the expression of a target gene and introducing a gene sequence into a host cell, which can be achieved by genomic integration of all or part of the vector, or by the transient or heritability of the vector as an extrachromosomal element. Therefore, this term includes cloning vectors and expression vectors, as well as integration vectors. Examples of vectors used herein include plasmids, phages, phagemids, adenoviruses, adeno-associated viruses (AAVs), and lentiviruses.

[0131] In one embodiment, the nucleic acid construct is contained in a recombinant vector in which a nucleic acid molecule encoding a uricase fusion protein is operably linked to an expression regulatory sequence that enables the expression of the uricase fusion protein in prokaryotic or eukaryotic cells.

[0132] Nucleic acid molecule expression involves the transcription of the nucleic acid molecule into translatable mRNA. Regulatory elements that enable expression in prokaryotic host cells include, for example, the lambda PL, lac, trp, tac, tet, or T7 promoters in E. coli. Regulatory elements that can ensure expression in eukaryotic cells, such as mammalian cells or yeast, are well known to those skilled in the art. In one embodiment, the regulatory element includes a regulatory sequence that ensures the initiation of transcription, and optionally, a polyA signal that ensures the termination of transcription and the stabilization of the transcript. Further regulatory elements for use herein include transcriptional enhancers and translational enhancers, and / or naturally occurring or heterologous promoter regions. Examples of regulatory elements that enable expression in eukaryotic host cells include the AOXI or GALI promoter in yeast, or the CMV, SV40, RSV promoters (Roussarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian cells and other animal cells. Apart from the elements responsible for transcription initiation, such regulatory elements may also include transcription termination signals (e.g., SV40-polyA moiety or tk-polyA moiety) downstream of the coding region.

[0133] Recombinant vectors can be constructed using methods well known to those skilled in the art (see, for example, Sambrook (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory NY and Ausubel (1989), Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY). In this regard, suitable expression vectors are known in the art and include, for example, Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDNA3, pPICZalpha A (Invitrogen), or pSPORT1 (GIBCO BRL). Furthermore, depending on the expression system used, a leader sequence that can guide polypeptides into intracellular compartments or secrete them into the culture medium can be added to the coding sequence of the nucleic acid molecule of the present invention.

[0134] The present invention also relates, in certain embodiments, to vectors conventionally used in genetic engineering, particularly plasmids, cosmids, viruses, and bacteriophages, including nucleic acid constructs encoding fusion proteins provided herein. In one embodiment, the vector is an expression vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpesviruses, or bovine papillomaviruses can be used to deliver the polynucleotide or vector of the present invention to a target cell population. Vectors containing nucleic acid molecules of the present invention can be introduced into host cells by different well-known methods depending on the type of cell host. Therefore, the present invention further relates to cells containing nucleic acid molecules or vectors.

[0135] In one embodiment, host cells are transfected with one of the nucleic acid molecules / vectors provided herein using calcium chloride transfection, which is commonly used for prokaryotic cells. In another embodiment, depending on the cell host, calcium phosphate treatment or electroporation is used. As a further alternative, the nucleic acid molecules and vectors of the present invention can be reconstituted in liposomes for delivery to target cells. The nucleic acid molecules or vectors of the present invention present in host cells may be integrated into the genome of the host cell or maintained outside the chromosome. Thus, the present invention also relates in part to host cells containing the nucleic acid molecules and / or vectors of the present invention. Host cells for polypeptide expression are well known in the art and include prokaryotic cells as well as eukaryotic cells, such as E. coli cells, yeast cells, invertebrate cells, melanoma cells such as CHO cells, CHO-K1 cells, Hela cells, COS-1 monkey cells, Bowes cells, mouse L-929 cells, 3T3 lines from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines, etc.

[0136] In a further embodiment, the present invention includes a method for preparing the uricase fusion protein of the present invention, comprising culturing host cells and isolating the uricase fusion protein from the culture. The uricase fusion protein, comprising a uricase domain and a random coil polypeptide domain, may be produced by recombinant DNA technology, for example, by culturing cells containing a nucleic acid construct or vector encoding the uricase fusion protein and isolating the uricase fusion protein from the culture. The uricase fusion protein may be produced in any suitable cell culture system, including prokaryotic cells, e.g., E. coli, BL21, or JM83, or eukaryotic cells, e.g., Pichia pastoris yeast strain X-33 or CHO cells. Further suitable cell lines known in the art are available from cell line depositories, such as the American Type Culture Collection (ATCC). The term “prokaryotic” includes bacterial cells, and the term “eukaryotic” includes yeast, higher plant, insect, and mammalian cells. The transformed host can be grown in a fermenter and cultured according to techniques known in the art to achieve optimal cell proliferation. In further embodiments, the present invention relates in part to a process for preparing the above-mentioned uricase fusion protein, comprising culturing the cells of the present invention under conditions suitable for the expression of the uricase fusion protein and isolating the uricase fusion protein from the cells or culture medium.

[0137] The uricase fusion protein can be isolated from growth medium, cell lysate, or cell membrane fraction. Isolation and purification of the expressed polypeptide of the present invention can be carried out by any conventional means, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc., and may include, for example, the use of monoclonal or polyclonal antibodies against the tag fused with the biologically active protein of the present invention. For example, the protein can be purified via Strep-tagII using streptavidin affinity chromatography (Skerra (2000). Methods Enzymol 326, pp. 271-304).

[0138] The cDNA encoding the conjugate can be cloned and inserted into a suitable vector for expression in a suitable host, such as E. coli or Saccharormyces cerevisiae.

[0139] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a uricase conjugate, which in some embodiments is a uricase fusion protein disclosed herein. In one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other substance well known to those skilled in the art. Such substances must be non-toxic and not interfere with the efficacy of the uricase enzyme. Examples of such substances include solvents, dispersions, antimicrobial and antifungal agents, isotonic agents and absorption retarders. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate-buffered saline, dextrose, glycerol, and ethanol, and combinations thereof. In one embodiment, the pharmaceutical composition comprises an isotonic agent, such as a sugar, and / or a polyhydric alcohol such as mannitol or sorbitol, or sodium chloride. Further examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifiers, preservatives, or buffers, which increase shelf life or efficacy.

[0140] Pharmaceutical compositions can be formulated in liquid, semi-solid, or solid forms, such as liquid solutions (e.g., injectable and injectable solutions), dispersions or suspensions, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration, therapeutic use, physicochemical properties of the uricase conjugate, and delivery route. Formulations may contain excipients, or combinations of excipients, such as sugars, amino acids, and surfactants. Liquid formulations may encompass a wide range of protein concentrations and pH. Solid formulations can be produced, for example, by freeze-drying, spray-drying, or drying using supercritical fluid technology.

[0141] For intravenous or pain site injection, the active ingredient may be in a parenterally acceptable aqueous solution that is pyrogenic and has a suitable pK, isotonicity, and stability. Those skilled in the art can easily prepare suitable solutions using isotonic vehicles such as sodium chloride solution, Ringer's solution, and lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included.

[0142] In some embodiments, the pharmaceutical composition is formulated as a suitable solution, microemulsion, dispersion, liposome dispersion, or other ordered structure containing the uricase conjugate (e.g., uricase fusion protein) described herein. A sterile injection solution may be prepared by incorporating the uricase conjugate, along with one or a combination of the components listed above, into a suitable solvent, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the uricase conjugate into a sterile vehicle containing a dispersion medium and other components from those listed above. For sterile powders for the preparation of sterile injection solutions, preparation methods include vacuum drying and freeze-drying, from a pre-sterilized filtered solution to obtain a powder of the active ingredient and any additional desired components. Appropriate fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the particle size of the dispersion, or by using a surfactant. Sustained absorption of the injection composition may be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition. In one embodiment, the uricase conjugate in the composition is a uricase fusion protein, as described herein.

[0143] In one embodiment, the pharmaceutical composition is a solution of uricase conjugate, for example, a phosphate-buffered saline solution containing one of the uricase fusion proteins described herein. In further embodiments, the solution is sterile and suitable for injection, for example, intravenous or subcutaneous injection.

[0144] In another embodiment, the disclosure provides a method for reducing elevated uric acid levels, i.e., a method for treating hyperuricemia, in subjects requiring such treatment. The method comprises administering an effective amount of uricase conjugate or a pharmaceutical composition containing the same to a subject.

[0145] As used herein, the term “subject” refers to vertebrates such as mammals. Mammals may be, for example, mice, rats, rabbits, cats, dogs, pigs, sheep, horses, non-human primates (e.g., cynomolgus macaques, chimpanzees), or humans. Human subjects may be adults, teenagers, children (2 to 14 years), infants (1 to 24 months), or neonates (up to 1 month). In one embodiment, the subject is an adult subject. In a further embodiment, the subject is an adult human subject or patient.

[0146] In one embodiment of this method, the uric acid level in the plasma or blood of a subject is reduced. In one embodiment, a preferred indicator for evaluating the effectiveness of this method includes normalization or reduction of plasma uric acid (PUA) levels, for example, reducing or maintaining PUA levels in human subjects to 6.8 mg / dL or less, or 6 mg / dL or less.

[0147] In some embodiments of this method, the administration of uricase conjugate or a pharmaceutical composition containing the same is carried out parenterally, for example, by intramuscular, intrathecal, subcutaneous, or intravenous administration. In one embodiment, the administration is intravenous. In another embodiment, the administration is subcutaneous. In yet another embodiment, the administration is rectal, topical, or pulmonary.

[0148] In one embodiment, the subject is a patient with gout. In one embodiment, the gout is recurrent gout. In another embodiment, the gout is advanced gout, accompanied by the deposition of uric acid crystals that form nodules called tophi under the skin. In human subjects, tophi can develop in several areas, such as the fingers, hands, feet, elbows, or Achilles tendons along the back of the ankles. In another embodiment, the subject with gout has kidney stones, which are uric acid crystals that have accumulated in the urinary tract.

[0149] In one embodiment, the subjects are patients with refractory gout, i.e., patients who have been resistant to different previous treatments. In one embodiment, the previous treatment is selected from nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, corticosteroids, allopurinol, febuxostat, probenecid, KRYSTEXXA® (pegroticase), rasburicase (Elitek®), or a combination of the above. In one embodiment, refractory gout is a chronic condition characterized by high serum uric acid levels, recurrent gout flare, chronic arthritis, and progressive nodular deposition. In another embodiment, refractory gout is associated with a high rate of cardiovascular and renal comorbidities.

[0150] In one embodiment, the subject is diagnosed with tumor lysis syndrome. In a further embodiment, the subject has lymphoma (e.g., Burkitt lymphoma, non-Hodgkin lymphoma), acute lymphoblastic leukemia, or acute myeloid leukemia. In one embodiment, the subject diagnosed with tumor lysis syndrome is a human subject with a plasma uric acid concentration >8 mg / dL. In another embodiment, the subject diagnosed with tumor lysis syndrome is a human subject with a plasma uric acid concentration >15 mg / dL (hyperuricemia).

[0151] In one embodiment, the disclosure provides a method for treating gout in a subject requiring treatment. The method comprises administering an effective amount of the uricase conjugate described herein or a pharmaceutical composition containing the same to the subject. In one embodiment, the gout is refractory gout. In one embodiment, the gout is recurrent gout. In another embodiment, the gout is advanced gout, characterized by the deposition of uric acid crystals forming nodules called tophi under the skin. In another embodiment, the subject has kidney stones. In one embodiment, the subject is an adult subject. In further embodiments, the subject is an adult human subject or patient.

[0152] In one embodiment, a method for treating gout is administered parenterally, for example, via intramuscular, intrathecal, subcutaneous, or intravenous administration. In one embodiment, the administration is intravenous. In another embodiment, the administration is subcutaneous. In yet another embodiment, the administration is rectal, topical, or pulmonary.

[0153] In yet another embodiment of the therapeutic method using uricase conjugate described herein, the method is a method for treating tumor lysis syndrome in a subject requiring treatment. The method comprises administering an effective amount of uricase conjugate or a pharmaceutical composition containing the same to the subject. In one embodiment, the subject is an adult subject. In further embodiments, the subject is an adult human subject or patient. In some embodiments of the method, the administration of uricase conjugate or a pharmaceutical composition containing the same is carried out parenterally, for example, by intramuscular, intrathecal, subcutaneous, or intravenous administration. In one embodiment, the administration is intravenous. In another embodiment, the administration is subcutaneous. In another embodiment, the administration is rectal, topical, or pulmonary. In some embodiments, the subject having tumor lysis syndrome has lymphoma (e.g., Burkitt lymphoma, non-Hodgkin lymphoma), acute lymphoblastic leukemia, or acute myeloid leukemia. In one embodiment, the subject having tumor lysis syndrome is a human subject with a plasma uric acid concentration >8 mg / dL. In another embodiment, subjects with tumor lysis syndrome are human subjects with a plasma uric acid concentration of >15 mg / dL (hyperuricemia). [Examples]

[0154] The present invention will be further described with reference to the following embodiments. However, it should be noted that these embodiments, like the embodiments described above, are illustrative and should not be construed as limiting the scope of the present invention in any way.

[0155] Example 1 - Preparation and characterization of uricase fusion proteins containing the uricase domain CPB40 or CPB41 uricase in combination with a PAS or XTEN polypeptide domain. This example describes the preparation and characterization of the uricase fusion proteins shown in Table 4. The uricase fusion proteins CPB40 uricase-CPAS10h (SEQ ID NO: 63), CPB40 uricase-CPAS20h (SEQ ID NO: 64), and CPB40 uricase-CPAS30h (SEQ ID NO: 65) each contain the amino acid sequence of the CPB40 uricase domain and one C-terminal PAS polypeptide domain. The uricase fusion protein CPB40 uricase-CXTENh (SEQ ID NO: 66) contains the amino acid sequence of the CPB40 uricase domain and one C-terminal XTEN polypeptide domain. The uricase fusion proteins NPAS20h-CPB41uricase-CPAS20h (SEQ ID NO: 75) and NPAS20h-CPB41uricase-CPAS30h (SEQ ID NO: 76) each contain the amino acid sequences of the CPB41 uricase domain and two PAS polypeptide domains. In each uricase fusion protein, one of the two PAS polypeptide domains is at the N-terminus and the other is at the C-terminus, and the uricase domain is located between the two PAS polypeptide domains. Since the amino acid sequences of CPB40 or CPB41 uricase are present in each protein monomer of the PEGylated homotetramer KRYSTEXXA® (pegroticase), KRYSTEXXA® (pegroticase) was used as a comparative standard in some of the tests described in this example. KRYSTEXXA® (Pegroticase) is referred to simply as "Pegroticase" in this embodiment for brevity.

[0156] [Table 5]

[0157] method 1. Production and expression of uricase fusion proteins The DNA encoding the CPB40 or CPB41 uricase domain of the fusion proteins listed in Table 4, and the DNA encoding the PAS polypeptide domain (i.e., PAS10, PAS20, and PAS30) or XTEN polypeptide domain, with or without a his tag and / or GS spacer, were synthesized by Synbio Technologies (Monmouth Junction, NJ, USA). The obtained DNA fragments encoding the uricase domain, PAS, or XTEN polypeptide domain were cloned into the expression vector pET-26b(+) (MilliporeSigma, MA, USA), digested with NdeI and BamHI using the NEBuilder® HiFi DNA Assembly Kit (New England Biolabs, Ipswich, MA, USA), to assemble the full-length coding sequences of each uricase fusion protein in the vector. The expression vector pET-26b(+) encodes a his tag containing six histidine residues at the C-terminus of the assembled full-length uricase fusion protein coding sequence. Expression of the uricase fusion protein was achieved by growing E. coli cultures transformed with the expression vector pET-26b(+) containing the full-length uricase fusion protein coding sequence, and then inducing protein expression with 100 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0158] 2. Purification The uricase fusion protein expressed in E. coli was purified to obtain a soluble (non-aggregated) tetramer by the following procedure. (1) Pelletize 0.5 L of IPTG-induced E. coli culture by centrifuging at 5,000 × g for 10 minutes. (2) Resuspend the E. coli pellet in 150 mL of 20 mM sodium borate buffer (pH 9.5). (3) The resuspended pellets are subjected to ultrasonic treatment. (4) The sonicated suspension is centrifuged at 17,000 × g for 20 minutes, and the soluble fraction is decanted. (5) Dilute the soluble fraction with 20 mM sodium borate buffer (pH 9.5) to 150 mL and stir at 4°C. (6) While stirring, slowly add 19.5 g of ammonium sulfate solid. (7) Incubate the sample from step (6) at 4°C for 2 hours. (8) The sample from step (7) is pelletized by centrifugation at 17,000 × g for 20 minutes and decanting the soluble fraction. (9) Resuspend the protein pellet from step (8) in 40 mL of 20 mM sodium borate buffer (pH 9.5). (10) Desalt the suspension from step (9) using a Zeba® spin desalting column (ThermoFisher Scientific). (11) The desalted sample from step (10) is loaded onto a 70 mL Toyopearl® NH2-750F anion exchange column (Tosoh Bioscience), and the purified sample is eluted with 140 mL of 20 mM sodium borate buffer (pH 9.5) to further purify it. (12) Using a centrifuge spin filter, the eluate from step (11) is exchanged for 1×PBS buffer. (13) The buffer-exchanged sample from step (12) is loaded onto a Superose® 6-size exclusion chromatography (SEC) column (Cytiva) to separate aggregates from the soluble tetramer (target) protein.

[0159] 3. Determination of the enzymatic activity of uricase fusion protein and pegroticase The uricase enzyme activity of uricase fusion protein and pegroticase (serial number 264790457565, lot number 0263A) was reported as specific activity at a concentration of 72.8 nM in units of μmol substrate (i.e., uric acid) / min / mg enzyme, and was determined and calculated by the following procedure. (1) Dilute the uricase fusion protein or pegroticase sample to 1456.7 nM with 1×PBS (pH 7.4). (2) In a 96-well plate, divide the diluted uricase fusion protein or pegroticase sample equally into 10 μL portions per well. (3) Add 190 μL of uric acid substrate solution containing 0.125 mM uric acid to each well to bring the final concentration of uricase fusion protein or pegroticase in the reaction mixture to 72.8 nM. (4) Load the 96-well plate into the BIOTEK Synergy® Neo2 plate reader and shake the plate for 30 seconds; (5) Using a plate reader, measure the absorbance at 293 nm every 30 seconds for 10 minutes under a controlled temperature of 25°C. (6) Calculate the Vmax value using BIOTEK Gen5 software.

[0160] 4. Differential scanning fluorescence quantification (DSF) Differential scanning fluorescence (DSF) uses the SYPRO® Orange fluorescent dye to measure the melting temperature of naturally folded protein samples. As the temperature is gradually increased using a real-time PCR instrument, the fluorescence signal at λem 570 nm also increases, indicating the exposure of hydrophobic residues and denaturation of the protein sample. By analyzing the inflection point of the resulting curve, the melting temperature (T), a measure of the protein's thermal stability, can be determined. m ) can be obtained.

[0161] T by DSF of uricase fusion protein m To determine the final concentration, the protein was diluted to a final concentration of 66–132 μg / mL in PBS buffer containing SYPRO® Orange dye (Invitrogen, catalog no. S6650) at a 5× final concentration. 30 μl of the protein-SYPRO® Orange dye mixture was added to the wells of an optical plate. The optical plate was then loaded into a BioRad C1000 Touch® Thermal Cycler with a CFX96® Real-Time System and exposed to a temperature gradient of 10°C–100°C in 0.5°C increments. Using PRISM software, Tm This was calculated using the increase and disappearance of fluorescence in SYPRO(trademark) orange.

[0162] 5. Dynamic light scattering method Uricase fusion proteins were subjected to dynamic light scattering (DLS) using a DynaPro® Plate Reader III (Waters Corporation) to determine the hydrodynamic radius and aggregation state (by polydispersity measurement) of each protein, as well as the mass percentage in various mass ranges indicating whether the protein was a soluble tetramer, oligomerized, or aggregated. Three mass ranges were selected: mass range 1 corresponds to a hydrodynamic radius of 0.5–10 nm, mass range 2 corresponds to a hydrodynamic radius of 10–100 nm, and mass range 3 corresponds to a hydrodynamic radius of 100–1000 nm. Briefly, 100 μL of protein sample with a concentration of 0.1–1 mg / mL was added to the wells of a 96-well plate. The plate was centrifuged at 2,000 × g for 5 minutes and loaded into a DynaPro® Plate Reader III. DLS data were collected with the following parameters. (1) Experiment type - isothermal, (2) Enable automatic damping - Yes (3) Imaging of each well - Yes, (4) DLS acquisition time - 10 seconds, (5) DLS acquisition for each measurement - 10, (6) SLS measurement - None (7) Measurement per well in the scan - 1, (8) Wait time between measurements within a scan - 0 minutes, (9) Number of scans - 1, (10) Wait time between scans - 0 minutes, (11) Starting temperature -25℃; (12) Plate sealant - No sealant, (13) Initial temperature lock standby - Yes (14) Set temperature -25℃, (15) Laser ON - Yes.

[0163] 6. In vivo pharmacokinetic (PK) and efficacy analysis of uricase fusion proteins and pegroticases Three PK tests were performed. The purified soluble tetrameric uricase fusion protein prepared as described in Section 2 above, as well as the pegroticase used as the control group in PK tests 1 and 2, were subjected to in vivo PK and efficacy analysis in Wistar rats.

[0164] 6.1. Administration of uricase fusion protein or pegroticase to Wistar rats and collection of rat blood samples. Female Wistar rats, each weighing approximately 220g, were purchased from Charles River. In the PK studies described herein, rats were either left untreated or treated intravenously with a single dose of 1 mg / kg body weight of pegroticase or an equimolar single dose of uricase fusion protein (equivalent to 1 mg / kg body weight of pegroticase) (n=3 rats per group). 120 μL of blood was collected from each rat at various time points before and after administration. Tables 5A-5C show the single doses (mg / kg rat body weight) of pegroticase or uricase fusion protein administered to Wistar rats, and the selected blood sample collection times for each of the three PK studies.

[0165] [Table 6]

[0166] [Table 7]

[0167] [Table 8]

[0168] 6.2. Determination of uric acid concentration in rat plasma Rat blood samples were collected as described in Section 6.1 above to obtain rat plasma, and then the plasma uric acid concentration at each time point was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Details of the quantification of uric acid concentration in rat plasma are as follows.

[0169] 6.2.1 Preparation of standard uric acid samples using artificial human plasma to establish artificial human plasma and standard curves Because endogenous uric acid is present in rat plasma, artificial human plasma was used as a surrogate matrix for preparing standard uric acid samples to establish a standard curve. Artificial human plasma was prepared by dissolving 2 g of human serum albumin (Sigma-Aldrich) in 50 mL of PBS, followed by adjusting the pH to 7.4 with 1 M sodium hydroxide or 1 M phosphoric acid. Standard uric acid samples containing 0.1, 0.2, 1, 5, 10, 60, 80, and 100 μg / mL of uric acid were prepared in the artificial human plasma and subjected to further processing and LC-MS / MS analysis in parallel with the test rat plasma samples, as described below.

[0170] 6.2.2. Processing of standard uric acid samples and rat plasma samples for LC-MS / MS The standard uric acid samples and test rat plasma samples from Section 6.2.1 were processed according to the following procedure. (1) Add 20 μl of 500 ng / mL uric acid-1,3- to a 20 μl standard uric acid sample or rat plasma sample. 15 Add an N2 aqueous solution (Sigma-Aldrich) and 40 μl of 0.4N perchloric acid. (2) Mix the samples from step (1) and centrifuge using an Eppendorf 5424R centrifuge at 15,000 rpm at 4°C for 15 minutes. (3) Take 50 μL of supernatant from each sample after centrifugation in step (2) using a pipette, and add 50 μL of water to the supernatant. (4) Mix the samples obtained in step (3) for the LC-MS / MS analysis described below.

[0171] 6.2.3. LC-MS / MS analysis of standard uric acid samples and rat plasma samples for quantitative determination of uric acid concentration. Ten μl of each standard uric acid sample or rat plasma sample, processed as described in Section 6.2.2, was injected into an Acquity UPLC® BEH amide column on a Shimadzu Nexera® LC (Sciex), followed by gradient elution. Uric acid was quantified using a triple-quad API 4500 mass spectrometer (Sciex) with ESI operating in negative mode. The parameters for the mass spectrometer and LC are shown in Tables 6A and 6B, respectively.

[0172] [Table 9]

[0173] [Table 10]

[0174] LC-MS / MS data were collected using data acquisition software Analyst® 1.7 and MultiQuant® 3.0 (Sciex) or equivalents. Standard curves were created using data from standard uric acid samples. Calibration curves were created by plotting the peak area ratio of the reference standard and the internal standard against the nominal concentration of the existing reference standard. The calibration curves were fitted using least-squares regression analysis to obtain information on the slope of the calibration curve, the y-intercept, the correlation coefficient, and the inversely calculated calibration standard concentration.

[0175] Calibration curves obtained using standard uric acid samples prepared with artificial human plasma were shown using the standard addition method to accurately measure uric acid concentrations in rat plasma. In the standard addition method, untreated rat plasma samples with uric acid added at three levels (5, 25, and 80 μg / mL), as well as a blank untreated rat plasma sample (BioIVT), were processed and analyzed by LC-MS / MS as described above. Using the standard curves obtained from the standard uric acid samples prepared with artificial human plasma, the endogenous uric acid levels in the blank untreated rat plasma and the uric acid levels in the three standard-added (i.e., uric acid-added) rat plasma samples were determined. The adjusted uric acid levels in the three standard-added samples, after subtracting the endogenous uric acid level, were then compared to their target (added) concentrations. The percentage difference between the adjusted uric acid concentration and the corresponding target uric acid concentration was found to be less than 15%, indicating that the acceptable standard was met.

[0176] 6.3 Determination of rat plasma concentrations of uricase fusion protein and pegroticase Rat blood samples collected as described in Section 6.1 above were processed to obtain rat plasma, and subsequently, the plasma concentrations of uricase fusion protein or pegroticase at each time point were determined using the Amplex® Red Uric Acid / Uricase Assay Kit (ThermoFisher Scientific, catalog number A22181). This assay kit provides a two-step fluorescence quantification method for quantifying uricase fusion protein or pegroticase based on uricase enzyme activity. First, the uricase enzyme activity of uricase fusion protein or pegroticase converts uric acid into allantoin, hydrogen peroxide (H2O2), and carbon dioxide. Second, in the presence of horseradish peroxidase (HRP), H2O2 reacts stoichiometrically with the Amplex Red reagent to produce resorphine, a red fluorescent oxidation product. In this example, the rat plasma concentrations of uricase fusion protein or pegroticase were indirectly quantified by measuring the fluorescence of resolphins captured by a SpectraMax® miniplate reader (Molecular Devices) in kinetic mode for 30 minutes. A standard curve for quantifying uricase fusion protein or pegroticase was created using rat plasma from the same strain. Using the IDBS Excel add-in software XLift®, standard curve regression was performed using an equilibrium-state model to inversely calculate the concentrations of uricase fusion protein or pegroticase in the rat plasma samples.

[0177] Rat plasma contained endogenous uricase enzymes, which also produced a fluorescent signal in the uricase assay. Furthermore, endogenous rat plasma uricase levels varied among rats and over time within a single rat, making it impossible to determine the true background uricase for a given sample. Therefore, the determination of the plasma concentration of exogenously administered uricase fusion protein or pegroticase in the test sample was approximated by subtracting the mean pre-administration background (endogenous) plasma uricase concentration for each uricase fusion protein or pegroticase-treated rat group. The corresponding plasma concentrations of endogenous uricase in the untreated control rat group at pre-administration (time 0) and post-administration time points were similarly adjusted by subtracting the mean pre-administration endogenous plasma uricase concentration of the control rat group.

[0178] result 1. Analysis by sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) The uricase fusion proteins listed in Table 4 were prepared as described in "Methods," expressed in E. coli, and purified as soluble tetramers. The purified uricase fusion proteins were analyzed together with pegroticase by SDS-PAGE under non-reducing conditions. Figures 2A and 2B are representative images of SDS-PAGE gels showing bands representing monomeric uricase fusion protein or monomeric pegroticase detected by Coomassie brilliant blue staining.

[0179] Figure 2A shows the protein bands detected in samples containing NPAS20h-CPB41 uricase-CPAS20h, NPAS20h-CPB41 uricase-CPAS30h, and pegroticase, respectively, in lanes 1, 2, and 3. These samples were used in PK test 2 of this example.

[0180] Figure 2B shows the protein bands detected in lanes 1 and 2, respectively, for samples containing CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h. These samples were used in PK test 3 of this example.

[0181] NPAS20h-CPB41 uricase-CPAS20h has an expected monomer molecular weight of 102650 Da. NPAS20h-CPB41 uricase-CPAS30h has an expected monomer molecular weight of 119168 Da. CPB40 uricase-CPAS20h has an expected monomer molecular weight of 68328 Da. Including the molecular weight standards shown in the rightmost lane, the SDS-PAGE gels in Figures 2A and 2B revealed that the apparent size of the monomeric uricase fusion proteins, based on the protein migration position, was significantly larger than their corresponding expected sizes. This observation is consistent with the findings by Breibeck et al. that "the [PAS polypeptide] fusion proteins with IL-1Ra [interleukin-1 receptor antagonist] and TrxA [E. coli thioredoxin] migrated to positions corresponding to much higher molecular weights than normally expected." Breibeck et al. hypothesized that "[the delay in the electrophoretic mobility of PAS-modified proteins] is largely explained by the weak binding of SDS, which provides a driving force in the electric field, likely due to the absence of hydrophobic amino acid side chains. See Breibeck et al., "The polypeptide biophysics of proline / alanine-rich sequences (PAS): Recombinant biopolymers with PEG-like properties," Biopolymers. 2018 Jan;109(1):e23069, p. 3, right column, first paragraph.

[0182] 2. In vivo PK and efficacy analysis of uricase fusion protein compared to pegroticase 2.1. Effectiveness analysis of plasma uric acid concentration in rat blood samples from PK test 1 and the test In PK Test 1, female Wistar rats in groups of three rats were either left untreated or treated via single intravenous administration of 1 mg / kg body weight pegroticase, 1.51 mg / kg body weight CPB40 uricase-CPAS10h, 1.99 mg / kg body weight CPB40 uricase-CPAS20h, 2.47 mg / kg body weight CPB40 uricase-CPAS30h, or 2.16 mg / kg body weight CPB40 uricase-CXTENh (Table 5A). Rat blood samples were collected before administration and at 0.5, 2, 6, 24, 72, and 96 hours after administration. Plasma concentrations of endogenous uricase in untreated control rats, or plasma concentrations of pegroticase and uricase fusion protein, and plasma concentrations of uric acid in treated rats were determined as described in "Methods". The results are shown in Figures 3A to 3F, where the endogenous uricase in rat plasma or the exogenously administered pegroticase and uricase fusion protein are generally referred to as "plasma uricase."

[0183] Figure 3A shows the plasma concentrations of endogenous uricase and uric acid in untreated control rats at the corresponding pre-administration time point (0 hours) and various corresponding post-administration time points up to 96 hours. The plasma concentration of endogenous uricase at time 0 was adjusted to 0 μg / mL by subtracting the mean pre-administration endogenous plasma uricase concentration, as described in the "Methods" section. At the corresponding post-administration time points, the similarly adjusted plasma concentrations of endogenous uricase varied, either below or above 0 μg / mL, as described in the "Methods" section. Plasma concentrations of uric acid in untreated control rats also varied and were greater than 0 μg / mL at all time points.

[0184] Figure 3B shows the plasma concentrations of pegroticase and uric acid in pegroticase-treated rats at various time points before administration (time 0) and up to 96 hours after administration. Plasma concentrations of pegroticase plateaued between 0.5 and 6 hours post-administration and remained above detectable levels until at least 96 hours post-administration, while plasma concentrations of uric acid decreased to 0 μg / ml at all time points tested post-administration, i.e., between 0.5 and 96 hours post-administration, and remained at 0 μg / ml.

[0185] Figure 3C shows the plasma concentrations of CPB40 uricase-CPAS10h and uric acid at various time points before administration (time 0) and up to 96 hours after administration in rats treated with CPB40 uricase-CPAS10h. Plasma concentrations of CPB40 uricase-CPAS10h peaked by 0.5 hours after administration and then decreased to below the detection limit by 24 hours after administration. Plasma concentrations of uric acid decreased to 0 μg / ml from 0.5 hours to 24 hours after administration, remained at 0 μg / ml, and then rebounded to values ​​above 0 μg / ml between 24 hours and 72 hours after administration.

[0186] Figure 3D shows the plasma concentrations of CPB40 uricase-CPAS20h and uric acid at various time points before administration (time 0) and up to 96 hours after administration in CPB40 uricase-CPAS20h-treated rats. Plasma concentrations of CPB40 uricase-CPAS20h peaked by 0.5 hours post-administration, remained at high levels until 24 hours post-administration, and then decreased to below the detection limit by 72 hours post-administration. Plasma concentrations of uric acid decreased to 0 μg / ml from 0.5 hours to 24 hours post-administration, were maintained at 0 μg / ml, and then rebounded to values ​​above 0 μg / ml between 24 hours and 72 hours post-administration, similar to the plasma uric acid profile in CPB40 uricase-CPAS10h-treated rats.

[0187] Figure 3E shows the plasma concentrations of CPB40 uricase-CPAS30h and uric acid at various time points before administration (time 0) and up to 96 hours after administration in CPB40 uricase-CPAS30h-treated rats. The changes in plasma concentrations of CPB40 uricase-CPAS30h and uric acid after administration were similar to those in CPB40 uricase-CPAS20h-treated rats. Specifically, the plasma concentration of CPB40 uricase-CPAS30h peaked by 0.5 hours after administration, remained at high or detectable levels until 24 hours after administration, and then decreased to below the detection limit by 72 hours after administration. The plasma concentration of uric acid decreased to 0 μg / ml from 0.5 hours to 24 hours after administration, was maintained at 0 μg / ml, and then rebounded to values ​​above 0 μg / ml between 24 hours and 72 hours after administration.

[0188] Figure 3F shows the plasma concentrations of CPB40 uricase-CXTENh and uric acid at various time points before administration (0 hours) and up to 96 hours after administration in CPB40 uricase-CXTENh-treated rats. Plasma concentrations of CPB40 uricase-CXTENh peaked by 0.5 hours after administration and then continuously decreased to below the detection limit by 72 hours after administration. The rate of decrease was faster than that observed in CPB40 uricase-CPAS20h-treated rats (see Figure 3D). Plasma concentrations of uric acid decreased to 0 μg / ml from 0.5 hours to 24 hours after administration, remained at 0 μg / ml, and then rebounded to values ​​above 0 μg / ml between 24 hours and 72 hours after administration, similar to the plasma uric acid profile in CPB40 uricase-CPAS20h-treated rats.

[0189] 2.2. Effectiveness analysis of plasma uric acid concentration in rat blood samples from PK test 2 and the test In PK Test 2, female Wistar rats in groups of three rats were either left untreated or treated via single intravenous administration of 1 mg / kg body weight pegroticase, 2.99 mg / kg body weight NPAS20h-CPB41 uricase-CPAS20h, or 3.47 mg / kg body weight NPAS20h-CPB41 uricase-CPAS30h (Table 5B). Rat blood samples were collected before administration and at 0.5, 1, 2, 6, 24, 48, 72, 96, 120, and 168 hours after administration. Plasma concentrations of endogenous uricase in untreated control rats, or plasma concentrations of pegroticase and uricase fusion protein, as well as plasma uric acid concentrations in treated rats, were determined as described in "Methods". The results are shown in Figures 4A to 4D, where the endogenous uricase in rat plasma or the exogenously administered pegroticase and uricase fusion protein are generally referred to as "plasma uricase."

[0190] Figure 4A shows the plasma concentrations of endogenous uricase and uric acid in untreated control rats at the corresponding pre-administration time point (time 0) and various corresponding post-administration time points up to 168 hours. The plasma concentration of endogenous uricase at time 0 was adjusted to approximately 0 μg / mL by subtracting the mean pre-administration endogenous plasma uricase concentration, as described in "Methods". At the corresponding post-administration time points, the similarly adjusted plasma concentrations of endogenous uricase fluctuated relative to the baseline concentration at time 0. Plasma concentrations of uric acid in untreated control rats also fluctuated and were greater than 0 μg / mL at all time points. These results were similar to those observed in untreated control rats in PK Test 1, shown in Figure 3A.

[0191] Figure 4B shows the plasma concentrations of pegroticase and uric acid in pegroticase-treated rats at various time points before administration (time 0) and up to 168 hours after administration. Plasma concentrations of pegroticase peaked at 0.5 hours after administration and remained above detectable levels until at least 168 hours after administration. In two of the three pegroticase-treated rats, plasma uric acid concentrations decreased to 0 μg / ml up to 168 hours after administration and remained at 0 μg / ml. However, in the third pegroticase-treated rat, plasma uric acid concentrations rebounded to low levels above 0 μg / ml only at 168 hours, and did not rebound at 120 hours.

[0192] Figure 4C shows the plasma concentrations of NPAS20h-CPB41 uricase-CPAS20h and uric acid at various time points before administration (time 0) and up to 168 hours after administration in NPAS20h-CPB41 uricase-CPAS20h-treated rats. The changes in plasma concentrations of NPAS20h-CPB41 uricase-CPAS20h and uric acid after administration were similar to those in pegroticase-treated rats. Specifically, the plasma concentration of NPAS20h-CPB41 uricase-CPAS20h plateaued between 0.5 and 2 hours after administration and remained above detectable levels until at least 168 hours after administration. In two of the three NPAS20h-CPB41 uricase-CPAS20h treated rats, plasma uric acid concentrations decreased to 0 μg / ml up to 168 hours after administration and remained at 0 μg / ml. However, in the third NPAS20h-CPB41 uricase-CPAS20h treated rat, plasma uric acid concentrations rebounded to a low level above 0 μg / ml only at 168 hours, and did not rebound at 120 hours.

[0193] Figure 4D shows the plasma concentrations of NPAS20h-CPB41 uricase-CPAS30h and uric acid at various time points before administration (time 0) and up to 168 hours after administration in NPAS20h-CPB41 uricase-CPAS30h treated rats. The plasma concentration of NPAS20h-CPB41 uricase-CPAS30h peaked approximately 1 hour after administration, remained at a high level until 6 hours after administration, and then decreased to a low steady state level near the limit of detection at 72 hours and thereafter. The plasma concentration of uric acid decreased to 0 μg / ml up to 72 hours after administration, remained at 0 μg / ml, and then rebounded to above 0 μg / ml at 96 hours and thereafter.

[0194] 2.3. Effectiveness analysis of plasma uric acid concentration in rat blood samples from PK test 3 and the test In PK Test 3, the uricase fusion proteins CPB40 uricase-CPAS20h tested in PK Test 1 and NPAS20h-CPB41 uricase-CPAS20h tested in PK Test 2 were compared side by side. Specifically, female Wistar rats from each group of three rats were either left untreated or treated via a single intravenous dose of either 1.99 mg / kg body weight of CPB40 uricase-CPAS20h or 2.99 mg / kg body weight of NPAS20h-CPB41 uricase-CPAS20h (Table 5C). Rat blood samples were collected before administration and at 1, 6, 24, 48, 72, 96, 120, 168, 192, 216, and 240 hours after administration. As described in "Methods," plasma concentrations of endogenous uricase in untreated control rats, or uricase fusion proteins (i.e., CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h) in treated rats, as well as plasma concentrations of uric acid, were determined. The results are shown in Figures 5A to 5C, where the above-mentioned endogenous uricase or exogenously administered uricase fusion proteins in rat plasma are generally referred to as "plasma uricase."

[0195] Figure 5A shows the plasma concentrations of endogenous uricase and uric acid in untreated control rats at the corresponding pre-administration time point (time 0) and various corresponding post-administration time points up to 240 hours. The plasma concentration of endogenous uricase at time 0 was adjusted to approximately 0 μg / mL by subtracting the mean pre-administration endogenous plasma uricase concentration, as described in "Methods." At the corresponding post-administration time points, the similarly adjusted plasma concentrations of endogenous uricase fluctuated relative to the baseline concentration at time 0. Plasma concentrations of uric acid in untreated control rats also fluctuated and were greater than 0 μg / mL at all time points. These results were similar to those observed in untreated control rats in PK studies 1 and 2, shown in Figures 3A and 4A, respectively.

[0196] Figure 5B shows the plasma concentrations of CPB40 uricase-CPAS20h and uric acid at various time points before administration (time 0) and up to 240 hours after administration in CPB40 uricase-CPAS20h-treated rats. Plasma concentrations of CPB40 uricase-CPAS20h peaked at 1 hour post-administration (the earliest post-administration time point tested in this study), remained at high levels until 24 hours post-administration, and then decreased to below the detection limit between 72 and 96 hours post-administration. Plasma concentrations of uric acid decreased to 0 μg / ml from 1 to 72 hours post-administration, remained at 0 μg / ml, and then rebounded to above 0 μg / ml between 72 and 96 hours post-administration. The performance of CPB40 uricase-CPAS20h at 72 hours post-administration appeared to be improved in this study compared to its performance at the same time point (i.e., 72 hours post-administration) in PK Study 1 (see Figure 3D), which is likely due to the higher quality of the uricase fusion protein produced for this PK Study (i.e., PK Study 3).

[0197] Figure 5C shows the plasma concentrations of NPAS20h-CPB41 uricase-CPAS20h and uric acid at various time points before administration (time 0) and up to 240 hours after administration in NPAS20h-CPB41 uricase-CPAS20h-treated rats. Plasma concentrations of NPAS20h-CPB41 uricase-CPAS20h plateaued between 1 hour (the earliest post-administration time point tested in this study) and 6 hours after administration and remained above detectable levels until at least 168 hours after administration. Plasma concentrations of uric acid decreased to 0 μg / ml up to 120 hours after administration in all three NPAS20h-CPB41 uricase-CPAS20h-treated rats and remained thereafter. 168 hours after administration, plasma uric acid concentration remained at 0 μg / ml in one of the three treated rats, but rebounded to low levels above 0 μg / ml in the other two treated rats in the group. Therefore, the PK and efficacy results for NPAS20h-CPB41 uricase-CPAS20h in this study were generally similar to those observed in PK Study 2 (see Figure 4C).

[0198] In summary, the results of the three PK and efficacy studies described above indicate that plasma uric acid concentrations in untreated control rats varied from rat to rat and at different time points in the study. Therefore, it was not useful to interpret precise plasma uric acid concentration values ​​after treatment with uricase fusion protein or pegroticase. However, the test results demonstrate a binary correlation between plasma uric acid concentrations of 0 μg / ml versus >0 μg / ml and between the presence of circulating exogenous uricase enzyme (i.e., uricase fusion protein or pegroticase) versus its clearance. Specifically, plasma uric acid concentrations of 0 μg / ml correlated with or indicated a detectable level of circulating exogenous uricase enzyme, while plasma uric acid concentrations rebounding above 0 μg / ml correlated with or indicated the elimination of exogenously administered uricase enzyme.

[0199] Aliquots of purified soluble tetrameric uricase fusion protein samples for use in PK tests 2 and 3 should be prepared as described below for uricase enzyme activity assays and protein melting temperature (T m Further in vitro characterization was performed, including differential scanning fluorescence (DSF) to determine the protein's properties, and dynamic light scattering (DLS) to determine the protein's hydrodynamic radius and aggregation state.

[0200] 3. Uricase enzyme activity of uricase fusion protein compared to pegroticase Aliquotes of two purified soluble tetrameric uricase fusion protein samples, each containing NPAS20h-CPB41uricase-CPAS20h and NPAS20h-CPB41uricase-CPAS30h, respectively, for use in PK Test 2, as well as an aliquot of pegroticase, were subjected to a uricase enzyme activity assay at a protein concentration of 72.8 nM, as described in "Methods," and the results are shown in Table 7A. The uricase enzyme activity of each sample was measured three times. The mean and standard deviation (SD) of the three measurements were calculated for each sample and are shown in Table 7A.

[0201] [Table 11]

[0202] The data in Table 7A show that the uricase fusion proteins NPAS20h-CPB41uricase-CPAS20h and NPAS20h-CPB41uricase-CPAS30h, each tested in PK Test 2, exhibited over 80% of the pegroticase activity in vitro.

[0203] Aliquotes of two purified soluble tetrameric uricase fusion protein samples, each containing CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h, respectively, for use in PK Test 3, as well as an aliquot of pegroticase, were subjected to a uricase enzyme activity assay at a protein concentration of 72.8 nM, as described in "Methods," and the results are shown in Table 7B. The uricase enzyme activity of each sample was measured three times. The mean and standard deviation (SD) of the three measurements were calculated for each sample and are shown in Table 7B.

[0204] [Table 12]

[0205] The data in Table 7B show that each of the uricase fusion proteins tested in PK Test 3, CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h, also exhibited more than 80% of the pegroticase activity in vitro.

[0206] 4. Melting temperature of uricase fusion protein (T m ) Aliquots of two purified soluble tetrameric uricase fusion protein samples, each containing NPAS20h-CPB41uricase-CPAS20h and NPAS20h-CPB41uricase-CPAS30h, respectively, for use in PK Test 2, were subjected to DSF as described in "Methods" to determine the melting temperature of the uricase fusion protein. The results are shown in Table 8A. Melting temperature T of each sample m The measurement was performed twice. For each sample, the mean and standard deviation (SD) of the two measurements were calculated and are shown in Table 8A.

[0207] [Table 13]

[0208] The data in Table 8A shows that the urease fusion proteins NPAS20h-CPB41 urease-CPAS20h and NPAS20h-CPB41 urease-CPAS30h, which were tested in PK test 2, had the same T of 63.5 °C, m higher than the 61 °C T of the historical PEGylated CPB40 urease determined in previous tests. m

[0209] Aliquots of two purified soluble tetrameric urease fusion protein samples containing CPB40 urease-CPAS20h and NPAS20h-CPB41 urease-CPAS20h respectively, for use in PK test 3, were similarly subjected to DSF as described in the "Method" to determine the melting temperature of the urease fusion proteins, and the results are shown in Table 8B. The melting temperature T m of each sample was measured twice. The mean and standard deviation (SD) of the two measurements for each sample were calculated and shown in Table 8B.

[0210]

Table 14

[0211] The data in Table 8B shows that the urease fusion proteins CPB40 urease-CPAS20h and NPAS20h-CPB41 urease-CPAS20h, which were tested in PK test 3, had melting temperatures of 63.00 °C and 63.50 °C respectively, and both of these were higher than the 61 °C T m of the historical PEGylated CPB40 urease.

[0212] 5. DLS analysis of urease fusion proteins Aliquots of two purified soluble tetrameric uricase fusion protein samples, each containing NPAS20h-CPB41uricase-CPAS20h and NPAS20h-CPB41uricase-CPAS30h, respectively, for use in PK Test 2, were subjected to DLS as described in "Methods" to determine the hydrodynamic radius, polydispersity, and mass percentage of the uricase fusion protein over various mass ranges. The results are shown in Table 9A.

[0213] [Table 15]

[0214] The DLS data in Table 9A shows that NPAS20h-CPB41 uricase-CPAS20h had a hydrodynamic radius of 17.65 nm (with a polydispersity of 7.3%), and NPAS20h-CPB41 uricase-CPAS30h had a hydrodynamic radius of 17.2 nm (with a polydispersity of 14.3%).

[0215] Furthermore, NPAS20h-CPB41 uricase-CPAS30h appeared to possess two distinct species: one with the expected hydrodynamic radius (approximately 17 nm, 46.85% mass in mass range 2), and another smaller than the tetrameric species (52.45% mass in mass range 1). These data suggest that the NPAS20h-CPB41 uricase-CPAS30h sample may contain a population of stabilized monomeric proteins distinct from the tetrameric population, and that the presence of this monomeric protein population may explain the shorter half-life of the NPAS20h-CPB41 uricase-CPAS30h sample compared to the NPAS20h-CPB41 uricase-CPAS20h sample observed in PK test 2 (see comparison with Figures 4C and 4D).

[0216] Aliquots of two purified soluble tetrameric uricase fusion protein samples, each containing CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h, respectively, for use in PK Test 3, were subjected to DLS as described in "Methods" to determine the hydrodynamic radius, polydispersity, and mass percentage of the uricase fusion protein in mass range 2 (corresponding to a hydrodynamic radius of 10–100 nm), and the results are shown in Table 9B. Each DLS parameter for each sample was measured once or twice (indicated as "1" and "2" in Table 9B), and the average of the two measurements for each parameter was calculated and is shown in Table 9B.

[0217] [Table 16]

[0218] The DLS data in Table 9B shows that both the CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h samples had >99% mass and <1% aggregation in mass range 2, which corresponds to a hydrodynamic radius of 10–100 nm. Furthermore, the finding that the hydrodynamic radius of NPAS20h-CPB41 uricase-CPAS20h is larger than that of CPB40 uricase-CPAS20h indicates that the hydrodynamic radius increases with increasing length of the PAS domain in the uricase fusion protein.

[0219] While the described invention is described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications can be made to adopt specific circumstances, materials, substance compositions, processes, and process steps to suit the spirit and scope of the described invention. All such modifications are intended to fall within the claims appended herein.

[0220] The patents, patent applications, patent publications, academic papers, and protocols referenced herein are incorporated in their entirety for all purposes.

Claims

1. A uricase conjugate comprising a first domain and a second domain, wherein the first domain comprises a uricase polypeptide or an amino acid variant thereof, and the second domain is a first random coil polypeptide domain comprising at least about 100 amino acids.

2. The uricase conjugate according to claim 1, wherein the uricase conjugate is a fusion protein of the first domain and the second domain.

3. The uricase conjugate according to claim 2, wherein the first domain is located on the C-terminal side of the second domain.

4. The uricase conjugate according to claim 2, wherein the first domain is located at the N-terminus of the second domain.

5. The uricase conjugate according to any one of claims 2 to 4, wherein an amino acid linker is present between the first domain and the second domain.

6. The uricase conjugate according to claim 5, wherein the amino acid linker is approximately 2 amino acid lengths to approximately 5 amino acid lengths.

7. The uricase conjugate according to claim 6, wherein the amino acid linker is 2 amino acid length.

8. The uricase conjugate according to claim 7, wherein the amino acid linker is Gly-Ser.

9. The uricase conjugate according to any one of claims 2 to 8, further comprising a third domain, wherein the third domain comprises a second random coil polypeptide domain comprising at least about 100 amino acids.

10. The uricase conjugate according to claim 9, wherein the first domain is located on the N-terminal side of the second domain and on the C-terminal side of the third domain.

11. The uricase conjugate according to claim 10, wherein an amino acid linker is present between the first domain and the second domain.

12. The uricase conjugate according to claim 10 or 11, wherein an amino acid linker is present between the second domain and the third domain.

13. The uricase conjugate according to claim 11 or 12, wherein the amino acid linker is approximately 2 amino acid lengths to approximately 5 amino acid lengths.

14. The uricase conjugate according to claim 13, wherein the amino acid linker is 2 amino acid length.

15. The uricase conjugate according to claim 14, wherein the amino acid linker is Gly-Ser.

16. The uricase conjugate according to any one of claims 1 to 15, wherein the second domain comprises a Pro-Ala-Ser (PAS) polypeptide.

17. The uricase conjugate according to any one of claims 9 to 15, wherein the third domain comprises a Pro-Ala-Ser (PAS) polypeptide.

18. The uricase conjugate according to claim 16 or 17, wherein the PAS polypeptide has the amino acid sequence described in SEQ ID NO:

60.

19. The uricase conjugate according to claim 16 or 17, wherein the PAS polypeptide has the amino acid sequence described in SEQ ID NO:

61.

20. The uricase conjugate according to claim 16 or 17, wherein the PAS polypeptide has the amino acid sequence described in SEQ ID NO:

62.

21. The uricase conjugate according to claim 16 or 17, wherein the PAS polypeptide comprises the amino acid sequence described in SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, or SEQ ID NO:

58.

22. The uricase conjugate according to claim 21, wherein the PAS polypeptide comprises the amino acid sequence of SEQ ID NO:

48.

23. The uricase conjugate according to claim 22, wherein the amino acid sequence of SEQ ID NO: 48 is encoded by a nucleotide sequence selected from SEQ ID NOs: 81 to 111.

24. The uricase conjugate according to any one of claims 1 to 15, wherein the second domain comprises an elongated recombinant (XTEN) polypeptide.

25. The uricase conjugate according to any one of claims 9 to 15, wherein the third domain comprises an elongated recombinant (XTEN) polypeptide.

26. The uricase conjugate according to claim 24 or 25, wherein the XTEN polypeptide has the amino acid sequence described in SEQ ID NO:

74.

27. The uricase conjugate according to any one of claims 1 to 26, wherein the first random coil polypeptide comprises about 100 amino acids to about 800 amino acids.

28. The uricase conjugate according to any one of claims 9 to 27, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 800 amino acids.

29. The uricase conjugate according to any one of claims 1 to 28, wherein the first random coil polypeptide comprises about 100 amino acids to about 700 amino acids.

30. The uricase conjugate according to any one of claims 9 to 29, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 700 amino acids.

31. The uricase conjugate according to any one of claims 1 to 30, wherein the first random coil polypeptide comprises about 100 amino acids to about 600 amino acids.

32. The uricase conjugate according to any one of claims 9 to 31, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 600 amino acids.

33. The uricase conjugate according to any one of claims 1 to 32, wherein the first random coil polypeptide comprises about 100 amino acids to about 500 amino acids.

34. The uricase conjugate according to any one of claims 9 to 33, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 500 amino acids.

35. The uricase conjugate according to any one of claims 1 to 34, wherein the first random coil polypeptide comprises about 100 amino acids to about 400 amino acids.

36. The uricase conjugate according to any one of claims 9 to 35, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 400 amino acids.

37. The uricase conjugate according to any one of claims 1 to 36, wherein the first random coil polypeptide comprises about 100 to about 300 amino acids.

38. The uricase conjugate according to any one of claims 9 to 37, wherein the second random coil polypeptide domain comprises about 100 amino acids to about 300 amino acids.

39. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

63.

40. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

64.

41. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

65.

42. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

66.

43. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

67.

44. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

68.

45. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

69.

46. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

70.

47. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

75.

48. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

76.

49. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

77.

50. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

78.

51. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

79.

52. The uricase conjugate according to claim 1, comprising the amino acid sequence described in Sequence ID No.

80.

53. The uricase conjugate according to claim 1, comprising the amino acid sequence described in SEQ ID NO:

112.

54. The uricase conjugate according to claim 1, comprising the amino acid sequence described in SEQ ID NO:

113.

55. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in Sequence ID No.

1.

56. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

2.

57. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

3.

58. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

4.

59. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

5.

60. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises an amino acid sequence selected from SEQ ID NOs: 6 to 39.

61. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises an amino acid sequence selected from SEQ ID NOs: 40 to 44.

62. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

40.

63. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

41.

64. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

42.

65. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

43.

66. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

44.

67. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

45.

68. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

46.

69. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises the amino acid sequence described in SEQ ID NO:

47.

70. The uricase conjugate according to any one of claims 1 to 38, wherein the uricase domain comprises an amino acid variant of the uricase polypeptide.

71. The uricase conjugate according to claim 70, wherein the amino acid variant has an amino acid sequence that is at least about 75% identical to the uricase polypeptide.

72. The uricase conjugate according to claim 70, wherein the amino acid variant has an amino acid sequence that is at least about 80% identical to the uricase polypeptide.

73. The uricase conjugate according to claim 70, wherein the amino acid variant has an amino acid sequence that is at least about 85% identical to the uricase polypeptide.

74. The uricase conjugate according to claim 70, wherein the amino acid variant has an amino acid sequence that is at least about 90% identical to the uricase polypeptide.

75. The uricase conjugate according to claim 70, wherein the amino acid variant has an amino acid sequence that is at least about 95% identical to the uricase polypeptide.

76. The uricase conjugate according to any one of claims 70 to 75, wherein the amino acid variant is a variant of the uricase polypeptide having the amino acid sequence described in SEQ ID NO:

40.

77. The uricase conjugate according to any one of claims 70 to 75, wherein the amino acid variant is a variant of the uricase polypeptide having the amino acid sequence described in SEQ ID NO:

41.

78. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 10 to about 20 amino acid substitutions in the uricase polypeptide.

79. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 10 to about 18 amino acid substitutions in the uricase polypeptide.

80. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 10 to about 16 amino acid substitutions in the uricase polypeptide.

81. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 10 to about 14 amino acid substitutions in the uricase polypeptide.

82. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 10 to about 13 amino acid substitutions in the uricase polypeptide.

83. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 10 to about 12 amino acid substitutions in the uricase polypeptide.

84. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 11 to about 20 amino acid substitutions in the uricase polypeptide.

85. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 12 to about 20 amino acid substitutions in the uricase polypeptide.

86. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 13 to about 20 amino acid substitutions in the uricase polypeptide.

87. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 14 to about 20 amino acid substitutions in the uricase polypeptide.

88. The uricase conjugate according to claim 70, wherein the amino acid variant comprises about 15 to about 20 amino acid substitutions in the uricase polypeptide.

89. The uricase conjugate according to any one of claims 78 to 88, wherein the amino acid variant is a variant of the uricase polypeptide having the amino acid sequence described in SEQ ID NO:

40.

90. The uricase conjugate according to any one of claims 78 to 88, wherein the amino acid variant is a variant of the uricase polypeptide having the amino acid sequence described in SEQ ID NO:

41.

91. The uricase conjugate according to any one of claims 2 to 90, wherein the uricase domain does not contain an N-terminal methionine residue.

92. The uricase conjugate according to any one of claims 2 to 91, wherein the first random coil polypeptide domain does not contain an N-terminal methionine residue.

93. The uricase conjugate according to any one of claims 9 to 92, wherein the second random coil polypeptide domain does not contain an N-terminal methionine residue.

94. The uricase conjugate according to any one of claims 2 to 93, further comprising a purification tag at the C-terminus.

95. The uricase conjugate according to any one of claims 2 to 94, further comprising a purification tag at the N-terminus.

96. The uricase conjugate according to claim 94 or 95, wherein the purification tag is a polyhistidine tag.

97. The uricase conjugate according to any one of claims 1 to 96, wherein the uricase conjugate is a monomer.

98. The uricase conjugate according to any one of claims 1 to 97, wherein the uricase conjugate is present in a homotetramer.

99. The uricase conjugate according to any one of claims 1 to 96, wherein the uricase conjugate is a homotetramer.

100. A nucleic acid encoding a uricase conjugate according to any one of claims 1 to 99.

101. A nucleic acid vector comprising the nucleic acid described in claim 100.

102. A host cell comprising the nucleic acid vector according to claim 101.

103. A pharmaceutical composition comprising uricase conjugate according to any one of claims 1 to 99.

104. A method for treating hyperuricemia in a subject requiring treatment, comprising administering to the subject an effective amount of uricase conjugate according to any one of claims 1 to 99, or the pharmaceutical composition according to claim 103.

105. The method according to claim 104, wherein the uric acid level decreases in the plasma of the subject.

106. The method according to claim 104 or 105, wherein the subject is a patient with gout.

107. The method according to claim 106, wherein the subject is a patient with intractable gout.

108. The method according to any one of claims 104 to 107, wherein the subject is diagnosed with tumor lysis syndrome.

109. A method for treating gout in a subject requiring treatment, comprising administering to the subject an effective amount of uricase conjugate according to any one of claims 1 to 99 or the pharmaceutical composition according to claim 103.

110. The method according to claim 109, wherein the gout is intractable gout.

111. A method for treating tumor lysis syndrome in a subject requiring treatment, comprising administering to the subject an effective amount of uricase conjugate according to any one of claims 1 to 99 or the pharmaceutical composition according to claim 103.

112. The method according to any one of claims 104 to 111, wherein the subject is a human patient.

113. The method according to claim 112, wherein the human patient is an adult human patient.

114. The method according to any one of claims 104 to 113, wherein administration includes parenteral administration.

115. The method according to claim 114, wherein the parenteral administration includes intravenous administration.

116. The method according to claim 114, wherein the parenteral administration includes subcutaneous administration.

117. A method for recombinantly producing a uricase conjugate according to any one of claims 1 to 99, (i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding a uricase conjugate according to any one of claims 1 to 99, wherein the nucleic acid sequence is operably linked to a heterologous promoter under conditions that enable expression of the nucleic acid sequence encoding the uricase conjugate and recombinant production of the uricase conjugate by the host cell, (ii) A method comprising isolating a uricase conjugate produced by recombinant.

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