Uricase conjugates and methods of use thereof

Abstract

The present invention provides novel uricase conjugates 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. The invention also provides methods of using the uricase conjugates of the invention, e.g., for the treatment of tumor lysis syndrome and / or gout.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 386,660, filed on December 8, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Reference to Electronic Sequence Listing

[0004] The content of the electronic sequence listing (INMD_18601WO_SeqList_ST26.xml; size: 184,960 bytes; creation date: December 7, 2023) is incorporated herein by reference in its entirety. Background Art

[0005] In humans, uric acid is produced after the breakdown of purines. The accumulation of uric acid in the blood (hyperuricemia) manifests in diseases such as gout and tumor lysis syndrome.

[0006] Gout is a common and complex form of arthritis, characterized by sudden, severe pain attacks, swelling, redness, and tenderness in one or more joints. Gout is caused by the accumulation of uric acid crystals in the joints, leading to inflammation with intense pain. Uric acid crystals form when there are high levels of uric acid in the blood.

[0007] Tumor lysis syndrome is a complication from cancer treatment, such as lymphoma, leukemia, including non - Hodgkin lymphoma, acute myeloid leukemia, and acute lymphoblastic leukemia. Tumor lysis syndrome occurs when a large number of tumor cells lyse, releasing their contents into the bloodstream. Tumor lysis syndrome is characterized by hyperuricemia (high blood uric acid), as well as hyperkalemia (high blood potassium), hyperphosphatemia (high blood phosphorus), hypocalcemia (low blood calcium), and elevated blood urea nitrogen (BUN) above normal levels. The metabolic abnormalities seen in tumor lysis syndrome can ultimately lead to severe complications such as acute uric acid nephropathy, acute renal failure, seizures, arrhythmias, and death.

[0008] Uricase (sometimes called urate oxidase) is an enzyme that catalyzes the oxidation of uric acid to a more soluble product, allantoin, which is a more easily excreted purine metabolite. Since humans have undergone multiple mutations in the uricase gene during the evolution into higher primates, resulting in the inability to produce enzymatically active uricase, exogenous administration of uricase can treat diseases manifested as hyperuricemia, such as gout and tumor lysis syndrome.

[0009] 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 of using them to treat the above - mentioned diseases. Summary of the Invention

[0010] In one aspect of the present invention, there is provided a uricase conjugate comprising a first domain and a second domain. 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.

[0011] In one embodiment of the uricase conjugate provided herein, the uricase conjugate is a fusion protein of the first domain and the second domain. In another embodiment, the first domain (uricase polypeptide) is at the C-terminus of the second domain (first random coil polypeptide).

[0012] In one embodiment of the uricase fusion protein, an amino acid linker (e.g., an amino acid linker comprising about 2 to about 5 amino acids) is present between the first domain and the second domain. In another embodiment, the linker is Gly-Ser.

[0013] In another embodiment, the uricase conjugate is a uricase fusion protein comprising three domains: a uricase polypeptide 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 can be the same or different. For each of the (first and second) random coil polypeptide domains, there are at least about 100 amino acids within the corresponding domain.

[0014] In the embodiments described herein, the random coil polypeptide domain comprises from about 100 amino acids to about 600 amino acids, such as from about 11 amino acids to about 300 amino acids.

[0015] In one embodiment, the random coil polypeptide domain comprises a Pro-Ala-Ser (PAS) polypeptide. In another embodiment, the PAS polypeptide has the amino acid sequence shown in SEQ ID NO: 60. In another embodiment, the PAS polypeptide has the amino acid sequence shown in SEQ ID NO: 61. In yet another embodiment, the PAS polypeptide has the amino acid sequence shown in SEQ ID NO: 62.

[0016] In yet another embodiment, the PAS polypeptide has the amino acid sequence shown 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 another embodiment, the PAS polypeptide has the amino acid sequence shown 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 - 111.

[0017] In one embodiment, the random coil polypeptide domain comprises an extended recombinant (XTEN) polypeptide. In another embodiment, the XTEN polypeptide has the amino acid sequence shown in SEQ ID NO: 74.

[0018] In one embodiment, the uricase domain of the uricase conjugate described herein comprises the amino acid sequence shown in SEQ ID NO: 1.

[0019] In another embodiment, the uricase domain of the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO: 2.

[0020] In another embodiment, the uricase domain of the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO: 3.

[0021] In another embodiment, the uricase domain of the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO: 4.

[0022] In yet another embodiment, the uricase domain of the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO: 5.

[0023] In another embodiment, the uricase domain of the uricase conjugate described herein comprises an amino acid sequence selected from SEQ ID NOs: 6 - 39.

[0024] In another embodiment, the uricase domain of the uricase conjugate described herein comprises an amino acid sequence selected from SEQ ID NOs: 40 - 44.

[0025] In yet another embodiment, the uricase domain of the uricase conjugate described herein comprises the amino acid sequence shown in SEQ ID NO: 40.

[0026] In yet another embodiment, the uricase domain of the uricase conjugate described herein comprises the amino acid sequence shown in SEQ ID NO: 41.

[0027] In yet another embodiment, the uricase domain of the uricase conjugate described herein comprises the amino acid sequence set forth in SEQ ID NO: 45.

[0028] In yet another embodiment, the uricase domain of the uricase conjugate described herein comprises the amino acid sequence set forth in SEQ ID NO: 46.

[0029] In another embodiment, the uricase domain of the uricase conjugate comprises the amino acid sequence set forth in SEQ ID NO: 47.

[0030] In yet another embodiment, the uricase domain is an amino acid variant of SEQ ID NO: 40. In another embodiment, the amino acid variant comprises from about 10 to about 20 amino acid substitutions. In another embodiment, the amino acid variant comprises from about 10 to about 16 or from about 10 to about 15 amino acid substitutions.

[0031] In yet another embodiment, the uricase domain is an amino acid variant of SEQ ID NO: 41. In another embodiment, the amino acid variant comprises from about 10 to about 20 amino acid substitutions. In another embodiment, the amino acid variant comprises from about 10 to about 16 or from about 10 to about 15 amino acid substitutions.

[0032] In one embodiment of the uricase conjugate described herein, the uricase conjugate is a fusion protein. In another embodiment, the fusion protein forms a homotetramer upon expression.

[0033] In another embodiment of the uricase fusion protein described herein, the uricase 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.

[0034] In another aspect of the invention, there is provided a nucleic acid molecule encoding a uricase conjugate or a domain thereof, as described herein. In one embodiment, the nucleic acid molecule is present in a vector to permit in vitro expression of the uricase conjugate.

[0035] In yet another aspect of the invention, there is provided a method of treatment comprising administering to a subject in need thereof an effective amount of one of the uricase conjugates described herein or a pharmaceutical composition comprising the conjugate. In one embodiment, the method of treatment is a method of treating hyperuricemia. Thus, in one embodiment provided herein, when an effective amount of the uricase conjugate of the invention or a pharmaceutical composition comprising the conjugate is administered, the uric acid level in the subject (e.g., in the blood or plasma of the subject) is reduced.

[0036] In another embodiment, the method of treatment is a method of treating gout. In another embodiment, the gout is refractory gout. In another embodiment, when an effective amount of the urate oxidase conjugate of the present invention or a pharmaceutical composition comprising the conjugate is administered, the uric acid level in a subject (e.g., in the blood or plasma of the subject) is reduced.

[0037] In another embodiment, the method of treatment is a method of treating tumor lysis syndrome. In another embodiment, when an effective amount of the urate oxidase conjugate of the present invention or a pharmaceutical composition comprising the conjugate is administered, the uric acid level in a subject (e.g., in the blood or plasma of the subject) is reduced.

[0038] In one embodiment of the method of treating a subject provided herein, the subject is an adult subject.

[0039] In one embodiment of the method provided herein, the method comprises parenteral administration to the subject. In another embodiment, the parenteral administration is intravenous administration. In yet another embodiment, the method comprises subcutaneous administration to the subject.

[0040] In another aspect, the present disclosure provides a method for recombinantly producing the urate oxidase conjugate (e.g., recombinant urate oxidase fusion protein) disclosed herein. The method comprises: (i) culturing a host cell comprising a nucleic acid vector that comprises a nucleic acid sequence encoding the urate oxidase conjugate (e.g., recombinant urate oxidase fusion protein) disclosed herein, wherein the nucleic acid sequence is operably linked to a heterologous promoter under conditions that permit expression of the nucleic acid sequence encoding the urate oxidase conjugate (e.g., recombinant urate oxidase fusion protein) and recombinant production of the urate oxidase conjugate (e.g., urate oxidase fusion protein) by the host cell; and (ii) isolating the recombinantly produced urate oxidase conjugate (e.g., recombinant urate oxidase fusion protein). BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a schematic diagram showing various configurations of the urate oxidase fusion protein of the present invention.

[0042] Figure 2A is an image of an SDS-PAGE gel that shows bands detected by Coomassie blue staining that represent monomeric NPAS20h-CPB41 urate oxidase-CPAS20h, NPAS20h-CPB41 urate oxidase-CPAS30h, and pegloticase in lanes 1, 2, and 3, respectively.

[0043] Figure 2BIt is an image of an SDS-PAGE gel that shows bands detected by Coomassie Brilliant Blue staining, which represent monomeric CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h in lanes 1 and 2, respectively.

[0044] Figure 3A For pharmacokinetic (PK) study 1, it is a graph showing the changes in the concentrations of endogenous uricase and uric acid in the plasma of untreated control Wistar rats (n = 3) at the corresponding pre-dose time point (at time 0) and each corresponding post-dose time point (up to 96 hours). Each data point is represented as mean ± SD.

[0045] Figure 3B For PK study 1, it is a graph showing the changes in the concentrations of polyethylene glycol recombinant uricase and uric acid in the plasma of Wistar rats (n = 3) intravenously administered a single dose of 1 mg / kg body weight of polyethylene glycol recombinant uricase before dosing (at time 0) and each post-dose time point (up to 96 hours). Each data point is represented as mean ± SD.

[0046] Figure 3C For PK study 1, it is a graph showing the changes in the concentrations of CPB40 uricase-CPAS10h and uric acid in the plasma of Wistar rats (n = 3) intravenously administered a single dose of 1.51 mg / kg body weight of CPB40 uricase-CPAS10h before dosing (at time 0) and each post-dose time point (up to 96 hours). Each data point is represented as mean ± SD.

[0047] Figure 3D For PK study 1, it is a graph showing the changes in the concentrations of CPB40 uricase-CPAS20h and uric acid in the plasma of Wistar rats (n = 3) intravenously administered a single dose of 1.99 mg / kg body weight of CPB40 uricase-CPAS20h before dosing (at time 0) and each post-dose time point (up to 96 hours). Each data point is represented as mean ± SD.

[0048] Figure 3E For PK study 1, it is a graph showing the changes in the concentrations of CPB40 uricase-CPAS30h and uric acid in the plasma of Wistar rats (n = 3) intravenously administered a single dose of 2.47 mg / kg body weight of CPB40 uricase-CPAS30h before dosing (at time 0) and each post-dose time point (up to 96 hours). Each data point is represented as mean ± SD.

[0049] Figure 3FFor PK study 1, in Wistar rats (n = 3) intravenously administered a single dose of 2.16 mg / kg body weight of CPB40 uricase-CXTENh, the concentration change curves of CPB40 uricase-CXTENh and uric acid in plasma before dosing (at time 0) and at each post-dosing time point (up to 96 hours) are shown. Each data point is represented as mean ± SD.

[0050] Figure 4A For PK study 2, in untreated control Wistar rats (n = 3), the concentration change curves of endogenous uricase and uric acid in plasma at the corresponding pre-dosing time point (at time 0) and at each corresponding post-dosing time point (up to 168 hours) are shown. Each data point is represented as mean ± SD.

[0051] Figure 4B For PK study 2, in Wistar rats (n = 3) intravenously administered a single dose of 1 mg / kg body weight of polyethylene glycol recombinant uricase, the concentration change curves of polyethylene glycol recombinant uricase and uric acid in plasma before dosing (at time 0) and at each post-dosing time point (up to 168 hours) are shown. Each data point is represented as mean ± SD.

[0052] Figure 4C For PK study 2, in Wistar rats (n = 3) intravenously administered a single dose of 2.99 mg / kg body weight of NPAS20h-CPB41 uricase-CPAS20h, the concentration change curves of NPAS20h-CPB41 uricase-CPAS20h and uric acid in plasma before dosing (at time 0) and at each post-dosing time point (up to 168 hours) are shown. Each data point is represented as mean ± SD.

[0053] Figure 4D For PK study 2, in Wistar rats (n = 3) intravenously administered a single dose of 3.47 mg / kg body weight of NPAS20h-CPB41 uricase-CPAS30h, the concentration change curves of NPAS20h-CPB41 uricase-CPAS30h and uric acid in plasma before dosing (at time 0) and at each post-dosing time point (up to 168 hours) are shown. Each data point is represented as mean ± SD.

[0054] Figure 5A For PK study 3, in untreated control Wistar rats (n = 3), the concentration change curves of endogenous uricase and uric acid in plasma at the corresponding pre-dosing time point (at time 0) and at each corresponding post-dosing time point (up to 240 hours) are shown. Each data point is represented as mean ± SD.

[0055] Figure 5BFor PK study 3, in Wistar rats (n = 3) intravenously administered a single dose of 1.99 mg / kg body weight of CPB40 uricase-CPAS20h, the concentration change curves of CPB40 uricase-CPAS20h and uric acid in plasma before dosing (at time 0) and at each post-dosing time point (up to 240 hours) are shown. Each data point is represented as mean ± SD.

[0056] Figure 5C For PK study 3, in Wistar rats (n = 3) intravenously administered a single dose of 2.99 mg / kg body weight of NPAS20h-CPB41 uricase-CPAS20h, the concentration change curves of NPAS20h-CPB41 uricase-CPAS20h and uric acid in plasma before dosing (at time 0) and at each post-dosing time point (up to 240 hours) are shown. Each data point is represented as mean ± SD. Detailed implementation mode

[0057] In one aspect, the present invention relates to a novel conjugate molecule comprising two domains: (i) uricase conjugated to (ii) a random coil polypeptide. As described in the preferred embodiments, the conjugation is accomplished at the DNA level by operably linking the uricase DNA sequence to the DNA sequence encoding the random coil polypeptide and then recombinantly expressing the continuous DNA sequence. Linking these two domains at the DNA level avoids in vitro coupling or modification steps to achieve the synthesis of the conjugate. For example, in the approved uricase molecule (PEGylated recombinant uricase), coupling polyethylene glycol (PEG) to uricase requires these coupling or modification steps.

[0058] In the preferred embodiments described herein, the random coil polypeptide comprises three amino acids (proline (Pro), alanine (Ala), and serine (Ser)). In another embodiment, the random coil polypeptide consists of three amino acids (proline (Pro), alanine (Ala), and serine (Ser)). Such a polypeptide is referred to herein as a "PAS polypeptide" when all of the amino acid residues in the polypeptide are Pro, Ala, and Ser, or when substantially all of the amino acids in the polypeptide are Pro, Ala, and Ser.

[0059] In another embodiment, the random coil polypeptide is an extended 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 content of which is incorporated herein by reference in its entirety for all purposes. In one embodiment, the XTEN polypeptide is at least about 800 amino acids in length and is 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 in length. In another embodiment, the XTEN polypeptide is a fragment of the 864-amino acid XTEN polypeptide. In one embodiment, the XTEN polypeptide comprises the amino acid sequence shown in SEQ ID NO: 74. The XTEN polypeptide can be conjugated to the uricase described herein via chemical conjugation or produced as a fusion protein with uricase.

[0060] In embodiments where the uricase conjugate is produced by recombinant expression from 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 that, while not normally linked in their native state, are joined by peptide bonds from their respective N-termini and C-termini to form a single continuous polypeptide. The uricase fusion protein can be a combination of two, three, four, or more different proteins. The uricase fusion protein can also include fusions with heterologous and homologous leader sequences, with or without an N-terminal methionine residue; and fusion proteins that contain additional sequences (e.g., polyhistidine tags) for purifying the fusion protein.

[0061] In one embodiment, the uricase conjugate comprises at least two domains. The first domain of the at least two domains comprises uricase, and the second domain of the at least two domains comprises a random coil polypeptide containing at least about 100 amino acid residues, such as a PAS polypeptide or an XTEN polypeptide. Without being bound by theory, the random coil conformation mediates an increase in the in vivo and / or in vitro stability of uricase. Furthermore, without being bound by theory, since the random coil polypeptide domain itself is not thought to adopt a stable structure or function, the biological activity mediated by the uricase conjugated thereto is substantially retained.

[0062] In another aspect of the invention, nucleic acid molecules are 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 comprising the nucleic acid molecule encoding the uricase fusion protein are also provided. Other aspects of the invention relate to compositions comprising the conjugates of the invention and specific uses of such compositions, such as for the treatment of gout and tumor lysis syndrome.

[0063] As used herein, the term "domain" refers to any region / portion of an amino acid sequence that is capable of autonomously adopting a specific structure and / or function. Thus, in the context of the present invention, a "domain" can 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 / portion that forms a random coil conformation (e.g., a PAS polypeptide domain). The uricase conjugates of the present invention can 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. In addition, the fusion proteins of the present invention can comprise, for example, an additional linker structure between two domains / portions as defined herein, or another domain / portion, such as a protease-sensitive cleavage site, an affinity tag (such as a polyhistidine tag or a Strep tag), a signal peptide, a retention peptide, a targeting peptide (such as a membrane translocation peptide), or another effector domain (such as an antibody fragment for targeting tumors associated with an anti-tumor toxin or an enzyme for prodrug activation), etc. In another embodiment of the uricase fusion protein, the uricase domain is a uricase monomer, and the fusion protein further comprises a random coil domain at the C-terminus of the uricase domain and a random coil domain at the N-terminus of the uricase domain. In another embodiment, the random coil domain comprises a PAS polypeptide.

[0064] Uricase (EC 1.7.3.3) is present in microorganisms (such as Bacillus fastidiosus, Candida mycoderma, and Aspergillus flavus), plants (such as beans and chickpeas), and animals (such as pigs, cows, dogs, and baboons) (Suzuki K et al., J. Biosci. Bioeng., 2004, 98: 153 - 158). This enzyme initiates a series of reactions that convert uric acid (UA) into a more soluble and more excretable product, allantoin. Briefly, uricase catalyzes the reaction of UA with O2 and water (H2O) to form 5-hydroxyisouric acid (HIU) and release hydrogen peroxide (H2O2). Then, HIU undergoes non-enzymatic hydrolysis to generate 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.

[0065] Due to three mutations that completely silence the human urate oxidase gene, humans lack functional urate oxidase. Although it has been hypothesized that the lack of urate oxidase may be beneficial from an evolutionary perspective, in modern humans, high uric acid can have negative consequences due to increased uric acid deposition and gout. Thus, in one aspect of the present invention, the urate oxidase conjugates described herein are provided as therapeutic agents to treat patients with elevated UA levels.

[0066] Active urate oxidase is a tetrameric protein with four identical subunits (i.e., urate oxidase is a homotetramer), each subunit having a molecular weight of approximately 34 kD and consisting of 301 to 304 amino acids. Urate oxidase has maximum enzymatic activity at pH 8.0 (Bayol A et al., Biophys. Chem. 1995, 54: 229-235). Among all sources, urate oxidase from Aspergillus flavus has the highest activity, up to 27 IU / mg; the second highest activity comes from Bacillus fastidiosus, with an activity of 13 IU / mg (Huang SH et al., Eur. J. Biochem., 2004, 271: 517-523). Additionally, urate oxidase from legume sources has an activity of only 2 IU / mg to 6 IU / mg. For recombinantly expressed mammalian urate oxidase, the activity of porcine urate oxidase can reach 5 IU / mg, and baboon urate oxidase is only 1 IU / mg (Michael H et al., 2006, U.S. Patent No. 7,056,713, which is hereby incorporated by reference in its entirety); while human urate oxidase has no activity.

[0067] The present invention is not limited by the source or amino acid sequence of the urate oxidase domain of the conjugate molecule. For example, the urate oxidase provided in the conjugates of the present invention is wild-type urate oxidase or an amino acid variant thereof. For example, in one embodiment, the urate oxidase is dog, pig, cow, goat, or baboon urate oxidase, or is derived from dog, pig, cow, goat, or baboon urate oxidase. In one embodiment, the urate oxidase provided in the conjugates of the present invention is an amino acid variant of dog, pig, cow, goat, or baboon urate oxidase. As used herein, a urate oxidase amino acid variant differs from the corresponding wild-type urate oxidase in amino acid sequence but still retains urate oxidase activity.

[0068] In one embodiment, the uricase is an amino acid variant of a known uricase. In another embodiment, the uricase amino acid variant has an amino acid sequence having at least about 75%, such as 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% identity to the amino acid sequence of the known uricase, including all values and sub-ranges therebetween. In another embodiment, the known uricase has the amino acid sequence shown in SEQ ID NO: 40 or SEQ ID NO: 41. Each of the uricases in SEQ ID NO: 40 and SEQ ID NO: 41 is a porcine-baboon chimeric uricase, which porcine-baboon chimeric uricase comprises amino acids (aa) 8 to 266 of porcine uricase (SEQ ID NO: 2) and amino acids 267 to 304 of baboon uricase (SEQ ID NO: 3). Accordingly, in the present application, the uricases of SEQ ID NO: 40 and SEQ ID NO: 41 are also referred to as "CPB40 uricase" and "CPB41 uricase", respectively. The amino acid sequence of SEQ ID NO: 41 is identical to the amino acid sequence of SEQ ID NO: 40 in other respects except for the lack of an N-terminal methionine. (Pegloticase) is a super PEGylated homotetrameric protein, each monomer of which is a single polypeptide chain composed of a porcine-baboon chimeric uricase having the amino acid sequence of SEQ ID NO: 40 or 41.

[0069] In one embodiment, the uricase is a uricase amino acid variant. The uricase amino acid variant comprises from about 10 to about 20 amino acid substitutions, such as 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 comprises from about 10 to about 20 amino acid substitutions, such as from about 10 to about 18, from about 10 to about 16, from about 10 to about 14, from about 10 to about 13 or from about 10 to about 12 amino acid substitutions.

[0070] In one embodiment, the urate oxidase amino acid variant is an amino acid variant of a porcine-baboon chimeric urate oxidase. In another embodiment, the urate oxidase amino acid variant is a variant of the urate oxidase polypeptide sequence shown in SEQ ID NO: 40. In another embodiment, the urate oxidase amino acid variant of SEQ ID NO: 40 comprises from about 10 to about 20 amino acid substitutions, such as from about 10 to about 18, from about 10 to about 16, from about 10 to about 14, from about 10 to about 13, or from about 10 to about 12 amino acid substitutions. In another embodiment, the urate oxidase amino acid variant is a variant of the urate oxidase polypeptide sequence shown in SEQ ID NO: 41. In another embodiment, the urate oxidase amino acid variant of SEQ ID NO: 41 comprises from about 10 to about 20 amino acid substitutions, such as from about 10 to about 18, from about 10 to about 16, from about 10 to about 14, from about 10 to about 13, or from about 10 to about 12 amino acid substitutions.

[0071] In a preferred embodiment, the urate oxidase conjugate provided herein is a recombinant urate oxidase fusion protein. As used herein, a "recombinant protein" refers to any artificially produced protein and is distinct from a naturally produced protein (i.e., a protein produced in an animal tissue having only the natural gene of the particular protein of interest). As described herein, the recombinant urate oxidase fusion protein comprises a urate oxidase domain that is bound, either directly or through an amino acid linker, by a peptide bond to one or more random coil polypeptide domains.

[0072] In one embodiment, the urate oxidase domain comprises a recombinant molecule that comprises fragments of porcine and baboon liver urate oxidase proteins. In another embodiment, the urate oxidase domain comprises a recombinantly modified baboon urate oxidase. In one embodiment, the urate oxidase domain comprises a porcine-baboon chimeric urate oxidase (PBC urate oxidase, SEQ ID NO: 1) that comprises amino acids (aa) 1 to 225 of porcine urate oxidase (SEQ ID NO: 2) and amino acids 226 to 304 of baboon urate oxidase (SEQ ID NO: 3). In another embodiment, the urate oxidase is a porcine-baboon chimeric urate oxidase that comprises amino acids 1 to 288 of porcine urate oxidase and amino acids 289 to 304 of baboon urate oxidase (PKS urate oxidase, SEQ ID NO: 4), or a variant of one of the foregoing, or a truncated form of one of the foregoing.

[0073] In one embodiment, the urate oxidase is a porcine-baboon chimeric urate oxidase.

[0074] In one embodiment, the urate oxidase is a porcine urate oxidase having the amino acid sequence shown in SEQ ID NO: 5.

[0075] In one embodiment, the uricase is a humanized uricase. For example, the uricase may comprise amino acids from an inactive human uricase substituted into the amino acid sequence of a non-human mammalian uricase to retain the original activity and improve homology to human uricase, thereby reducing immunogenicity in the human body. Such humanized uricases are disclosed in U.S. Patent No. 8,586,535, the disclosure of which is incorporated herein by reference in its entirety.

[0076] In one embodiment, the uricase is Aspergillus flavus uricase or is derived from Aspergillus flavus uricase.

[0077] 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: WP013581210.1), 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 foregoing uricases.

[0078] 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|6678509|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 the public databases accessible through the National Center for Biotechnology Information (NCBI) of the United States are incorporated herein by reference in their entirety for all purposes.

[0079] 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 another embodiment, the mammalian uricase comprises the amino acid sequence of porcine, bovine, ovine, or baboon liver uricase. In one embodiment of the present invention, the uricase is a chimeric uricase of two or more mammalian uricases. In another embodiment, the mammalian uricases of the chimeric uricase are selected from porcine, bovine, ovine, or baboon liver uricase.

[0080] In one embodiment, the uricase domain of the uricase conjugate comprises a fungal or microbial uricase. In another 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 comprises an invertebrate uricase. In another embodiment, the invertebrate uricase is Drosophila melanogaster or Drosophila pseudoobscura uricase.

[0081] In another embodiment, the uricase domain of the uricase conjugate comprises Candida utilis uricase.

[0082] In another embodiment, the uricase domain of the uricase conjugate comprises a plant uricase. In another embodiment, the plant uricase is the soybean uricase of Rhizobium.

[0083] In another embodiment, the uricase moiety of the conjugate has an amino acid sequence selected from one of SEQ ID NOs: 6-39 or an amino acid variant thereof (Table 1).

[0084]

[0085]

[0086] In yet another embodiment, the uricase is a synthetic uricase produced from a consensus uricase amino acid sequence that is derived from the alignment of 50 uricase sequences having the greatest identity to Arthrobacter globiformis uricase (SEQ ID NO: 39).

[0087] In one embodiment, the uricase can be a wild-type uricase or an engineered variant thereof. For example, in one embodiment, the uricase is a recombinant mammalian uricase.

[0088] In one embodiment, the uricase has the amino acid sequence disclosed in U.S. Patent No. 10,731,139, the content of which is incorporated herein by reference in its entirety for all purposes.

[0089] Methods for mutating amino acids are well known in the art and can be used to mutate one or more amino acids of a wild-type uricase to produce uricase amino acid variants. For example, Arg-Gly-Asp (RGD), a tripeptide reported to mediate cell adhesion through integrin binding, can be mutated to a Ser-Gly-Asp (SGD) sequence so that the uricase domain does not contain the RGD sequence.

[0090] In one embodiment, the uricase moiety of the conjugate is a truncated uricase. As used herein, "truncated uricase" refers to a uricase molecule having a shortened primary amino acid sequence as compared to the amino acid sequence of a known uricase (e.g., the wild-type enzyme). In one embodiment, the truncation is at or near the N-terminus and / or C-terminus of the uricase. In one embodiment, the uricase is truncated at the N-terminus. In another embodiment, the N-terminal truncation begins at position 1, 2, 3, 4, 5, or 6. In one embodiment, the amino-terminal truncation begins at position 2, thereby removing the amino-terminal methionine (Met). In one embodiment, the amino-terminal Met can be removed by post-translational modification. In another embodiment, the amino-terminal Met is removed after the uricase is produced as one of the fusion proteins described herein. In another embodiment, the Met is removed by an endogenous bacterial aminopeptidase.

[0091] In one embodiment of the truncated uricase, the uricase is truncated at its N-terminus by 4 to 13 amino acids. In another embodiment, the uricase is truncated at its C-terminus by 4 to 13 amino acids. In one embodiment of the truncated uricase, the uricase is truncated at both its C-terminus and N-terminus by 4 to 13 amino acids. In another embodiment, the uricase is truncated at its N-terminus by 6 amino acids. In another embodiment, the uricase for one of the conjugates (e.g., fusion protein) described herein is truncated at its C-terminus by 6 amino acids. In one embodiment of the present invention, the uricase for one of the conjugates (e.g., fusion protein) described herein is truncated at both its carboxyl terminus and amino terminus by 6 amino acids.

[0092] In one embodiment of the uricase conjugate described herein, the uricase domain of the conjugate comprises the amino acid sequence shown in the sequences selected from SEQ ID NOs: 40-44. Like the uricases of SEQ ID NO: 40 and SEQ ID NO: 41, each of the uricases of SEQ ID NO: 42 and SEQ ID NO: 43 is a porcine-baboon chimeric uricase comprising amino acids 8 to 220 of porcine uricase (SEQ ID NO: 2) and amino acids 221 to 301 of baboon uricase (SEQ ID NO: 3). Except for the lack of the N-terminal methionine, the amino acid sequence of SEQ ID NO: 43 is identical to the amino acid sequence of SEQ ID NO: 42 in other respects.

[0093] In another embodiment, the uricase protein comprises the amino acid sequence shown in SEQ ID NO: 1. In another embodiment, the uricase protein comprises the amino acid sequence shown in SEQ ID NO: 40 or 41.

[0094] In one embodiment, the uricase protein comprises the amino acid sequence shown in SEQ ID NO:7. In yet another embodiment, the uricase protein comprises the amino acid sequence shown in SEQ ID NO:40, 41, 42, 43 or 44. In one embodiment of the present invention, the uricase comprises the amino acid sequence of SEQ ID NO.41. In another embodiment of the present invention, the uricase comprises the amino acid sequence of SEQ ID NO.43.

[0095] In yet another embodiment, the uricase domain comprises a uricase polypeptide among the uricase polypeptides disclosed in PCT Publication No. 2016 / 187026, the entire content of which patent application is incorporated herein by reference.

[0096] In one embodiment, the uricase domain comprises rasburicase (sold under the trade name ), and has the amino acid sequence shown in SEQ ID NO:45.

[0097] In another embodiment, the uricase domain comprises a uricase having the amino acid sequence shown in SEQ ID NO:46, which uricase is also referred to as HZN-003.

[0098] In yet another embodiment, the uricase domain comprises a uricase having the amino acid sequence shown in SEQ ID NO:47, which uricase is also referred to as SEL-212.

[0099] Disadvantages of prior art uricase therapeutic agents include: (i) lack of solubility, (ii) immunogenicity, and (iii) rapid clearance from the circulatory system by renal filtration, the latter severely hindering its efficacy in animal studies and human therapy. To this end, the present invention provides uricase conjugates that comprise a conformationally disordered (random coil) polypeptide domain that comprises the amino acid residues Pro, Ala, and Ser (PAS). The PAS sequence is a hydrophilic, uncharged biopolymer whose biophysical properties are similar to those of polyethylene glycol (PEG), and the chemical conjugation of which to a drug is an established method for prolonging plasma half-life and reducing immunogenicity (e.g., (pegylated recombinant uricase)). In contrast to PEG modification, PAS polypeptides provide fusion with uricase at the genetic level, i.e., they are genetically encodable polypeptides and are capable of producing fully active uricase fusion proteins in a host (e.g., Escherichia coli, etc.), thereby avoiding in vitro conjugation or modification steps.

[0100] As described above, the uricase conjugate provided herein comprises at least two domains. The uricase domain is as described above. In some embodiments, the second domain is referred to herein as the PAS domain and 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 that form the random coil contain 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. Without being bound by theory, the random coil conformation mediates an increase in the in vivo and / or in vitro stability of uricase. Details regarding the various types of PAS polypeptides and nucleic acids encoding such PAS polypeptides for use in the present invention can be found in PCT Publication No. WO 2008 / 155134, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0101] As used herein, the term "random coil" or "random coil polypeptide domain" refers to the conformation of a polymeric molecule, including an amino acid polymer, in which the individual monomeric elements that form the polymeric structure are oriented substantially randomly with respect to adjacent monomeric elements while still being chemically bonded to said adjacent monomeric elements. In particular, a polypeptide or amino acid polymer that adopts / has / forms a "random coil" conformation is substantially devoid of a defined secondary and tertiary structure. The nature of polypeptide random coils and methods for their experimental identification are known to those of skill in the art.

[0102] A random coil is formed under physiological conditions. For example, in one embodiment, physiological conditions refer to parameters that are typically effective for higher life forms, particularly mammals, and most preferably humans. Thus, physiological conditions can be those typically found in mammalian body fluids. Physiological conditions can refer to the corresponding parameters found in a healthy body as well as those found in a diseased mammal or human patient. For example, when a diseased mammal or human patient has a fever, the body temperature of the mammal or human patient may increase, but still fall within the range of physiological temperature conditions.

[0103] Several buffers in the experimental environment (e.g., for determining protein structure, especially for circular dichroism (CD) measurements and other methods for determining the structural properties of protein / amino acid segments), solvents and / or excipients for pharmaceutical compositions are considered to represent in vitro physiological solutions and / or in vitro physiological conditions. Examples of such buffers are, for example, phosphate buffered saline, Tris buffer, acetate buffer, citrate buffer or similar buffers. Generally, the pH of the buffer representing physiological solution conditions is in the range of 6.5 to 8.5, for example in the range of 7.0 to 8.0, for example in the range of 7.2 to 7.7, and the osmolality can be in the range of 10 mmol / kg H2O to 1000 mmol / kg H2O, more particularly in the range of 50 mmol / kg H2O to 500 mmol / kg H2O, for example in the range of 200 mmol / kg H2O to 350 mmol / kg H2O.

[0104] 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 a light absorption spectroscopy for measuring the difference in the absorption rates of right-handed circularly polarized light and left-handed circularly polarized light by a substance. The secondary structure of a protein can be determined by CD spectroscopy using the far-ultraviolet spectrum with wavelengths between about 190 nm and 250 nm. At these wavelengths, the different secondary structures common in polypeptides can be analyzed because the α-helix, parallel and antiparallel β-sheets, and random coil conformations each produce characteristic shapes and sizes of the CD spectrum. Thus, 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, measuring the hydrodynamic volume by size exclusion chromatography, analytical ultracentrifugation or dynamic / static light scattering, and measuring the friction coefficient or intrinsic viscosity.

[0105] 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 at most about 1000 amino acid residues, at most about 900 amino acid residues, at most about 800 amino acid residues, at most about 700 amino acid residues or at most about 600 amino acid residues. In one embodiment, the random coil domain contains at most about 500 amino acid residues or at most about 450 amino acid residues.

[0106] In one embodiment, the random coil polypeptide domain comprises from about 100 to about 3000 amino acid residues. In another embodiment, the random coil domain comprises from about 100 to 1000 amino acid residues. In one embodiment, the random coil polypeptide domain comprises an amino acid sequence in which Pro residues account for from about 4% to about 40% of the random coil polypeptide domain. In another embodiment, alanine and serine residues constitute the remaining from about 60% to about 96% of the random coil polypeptide. In some embodiments, the random coil polypeptide domain comprises other amino acids different from Ala, Ser, and Pro, i.e., as minor components. As used in the context herein, the term "minor component" means that up to 10% of the amino acids in the random coil polypeptide domain are different from alanine, serine, and proline, e.g., up to 8% of the amino acids in the random coil polypeptide domain, e.g., up to 6% of the amino acids in the random coil polypeptide domain, e.g., up to 5% of the amino acids in the random coil polypeptide domain, up to 4% of the amino acids in the random coil polypeptide domain, up to 3% of the amino acids in the random coil polypeptide domain, up to 2% of the amino acids in the random coil polypeptide domain, 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 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 include one or more non-natural amino acids.

[0107] In another embodiment, the random coil polypeptide is an extended 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 content of which is incorporated herein by reference in its entirety for all purposes. In one embodiment, the XTEN polypeptide has a length of at least about 800 amino acids and is 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 in length. In another embodiment, the XTEN polypeptide is a fragment of the 864 amino acid XTEN polypeptide. The XTEN polypeptide can be conjugated to the uricase described herein via chemical conjugation or produced as a fusion protein with uricase.

[0108] In another embodiment, the random coil polypeptide domain comprises a plurality of "amino acid repeats", i.e., the same amino acid sequence that occurs two or more times within the domain, wherein the "amino acid repeat" is composed of Ala, Ser, and Pro residues (described herein as "PAS" or "APS"). In another embodiment, no more than 6 consecutive amino acid residues within the random coil polypeptide domain are the same, and Pro residues constitute more than about 4% and less than about 40% of the amino acids of the random coil polypeptide domain. Non-limiting examples of "amino acid repeats" composed of Ala, Ser, and Pro residues are provided herein; see, e.g., SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, and SEQ ID NO: 58 (Table 2A). Fragments and / or multimers of these sequences are used in some embodiments. A "fragment" comprises at least 3 amino acids and comprises at least one Ala, one Ser, and / or one Pro.

[0109]

[0110]

[0111] The foregoing repeat sequences may be encoded by nucleic acid molecules having the sequences shown 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).

[0112] In one embodiment, a nucleotide sequence encoding the amino acid repeat of SEQ ID NO: 48 is provided. In another embodiment, the nucleotide sequence is selected from one of the nucleotide sequences shown in Table 2B, i.e., one of SEQ ID NOs: 81 - 111.

[0113]

[0114]

[0115] In one embodiment, the amino acid repeats in the random coil PAS polypeptide domain contain 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, wherein the amino acid repeats contain at least one Ala, Ser and Pro residue. In one embodiment, the amino acid repeats do not contain more than 100 amino acid residues. In one embodiment, the amino acid repeats contain 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 another embodiment, the amino acid repeats contain less than about 40%, such as less than about 35% Pro residues.

[0116] In one embodiment, the random coil polypeptide domain contains no more than 5 identical consecutive amino acid residues, such as no more than 4 identical consecutive amino acid residues, such as no more than 3 identical consecutive amino acid residues.

[0117] In one embodiment, the random coil polypeptide domain contains more than about 4% Ala residues but less than about 50% Ala residues, such as more than about 10% Ala residues but less than about 50% Ala residues, such as more than about 20% Ala residues but less than about 50% Ala residues.

[0118] In another embodiment, the random coil polypeptide domain contains more than about 4% Ser residues but less than about 50% Ser residues, such as more than about 10% Ser residues but less than about 50% Ser residues, such as more than about 20% Ser residues but less than about 50% Ser residues.

[0119] In one embodiment, the random coil polypeptide domain contains about 35% Pro residues, about 50% Ala residues and about 15% Ser residues. Alternatively, the random coil polypeptide domain contains about 35% Pro residues, about 15% Ala residues and about 50% Ser residues.

[0120] In one embodiment of the present invention, the uricase conjugate contains a random coil domain having the amino acid sequence shown in SEQ ID NO: 60.

[0121] In one embodiment of the present invention, the uricase conjugate contains a random coil domain having the amino acid sequence shown in SEQ ID NO: 61.

[0122] In one embodiment of the present invention, the uricase conjugate contains a random coil domain having the amino acid sequence shown in SEQ ID NO: 62.

[0123] In one embodiment of the intrinsically disordered domain provided herein, where the intrinsically disordered domain is present at the N-terminus of a fusion protein, the intrinsically disordered domain comprises an N-terminal Met residue. In another embodiment, the fusion protein does not comprise an N-terminal Met residue, for example because the N-terminal Met residue is removed post-translationally.

[0124] 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 intrinsically disordered polypeptide domain in a multi-domain polypeptide. In an alternative embodiment, the uricase is bound to the intrinsically disordered polypeptide domain by a non-peptide bond. Non-peptide bonds that can be used to crosslink proteins are known in the art and can include disulfide bonds, such as disulfide bonds between Cys side chains, thioether bonds, or non-peptide covalent bonds induced by chemical crosslinkers, such as disuccinimidyl suberate (DSS) or sulfo-succinimidyl 4-[p-maleimidophenyl]butyrate (Sulfo-SMPB), as well as non-covalent protein-protein interactions.

[0125] For the fusion protein embodiment, the two domains can be arranged in an order selected by one of ordinary skill in the art. For example, in one embodiment of a uricase conjugate that is a fusion protein, the uricase domain is located at the amino (N)-terminus of the fusion protein, and the intrinsically disordered polypeptide domain is located at the carboxyl (C)-terminus of the fusion protein. However, the order can also be reversed. For example, in one embodiment, the uricase domain is located at or in the carboxyl (C)-terminus of the fusion protein, and the intrinsically disordered polypeptide domain is located at or in the amino (N)-terminus of the fusion protein. In yet another embodiment, the intrinsically disordered polypeptide domain is located at both the C-terminus and the N-terminus of the fusion protein, and the uricase domain is located between the two intrinsically disordered domains.

[0126] In one embodiment, the uricase fusion protein comprises an N-terminal Met residue. In another embodiment, the uricase fusion protein does not comprise an N-terminal Met residue, for example because the N-terminal Met residue is removed post-translationally. Thus, for the sequences provided herein, if an N-terminal Met is present, alternative embodiments include fusion proteins of the same sequence that do not comprise an N-terminal Met. Similarly, if an N-terminal Met is not present in the fusion proteins provided herein, alternative embodiments include fusion proteins of the same sequence that have an N-terminal Met.

[0127] In one embodiment of the uricase fusion protein, there is an amino acid spacer sequence between the uricase domain and the random coil (e.g., PAS) domain. In one embodiment, the amino acid spacer sequence is one amino acid long, two amino acids long, three amino acids long, or four amino acids long. In another embodiment, the amino acid spacer sequence is two amino acids long. In another embodiment, the spacer sequence is Gly-Ser.

[0128] In one embodiment of the uricase conjugate described herein, the conjugate comprises 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., the uricase fusion protein) from an in vitro expression system, for example, by using immobilized metal affinity chromatography (IMAC). In one embodiment, the purification tag is present at the C-terminus of the uricase fusion protein. In another embodiment, the purification tag is a polyhistidine tag (also referred to as a "his-tag"). In one embodiment, the his-tag comprises six (6) histidine residues.

[0129] Alternative purification tags can also be employed 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 another embodiment, the HQ tag has the amino acid sequence HQHQHQ (SEQ ID NO: 71). In another embodiment, the uricase conjugate comprises a his-asp tag (HN tag) at the C-terminus to allow 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 comprises a HAT peptide tag at the C-terminus to allow purification of the conjugate. In one embodiment, the HAT peptide tag has the amino acid sequence: KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 73).

[0130] For the fusion protein embodiment of the uricase conjugate of the present disclosure, Figure 1Exemplary configurations of recombinant uricase fusion proteins comprising a uricase domain and one or two intrinsically disordered PAS polypeptides or XTEN polypeptide domains are provided. In one embodiment, the uricase fusion protein comprises a uricase domain and an intrinsically disordered PAS polypeptide or XTEN polypeptide domain. In the uricase fusion protein, the uricase domain can be at the C-terminus of an intrinsically disordered PAS polypeptide or XTEN polypeptide domain, or can be at the N-terminus of an intrinsically disordered PAS polypeptide or XTEN polypeptide domain. In another embodiment, the uricase fusion protein comprises a uricase domain and two intrinsically disordered PAS polypeptide domains or two XTEN polypeptide domains. In the uricase fusion protein, one of the two intrinsically disordered PAS polypeptides or XTEN polypeptide domains is at the N-terminus, and the other is at the C-terminus, and the uricase domain is between the two intrinsically disordered PAS polypeptides or XTEN polypeptide domains. In some embodiments, as Figure 1 depicted in, the spacer sequence Gly-Ser (GS) between the uricase domain and the C-terminal intrinsically disordered PAS polypeptide (e.g., PAS10, PAS20, or PAS30) or XTEN polypeptide domain is optional and can be absent. In one embodiment, the uricase fusion protein has polyhistidine tags (also referred to as "his tags") at the C-terminus, N-terminus, or both the C-terminus and N-terminus, the polyhistidine tags comprising, for example, 6 histidine residues, for purifying the uricase fusion protein. In some embodiments, the his tag is separated from its adjacent domain by a spacer sequence Gly-Ser (GS).

[0131] PAS10, PAS20, and PAS30 denote intrinsically disordered PAS polypeptide domains comprising 10, 20, and 30 tandem copies, respectively, of a PAS sequence that contains Pro, Ala, and Ser, such as the PAS sequences shown 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.

[0132] In one embodiment, PAS10, PAS20, and PAS30 denote intrinsically disordered PAS polypeptide domains comprising 10, 20, and 30 tandem copies, respectively, of the PAS sequence of SEQ ID NO: 48, and thus having the amino acid sequences shown in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively.

[0133] 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 respectively contain a total of 10, 20, and 30 tandem copies of one or more nucleotide sequences selected from SEQ ID NO: 49 and SEQ ID NOs: 81 - 111.

[0134] 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 respectively contain a total of 10, 20, and 30 tandem copies of the same (one) nucleotide sequence selected from SEQ ID NO: 49 and SEQ ID NOs: 81 - 111.

[0135] 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 respectively contain a total of 10, 20, and 30 tandem copies of two or more nucleotide sequences selected from SEQ ID NO: 49 and SEQ ID NOs: 81 - 111.

[0136] 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 respectively contain 10, 20, and 30 unique (i.e., different) nucleotide sequences selected from SEQ ID NO: 49 and SEQ ID NOs: 81 - 111 that are tandemly linked together via 3′,5′-phosphodiester bonds.

[0137] In an exemplary embodiment, the DNA sequence encoding PAS10 of SEQ ID NO: 60 contains each of SEQ ID NOs: 102 - 111 in the 5′ to 3′ direction, which are tandemly linked together via 3′,5′-phosphodiester bonds in ascending ID number order.

[0138] In an exemplary embodiment, the DNA sequence encoding PAS20 of SEQ ID NO: 61 contains each of SEQ ID NOs: 92 - 111 in the 5′ to 3′ direction, which are tandemly linked together via 3′,5′-phosphodiester bonds in ascending ID number order.

[0139] In an exemplary embodiment, the DNA sequence encoding PAS30 of SEQ ID NO: 62 comprises each of SEQ ID NOs: 82 - 111 in the 5′ to 3′ direction, which are serially linked together by 3′,5′-phosphodiester bonds in ascending ID number order.

[0140] In another exemplary embodiment, the DNA sequence encoding PAS30 of SEQ ID NO: 62 comprises SEQ ID NO: 81 and each of SEQ ID NOs: 83 - 111 in the 5′ to 3′ direction, which are serially linked together by 3′,5′-phosphodiester bonds in ascending ID number order.

[0141] In one embodiment, the XTEN polypeptide domain of the uricase fusion protein comprises the amino acid sequence of SEQ ID NO: 74.

[0142] In one embodiment, the uricase domain of the uricase fusion protein comprises the porcine - baboon chimeric uricase of SEQ ID NO: 1. In one embodiment, the uricase domain of the uricase fusion protein comprises the porcine - baboon chimeric uricase of SEQ ID NO: 4. In one embodiment, the uricase domain of the uricase fusion protein comprises the porcine - baboon chimeric uricase of SEQ ID NO: 40 (also referred to herein as "CPB40 uricase"). In one embodiment, the uricase domain of the uricase fusion protein comprises the porcine - baboon chimeric uricase of SEQ ID NO: 41 (also referred to herein as "CPB41 uricase"). In one embodiment, the uricase domain of the uricase fusion protein comprises the porcine - baboon chimeric uricase of SEQ ID NO: 42. In one embodiment, the uricase domain of the uricase fusion protein comprises the porcine - baboon chimeric uricase of SEQ ID NO: 43.

[0143] Table 3 shows the code names, SEQ ID NOs and domain compositions of exemplary uricase fusion proteins produced and generated by the inventors of the present application in Escherichia coli. Table 3 also describes the DNA sequences encoding PAS10 of SEQ ID NO: 60, PAS20 of SEQ ID NO: 61 and PAS30 of SEQ ID NO: 62. According to Figure 1 the configurations or variations thereof disclosed above, the exemplary uricase fusion proteins comprise a combination of the amino acid sequences of CPB40 uricase or CPB41 uricase of the uricase domain with one or two random coil PAS polypeptide domains or one random coil XTEN polypeptide domain. Details of the production and characterization of some exemplary uricase fusion proteins are described in the "Examples".

[0144]

[0145]

[0146]

[0147]

[0148] In one aspect, the present invention relates to nucleic acid constructs encoding the uricase conjugates (fusion proteins) of the present invention. The nucleic acid molecule can be operably linked to suitable expression control sequences known in the art to ensure correct transcription and translation of the polypeptide, and can be operably linked to a signal sequence to ensure cellular secretion or targeting to an organelle. Such vectors can contain additional genes, such as marker genes that allow selection of the vector in a suitable host cell and under suitable conditions.

[0149] ″Nucleic acid construct″ refers to a constructed nucleic acid sequence that contains one or more functional units that do not occur together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences from bacteriophage λ), viral genomes containing non-natural nucleic acid sequences, two or more operably linked proteins (e.g., to produce a fusion protein), etc.

[0150] ″Operably linked″ refers to the arrangement of elements where the components so described are configured to perform their normal functions. In the case of a promoter, a promoter operably linked to a coding sequence will affect the expression of the coding sequence. The promoter or other control element need not be adjacent to the coding sequence so long as they are able to function to direct the expression of that coding sequence. For example, there can be intervening untranslated but transcribed sequences between the promoter sequence and the coding sequence, and the promoter sequence can still be considered ″operably linked″ to the coding sequence. In the case of the different domains of the uricase conjugate described herein, the domains can be operably linked in a single DNA sequence to allow expression of the uricase fusion protein.

[0151] A ″vector″ is capable of transferring a gene sequence into a host cell. A ″vector″ refers to a nucleic acid construct that can direct the expression of a gene of interest and can transfer the gene sequence into a host cell, and this transfer can be achieved by integrating all or part of the genome of the vector, or by transient or heritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning and expression vectors, as well as integrating vectors. Vectors used herein include, for example, plasmids, phages, phagemids, adenoviruses, adeno-associated viruses (AAV), lentiviruses.

[0152] In one embodiment, the nucleic acid construct is present in a recombinant vector, wherein the nucleic acid molecule encoding the urate oxidase fusion protein is operably linked to an expression control sequence, thereby allowing the expression of the urate oxidase fusion protein in prokaryotic or eukaryotic cells.

[0153] The expression of the nucleic acid molecule includes transcribing the nucleic acid molecule into translatable mRNA. Regulatory elements that allow expression in prokaryotic host cells include, for example, the λPL, lac, trp, tac, tet, or T7 promoters in Escherichia coli. Possible regulatory elements that ensure expression in eukaryotic cells (e.g., in mammalian cells or yeast) are well known to those skilled in the art. In one embodiment, the regulatory element contains a regulatory sequence that ensures transcription initiation and optionally a polyadenylation (poly-A) signal that ensures transcription termination and transcript stability. Additional regulatory elements used herein include transcriptional and translational enhancers, and / or native or heterologous promoter regions. Examples of regulatory elements that allow expression in eukaryotic host cells are the AOXI or GAL1 promoters in yeast or the CMV, SV40, RSV promoters (Rous sarcoma virus), CMV enhancer, SV40 enhancer, or globin intron in mammalian and other animal cells. In addition to the elements responsible for transcription initiation, such regulatory elements may also contain transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site downstream of the coding region.

[0154] Methods well known to those skilled in the art can be used to construct recombinant vectors (see, for example, the techniques described in Sambrook (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory N.Y. and Ausubel (1989), Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y). In the present context, suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNA1, pcDNA3, pPICZalpha A (Invitrogen), or pSPORT1 (GIBCO BRL). In addition, depending on the expression system used, a leader sequence capable of directing the polypeptide to a cellular compartment or secreting it into the culture medium can be added to the coding sequence of the nucleic acid molecule of the present invention.

[0155] In certain embodiments, the invention also relates to vectors comprising nucleic acid constructs encoding the fusion proteins provided herein, particularly plasmids, cosmids, viruses, and bacteriophages, which are routinely used in genetic engineering. In one embodiment, the vector is an expression vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses can be used to deliver the polynucleotides or vectors of the invention into a target cell population. Vectors containing the nucleic acid molecules of the invention can be transferred into host cells by well-known methods, which vary according to the type of cell host. Accordingly, the invention further relates to cells comprising the nucleic acid molecule or vector.

[0156] In one embodiment, a nucleic acid molecule / vector among the nucleic acid molecules / vectors provided herein is transfected into a host cell using the calcium chloride transfection method commonly used for prokaryotic cells. In another embodiment, calcium phosphate treatment or electroporation is used, depending on the cell host. As an alternative, the nucleic acid molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells. The nucleic acid molecule or vector of the invention present in the host cell can be integrated into the genome of the host cell or can remain episomal. Accordingly, the invention also partly relates to host cells comprising the nucleic acid molecule and / or vector of the invention. Host cells for expressing polypeptides are well known in the art and include prokaryotic cells as well as eukaryotic cells, such as Escherichia coli cells, yeast cells, invertebrate cells, CHO cells, CHO-K1 cells, Hela cells, COS-1 monkey cells, melanoma cells such as Bowes cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines, etc.

[0157] On the other hand, the present invention includes a method for preparing the urate oxidase fusion protein of the present invention, which method comprises culturing a host cell and isolating the urate oxidase fusion protein from the culture. The urate oxidase fusion protein comprising a urate oxidase domain and a random coil polypeptide domain can be produced by recombinant DNA techniques, for example by culturing a cell comprising a nucleic acid construct or vector encoding the urate oxidase fusion protein and isolating the urate oxidase fusion protein from the culture. The urate oxidase fusion protein can be produced in any suitable cell culture system, including prokaryotic cells, such as Escherichia coli (BL21 or JM83), or eukaryotic cells, such as the yeast strain X-33 of Pichia pastoris or CHO cells. Other suitable cell lines known in the art can be obtained from cell line depositories, such as the American Type Culture Collection (ATCC). The term "prokaryotic" is intended to include bacterial cells, while the term "eukaryotic" is intended to include yeast, higher plants, 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 growth. In another embodiment, the present invention partly relates to a method for preparing the above-mentioned urate oxidase fusion protein, which method comprises culturing the cells of the present invention under conditions suitable for the expression of the urate oxidase fusion protein and isolating the urate oxidase fusion protein from the cells or the culture medium.

[0158] The urate oxidase fusion protein can be isolated from the growth medium, cell lysates or cell membrane fractions. The isolation and purification of the expressed polypeptide of the present invention can be carried out by any conventional methods, which methods include ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc., and may include the use of monoclonal or polyclonal antibodies directed against, for example, a tag fused to the bioactive protein of the present invention. For example, streptavidin affinity chromatography can be used to purify the protein via Strep tag II (Skerra (2000). Methods Enzymol 326, pp. 271-304).

[0159] The cDNA encoding the conjugate can be cloned and inserted into a suitable vector, for example for expression in a suitable host, such as Escherichia coli or Saccharomyces cerevisiae.

[0160] In one aspect, 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 materials well known to those skilled in the art. These materials are non-toxic and should not interfere with the efficacy of uricase. These materials may include, for example, solvents, dispersion media, antibacterial and antifungal agents, isotonic agents and absorption delaying agents. 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 polyol (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 the shelf life or effectiveness.

[0161] The pharmaceutical composition can be formulated in liquid, semi-solid or solid forms such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration, therapeutic application, physicochemical properties of the uricase conjugate and the delivery route. The formulation may include an excipient or a combination of excipients, such as: sugars, amino acids and surfactants. Liquid formulations can include a wide range of protein concentrations and pH values. Solid formulations can be produced, for example, by freeze drying, spray drying or drying by supercritical fluid technology.

[0162] When administered intravenously or at the site of disease, the active ingredient can be a parenterally acceptable aqueous solution that is pyrogen-free and has a suitable pK, isotonicity and stability. Those skilled in the relevant art can well prepare a suitable solution using, for example, isotonic media such as sodium chloride solution, Ringer's solution and lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included.

[0163] In some embodiments, the pharmaceutical composition is formulated as a solution, microemulsion, dispersion, liposomal dispersion, or other ordered structure suitable for containing the urate oxidase conjugate (e.g., urate oxidase fusion protein) described herein. A sterile injectable solution can be prepared by incorporating the urate oxidase conjugate into a suitable solvent with one or a combination of the ingredients listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the urate oxidase conjugate into a sterile vehicle that contains a dispersion medium and other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods include vacuum drying and freeze drying, from which powders of the active ingredient plus any additional desired ingredients can be produced from its previously sterile filtered solution. For example, the proper fluidity of the solution can be maintained by using coatings such as lecithin, by maintaining the particle size of the dispersion, or by using surfactants. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption (e.g., monostearate and gelatin). In one embodiment, the urate oxidase conjugate in the composition is a urate oxidase fusion protein as described herein.

[0164] In one embodiment, the pharmaceutical composition is a solution of a urate oxidase conjugate, such as a phosphate buffered saline solution containing one of the urate oxidase fusion proteins described herein. In another embodiment, the solution is sterile and suitable for injection, such as intravenous injection or subcutaneous injection.

[0165] In another aspect, the present disclosure provides a method for reducing elevated uric acid levels in a subject in need thereof, i.e., a method for treating hyperuricemia. The method includes administering to the subject an effective amount of a urate oxidase conjugate or a pharmaceutical composition containing the conjugate.

[0166] As used herein, the term "subject" refers to a vertebrate, such as a mammal. The mammal can be, for example, a mouse, rat, rabbit, cat, dog, pig, sheep, horse, non-human primate (e.g., cynomolgus monkey, chimpanzee), or a human. The human subject can be an adult, adolescent, child (2 to 14 years old), infant (1 to 24 months old), or neonate (up to 1 month old). In one embodiment, the subject is an adult subject. In another embodiment, the subject is an adult subject or a patient.

[0167] In one embodiment of the method, the uric acid level in the plasma or blood of the subject is reduced. In one embodiment, suitable indicators for evaluating the effectiveness of the method include normalization or reduction of the plasma uric acid level (PUA), e.g., in a human subject, the PUA is reduced or maintained at 6.8 mg / dL or lower, or 6 mg / dL or lower.

[0168] In some embodiments of the method, administration of the uricase conjugate or the pharmaceutical composition comprising the conjugate is carried out parenterally, such as by intramuscular, intrathecal, subcutaneous or intravenous administration. In one embodiment, the administration is intravenous administration. In another embodiment, the administration is subcutaneous administration. In another embodiment, the administration is rectal, topical or pulmonary administration.

[0169] 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, with uric acid crystal deposits in the form of nodules called tophi forming under the skin. In human subjects, tophi can form in multiple locations, such as the fingers, hands, feet, elbows or the Achilles tendon along the back of the ankle. In another embodiment, the subject with gout has kidney stones, which are uric acid crystals collected in the urinary tract.

[0170] In one embodiment, the subject is a patient with refractory gout, i.e., refractory to previous different treatments. In one embodiment, the previous treatments are selected from treatment with non-steroidal anti-inflammatory drugs (NSAIDs), colchicine, corticosteroids, allopurinol, febuxostat, probenecid, (pegylated recombinant uricase), rasburicase or a combination of the foregoing. In one embodiment, refractory gout is a chronic condition characterized by high serum uric acid levels, recurrent gout attacks, chronic arthritis and progressive tophi deposition. In another embodiment, refractory gout is associated with a high rate of cardiovascular and renal comorbidities.

[0171] In one embodiment, the subject is diagnosed with tumor lysis syndrome. In another embodiment, the subject has lymphoma (e.g., Burkitt's 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).

[0172] In one embodiment, the present disclosure provides a method of treating gout in a subject in need thereof. The method comprises administering to the subject an effective amount of the urate oxidase conjugate described herein or a pharmaceutical composition comprising the conjugate. 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 formation of subcutaneous nodular deposits of uric acid crystals called tophi. In another embodiment, the subject has kidney stones. In one embodiment, the subject is an adult subject. In another embodiment, the subject is an adult subject or patient.

[0173] In one embodiment, the method of treating gout is carried out parenterally, such as by intramuscular, intrathecal, subcutaneous or intravenous administration. In one embodiment, the administration is intravenous administration. In another embodiment, the administration is subcutaneous administration. In another embodiment, the administration is rectal, topical or pulmonary administration.

[0174] In yet another embodiment of the method of treating with the urate oxidase conjugate described herein, the method is a method of treating tumor lysis syndrome in a subject in need thereof. The method comprises administering to the subject an effective amount of the urate oxidase conjugate or a pharmaceutical composition comprising the conjugate. In one embodiment, the subject is an adult subject. In another embodiment, the subject is an adult subject or patient. In some embodiments of the method, the administration of the urate oxidase conjugate or a pharmaceutical composition comprising the conjugate is carried out parenterally, such as by intramuscular, intrathecal, subcutaneous or intravenous administration. In one embodiment, the administration is intravenous administration. In another embodiment, the administration is subcutaneous administration. In another embodiment, the administration is rectal, topical or pulmonary administration. In some embodiments, the subject with tumor lysis syndrome has lymphoma (e.g., Burkitt's lymphoma, non-Hodgkin lymphoma), acute lymphoblastic leukemia or acute myeloid leukemia. In one embodiment, the subject with tumor lysis syndrome is a human subject with a plasma uric acid concentration > 8 mg / dL. In another embodiment, the subject with tumor lysis syndrome is a human subject with a plasma uric acid concentration > 15 mg / dL (hyperuricemia).

[0175] Example

[0176] The present invention is further illustrated by reference to the following examples. However, it should be noted that, like the above embodiments, the examples are illustrative and should not be construed as limiting the scope of the present invention in any way.

[0177] Example 1 - Generation and Characterization of Uricase Fusion Proteins Comprising Combinations of CPB40 or CPB41 Uricases Directed Against Uricase Domains with PAS or XTEN Polypeptide Domains Method 。

[0178] This example describes the production and characterization of the uricase fusion proteins shown in Table 4. The uricase fusion proteins of 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 CPB40 uricase of the uricase domain and a C-terminal PAS polypeptide domain. The uricase fusion protein of CPB40 uricase-CXTENh (SEQ ID NO: 66) contains the amino acid sequence of CPB40 uricase of the uricase domain and a C-terminal XTEN polypeptide domain. The uricase fusion proteins of NPAS20h-CPB41 uricase-CPAS20h (SEQ ID NO: 75) and NPAS20h-CPB41 uricase-CPAS30h (SEQ ID NO: 76) each contain the amino acid sequence of CPB41 uricase of the uricase domain and two PAS polypeptide domains. In each uricase fusion protein, one of the two PAS polypeptide domains is at the N-terminus, the other PAS polypeptide domain is at the C-terminus, and the uricase domain is between the two PAS polypeptide domains. Since the amino acid sequence of CPB40 or CPB41 uricase is present in each protein monomer of the pegylated homotetramer (pegylated recombinant uricase), it was used in some of the studies described in this example (pegylated recombinant uricase) as a comparator. In this example, for simplicity, (pegylated recombinant uricase) is referred to as "pegylated recombinant uricase".

[0179]

[0180]

[0181]

[0182] 1. Generation and Expression of Uricase Fusion Proteins

[0183] 2. Purification

[0184] DNA encoding CPB40 or CPB41 uricase of the uricase domain of the fusion proteins shown in Table 4, and DNA encoding the PAS polypeptide domains (i.e., PAS10, PAS20, and PAS30) or the XTEN polypeptide domain (with or without a his tag and / or GS spacer) was synthesized by Synbio Technologies (Monmouth Junction, NJ, USA). Using The HiFi DNA Assembly Kit (New England Biolabs, Ipswich, MA, USA) was used to clone the resulting DNA fragments encoding the uricase domain, PAS or XTEN polypeptide domain into the NdeI and BamHI digested expression vector pET-26b(+) (MilliporeSigma, 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 that contains 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 culturing an E. coli culture and inducing protein expression with 100 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), which was transformed with the expression vector pET-26b(+) containing the full-length uricase fusion protein coding sequence.

[0185] 3. Enzymatic Activity Assays of Uricase Fusion Proteins and PEGylated Recombinant Uricases

[0186] The uricase fusion protein expressed in E. coli was purified by the following steps to obtain a soluble (non-aggregated) tetramer:

[0187] (1) Pellet 0.5 L of IPTG-induced E. coli culture by centrifugation at 5,000×g for 10 minutes;

[0188] (2) Resuspend the E. coli pellet in 150 mL of 20 mM sodium borate buffer (pH 9.5)

[0189] ;

[0190] (3) Sonicate the resuspended pellet;

[0191] (4) Centrifuge the sonicated suspension at 17,000×g for 20 minutes and decant the soluble fraction;

[0192] (5) Dilute the soluble fraction to 150 mL with 20 mM sodium borate buffer (pH 9.5) and stir at 4 °C;

[0193] (6) Slowly add 19.5 g of solid ammonium sulfate during stirring;

[0194] (7) Incubate the sample from step (6) at 4 °C for 2 hours;

[0195] (8) Pellet the sample from step (7) by centrifugation at 17,000×g for 20 minutes and decant the soluble fraction;

[0196] (9) Resuspend the protein pellet from step (8) in 40 mL of 20 mM sodium borate buffer (pH 9.5);

[0197] (10) Desalt the suspension from step (9) using a Zeba TM spin desalting column (ThermoFisher Scientific);

[0198] (11) Purify the desalted sample from step (10) by loading the sample onto a 70 mL NH2-750F anion exchange column (Tosoh Bioscience) and eluting the purified sample with 140 mL of 20 mM sodium borate buffer (pH 9.5);

[0199] (12) Use a centrifugal filter to buffer exchange the eluate from step (11) into 1x PBS;

[0200] (13) Load the buffer-exchanged sample from step (12) onto a Superose TM 6 size exclusion chromatography (SEC) column (Cytiva) to separate aggregates from soluble tetramer (target) protein.

[0201] 4. Differential Scanning Fluorimetry (DSF)

[0202] The uricase enzyme activity of the uricase fusion protein and polyethylene glycol recombinant uricase (serial number 264790457565, lot number 0263A), reported as specific activity at a concentration of 72.8 nM, in units of pmol substrate (i.e., uric acid) / min / mg enzyme, was determined and calculated by the following steps:

[0203] (1) Dilute the uricase fusion protein or polyethylene glycol recombinant uricase sample in 1x PBS (pH 7.4)

[0204] to 1456.7 nM;

[0205] (2) Aliquot 10 μL of the diluted uricase fusion protein or polyethylene glycol recombinant uricase sample into each well of a 96-well plate;

[0206] (3) Add 190 μL of uric acid substrate solution containing 0.125 mM uric acid to each well to give a final concentration of 72.8 nM of uricase fusion protein or polyethylene glycol recombinant uricase in the reaction mixture;

[0207] (4) Load the 96-well plate onto a BIOTEK Synergy TM Neo2 microplate reader and shake the plate for 30 seconds;

[0208] (5) Measure the absorbance at 293 nm every 30 seconds on the microplate reader at a controlled temperature of 25 °C for 10 minutes;

[0209] (6) Calculate the Vmax value using BIOTEK Gen5 software.

[0210] 5. Dynamic Light Scattering

[0211] Differential scanning fluorimetry (DSF) uses SYPRO TM Orange fluorescent dye to measure the melting temperature of a native folded protein sample. As the temperature gradually increases on a real-time PCR instrument, the fluorescence signal at λem 570 nm also increases, indicating the exposure of hydrophobic residues and the denaturation of the protein sample. The melting temperature (T m ) of the protein is obtained by analyzing the inflection point of the resulting curve, which is a measure of thermal stability.

[0212] To determine the T of the uricase fusion protein by DSF m , the protein was diluted to a final concentration of 66 μg / mL - 132 μg / mL in PBS buffer containing 5x the final concentration of SYPRO TM Orange dye (Invitrogen, catalog number S6650). 30 μl of the protein-SYPRO TM Orange dye mixture was added to the wells of an optical plate. The optical plate was then loaded onto a BioRad C1000 Touch TM thermal cycler with a CFX96 TM real-time system and exposed to a temperature gradient from 10 °C to 100 °C in 0.5 °C increments. The fluorescence gain of SYPRO TM orange was used and the fluorescence loss of the PRISM software was used to calculate T m .

[0213] 6. In Vivo Pharmacokinetics (PK) and Efficacy Analysis of Uricase Fusion Proteins and PEGylated Recombinant Uricases

[0214] Use a microplate reader III ( Plate ReaderIII, Waters Corporation) to perform dynamic light scattering (DLS) on the uricase fusion protein to determine the hydrodynamic radius and aggregation state (measured by polydispersity) of each protein, as well as the mass percentage within different mass ranges indicating whether the protein is a soluble tetramer or oligomeric or aggregated. Three mass ranges were selected: mass range 1, corresponding to a hydrodynamic radius of 0.5 nm - 10 nm; mass range 2, corresponding to a hydrodynamic radius of 10 nm - 100 nm; and mass range 3, corresponding to a hydrodynamic radius of 100 nm - 1000 nm. Briefly, 100 μL of a protein sample with a concentration of 0.1 mg / mL to 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 onto On the microplate reader III. Collect DLS data with the following parameters:

[0215] (1) Experiment type - Isothermal;

[0216] (2) Enable auto - attenuation - Yes;

[0217] (3) Image each well - Yes;

[0218] (4) DLS acquisition time - 10 seconds;

[0219] (5) Number of DLS acquisitions per measurement - 10;

[0220] (6) Measure SLS - No;

[0221] (7) Number of measurements per well within a scan - 1;

[0222] (8) Wait time between measurements within a scan - 0 minutes;

[0223] (9) Number of scans - 1;

[0224] (10) Wait time between scans - 0 minutes;

[0225] (11) Starting temperature - 25 °C;

[0226] (12) Plate sealer - No sealer;

[0227] (13) Wait for initial temperature lock - Yes;

[0228] (14) Set temperature - 25 °C;

[0229] (15) Laser on - Yes.

[0230] 6.1. Administration of Uricase Fusion Proteins or PEGylated Recombinant Uricases to Wistar Rats and Collection of Rat Blood Samples

[0231] Three PK studies were conducted. The purified soluble tetrameric uricase fusion protein prepared as described in Section 2 above and the polyethylene glycol - recombinant uricase used as a comparator in PK studies 1 and 2 were subjected to PK and efficacy analysis in Wistar rats.

[0232] 6.2. Determination of Uric Acid Concentration in Rat Plasma 6.2.1 Preparation of Artificial Human Plasma and Preparation of Standard Uric Acid Samples for Establishing Standard Curves Using Artificial Human Plasma

[0233] Female Wistar rats weighing approximately 220 g each were purchased from Charles River. In the PK studies described herein, the rats were either untreated or treated by intravenous administration of a single dose of 1 mg / kg body weight of pegylated recombinant uricase or an equimolar single dose (equivalent to 1 mg / kg body weight of pegylated recombinant uricase) of uricase fusion protein (n = 3 rats per group). At different time points before and after dosing, 120 μL of blood was collected from each rat. Tables 5A to 5C show the single doses (in mg / kg rat body weight) of pegylated recombinant uricase or uricase fusion protein administered to the Wistar rats and the blood sample collection time points selected in each of the three PK studies.

[0234]

[0235]

[0236]

[0237] 6.2.2. Processing of Standard Uric Acid Samples and Rat Plasma Samples for LC-MS / MS

[0238] The rat blood samples collected as described in Section 6.1 above were processed 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). The details of the quantification of uric acid concentration in rat plasma are as follows.

[0239] 6.2.3. LC-MS / MS Analysis of Standard Uric Acid Samples and Rat Plasma Samples to Quantify Uric Acid Concentration 6.3. Determination of Uricase Fusion Protein and PEGylated Recombinant Uricase Concentrations in Rat Plasma

[0240] Due to the presence of endogenous uric acid in rat plasma, artificial human plasma was used as a surrogate matrix to prepare standard uric acid samples for establishing a standard curve. Artificial human plasma was prepared by dissolving 2 g of human serum albumin (Sigma - Aldrich) in 50 mL of PBS and then adjusting the pH to 7.4 with 1 M sodium hydroxide or 1 M phosphoric acid. Standard uric acid samples containing 0.1 μg / mL, 0.2 μg / mL, 1 μg / mL, 5 μg / mL, 10 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL uric acid in artificial human plasma were prepared and further processed and analyzed by LC - MS / MS in parallel with the study rat plasma samples as described below.

[0241] Results

[0242] The standard uric acid samples and study rat plasma samples in Section 6.2.1 were processed according to the following procedure:

[0243] (1) Add 20 μl of 500 ng / mL uric acid-1,3- 15 N2 aqueous solution (Sigma-Aldrich) and 40 μl of 0.4 N perchloric acid to 20 μl of a standard uric acid sample or a rat plasma sample;

[0244] (2) Centrifuge the sample from step (1) at 4 °C in an Eppendorf 5424R centrifuge at 15,000 rpm for 15 minutes;

[0245] (3) Pipette out 50 μL of the supernatant from each sample after centrifugation in step (2), and add 50 μL of water to the supernatant;

[0246] (4) Mix the samples obtained in step (3) for the following LC-MS / MS analysis.

[0247] 1. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) Analysis

[0248] Inject 10 μl of each standard uric acid sample or rat plasma sample processed as described in Section 6.2.2 into the Acquity TM BEH amide column of a Shimadzu Nexera LC (Sciex), and then perform gradient elution. Uric acid quantification is carried out using a triple quadrupole AP 14500 mass spectrometer (Sciex) equipped with ESI operating in the negative mode. The parameters of the mass spectrometer and the LC are shown in Table 6A and Table 6B, respectively.

[0249]

[0250]

[0251]

[0252] Use the data acquisition software Analyst TM 1.7 and MultiQuant TM 3.0 (Sciex) or its equivalent software to collect LC-MS / MS data. Generate a standard curve using the data of the standard uric acid samples. Construct a calibration curve by plotting the ratio of the peak areas of the reference standard to the internal standard against the nominal concentration of the reference standard. Fit the calibration curve by least squares regression analysis to provide information on the slope of the calibration curve, the y-axis intercept, the correlation coefficient, and the back-calculated calibration standard concentration.

[0253] The standard addition method was used to show the standard curve obtained using a standard uric acid sample prepared with human artificial plasma to accurately measure the uric acid concentration in rat plasma. In the standard addition method, as described above, untreated rat plasma samples spiked with three uric acid concentration levels of 5 μg / mL, 25 μg / mL, and 80 μg / mL, respectively, and blank untreated rat plasma samples (BioIVT) were processed and analyzed by LC-MS / MS. Using the standard curve obtained from the standard uric acid sample prepared with human artificial plasma, the endogenous uric acid level in the blank untreated rat plasma and the uric acid levels in the three standard addition (i.e., spiked with uric acid) rat plasma samples were determined. Then, the adjusted uric acid levels after subtracting the endogenous uric acid level in the three standard addition samples were compared with their target (spiked) concentrations. The percentage difference between the adjusted uric acid concentration and the corresponding target uric acid concentration was found to be less than 15%, meeting the acceptance criteria.

[0254] Figure 2A

[0255] The rat blood samples collected as described in Section 6.1 above were processed to obtain rat plasma, and then Amplex TM Red Uric Acid / Urease Assay Kit (ThermoFisher Scientific, catalog number A22181) was used to determine the concentration of uricase fusion protein or polyethylene glycol recombinant uricase in the plasma at each time point. This assay kit provides a fluorescence assay method that includes a two-step reaction to quantify the uricase fusion protein or polyethylene glycol recombinant uricase based on the uricase enzyme activity. First, the uricase enzyme activity of the uricase fusion protein or polyethylene glycol recombinant uricase converts uric acid into allantoin, hydrogen peroxide (H2O2), and carbon dioxide. Second, in the presence of horseradish peroxidase (HRP), H2O2 reacts stoichiometrically with Amplex TM Red reagent to generate the red fluorescent oxidation product resorufin. In the study of this example, the concentration of uricase fusion protein or polyethylene glycol recombinant uricase in rat plasma was indirectly quantified by measuring the fluorescence of resorufin captured by a microplate reader (Molecular Devices) in kinetic mode for 30 minutes. A standard curve for quantifying the uricase fusion protein or polyethylene glycol recombinant uricase was constructed using rat plasma of the same strain. IDBS's Excel add-in software was used to process the standard curve regression with a weighted model and back-calculate the concentration of uricase fusion protein or polyethylene glycol recombinant uricase in the rat plasma samples.

[0256] Rat plasma has endogenous uricase, which also produces a fluorescent signal in the uricase assay. Additionally, the levels of endogenous rat plasma uricase fluctuate between rats and over time within an individual rat, making it infeasible to determine the true background uricase for a given sample. Thus, the concentration of exogenously administered uricase fusion protein or PEGylated recombinant uricase in plasma of study samples was approximated by subtracting the background (endogenous) uricase concentration in the pre-dose plasma of each group of rats treated with uricase fusion protein or PEGylated recombinant uricase. The endogenous uricase concentration in plasma of the untreated control rat group at the corresponding pre-dose time point (at time 0) and post-dose time points was also adjusted by subtracting the mean pre-dose endogenous plasma uricase concentration of the control rat group.

[0257] Figure 2B

[0258] Figure 2A

[0259] As described in "Methods", the uricase fusion proteins shown in Table 4 were generated, expressed in E. coli, and purified as soluble tetramers. The purified uricase fusion proteins together with PEGylated recombinant uricase were analyzed by SDS-PAGE under non-reducing conditions. Figure 2B and Figure 2A are representative images of SDS-PAGE gels, where the bands represent monomeric uricase fusion protein or monomeric PEGylated recombinant uricase detected by Coomassie blue staining.

[0260] Figure 2B Shows the protein bands detected with samples containing NPAS20h-CPB41 uricase-CPAS20h, NPAS20h-CPB41 uricase-CPAS30h, and PEGylated recombinant uricase in lanes 1, 2, and 3, respectively. These samples were used for PK study 2 of this example.

[0261] 2. In Vivo PK and Efficacy Analysis Comparing Uricase Fusion Proteins with PEGylated Recombinant Uricases Shows the protein bands detected with samples containing CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h in lanes 1 and 2, respectively. These samples were used for PK study 3 of this example.

[0262] NPAS20h-CPB41 uricase-CPAS20h has an expected monomer molecular weight of 102,650 Da. NPAS20h-CPB41 uricase-CPAS30h has an expected monomer molecular weight of 119,168 Da. CPB40 uricase-CPAS20h has an expected monomer molecular weight of 68,328 Da. Including the molecular weight standards shown in the rightmost lane, 2.1. PK Study 1 and Efficacy Analysis of Plasma Uric Acid Concentration in Rat Blood Samples from This Study and Figures 3A to 3FSDS-PAGE gels showed that the apparent size of the monomeric uricase fusion protein based on the protein migration position was significantly larger than its corresponding expected size. This observation is consistent with the finding of Breibeck et al. that ″[the fusion protein of PAS polypeptide] with IL-1Ra [interleukin-1 receptor antagonist] and TrxA [Escherichia coli thioredoxin] migrated at a position corresponding to a much higher molecular weight than normally expected.″ Breibeck et al. have hypothesized that ″[the delayed electrophoretic mobility of PASylated proteins] is mainly due to poor binding of SDS, whose negatively charged head groups provide the driving force in the electric field, which may be caused by the lack of any 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. January 2018; 109(1): e23069, page 3, right column, first paragraph.

[0263] Figure 3A

[0264] Figure 3B

[0265] In PK study 1, female Wistar rats in groups of 3 rats per group were untreated or treated by intravenous administration of a single dose of 1 mg / kg body weight of polyethylene glycol recombinant uricase, 1.51 mg / kg body weight of CPB40 uricase-CPAS10h, 1.99 mg / kg body weight of CPB40 uricase-CPAS20h, 2.47 mg / kg body weight of CPB40 uricase-CPAS30h, or 2.16 mg / kg body weight of CPB40 uricase-CXTENh (Table 5A). Rat blood samples were collected before dosing and at 0.5 hour, 2 hours, 6 hours, 24 hours, 72 hours, and 96 hours after dosing. As described in ″Methods″, the concentration of endogenous uricase in the plasma of untreated control rats or the concentration of polyethylene glycol recombinant uricase and uricase fusion protein in the plasma of treated rats, as well as the concentration of uric acid in the plasma, were measured. The results are as Figure 3C shown, where the above-mentioned endogenous uricase or exogenously administered polyethylene glycol recombinant uricase and uricase fusion protein in rat plasma are commonly referred to as ″plasma uricase″.

[0266] Figure 3DShows the concentrations of endogenous uricase and uric acid in the plasma of untreated control rats at the corresponding pre - dosing time points (at time 0) and at each corresponding post - dosing time point (up to 96 hours). The concentration of endogenous uricase in the plasma at time 0 was adjusted to 0 μg / mL by subtracting the mean pre - dosing plasma endogenous uricase concentration as described in "Methods". At the corresponding post - dosing time points, the adjusted plasma endogenous uricase concentration fluctuated below or above 0 μg / mL as described in the "Methods" section. The uric acid concentration in the plasma of untreated control rats also fluctuated and was greater than 0 μg / mL at all time points.

[0267] Figure 3E Shows the concentrations of polyethylene glycol - recombinant uricase and uric acid in the plasma of rats treated with polyethylene glycol - recombinant uricase, before dosing (at time 0) and at each post - dosing time point (up to 96 hours). The concentration of polyethylene glycol - recombinant uricase in the plasma reached stability between 0.5 hour and 6 hours after dosing and remained above the detectable level for at least 96 hours after dosing, while the concentration of uric acid in the plasma decreased to and remained at 0 μg / ml at all time points tested after dosing (i.e., from 0.5 hour to 96 hours after dosing).

[0268] Figure 3F Shows the concentrations of CPB40 uricase - CPAS10h and uric acid in the plasma of rats treated with CPB40 uricase - CPAS10h, before dosing (at time 0) and at each time point up to 96 hours after dosing. The concentration of CPB40 uricase - CPAS10h in the plasma peaked at 0.5 hour after dosing and then dropped below the detection limit at 24 hours after dosing. The concentration of uric acid in the plasma decreased to and remained at 0 μg / ml from 0.5 hour to 24 hours after dosing, and then rebounded above 0 μg / ml between 24 hours and 72 hours after dosing.

[0269] Figure 3D Shows the concentrations of CPB40 uricase - CPAS20h and uric acid in the plasma of rats treated with CPB40 uricase - CPAS20h, before dosing (at time 0) and at each time point up to 96 hours after dosing. The concentration of CPB40 uricase - CPAS20h in the plasma peaked at 0.5 hour after dosing, remained at a high level at 24 hours after dosing, and then dropped below the detection limit at 72 hours after dosing. The concentration of uric acid in the plasma decreased to and remained at 0 μg / ml from 0.5 hour to 24 hours after dosing, and then rebounded above 0 μg / ml between 24 hours and 72 hours after dosing, similar to the uric acid curve in the plasma of rats treated with CPB40 uricase - CPAS10h.

[0270] 2.2. PK Study 2 and Efficacy Analysis of Plasma Uric Acid Concentration in Rat Blood Samples from This StudyShows the concentrations of CPB40 uricase-CPAS30h and uric acid in plasma of rats treated with CPB40 uricase-CPAS30h at various time points before dosing (at time 0) and up to 96 hours after dosing. The changes in the concentrations of CPB40 uricase-CPAS30h and uric acid in plasma after dosing were similar to those in rats treated with CPB40 uricase-CPAS20h. That is, the concentration of CPB40 uricase-CPAS30h in plasma reached a peak at 0.5 hour after dosing, remained at a high or detectable level at 24 hours after dosing, and then dropped below the limit of detection at 72 hours after dosing. The concentration of uric acid in plasma decreased to and remained at 0 μg / ml from 0.5 hour to 24 hours after dosing, and then rebounded to above 0 μg / ml between 24 hours and 72 hours after dosing.

[0271] Figures 4A to 4D Shows the concentrations of CPB40 uricase-CXTENh and uric acid in plasma of rats treated with CPB40 uricase-CXTENh at various time points before dosing (at time 0) and up to 96 hours after dosing. The concentration of CPB40 uricase-CXTENh in plasma reached a peak at 0.5 hour after dosing and then continuously dropped below the limit of detection at 72 hours after dosing. The rate of decline was faster than that observed in rats treated with CPB40 uricase-CPAS20h (see Figure 4A ). The concentration of uric acid in plasma decreased to and remained at 0 μg / ml from 0.5 hour to 24 hours after dosing, and then rebounded to above 0 μg / ml between 24 hours and 72 hours after dosing, similar to the uric acid curve in plasma of rats treated with CPB40 uricase-CPAS20h.

[0272] Figure 3A

[0273] In PK study 2, female Wistar rats in groups of 3 rats per group were either not treated or treated by intravenous administration of a single dose of 1 mg / kg body weight of polyethylene glycol recombinant uricase, 2.99 mg / kg body weight of NPAS20h-CPB41 uricase-CPAS20h, or 3.47 mg / kg body weight of NPAS20h-CPB41 uricase-CPAS30h (Table 5B). Rat blood samples were collected before dosing and at 0.5 hour, 1 hour, 2 hours, 6 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 168 hours after dosing. As described in "Methods", the concentration of endogenous uricase in plasma of untreated control rats, or the concentration of polyethylene glycol recombinant uricase and uricase fusion protein in plasma of treated rats, as well as the concentration of uric acid in plasma, were measured. The results are shown in Figure 4BAs shown, the above-mentioned endogenous uricase or exogenously administered polyethylene glycol recombinant uricase and uricase fusion protein in rat plasma are generally referred to as "plasma uricase".

[0274] Figure 4C Shows the concentrations of endogenous uricase and uric acid in the plasma of untreated control rats at the corresponding pre-dosing time points (at time 0) and at each corresponding post-dosing time point (up to 168 hours). The concentration of endogenous uricase in the plasma at time 0 was adjusted to be close to 0 μg / mL by subtracting the average pre-dosing plasma endogenous uricase concentration as described in "Methods". At the corresponding post-dosing time points, the concentration of the similarly adjusted endogenous uricase in the plasma fluctuated relative to the baseline concentration at time 0. The uric acid concentration in the plasma of untreated control rats also fluctuated and was greater than 0 μg / mL at all time points. These results are similar to those observed in the untreated control rats in Figure 4D PK study 1 as shown.

[0275] 2.3. PK Study 3 and Efficacy Analysis of Plasma Uric Acid Concentration in Rat Blood Samples from This Study Shows the concentrations of polyethylene glycol recombinant uricase and uric acid in the plasma of rats treated with polyethylene glycol recombinant uricase before dosing (at time 0) and at each post-dosing time point (up to 168 hours). The concentration of polyethylene glycol recombinant uricase in the plasma peaked at 0.5 hours after dosing and remained above the detectable level for at least 168 hours after dosing. In two of the three rats treated with polyethylene glycol recombinant uricase, the concentration of uric acid in the plasma decreased to and remained at 0 μg / ml at 168 hours after dosing, while in the third rat treated with polyethylene glycol recombinant uricase, the concentration of uric acid in the plasma rebounded to a low level above 0 μg / ml only at the 168-hour time point and not at the 120-hour time point.

[0276] Figures 5A to 5CShows the concentrations of NPAS20h-CPB41 uricase-CPAS20h and uric acid in plasma at various time points before dosing (at time 0) and up to 168 hours after dosing in rats treated with NPAS20h-CPB41 uricase-CPAS20h. The changes in the concentrations of NPAS20h-CPB41 uricase-CPAS20h and uric acid in plasma after dosing were similar to those in rats treated with pegylated recombinant uricase. That is, the concentration of NPAS20h-CPB41 uricase-CPAS20h in plasma reached a steady state between 0.5 hour and 2 hours after dosing and remained above the detectable level for at least 168 hours after dosing. In two of the three rats treated with NPAS20h-CPB41 uricase-CPAS20h, the concentration of uric acid in plasma decreased to and remained at 0 μg / ml at 168 hours after dosing, while the concentration of uric acid in plasma of the third rat treated with NPAS20h-CPB41 uricase-CPAS20h rebounded to a low level above 0 μg / ml only at the 168-hour time point and not at the 120-hour time point.

[0277] Figure 5A Shows the concentrations of NPAS20h-CPB41 uricase-CPAS30h and uric acid in plasma at various time points before dosing (at time 0) and up to 168 hours after dosing in rats treated with NPAS20h-CPB41 uricase-CPAS30h. The concentration of NPAS20h-CPB41 uricase-CPAS30h in plasma peaked at approximately 1 hour after dosing, remained at a high level until 6 hours after dosing, and then decreased to a low steady state level close to the limit of detection at 72 hours and later time points after dosing. The concentration of uric acid in plasma decreased to and remained at 0 μg / ml from 72 hours after dosing, and then rebounded to above 0 μg / ml at 96 hours and later time points after dosing.

[0278] Figure 3A

[0279] In PK study 3, the uricase fusion protein CPB40 uricase-CPAS20h studied in PK study 1 and NPAS20h-CPB41 uricase-CPAS20h studied in PK study 2 were compared side by side. Specifically, female Wistar rats in groups of 3 rats per group were left untreated or treated by intravenous administration of a single dose of 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 dosing and at 1 hour, 6 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 168 hours, 192 hours, 216 hours, and 240 hours after dosing. As described in "Methods", the concentration of endogenous uricase in the plasma of untreated control rats or the concentration of uricase fusion proteins (i.e., CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h) in the plasma of treated rats, as well as the concentration of uric acid in the plasma, were measured. The results are as Figure 4A shown, where the above endogenous uricase or exogenously administered uricase fusion protein in the rat plasma is generally referred to as "plasma uricase".

[0280] Figure 5B shown the concentrations of endogenous uricase and uric acid in the plasma of untreated control rats at the corresponding pre-dosing time point (at time 0) and at each corresponding post-dosing time point (up to 240 hours). The concentration of endogenous uricase in the plasma at time 0 was adjusted to be close to 0 μg / mL by subtracting the average pre-dosing plasma endogenous uricase concentration as described in "Methods". At the corresponding post-dosing time points, the similarly adjusted plasma endogenous uricase concentration fluctuated relative to the baseline concentration at time 0. The uric acid concentration in the plasma of untreated control rats also fluctuated and was greater than 0 μg / mL at all time points. These results are similar to those observed in the untreated control rats in PK studies 1 and 2 shown in Figure 3D and Figure 5C respectively.

[0281] Figure 4CShows the concentrations of CPB40 uricase - CPAS20h and uric acid in plasma at various time points before dosing (at time 0) and up to 240 hours after dosing in rats treated with CPB40 uricase - CPAS20h. The concentration of CPB40 uricase - CPAS20h in plasma reached a peak at 1 hour after dosing (the earliest post - dosing time point tested in the study), remained at a high level at 24 hours after dosing, and then dropped below the limit of detection between 72 hours and 96 hours after dosing. The concentration of uric acid in plasma decreased to and remained at 0 μg / ml from 1 hour to 72 hours after dosing, and then rebounded to above 0 μg / ml between 72 hours and 96 hours after dosing. In this study, the performance of CPB40 uricase - CPAS20h at 72 hours after dosing seemed to be improved compared to its performance at the same time point (i.e., 72 hours after dosing) in PK study 1 (see 3. Uricase Enzymatic Activity of Uricase Fusion Proteins Compared with PEGylated Recombinant Uricases ), which may be due to the higher quality of the uricase fusion protein produced in this PK study (i.e., PK study 3).

[0282] 5. DLS Analysis of Uricase Fusion Proteins Shows the concentrations of NPAS20h - CPB41 uricase - CPAS20h and uric acid in plasma at various time points before dosing (at time 0) and up to 240 hours after dosing in rats treated with NPAS20h - CPB41 uricase - CPAS20h. The concentration of NPAS20h - CPB41 uricase - CPAS20h in plasma reached stability between 1 hour and 6 hours after dosing (the earliest post - dosing time point tested in the study) and remained above the detectable level for at least 168 hours after dosing. In all three rats treated with NPAS20h - CPB41 uricase - CPAS20h, the concentration of uric acid in plasma decreased to and remained at 0 μg / ml at 120 hours after dosing. At 168 hours after dosing, although in one of the three treated rats, the concentration of uric acid in plasma remained at 0 μg / ml, in the other two treated rats in this group, the concentration rebounded to a low level above 0 μg / ml. Thus, the PK and efficacy results of NPAS20h - CPB41 uricase - CPAS20h in this study were generally similar to those seen in PK study 2 (see Figure 4C ).

[0283] In summary, the results from the above three PK and efficacy studies showed that the uric acid concentration in the plasma of untreated control rats varied between rats and at different time points of the study. Therefore, it was not meaningful to interpret accurate plasma uric acid concentration values after treatment with uricase fusion protein or PEGylated recombinant uricase. However, the study results showed a binary correlation between plasma uric acid concentrations of 0 μg / ml and greater than 0 μg / ml and the presence and clearance rate of circulating exogenous uricase (i.e., uricase fusion protein or PEGylated recombinant uricase). Specifically, a plasma uric acid concentration of 0 μg / ml was correlated with or indicative of the detectable level of circulating exogenous uricase, while the rebound of plasma uric acid concentration to above 0 μg / ml was correlated with or indicative of the elimination of exogenously administered uricase.

[0284] Aliquots of the purified soluble tetrameric uricase fusion protein samples used in PK studies 2 and 3 were further characterized in vitro, including uricase enzyme activity assays, differential scanning fluorimetry (DSF) to determine the protein's melting temperature (T m ) and dynamic light scattering (DLS) to determine the protein's hydrodynamic radius and aggregation state, as described below.

[0285] Figure 4D

[0286] Aliquots of two purified soluble tetrameric uricase fusion protein samples containing NPAS20h-CPB41 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS30h, respectively, used in PK study 2, and an aliquot of PEGylated recombinant uricase were assayed for uricase enzyme activity at a protein concentration of 72.8 nM as described in the "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 for each sample were calculated and are shown in Table 7A.

[0287]

[0288] The data in Table 7A showed that each of the uricase fusion proteins NPAS20h-CPB41 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS30h tested in PK study 2 had more than 80% of the in vitro activity of PEGylated recombinant uricase.

[0289] Aliquots of two purified soluble tetrameric uricase fusion protein samples containing CPB40 uricase - CPAS20h and NPAS20h - CPB41 uricase - CPAS20h, respectively, for PK study 3, and an aliquot of pegylated recombinant uricase were also assayed for uricase enzyme activity at a protein concentration of 72.8 nM as described in "Methods". 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 for each sample were calculated and are shown in Table 7B.

[0290]

[0291] The data in Table 7B show that each of the uricase fusion proteins CPB40 uricase - CPAS20h and NPAS20h - CPB41 uricase - CPAS20h tested in PK study 3 had more than 80% of the in vitro pegylated recombinant uricase activity.

[0292] 4. Melting temperature (T m )

[0293] Aliquots of two purified soluble tetrameric uricase fusion protein samples containing NPAS20h - CPB41 uricase - CPAS20h and NPAS20h - CPB41 uricase - CPAS30h, respectively, for PK study 2 were subjected to DSF to determine the melting temperature of the uricase fusion proteins as described in "Methods". The results are shown in Table 8A. The melting temperature (T m ) was measured twice for each sample. The mean and standard deviation (SD) of the two measurements for each sample were calculated and are shown in Table 8A.

[0294]

[0295] The data in Table 8A show that the uricase fusion proteins NPAS20h - CPB41 uricase - CPAS20h and NPAS20h - CPB41 uricase - CPAS30h tested in PK study 2 had the same T of 63.5 °C m , which is higher than the historical T of 61 °C for pegylated CPB40 uricase determined in previous studies. m

[0296] Aliquots of two purified soluble tetrameric uricase fusion protein samples containing CPB40 uricase - CPAS20h and NPAS20h - CPB41 uricase - CPAS20h, respectively, for PK study 3 were also subjected to DSF to determine the melting temperature of the uricase fusion proteins as described in "Methods". The results are shown in Table 8B. The melting temperature (T m)。The average value and standard deviation (SD) of the two measurements for each sample were calculated and are shown in Table 8B.

[0297]

[0298] The data in Table 8B show that the uricase fusion proteins CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h tested in PK study 3 had melting temperatures of 63.00 °C and 63.50 °C, respectively, both of which were higher than the historical T of 61 °C for pegylated CPB40 uricase. m 。

[0299] ​

[0300] Aliquots of two purified soluble tetrameric uricase fusion protein samples containing NPAS20h-CPB41 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS30h, respectively, for PK study 2 were subjected to DLS as described in "Methods" to determine the hydrodynamic radius, polydispersity, and mass percentages in various mass ranges of the uricase fusion proteins, and the results are shown in Table 9A.

[0301]

[0302] The DLS data in Table 9A show 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%).

[0303] In addition, NPAS20h-CPB41 uricase-CPAS30h appeared to have two different species: one at the expected hydrodynamic radius (about 17 nm, 46.85% mass in mass range 2), and the other 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 stable monomeric protein population different from the tetrameric population, and the presence of the monomeric protein population may be the reason for the shorter half-life of the NPAS20h-CPB41 uricase-CPAS30h sample compared to the NPAS20h-CPB41 uricase-CPAS20h sample observed in PK study 2 (see and compare ​ and ​ ).

[0304] Aliquots of two purified soluble tetrameric uricase fusion protein samples containing CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h, respectively, for PK study 3 were also subjected to DLS as described in "Methods" to determine the hydrodynamic radius, polydispersity, and mass percentage in mass range 2 (corresponding to hydrodynamic radius 10 nm - 100 nm) of the uricase fusion proteins. The results are shown in Table 9B. Each DLS parameter of each sample was measured one or two times (denoted as "1" and "2" in Table 9B), and the average of the two measurements of each parameter was calculated and shown in Table 9B.

[0305]

[0306] The DLS data in Table 9B show that both the CPB40 uricase-CPAS20h and NPAS20h-CPB41 uricase-CPAS20h samples have > 99% mass in mass range 2 corresponding to a hydrodynamic radius of 10 nm - 100 nm, indicating < 1% aggregation. Additionally, the finding that the hydrodynamic radius of NPAS20h-CPB41 uricase-CPAS20h is greater than that of CPB40 uricase-CPAS20h indicates that the hydrodynamic radius increases with the increase in the length of the PAS domain in the uricase fusion protein.

[0307] *********

[0308] Although the described invention has been described with reference to specific embodiments of the present invention, those skilled in the art will understand that various changes can be made and equivalents can be substituted without departing from the true spirit and scope of the present invention. In addition, various modifications can be made to adapt to a particular situation, material, composition of matter, process, one or more process steps, in accordance with the objective spirit and scope of the described invention. All such modifications are intended to fall within the scope of the appended claims.

[0309] The patents, patent applications, patent application publications, journal articles, and protocols cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A uricase conjugate, the 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 at the C-terminus of the second domain.

4. The uricase conjugate according to claim 2, wherein the first domain is 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 from about 2 amino acids to about 5 amino acids in length.

7. The uricase conjugate according to claim 6, wherein the amino acid linker is 2 amino acids in 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, the uricase conjugate 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 at the N-terminus of the second domain and the C-terminus 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 from about 2 amino acids to about 5 amino acids in length.

14. The uricase conjugate according to claim 13, wherein the amino acid linker is 2 amino acids in 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 shown in SEQ ID NO:

60.

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

61.

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

62.

21. The uricase conjugate according to claim 16 or 17, wherein the PAS polypeptide comprises the amino acid sequence shown 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 - 111.

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

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

26. The uricase conjugate according to claim 24 or 25, wherein the XTEN polypeptide has the amino acid sequence shown 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 from 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 from 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 from 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 from 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 from 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 from 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 from 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 from 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 from 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 from 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 from about 100 amino acids 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 from about 100 amino acids to about 300 amino acids.

39. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

63.

40. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

64.

41. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

65.

42. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

66.

43. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

67.

44. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

68.

45. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

69.

46. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

70.

47. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

75.

48. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

76.

49. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

77.

50. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

78.

51. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

79.

52. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

80.

53. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown in SEQ ID NO:

112.

54. The uricase conjugate according to claim 1, wherein the uricase conjugate comprises the amino acid sequence shown 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 shown in SEQ 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 shown 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 shown 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 shown 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 shown 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 - 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 - 44.

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

41.

78. The uricase conjugate according to claim 70, wherein the amino acid variant comprises from 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 from 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 from 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 from 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 from 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 from 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 from 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 from 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 from 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 from 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 from 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 shown 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 shown 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, the uricase conjugate further comprising a purification tag at the C-terminus.

95. The uricase conjugate according to any one of claims 2 to 94, the uricase conjugate 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 the uricase conjugate according to any one of claims 1 to 99.

101. A nucleic acid vector comprising the nucleic acid according to claim 100.

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

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

104. A method of treating hyperuricemia in a subject in need thereof, the method comprising administering to the subject an effective amount of the 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 in the plasma of the subject is decreased.

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 refractory gout.

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

109. A method for treating gout in a subject in need thereof, the method comprising administering to the subject an effective amount of the 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 refractory gout.

111. A method for treating tumor lysis syndrome in a subject in need thereof, the method comprising administering to the subject an effective amount of the 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 patient.

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

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

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

117. A method for recombinantly producing the uricase conjugate according to any one of claims 1 to 99, the method comprising: (i) culturing a host cell comprising a nucleic acid vector, the nucleic acid vector comprising a nucleic acid sequence encoding the 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 permit expression of the nucleic acid sequence encoding the uricase conjugate and recombinant production of the uricase conjugate by the host cell; and (ii) isolating the recombinantly produced uricase conjugate.

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