Uricase variant and its method of use

Abstract

This disclosure relates to uricase variants and their polymers, and to uricase conjugates, wherein the uricase conjugate comprises a first domain containing one of the uricase variants, and the first domain is conjugated to a second domain containing a random coil polypeptide domain. Methods for using them to treat gout, tumor lysis syndrome, and other diseases associated with elevated uric acid levels in body fluids, including blood, are provided. Methods for recombinantly producing the uricase variants or uricase conjugates disclosed herein are also provided.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 500,541, filed on 5 May 2023, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Reference to electronic sequence listings The contents of the electronic sequence listing (INMD_191_01WO_SeqList_ST26.xml, size: 3,082,460 bytes, and creation date: April 30, 2024) are incorporated herein by reference in their entirety. [Background technology]

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

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

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

[0006] Uricase is an enzyme that catalyzes the oxidation of uric acid, converting it into allantoin, a more soluble product of purine metabolites, which is more easily excreted. Humans do not produce enzymatically active uricase due to several mutations in the uricase gene acquired during the evolution of higher primates; therefore, exogenously administered uricase provides a therapy for diseases exhibiting hyperuricemia (e.g., gout and tumor lysis syndrome). [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] This invention addresses the need for deimmunizing uricases for the treatment of gout, tumor lysis syndrome, and other diseases associated with elevated uric acid levels in bodily fluids, including blood. [Means for solving the problem]

[0008] In one embodiment, the present disclosure relates to a uricase variant comprising one or more amino acid mutations relative to the corresponding uricase protein, wherein one or more amino acid mutations are located at one or more amino acid positions corresponding to one or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257, and the uricase variant comprises an amino acid sequence that is at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 1.

[0009] In one embodiment, the corresponding uricase protein is a chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1.

[0010] In another embodiment, the corresponding uricase protein contains an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or about 90% to about 100% identical to SEQ ID NO: 1. In a further embodiment, the corresponding uricase protein is a chimeric pig-baboon uricase containing an amino acid sequence that is at least about 70%, at least about 80%, at least about 90%, or about 90% to about 100% identical to SEQ ID NO: 1.

[0011] In one embodiment, the uricase variant contains two or more, three or more, four or more, five or more, seven or more, ten or more, fifteen or more, or about five to about 25 amino acid mutations relative to the corresponding uricase protein, the amino acid mutations being selected from mutations at amino acid positions corresponding to the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each of the amino acid mutations is an amino acid substitution. In further embodiments, the amino acid substitutions correspond to two or more, three or more, four or more, five or more, seven or more, ten or more, fifteen or more, or about five to about 25 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In further embodiments, the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1 that include one of the exemplary groups of the amino acid substitutions listed in Table B. In further embodiments, the uricase variant further includes amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In further embodiments, the uricase variant further includes amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1.

[0012] In one embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions in SEQ ID NO: 1: V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q in SEQ ID NO: 1. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117G or N117D in SEQ ID NO: 1.

[0013] In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions in SEQ ID NO: 1: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K. In a further embodiment, the uricase variant further comprises amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q in SEQ ID NO: 1. In a further embodiment, the uricase variant further comprises amino acid substitutions corresponding to N117G or N117D in SEQ ID NO: 1.

[0014] In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions in SEQ ID NO: 1: V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q in SEQ ID NO: 1. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117G or N117D in SEQ ID NO: 1.

[0015] In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions in SEQ ID NO: 1: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q in SEQ ID NO: 1. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117G or N117D in SEQ ID NO: 1.

[0016] In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions in SEQ ID NO: 1: V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q in SEQ ID NO: 1. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117G or N117D in SEQ ID NO: 1.

[0017] In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions in SEQ ID NO: 1: V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K. In a further embodiment, the uricase variant further comprises amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q in SEQ ID NO: 1. In a further embodiment, the uricase variant further comprises amino acid substitutions corresponding to N117G or N117D in SEQ ID NO: 1.

[0018] In one embodiment, the uricase variant includes an amino acid sequence selected from SEQ ID NOs: 25 to 154. In a further embodiment, the uricase variant includes an amino acid sequence selected from SEQ ID NOs: 28, 45, 48, 49, 53, 66, 67, 101, 104, 112, 114, 117, 119, 123, 125, 129, 139, 142, 149, 150, and 152. In yet another embodiment, the uricase variant includes an amino acid sequence selected from SEQ ID NOs: 45, 53, 66, 114, 129, and 149.

[0019] In one embodiment, the uricase variant includes an amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NOs. 25-154. In a further embodiment, the uricase variant includes an amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NOs. 28, 45, 48, 49, 53, 66, 67, 101, 104, 112, 114, 117, 119, 123, 125, 129, 139, 142, 149, 150, and 152. In a further embodiment, the uricase variant includes an amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NOs. 45, 53, 66, 114, 129, and 149.

[0020] In one embodiment, the uricase variant contains the amino acid sequence of SEQ ID NO: 53. In another embodiment, the uricase variant contains an amino acid sequence that is 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% identical to SEQ ID NO: 53. Exemplary uricase variants include those containing an amino acid sequence selected from SEQ ID NOs: 197-309.

[0021] In one embodiment, the uricase variant comprises the amino acid sequence of SEQ ID NO: 129. In another embodiment, the uricase variant comprises an amino acid sequence that is 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% identical to SEQ ID NO: 129. Exemplary uricase variants include those comprising an amino acid sequence selected from SEQ ID NOs: 187, 189, 191, 193, and 195, and SEQ ID NOs: 310-1566.

[0022] In one embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs: 971, 973, 976, 978, 979, 983, 985, 988, 991, 997, 1000, 1013, 1016, 1018, 1022, 1023, 1024, 1030, 1032, 1033, 1035, 1037, 1041, 1043, 1044, 1045, 1046, 1051, 1053, 1054, 1055, 1057, 1077, 1134, and 1142.

[0023] In one embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs: 1330-1474. In a further embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs: 1330, 1333, 1342, 1358, 1375, 1385, 1391, 1436, 1442, 1451, 1457, and 1465.

[0024] In one embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs: 1475-1566.

[0025] In some embodiments of the uricase variants provided herein, the uricase variant comprises a first set of amino acid substitutions and a second set of amino acid substitutions relative to the corresponding wild-type uricase protein. The first set of amino acid substitutions is in group (a) or group (b) below, (a) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K, or (b) corresponds to the amino acid substitutions of SEQ ID NO: 1 as set forth in V22L, Q26T, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

[0026] The second set of amino acid substitutions is a set of about 6 to about 10 (e.g., 6, 7, 8, 9, or 10) amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1: one of K30E and K30F, L52T, H53D, K79S, T83E, V86N, S94D, one of K97D, N117G and N117D, one of F172W and F172L, T174N, E177Q, R192T, G196D, R197D, Y225H, S228G, L239G, L279T, and N281Y. The urease variant comprises an amino acid sequence that is at least about 85%, at least about 90%, at least about 91%, or at least about 92% identical to SEQ ID NO: 1.

[0027] In one embodiment of the urease variants disclosed herein, the urease variant is not PEGylated. In another embodiment, the urease variant is PEGylated. In one embodiment, the urease variant is PASylated. In another embodiment, the urease variant is PASylated and PEGylated. In one embodiment, the urease variant is XTENylated. In yet another embodiment, the urease variant is XTENylated and PEGylated.

[0028] In one embodiment, the urease variants disclosed herein are monomers. In another embodiment, the urease variant is a homodimer. In a preferred embodiment, the urease variant is a homotetramer.

[0029] In another aspect of this disclosure, a uricase dimer is provided comprising a first uricase monomer and a second uricase monomer, wherein the first uricase monomer is one of the uricase variants disclosed herein. In one embodiment, the second uricase monomer is one of the uricase variants disclosed herein and forms either a homodimer or a heterodimer with the first uricase monomer. In a preferred embodiment, the uricase dimer is a homodimer comprising two identical uricase variant monomers disclosed herein.

[0030] In yet another aspect of this disclosure, a uricase tetramer comprising first, second, third, and fourth uricase monomers is provided, the first uricase monomer being one of the uricase variants disclosed herein. In one embodiment, the second uricase monomer is one of the uricase variants disclosed herein. In a further embodiment, the third uricase monomer is one of the uricase variants disclosed herein. In yet another embodiment, the fourth uricase monomer is one of the uricase variants disclosed herein. In one embodiment, the uricase tetramer is a heterotetramer. In a preferred embodiment, the uricase tetramer is a homotetramer comprising four identical uricase variant monomers disclosed herein.

[0031] In another embodiment, the disclosure relates to a uricase conjugate comprising at least two domains, wherein (i) a first domain comprising one of the uricase variants disclosed herein is conjugated to (ii) a second domain comprising a first random coil polypeptide domain comprising at least about 100 amino acids. In one embodiment, the uricase conjugate is a fusion protein of the first and second domains, and the fusion protein is referred to in this disclosure as a “uricase fusion protein” or “recombinant uricase fusion protein”. In one embodiment, the uricase conjugate resides in a uricase tetramer, which comprises four uricase conjugate subunits.

[0032] In one embodiment, the random coil polypeptide comprises a PAS polypeptide. In another embodiment, the random coil polypeptide comprises an elongated recombinant (XTEN) polypeptide. In yet another embodiment, the random coil polypeptide comprises a Pro-Ala (PA) polypeptide.

[0033] In some embodiments, the uricase conjugate is a uricase fusion protein whose monomeric form contains an amino acid sequence selected from SEQ ID NOs: 1568-2288.

[0034] In one embodiment, the uricase conjugate disclosed herein is a monomer. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein. In another embodiment, the uricase conjugate exists in a homotetramer. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein. In one embodiment, the uricase conjugate is a homotetramer. In a further embodiment, the homotetramer is a homotetramer recombinant uricase fusion protein. In one embodiment of the uricase conjugate disclosed herein, the uricase variant is not PEGylated. In another embodiment, the uricase conjugate is PEGylated.

[0035] In another aspect, the Disclosure provides isolated nucleic acids encoding uricase variants or uricase conjugates (e.g., recombinant uricase fusion proteins) as disclosed herein.

[0036] In another aspect, the Disclosure provides a nucleic acid vector comprising a nucleic acid encoding a uricase variant or uricase conjugate (e.g., a recombinant uricase fusion protein) as disclosed herein.

[0037] In another aspect, the disclosure provides a host cell comprising a nucleic acid vector disclosed herein.

[0038] In another embodiment, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a uricase conjugate, which is a uricase variant, uricase tetramer, or, in some embodiments, a recombinant uricase fusion protein, as disclosed herein. In one embodiment, the uricase tetramer is a homotetramer comprising four identical uricase variant monomers disclosed herein.

[0039] In another embodiment, the disclosure provides a method for reducing elevated uric acid levels in a subject requiring treatment. The method comprises administering an effective amount of the pharmaceutical composition disclosed herein to the subject. In one embodiment, the subject is a human patient. In a further embodiment, the human patient is an adult human patient. In one embodiment of the method, the uric acid levels in the subject's plasma or blood are reduced. In one embodiment of the method, the administration is carried out via intravenous administration. In another embodiment, the administration is carried out via subcutaneous administration. In one embodiment of the method, the subject is a patient with gout. In a further embodiment, the subject is a patient with refractory gout. In one embodiment of the method, the subject is diagnosed with tumor lysis syndrome.

[0040] In another embodiment, the Disclosure provides a method for treating gout in a subject requiring treatment. The method comprises administering an effective amount of the pharmaceutical composition disclosed herein to the subject. In one embodiment of the method, the gout being treated is refractory gout. In one embodiment, the subject is a human patient. In a further embodiment, the human patient is an adult human patient. In one embodiment of the method, the administration is carried out via intravenous administration. In another embodiment, the administration is carried out via subcutaneous administration.

[0041] In another embodiment, the present disclosure provides a method for treating tumor lysis syndrome in a subject requiring treatment. The method comprises administering to the subject an effective amount of the pharmaceutical composition disclosed herein. In one embodiment, the subject is a human patient. In a further embodiment, the human patient is an adult human patient. In one embodiment of the method, the administration is carried out via intravenous administration. In another embodiment, the administration is carried out via subcutaneous administration.

[0042] In another aspect, the present disclosure provides a method for recombinantly producing a uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) as disclosed herein. The method comprises (i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding a uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) as disclosed herein, wherein the nucleic acid sequence is operably linked to a heterologous promoter under conditions that enable the host cell to express the nucleic acid sequence encoding the uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) and to recombinantly produce the uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein); and (ii) isolating the recombinantly produced uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein). [Brief explanation of the drawing]

[0043] [Figure 1]This is a schematic diagram showing various configurations of the uricase fusion protein of the present invention. [Figure 2A] The first step in the uricase deimmunization workflow is shown, in which an immunodominant T cell epitope in wild-type chimeric pig-baboon uricase (WT) constituting pegroticase (top) was targeted for mutation silencing using substitutions predicted to maintain protein structure and function (center). The set of mutations was computationally optimized to obtain diverse combinatorial libraries, which differentially maintained a balance between reduction in immunogenicity and preservation of function (bottom). [Figure 2B] The second stage of the uricase deimmunization workflow is shown, in which one library design was selected and screened using a high-throughput halo assay reporting uricase activity (top). Promising clones were collected in deep 96-well plates (center) to enable medium-throughput analysis of normalized enzyme activity in solution (bottom). The fastest enzymes were purified and analyzed in more detail. [Figure 2C] The third stage of the uricase deimmunization workflow was demonstrated, in which a highly sensitive cell immunoassay quantified antigen-specific proliferation of T cells among peripheral blood mononuclear cells (PBMCs) from a panel of genetically diverse donors. The immunoassay validated computational predictions of reduced immunogenicity for each analyzed variant, and the data were used to guide lead candidate selection. [Figure 3A] This photograph shows the growth of E. coli expressing a combinatorial deimmunization uricase library on agar containing uric acid, and a halo created by an active clone that converts insoluble uric acid to soluble allantoin. [Figure 3B] This graph shows the absorbance at 293 nm over time for whole cell lysates from cultured colonies incubated with uric acid solution. The conversion of uric acid to allantoin results in a decrease in absorbance, and the specific activity is calculated from the slope. [Figure 4A]This is an image of an SDS-PAGE gel showing approximately 291 kDa MW bands representing PEGylated uricase species for wild-type chimeric pig-baboon uricase (WT) and the top six candidate chimeric pig-baboon uricase variants. [Figure 4B] This graph shows the percentage of soluble tetramers obtained using size exclusion chromatography (SEC) from one-step purification for non-PEGylated wild-type chimeric porcine-bahiuricase (WT) and the top six non-PEGylated chimeric porcine-bahiuricase variant candidates. [Figure 4C] This graph shows the specific activity measured by reaction rates for PEGylated wild-type chimeric porcine-hihiuricase (WT) and the top six candidate PEGylated chimeric porcine-hihiuricase variants. [Figure 4D] This graph shows the Michaelis-Menten kinetic parameter Vmax for PEGylated wild-type chimeric pig-hihiuricase (WT) and the top six candidate PEGylated chimeric pig-hihiuricase variants. [Figure 4E] This graph shows the apparent kcat / Km for PEGylated wild-type chimeric porcine-hihiuricase (WT) and the top six candidate PEGylated chimeric porcine-hihiuricase variants. [Figure 4F] This graph shows the melting temperature (Tm) determined by differential scanning fluorescence quantification for PEGylated wild-type chimeric porcine melon case (WT) and the top six candidate PEGylated chimeric porcine melon case variants. [Figure 5A] This is a schematic diagram of an ex vivo cell immunoassay for determining the immunogenicity of the top six non-PEGylated chimeric pig-hihiuricase variant candidates against non-PEGylated wild-type chimeric pig-hihiuricase (WT). [Figure 5B] This graph shows the median CD4+ T cell stimulation index determined by ex vivo cell immunoassay for the top six non-PEGylated chimeric pig-hihiuricase variant candidates and non-PEGylated wild-type chimeric pig-hihiuricase (WT). The dashed line shows the median stimulation index in vehicle control. [Figure 6A] This is a schematic diagram of a study design to evaluate the immunogenicity of the top six PEGylated chimeric pig-bahiuricase variant candidates in the humanized HLA transgenic mouse strain DR4 against PEGylated wild-type chimeric pig-bahiuricase. [Figure 6B] This line graph shows the relationship between anti-drug antibody titer, determined by ELISA absorbance, and plasma dilution on a logarithmic scale. It uses 42-day plasma collected from humanized DR4 HLA transgenic mice immunized with either a PEGylated chimeric porcine-baboon melon case variant candidate or PEGylated wild-type chimeric porcine-baboon melon case (WT). Ten mice were present in each group. Each data point is presented as mean ± SEM. Abs = absorbance. [Figure 6C] Figure 6B is a graph showing the area under the plasma dose-response curve (AUC) as a summary measure of the anti-drug immune response in the indicated PEGylated chimeric pig-bahiuricase variant candidate or PEGylated wild-type chimeric pig-bahiuricase (WT). [Figure 7A] This is a schematic diagram of a study design to evaluate the efficacy of repeated dosing of candidate PEGylated chimeric porcine-baboon melon case variants HF0536, HF0554, and HF0752 in the humanized HLA transgenic mouse strain DR4 compared to PEGylated wild-type chimeric porcine-baboon melon case (WT). [Figure 7B] This graph shows serum uric acid levels in humanized DR4 HLA transgenic mice at various weeks of either untreated (no Rx) reception or weekly repeated dose reception of one of the following: PEGylated chimeric porcine-hihiuricase variant candidates HF0536, HF0554, and HF0752, or PEGylated wild-type chimeric porcine-hihiuricase (WT). Nine mice were present in each group. Each data point is presented as mean ± SEM. [Figure 7C]This graph shows the percentage of humanized DR4 HLA transgenic mice with zero serum uric acid levels at various weeks, either receiving no treatment (no Rx) or receiving weekly repeated doses of one of the following: PEGylated chimeric porcine-hihiuricase candidate variants HF0536, HF0554, and HF0752, or PEGylated wild-type chimeric porcine-hihiuricase (WT). There were 9 mice in each group. [Figure 8] This is a schematic diagram of the PBMC (peripheral blood mononuclear cell) assay used to measure CD4+ T cell activity and epitopes for the top six non-PEGylated deimmunized chimeric pig-bahiuricase variant candidates and non-PEGylated wild-type chimeric pig-bahiuricase (WT). [Figure 9] This is a schematic diagram of the workflow for an MHC-related peptide proteomics (MAPP) assay used to identify peptides presented in HLA II monocyte-derived dendritic cells from the top six non-PEGylated chimeric pig-bahiuricase variant candidates or non-PEGylated wild-type chimeric pig-bahiuricase (WT). [Figure 10] This graph shows the CD4+ T cell epitope mapping of wild-type chimeric pig-baboon melon case protein using a PBMC assay. [Figure 11] This graph shows the results of MAPP analysis of wild-type chimeric pig-baboon melon case protein. [Figure 12] This graph shows the CD4+ T cell epitope mapping of deimmunized uricase variants compared to wild-type (WT) uricase, as determined by a PBMC assay. [Figure 13] This graph shows the CD4+ T cell response to WT uricase compared to a deimmunized variant, as determined by a PBMC assay. [Figure 14] This graph shows the results of MAPP analysis of wild-type uricase protein compared to non-immunized variants. [Figure 15A]This graph shows the plasma concentrations of endogenous uricase and uric acid in untreated control Wistar rats (n=3) at the corresponding pre-dose time point (hour 0) and various corresponding post-dose time points up to 96 hours, in the pharmacokinetic (PK) study according to Example 5. Each data point is displayed as mean ± SD. [Figure 15B] This graph shows the plasma concentrations of pegroticase and uric acid at various time points, from before administration (hour 0) to up to 96 hours after administration, in Wistar rats (n=3) that received a single dose of 1 mg / kg body weight of pegroticase intravenously in the PK study according to Example 5. Each data point is displayed as mean ± SD. [Figure 15C] This graph shows the plasma concentrations of WT-CPAS20h and uric acid at various time points, from before administration (hour 0) to up to 96 hours after administration, in Wistar rats (n=3) that received a single dose of 1.99 mg / kg body weight of WT-CPAS20h intravenously in the PK study according to Example 5. Each data point is displayed as mean ± SD. [Figure 15D] This graph shows the plasma concentrations of HF752-CPAS20h and uric acid at various time points from before administration (hour 0) to up to 96 hours after administration in Wistar rats (n=3) that received a single dose of HF752-CPAS20h at a dose of 1.99 mg / kg body weight in a PK study according to Example 5. Each data point is displayed as mean ± SD. [Figure 15E] This graph shows the plasma concentrations of PEGylated HF752 uricase variant and uric acid at various time points from before administration (hour 0) to up to 96 hours after administration in Wistar rats (n=3) that received a single dose of PEGylated HF752 uricase variant at a dose of 1 mg / kg body weight in a PK study according to Example 5. Each data point is shown as mean ± SD. [Modes for carrying out the invention]

[0044] Humans and higher primates lack uricase, an enzyme capable of oxidizing uric acid. As a result, humans have high serum uric acid levels. In some individuals, uric acid levels can rise beyond the solubility limit, leading to uric acid crystallization in the joints. Acute inflammation in response to these crystals causes severe pain, a condition known as gout. Treatment for severe gout involves injections of non-human uricase to reduce serum uric acid levels. Nyborg et al., PLOS One, 2016, DOI:10.1371 / journal.pone.0167935, which is incorporated herein by reference in its entirety.

[0045] The U.S. Food and Drug Administration (FDA) has approved KRYSTEXXA® (pegroticase) for use in the treatment of chronic refractory gout (Sundy et al., Jama, 2011, 306(7):p.711-20). Pegroticase is a homotetrameric protein, where each monomer is a single polypeptide chain composed of porcine-hihikimelauricase having the amino acid sequence of SEQ ID NO: 1. The homotetramer is highly PEGylated via random lysine conjugation, thereby reducing immunogenicity and extending half-life. Because pegroticase is inherently non-human, it elicits a potent immune response that leads to a black box warning, and in many pegroticase patients, there is a fairly rapid loss of therapeutic efficacy due to problems resulting from the development of anti-drug antibodies (ADAs) (Baraf et al., J Clin Rheumatol, 2014, 20(8): p.427-32, Garay, et al., Joint Bone Spine, 2012, 79(3): p.237-42, Verhoef et al., Drug Discov Today, 2014, 19(12): p.1945-52). 92% of patients develop ADA against pegroticase, which highlights the need for more effective uricase-based therapies. Generally, clinical complications associated with immunogenicity dramatically reduce the usefulness of uricases.

[0046] Aspects of this disclosure primarily relate to recombinant uricase variant proteins, which contain one or more amino acid mutations in one or more T cell epitopes, thereby depleting the T cell epitopes in the uricase variant protein compared to the counterpart uricase protein, e.g., wild-type (WT) uricase protein. Such uricase protein variants are referred herein, in some cases, to be “immune-deimmunized uricase variants” or “T cell epitope depletion” uricase variants. The use of such variants and methods for producing such variants are also described herein.

[0047] It should be understood that the terms used herein are for the sole purpose of describing specific embodiments and are not intended to be limiting.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which this application pertains. Any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this application, but representative methods and materials are described herein.

[0049] In accordance with long-standing patent law conventions, the terms “a,” “an,” and “the,” as used in this application, refer to “one or more” including the claims. Thus, for example, a reference to “carrier” includes one or more carriers and mixtures such as two or more carriers, and a reference to “method” includes equivalent steps and / or methods known to those skilled in the art.

[0050] Unless otherwise indicated, all numbers used herein and in the claims to represent quantities of ingredients, reaction conditions, etc., are understood in all cases to be modified by the term “approximately.” Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties sought to be obtained by this application. Generally, where used herein in reference to measurable values, such as quantities including weight, time, or dose, the term “approximately” means to encompass values ​​within a degree of variability acceptable in the art. In some embodiments, the degree of variability is based on FDA guidelines.

[0051] Furthermore, as used herein, “and / or” refers to and encompasses all possible combinations of any one or more of the related enumerated items, and, when interpreted in the choice ("or"), refers to and encompasses the absence of any combination.

[0052] When used in the context of protein sequences, the terms “substantial identity” or “substantially identical” refer to a sequence having at least about 60% sequence identity with a reference sequence. Alternatively, percent identity can be any integer from about 60% to about 100%. Exemplary embodiments include sequence identity of at least about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% compared to a reference sequence, using the standard parameters described herein, for example, BLAST.

[0053] For sequence comparison, typically one sequence acts as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, sub-sequence coordinates are specified as needed, and sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the sequence identity percentage for the test sequence compared to the reference sequence, based on the program parameters.

[0054] The algorithms preferred for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J.Mol.Biol.215:403-410 and Altschul et al. (1997) Nucleic Acids Res.25:3389-3402, respectively. Software for performing BLAST analysis is publicly available via the National Center for Biotechnology Information (NCBI) website. The algorithm first involves identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, where the short words, when aligned with words of the same length in the database sequence, either match or satisfy a certain positive threshold score T. T is referred to as the adjacent word score threshold (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). These initial adjacent word hits serve as a seed to initiate a search for longer HSPs containing them. Word hits are then extended in both directions along each sequence to the extent that the cumulative alignment score can increase. For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of word hits in each direction stops when the cumulative alignment score decreases by X from its maximum achieved value, or when the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. For amino acid sequences, the BLASTP program uses a word size (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix by default (see Henikoff & Henikoff. (1992). Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0055] The BLAST algorithm also performs statistical analysis of similarity between two sequences (see, for example, Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which represents the probability that the match between two nucleotide or amino acid sequences occurs by chance.

[0056] As used herein, “corresponding uricase protein” or “corresponding uricase” refers to a uricase protein that is compared with one of the uricase protein variants described herein, for example, by primary sequence comparison or functional comparison. In one embodiment, the corresponding uricase protein does not contain any mutation(s) in one or more T cell epitopes. In some embodiments, the corresponding uricase protein is a WT uricase protein, e.g., a naturally occurring mammalian uricase protein, e.g., wild-type dog, pig, cattle, sheep, or baboon uricase. In some embodiments, the corresponding uricase protein is a non-naturally occurring or recombinant uricase protein, e.g., a humanized uricase, e.g., a humanized porcine uricase, or a chimeric mammalian uricase, e.g., a chimeric pig-baboon uricase having the amino acid sequence of SEQ ID NO: 1. In this disclosure, the chimeric porcine-baboon melon case protein containing the amino acid sequence of Sequence ID No. 1 functions as a counterpart enzyme for the exemplary melon case variants and amino acid mutations disclosed herein and is referred to as wild-type (WT) chimeric porcine-baboon melon case for ease of description.

[0057] In one embodiment, the counterpart uricase protein is a WT chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein contains an amino acid sequence substantially identical to SEQ ID NO: 1. According to the definition of “substantially identical” as provided herein, in one embodiment, the counterpart uricase protein contains an amino acid sequence that is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to SEQ ID NO: 1. In another embodiment, the counterpart uricase protein contains an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to SEQ ID NO: 1. In another embodiment, the corresponding uricase protein contains an amino acid sequence that is approximately 90% to 100% identical to SEQ ID NO: 1.

[0058] In further embodiments, the counterpart uricase protein is a chimeric porcine-uricase containing an amino acid sequence that is at least about 70%, at least about 80%, or at least about 90% identical to SEQ ID NO: 1. An exemplary embodiment of such a counterpart uricase protein is a chimeric porcine-uricase containing different segments of porcine uricase, different segments of baby uricase, or both, compared to the wild-type chimeric porcine-uricase of SEQ ID NO: 1. In a particular embodiment, the counterpart uricase protein is a chimeric porcine-uricase containing the amino acid sequence of SEQ ID NO: 4, the amino acid sequence of SEQ ID NO: 4 containing amino acids (aa)1-266 of porcine uricase of SEQ ID NO: 2 and aa267-304 of baby uricase of SEQ ID NO: 3. In comparison, the wild-type chimeric pig-baby melon case of SEQ ID NO: 1 contains aa8-266 of the pig melon case of SEQ ID NO: 2 and aa267-304 of the pig melon case of SEQ ID NO: 3. In another specific embodiment, the corresponding melon case protein is a chimeric pig-baby melon case containing the amino acid sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 5 contains aa1-220 of the pig melon case of SEQ ID NO: 2 and aa221-304 of the pig melon case of SEQ ID NO: 3.

[0059] Other exemplary embodiments of the corresponding uricase protein containing an amino acid sequence substantially identical to SEQ ID NO: 1 include: (1) a mammalian uricase, e.g., canine uricase, whose amino acid sequence (having the NCBI reference number NP_001011886.1, which is incorporated herein by reference in its entirety) is 93% identical to SEQ ID NO: 1 when determined by the protein-protein BLAST algorithm (BLASTP); a porcine uricase of SEQ ID NO: 2, which is 99% identical to SEQ ID NO: 1 when determined by BLASTP; and a bovine uricase, whose amino acid sequence (having the NCBI reference number NP_001069116.1, which is incorporated herein by reference in its entirety) (1) Bovine melonicase, which, when determined by BLASTP, is 91% identical to SEQ ID NO: 1; sheep melonicase, whose amino acid sequence (having NCBI reference number XP_004002186.4, which is entirely incorporated herein by reference) is 90% identical to SEQ ID NO: 1 when determined by BLASTP; and baboon melonicase of SEQ ID NO: 3, which, when determined by BLASTP, is 91% identical to SEQ ID NO: 1; and (2) chimeric melonicases comprising two or more mammalian melonicase domains, including, but not limited to, chimeric pig-baboon melonicase (above), chimeric dog-baboon melonicase, and chimeric pig-bovine melonicase.

[0060] In one embodiment, the counterpart uricase protein is a cleaved uricase, i.e., uricase cleaved at either the C-terminus or the N-terminus. In one embodiment, the counterpart uricase protein is a cleaved uricase obtained by cleaving the corresponding full-length uricase at either the C-terminus or the N-terminus. In another embodiment, the counterpart uricase protein is a cleaved uricase obtained by further cleaving the corresponding uricase, which has been less previously cleaved, at either the C-terminus or the N-terminus. In a further embodiment, the cleaved uricase is a cleaved chimeric pig-baboon uricase. Thus, in one embodiment of the uricase variants of this disclosure, the variant further comprises amino acid cleavage in addition to the amino acid mutation(s) disclosed herein, compared to a counterpart uricase protein that does not have the corresponding amino acid cleavage. In one embodiment, the amino acid cleavage is located at or around the N and / or C-terminus of the counterpart uricase protein. In one embodiment, the amino acid cleavage is located at the N-terminus of the counterpart uricase protein. In a further embodiment, amino acid cleavage begins at position 1, 2, 3, 4, 5, or 6 of the corresponding uricase protein. In another embodiment, amino acid cleavage includes 4 to 13 amino acids at the N-terminus of the corresponding uricase protein. In yet another embodiment, amino acid cleavage includes 4 to 13 amino acids at the C-terminus of the corresponding uricase protein. In yet another embodiment, amino acid cleavage includes 4 to 13 amino acids at the C-terminus and 4 to 13 amino acids at the N-terminus of the corresponding uricase protein. In yet another embodiment, amino acid cleavage includes 6 amino acids at the N-terminus of the corresponding uricase protein. In yet another embodiment, amino acid cleavage includes 6 amino acids at the C-terminus of the corresponding uricase protein. In yet another embodiment, amino acid cleavage includes 6 amino acids at the C-terminus and 6 amino acids at the N-terminus of the corresponding uricase protein.

[0061] "Operatively linked" means an arrangement of elements configured so that the components described herein perform their normal functions. In the case of a promoter, a promoter operably linked to a coding sequence influences the expression of the coding sequence. Promoters or other regulatory elements do not need to be contiguous with the coding sequence insofar as they function to guide its expression. For example, there may be an intervening untranslated but transcribed sequence 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 uricase conjugates described herein, the domains can be operably linked in a single DNA sequence to enable the expression of a uricase fusion protein.

[0062] The term "pharmaceutically acceptable," unless otherwise noted, is used to characterize a part (e.g., a salt, dosage form, or excipient) as appropriate for use in accordance with sound medical judgment. Generally, a pharmaceutically acceptable part has one or more benefits that outweigh any potential adverse effects it may have. Adverse effects may include, for example, excessive toxicity, irritation, allergic reactions, and other problems and complications.

[0063] As used herein, “recombinant protein” refers to any artificially produced protein, which is distinguished from naturally produced proteins (i.e., proteins produced in the tissues of animals that have only the natural genes for the specific protein of interest). As described herein, a recombinant uricase fusion protein comprises a first (uricase) domain attached via peptide bonds (directly or via amino acid linkers) to one or more random coil polypeptide domains.

[0064] A “T cell epitope” refers to a peptide epitope that binds to an MHCII molecule and subsequently forms a ternary complex with a congeneral T cell receptor (TCR). As used herein, one or more T cell epitopes are “depleted” of the uricase variant if a peptide of uricase variant origin is unable to bind to the MHCII molecule, or if the peptide binds to the MHCII molecule but subsequently fails to bind to the TCR, for example, due to a lack of affinity for the MHCII molecule.

[0065] The terms “variant protein” and “protein variant” are terms of the art and are used interchangeably herein. Each refers to a protein distinguished from its counterpart protein, e.g., the WT form of the protein, based on the presence of one or more amino acid modifications, e.g., one or more amino acid substitutions, insertions, deletions, or combinations thereof. The term “mutant gene” is a term of the art and refers to a gene distinguished from its WT form of the gene, based on the presence of one or more nucleic acid modifications, e.g., one or more nucleic acid substitutions, insertions, deletions, or combinations thereof. In some embodiments, a mutant gene encodes a variant protein. However, mutations in a gene can also be silent mutations, i.e., mutations that do not affect the amino acid sequence in the protein encoded by the gene. Mutants / variants may occur naturally or may be engineered mutants / variants.

[0066] As used herein, the term “wild type” (abbreviated as “WT”) means, unless otherwise specified, the most frequently occurring form of an organism, strain, gene, protein, or characteristic in nature, and / or distinct from mutant or variant forms.

[0067] Amino acid modifications can be amino acid substitutions, amino acid deletions, and / or amino acid insertions. Amino acid substitutions can be conserved or non-conserved. Amino acid substitutions at specific locations in a protein sequence are indicated herein in the following format: "WT amino acid residue single-letter code - amino acid position - single-letter code of the amino acid residue that replaces this WT residue." For example, a uricase variant having the amino acid substitution W22L refers to a uricase variant protein in which the wild-type residue (W or tryptophan) at position 22 is replaced by L or leucine.

[0068] All amino acid position designations of uricase variants in this disclosure and the appended claims are relative to the wild-type chimeric pig-baboon uricase enzyme amino acid sequence (monomer) of SEQ ID NO: 1. It will be understood by those skilled in the art that the corresponding amino acid positions in the amino acid sequences of other corresponding uricase proteins can be readily identified by aligning one of the amino acid sequences of the other corresponding uricase proteins with SEQ ID NO: 1 using the BLAST, BLAST 2.0, or BLASTP algorithms described above, or by other similar methods or algorithms known to those skilled in the art.

[0069] This disclosure provides, in part, uricase protein variants (e.g., recombinant uricase protein variants), which are engineered variants of their counterpart uricase proteins, e.g., WT uricase proteins. In the embodiments described herein, the uricase variants are deimmunized compared to their counterpart uricase proteins, for example, because the variants are T-cell epitope depleted compared to their counterpart uricase proteins. In one embodiment, the uricase variant is a variant of WT chimeric pig-baboon uricase having the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricase variant is a variant of one of the counterpart uricase proteins having an amino acid sequence substantially identical to SEQ ID NO: 1 described above.

[0070] It should be noted that the amino acid positions and mutations described herein are provided for the uricase protein monomer. However, embodiments of the present invention also cover multimers, e.g., uricase in dimeric and tetrameric forms. In the case of homodimers and homotetratomers, the amino acid mutations described for the monomer protein are also present in the remaining protein subunits. However, in embodiments providing heterodimeric and / or heterotetrameric uricase variants, mutations in one subunit may or may not be present in the remaining uricase monomer subunits.

[0071] In one embodiment of the uricase variants provided herein, a chimeric porcine-baboon uricase variant is provided. Pegroticase is a homotetrameric protein, where each monomer is a single polypeptide chain composed of porcine-baboon chimeric uricase having the amino acid sequence of SEQ ID NO: 1. The homotetramer is highly PEGylated via random lysine conjugation, thereby reducing immunogenicity and extending half-life. As will be discussed in more detail herein, in one embodiment, the uricase variant of the present invention is PEGylated. In another embodiment, the uricase variant of the present invention is PAS-modified. In yet another embodiment, the uricase variant is PAS-modified and PEGylated. In yet another embodiment, the uricase variant of the present invention is XTEN-modified. In one embodiment, the chimeric porcine-baboon uricase variant is T-cell epitope depletion compared to its counterpart uricase (e.g., WT chimeric porcine-baboon uricase of SEQ ID NO: 1 or chimeric porcine-baboon uricase containing an amino acid sequence substantially identical to SEQ ID NO: 1). In a further embodiment, the chimeric porcine-baboon uricase variant has substantially the same stability and / or enzymatic activity as its counterpart uricase protein. In one embodiment, the uricase variant provided herein is a monomer. In another embodiment, the uricase variant is provided as a homodimer or homotetramer. In a preferred embodiment, the uricase variant is a homotetramer.

[0072] In some embodiments of the uricase variants provided herein, the uricase variant comprises one or more amino acid mutations relative to the counterpart uricase protein, where one or more amino acid mutations are located at one or more amino acid positions corresponding to one or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In further embodiments, one or more amino acid mutations are amino acid substitutions. In further embodiments, the amino acid substitutions correspond to one or more of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins containing an amino acid sequence substantially identical to SEQ ID NO: 1 (an unspecified example provided above).

[0073] In one embodiment, a mutation(s) at one or more of the above amino acid positions eliminates one or more dominant immunogenic T cell epitopes present in the counterpart uricase protein, thereby the variant being T cell epitope depleted compared to the counterpart uricase protein. Mutations of this disclosure include, but are not limited to, amino acid exchanges(s), insertions(s), deletions(s), additions(s), substitutions(s), inversions(s), and / or duplications(s). These mutations / modifications(s) also include conservative and / or homologous amino acid exchanges(s). In preferred embodiments of the present invention, the mutation(s) are amino acid substitutions.

[0074] In one embodiment, the uricase variant contains two or more, three or more, four or more, five or more, seven or more, ten or more, fifteen or more, about five, about ten, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid mutations relative to the corresponding uricase protein, wherein the amino acid mutations are the following amino acid residues of SEQ ID NO: 1. The mutations are selected from those at amino acid positions corresponding to D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In further embodiments, each mutation is an amino acid substitution. In further embodiments, the amino acid substitutions correspond to two or more, three or more, four or more, five or more, seven or more, ten or more, fifteen or more, about five, about ten, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 amino acid substitutions of SEQ ID NO: 1 as listed in Table A. In one embodiment, the corresponding uricase protein is a wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the corresponding uricase protein is one of the corresponding uricase proteins provided above containing an amino acid sequence substantially identical to SEQ ID NO: 1.

[0075] In another embodiment, the uricase variant has approximately 5 to 25 amino acid mutations, approximately 5 to 20 amino acid mutations, approximately 10 to 20 amino acid mutations, approximately 10 to 18 amino acid mutations, approximately 10 to 16 amino acid mutations, approximately 10 to 14 amino acid mutations, approximately 10 to 13 amino acid mutations, approximately 10 to 12 amino acid mutations, approximately 11 to 20 amino acid mutations, approximately 11 to 13 amino acid mutations, approximately 12 to 14 amino acid mutations, and approximately 13 to 20 amino acid mutations relative to the corresponding uricase protein. The mutations consist of approximately 14 to 20 amino acid mutations, or approximately 15 to 20 amino acid mutations, and the amino acid mutations are selected from mutations at amino acid positions corresponding to the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In further embodiments, each mutation is an amino acid substitution. In further embodiments, the amino acid substitutions correspond to about 5 to about 25, about 5 to about 20, about 10 to about 20, about 10 to about 18, about 10 to about 16, about 10 to about 14, about 10 to about 13, about 10 to about 12, about 11 to about 20, about 11 to about 13, about 12 to about 14, about 13 to about 20, about 14 to about 20, or about 15 to about 20 amino acid substitutions of SEQ ID NO: 1 as listed in Table A. In one embodiment, the corresponding uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the corresponding uricase protein is one of the corresponding uricase proteins provided above containing an amino acid sequence substantially identical to SEQ ID NO: 1.

[0076] In one embodiment, the uricase variant contains approximately 10 to 13 amino acid mutations relative to the counterpart uricase protein, with the amino acid mutations located at amino acid positions corresponding to approximately 10 to 13 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 10 to 13 amino acid substitutions correspond to approximately 10 to 13 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1.

[0077] In one embodiment, the uricase variant contains approximately 10 to 14 amino acid mutations relative to the counterpart uricase protein, the amino acid mutations being located at amino acid positions corresponding to approximately 10 to 14 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 10 to 14 amino acid substitutions correspond to approximately 10 to 14 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1.

[0078] In one embodiment, the uricase variant contains approximately 10 to 15 amino acid mutations relative to the counterpart uricase protein, the amino acid mutations being located at amino acid positions corresponding to approximately 10 to 15 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 10 to 15 amino acid substitutions correspond to approximately 10 to 15 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1.

[0079] In one embodiment, the uricase variant contains approximately 10 to 16 amino acid mutations relative to the counterpart uricase protein, with the amino acid mutations located at amino acid positions corresponding to approximately 10 to 16 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 10 to 16 amino acid substitutions correspond to approximately 10 to 16 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1.

[0080] In one embodiment, the uricase variant contains approximately 10 to 17 amino acid mutations relative to the counterpart uricase protein, the amino acid mutations being located at amino acid positions corresponding to approximately 10 to 17 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 10 to 17 amino acid substitutions correspond to approximately 10 to 17 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1.

[0081] In one embodiment, the uricase variant contains approximately 10 to 18 amino acid mutations relative to the counterpart uricase protein, with the amino acid mutations located at amino acid positions corresponding to approximately 10 to 18 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 10 to 18 amino acid substitutions correspond to approximately 10 to 18 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1.

[0082] In one embodiment, the uricase variant contains approximately 10 to 19 amino acid mutations relative to the counterpart uricase protein, the amino acid mutations being located at amino acid positions corresponding to approximately 10 to 19 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 10 to 19 amino acid substitutions correspond to approximately 10 to 19 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1.

[0083] In one embodiment, the uricase variant contains approximately 10 to 20 amino acid mutations relative to the counterpart uricase protein, the amino acid mutations being located at amino acid positions corresponding to approximately 10 to 20 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257 of SEQ ID NO: 1. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 10 to 20 amino acid substitutions correspond to approximately 10 to 20 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1.

[0084] In one embodiment, the uricase variant contains approximately 11 to 13 amino acid mutations relative to the counterpart uricase protein, the amino acid mutations being located at amino acid positions corresponding to approximately 11 to 13 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 11 to 13 amino acid substitutions correspond to approximately 11 to 13 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1.

[0085] In one embodiment, the uricase variant contains approximately 12 to 14 amino acid mutations relative to the counterpart uricase protein, the amino acid mutations being located at amino acid positions corresponding to approximately 12 to 14 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In a further embodiment, each mutation is an amino acid substitution. In a further embodiment, the approximately 12 to 14 amino acid substitutions correspond to approximately 12 to 14 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to the amino acid sequence of SEQ ID NO: 1.

[0086] In one embodiment, the uricase variant comprises one or more amino acid substitutions relative to the counterpart uricase protein, where the one or more amino acid substitutions correspond to one or more of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is a wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above, containing an amino acid sequence substantially identical to that of SEQ ID NO: 1. [Table A]

[0087] In one embodiment, the uricase variant includes amino acid substitutions relative to the corresponding uricase protein, where the amino acid substitutions correspond to two or more, three or more, four or more, five or more, seven or more, ten or more, fifteen or more, about 5, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 5 to about 25, about 5 to about 20, about 10 to about 20, about 10 to about 18, about 10 to about 16, about 10 to about 14, about 10 to about 13, about 10 to about 12, about 11 to about 20, about 11 to about 13, about 12 to about 14, about 13 to about 20, about 14 to about 20, or about 15 to about 20 of the amino acid substitutions of Sequence ID No. 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to approximately 10 to 13 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to approximately 10 to 14 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to approximately 10 to 15 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to approximately 10 to 16 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to approximately 10 to 17 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to approximately 10 to 18 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to approximately 10 to 19 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to approximately 10 to 20 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to approximately 11 to 13 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In another embodiment, the amino acid substitutions correspond to about 12 to about 14 of the amino acid substitutions of SEQ ID NO: 1 listed in Table A. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above containing an amino acid sequence substantially identical to SEQ ID NO: 1.

[0088] In one embodiment, the uricase variant comprises amino acid substitutions relative to the corresponding uricase protein, the amino acid substitutions corresponding to three or more, five or more, or ten or more of the following amino acid substitutions of SEQ ID NO: 1: V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K. In a further embodiment, the uricase variant further comprises amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In a further embodiment, the uricase variant further comprises amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1. In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions in SEQ ID NO: 1: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K. In a further embodiment, the uricase variant further comprises amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q in SEQ ID NO: 1. In a further embodiment, the uricase variant further comprises amino acid substitutions corresponding to N117G or N117D in SEQ ID NO: 1. In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions in SEQ ID NO: 1: V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q in SEQ ID NO: 1. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117G or N117D in SEQ ID NO: 1. In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions in SEQ ID NO: 1, V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K.In further embodiments, the uricase variant further comprises amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In further embodiments, the uricase variant further comprises amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1. In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions of SEQ ID NO: 1: V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K. In further embodiments, the uricase variant further comprises amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In further embodiments, the uricase variant further comprises amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1. In another embodiment, the amino acid substitutions correspond to three or more, five or more, or ten or more of the following amino acid substitutions of SEQ ID NO: 1: V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K. In further embodiments, the uricase variant further comprises amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In further embodiments, the uricase variant further comprises amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1. In one embodiment, the corresponding uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the corresponding uricase protein is one of the corresponding uricase proteins provided above, having an amino acid sequence substantially identical to SEQ ID NO: 1.

[0089] In some embodiments, the uricase variant includes an amino acid substitution relative to the corresponding uricase protein, the amino acid substitution corresponding to the amino acid substitution of SEQ ID NO: 1, which includes one of the exemplary amino acid substitution groups listed in Table B. In one embodiment, the uricase variant includes one amino acid substitution corresponding to one amino acid substitution selected from V22L, F75L, and Y127H listed in the exemplary amino acid substitution groups of SEQ ID NO: 1 in Table B. In another embodiment, the uricase variant includes two amino acid substitutions corresponding to a combination of two amino acid substitutions selected from V22L, F75L, and Y127H listed in the exemplary amino acid substitution groups of SEQ ID NO: 1 in Table B. In further embodiments, the uricase variant further includes an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In further embodiments, the uricase variant further includes an amino acid substitution corresponding to N117G or N117D of SEQ ID NO: 1. [Table B] TIFF2026517791000004.tif254170TIFF2026517791000005.tif224170

[0090] In one embodiment, the uricase variant contains multiple amino acid mutations relative to the corresponding uricase protein, and the multiple amino acid mutations are the following combinations of amino acid residues of SEQ ID NO: 1, (1a) V22, D28, S33, N70, F75, K79, A102, Y127, K155, R209, and I257, (2a) V22, D28, S33, N70, F75, K79, A102, F114, V119, Y127, K155, R209, and I257, (3a) V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (4a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257, (5a) V22, Q26, Y31, N70, F75, K79, A102, Y127, K155, R209, and I257, (6a) V22, D28, Y31, N70, F75, K79, A102, K112, Y127, K155, R209, and I257, (7a) V22, Q26, S33, N70, F75, K79, A102, K112, Y127, K155, R209, and I257, (8a) V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (9a)V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (10a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (11a) V22, Q26, S33, N70, F75, K79, A102, K112, Y127, K155, R209, and I257, (12a) V22, D28, Y31, N70, F75, K79, A102, V106, K116, Y127, K155, R209, and I257, (13a) V22, Q26, Y31, N70, F75, K76, A102, Y127, K155, R209, and I257, (14a) V22, Q26, S33, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (15a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257, (16a) V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257, (17a) V22, Q26, S33, N70, F75, K76, A102, K112, Y127, K155, R209, and I257, (18a) V22, D28, Y31, N70, F75, K79, A102, V106, K116, Y127, K155, R209, and I257, (19a) V22, D28, Y31, N70, F75, K79, A102, K116, Y127, K155, R209, and I257, (20a) V22, Q26, S33, N70, F75, K76, A102, K116, V119, Y127, K155, R209, and I257, and (21a) V22, Q26, S33, N70, F75, K79, A102, F114, V119, Y127, K155, R209, and I257 It is located at the amino acid position corresponding to one of the amino acids.

[0091] In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above containing an amino acid sequence substantially identical to SEQ ID NO: 1. In one embodiment, the uricase variant includes an amino acid mutation at one amino acid position corresponding to one amino acid residue selected from V22, F75, and Y127 as described in one of the corresponding amino acid residue combinations of SEQ ID NO: 1 described in (1a) to (21a) above. In another embodiment, the uricase variant includes two amino acid mutations at two amino acid positions corresponding to a combination of two amino acid residues selected from V22, F75, and Y127 as described in one of the corresponding amino acid residue combinations of SEQ ID NO: 1 described in (1a) to (21a) above.

[0092] In one embodiment, each amino acid mutation is an amino acid substitution. In a further embodiment, the amino acid substitution is the following combination of amino acid substitutions of SEQ ID NO: 1 (1b) V22L, D28H, S33D, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K, (2b) V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K, (3b) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209W, and I257T, (4b) V22L, D28T, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257T, (5b) V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K, (6b) V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K, (7b) V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K, (8b) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209F, and I257T, (9b) V22L, D28H, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209F, and I257T, (10b) V22L, D28T, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209F, and I257T, (11b) V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K, (12b) V22L, D28N, Y31H, N70L, F75L, K79G, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K, (13b) V22L, Q26R, Y31H, N70L, F75L, K76S, A102L, Y127H, K155G, R209F, and I257K, (14b) V22L, Q26R, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209W, and I257T, (15b) V22L, D28T, S33D, N70L, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209F, and I257K, (16b) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K, (17b) V22L, Q26R, S33D, N70H, F75L, K76S, A102V, K112S, Y127H, K155G, R209F, and I257K, (18b) V22L, D28N, Y31H, N70L, F75L, K79G, A102L, V106M, K116M, Y127H, K155G, R209F, and I257K, (19b) V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K. (20b) V22L, Q26R, S33D, N70L, F75L, K76S, A102L, K116M, V119T, Y127H, K155G, R209W, and I257K, and (21b) V22L, Q26R, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209F, and I257K It includes / corresponds to one of the following.

[0093] In one embodiment, the uricase variant includes one amino acid substitution corresponding to one amino acid substitution selected from V22L, F75L, and Y127H as described in one of the corresponding amino acid substitution combinations of SEQ ID NO: 1 in (1b) to (21b) above. In another embodiment, the uricase variant includes two amino acid substitutions corresponding to a combination of two amino acid substitutions selected from V22L, F75L, and Y127H as described in one of the corresponding amino acid substitution combinations of SEQ ID NO: 1 in (1b) to (21b) above. In some embodiments, the uricase variants (1b) to (21b) may further include amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In other embodiments, the uricase variants (1b) to (21b) may further include amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1.

[0094] In one embodiment, the amino acid substitutions in the uricase variant of the present disclosure include / correspond to the following amino acid substitutions of SEQ ID NO: 1, V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1. In one embodiment, the amino acid substitutions in the uricase variant of the present disclosure include / correspond to the following amino acid substitutions of SEQ ID NO: 1, V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1. In one embodiment, the amino acid substitutions in the uricase variant of the present disclosure include / correspond to the following amino acid substitutions of SEQ ID NO: 1, V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In a further embodiment, the uricase variant further includes amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1. In one embodiment, the amino acid substitutions in the uricase variant of the present disclosure include / correspond to the following amino acid substitutions of SEQ ID NO: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K.In further embodiments, the uricase variant further comprises amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In further embodiments, the uricase variant further comprises amino acid substitutions corresponding to N117G or N117D of SEQ ID NO: 1. In one embodiment, the amino acid substitutions in the uricase variant of the present disclosure include / correspond to the following amino acid substitutions of SEQ ID NO: 1: V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K. In further embodiments, the uricase variant further comprises amino acid substitutions corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In further embodiments, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of SEQ ID NO: 1. In one embodiment, the amino acid substitutions in the uricase variant of the present disclosure include / correspond to the following amino acid substitutions of SEQ ID NO: 1: V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K. In further embodiments, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO: 1. In further embodiments, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of SEQ ID NO: 1. In one embodiment, the corresponding uricase protein is a wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the corresponding uricase protein is one of the corresponding uricase proteins provided above, having an amino acid sequence substantially identical to SEQ ID NO: 1.

[0095] In one embodiment, the uricase variant contains one or more conserved amino acid mutations relative to the corresponding uricase protein, where one or more conserved amino acid mutations are located at one or more amino acid positions corresponding to one or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In another embodiment, one or more conserved amino acid mutations are located at one or more amino acid positions other than those corresponding to one or more of the following amino acid residues of SEQ ID NO: 1: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided above containing an amino acid sequence substantially identical to SEQ ID NO: 1. In one embodiment, one or more conservative amino acid mutations are conservative amino acid substitutions.

[0096] Conservative amino acid substitutions are generally defined as substitutions of amino acids that have similar properties (e.g., acidity, basicity, aromaticity, size, positive or negative charge, polarity, nonpolarity). Exemplary conservative substitutions that can be performed are from the following group of amino acids:

[0097] (i) Glycine (G), alanine (A), valine (V), leucine (L), and isoleucine (I), (ii) Aspartic acid (D) and glutamic acid (E), (iii) Alanine (A), serine (S), and threonine (T), (iv) Histidine (H), lysine (K), and arginine (R), (v) Asparagine (N) and glutamine (Q), (vi) Phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0098] In the embodiments described herein, a uricase variant having one or more conservative substitutions retains the structural stability of the corresponding uricase enzyme from which the variant is derived, i.e., the corresponding uricase variant or the corresponding wild-type uricase, and has enzymatic activity, although its protein and DNA sequence is not the same as that of the corresponding uricase without conservative substitutions.

[0099] In one embodiment, a uricase variant containing at least one conservative mutation has the same uricase activity as a uricase variant without such mutation. In another embodiment, a uricase variant containing at least one conservative mutation has substantially the same uricase activity as a uricase variant without such mutation, or uricase activity within 5%, 10%, or 30% of the activity of a uricase variant without such mutation.

[0100] In one embodiment, the uricase variant of the present disclosure is a humanized uricase variant. An exemplary method for generating a humanized uricase enzyme is disclosed in U.S. Patent No. 8,586,535, which is incorporated in whole by reference. In one embodiment, the humanized uricase variant comprises one or more humanized amino acid mutations relative to the non-human counterpart uricase protein, where one or more humanized amino acid mutations are located at one or more amino acid positions corresponding to one or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In another embodiment, one or more humanization amino acid mutations are located at one or more amino acid positions other than those corresponding to one or more of the following amino acid residues of SEQ ID NO: 1: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257. In one embodiment, the counterpart uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the above counterpart uricase proteins containing an amino acid sequence substantially identical to SEQ ID NO: 1.

[0101] In one embodiment, the humanization mutation is an amino acid substitution having a corresponding or homologous amino acid sequence in inactive human uricase, thereby the variant retains uricase activity while improving homology with human uricase, and therefore reduces immunogenicity. In one embodiment, the humanized uricase variant comprises one or more humanization amino acid mutations to one of the following: chimeric porcine-baboon uricase, e.g., wild-type chimeric porcine-baboon uricase containing the amino acid sequence of SEQ ID NO: 1, or the above chimeric porcine-baboon uricase containing an amino acid sequence substantially identical to SEQ ID NO: 1. In the humanized uricase variant, the humanization mutation may be an amino acid sequence substitution(s) in the porcine uricase segment, the baboon uricase segment, or both segments of the counterpart chimeric porcine-baboon uricase protein.

[0102] In some embodiments of the uricase variants provided herein, the uricase variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 1.

[0103] In one embodiment, the uricase variant includes an amino acid sequence selected from SEQ ID NOs: 25 to 154. In a further embodiment, the uricase variant includes an amino acid sequence selected from SEQ ID NOs: 28, 45, 48, 49, 53, 66, 67, 101, 104, 112, 114, 117, 119, 123, 125, 129, 139, 142, 149, 150, and 152. In yet another embodiment, the uricase variant includes an amino acid sequence selected from SEQ ID NOs: 45, 53, 66, 114, 129, and 149.

[0104] The disclosure also provides uricase variants comprising amino acid sequences that are 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% identical to amino acid sequences selected from SEQ ID NOs. Further embodiments include uricase variants comprising amino acid sequences that are 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% identical to amino acid sequences selected from SEQ ID NOs. 28, 45, 48, 49, 53, 66, 67, 101, 104, 112, 114, 117, 119, 123, 125, 129, 139, 142, 149, 150, and 152. In a further embodiment, the uricase variant includes an amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NOs. 45, 53, 66, 114, 129, and 149.

[0105] In one embodiment, the uricase variant contains the amino acid sequence of SEQ ID NO: 53. In another embodiment, the uricase variant contains an amino acid sequence that is 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% identical to SEQ ID NO: 53. Exemplary uricase variants include those containing an amino acid sequence selected from SEQ ID NOs: 197-309.

[0106] In one embodiment, the uricase variant contains the amino acid sequence of SEQ ID NO: 129. In another embodiment, the uricase variant contains an amino acid sequence that is 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% identical to SEQ ID NO: 129. Exemplary uricase variants include those containing amino acid sequences selected from SEQ ID NOs: 187, 189, 191, 193, and 195, as well as SEQ ID NOs: 310-1566.

[0107] In one embodiment, the uricase variant contains the amino acid sequence of SEQ ID NO: 187.

[0108] In one embodiment, the uricase variant contains the amino acid sequence of SEQ ID NO: 191.

[0109] In one embodiment, the uricase variant includes an amino acid sequence selected from SEQ ID NOs: 622, 639, 640, 644, 646, 649, 657, 660, 668, 671, 673, 675, 676, 677, 678, 679, 680, 689, 694, 695, 704, 774, and 791.

[0110] In one embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs: 971, 973, 976, 978, 979, 983, 985, 988, 991, 997, 1000, 1013, 1016, 1018, 1022, 1023, 1024, 1030, 1032, 1033, 1035, 1037, 1041, 1043, 1044, 1045, 1046, 1051, 1053, 1054, 1055, 1057, 1077, 1134, and 1142.

[0111] In one embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs: 1330, 1333, 1342, 1358, 1375, 1385, 1391, 1436, 1442, 1451, 1457, and 1465.

[0112] In one embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs: 1475-1566.

[0113] In some embodiments of the uricase variants provided herein, the uricase variant comprises a first set of amino acid substitutions and a second set of amino acid substitutions relative to the corresponding uricase protein. The first set of amino acid substitutions is group (a) or group (b) below: (a) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K, or (b) Corresponds to the amino acid substitutions of SEQ ID NO: 1 described in V22L, Q26T, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

[0114] The second set of amino acid substitutions is a set of about 6 to about 10 (e.g., 6, 7, 8, 9, or 10) amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions in SEQ ID NO: 1, one of K30E and K30F, one of L52T, H53D, K79S, T83E, V86N, S94D, K97D, one of N117G and N117D, one of F172W and F172L, T174N, E177Q, R192T, G196D, R197D, Y225H, S228G, L239G, L279T, and N281Y. In one embodiment, the corresponding uricase protein is wild-type chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins provided herein that has an amino acid sequence substantially identical to SEQ ID NO: 1. In some embodiments, the uricase variant further comprises one or more conserved amino acid substitutions and / or humanization amino acid mutations disclosed herein. In one embodiment, the uricase variant has an amino acid sequence that is at least about 85%, at least about 90%, at least about 91%, or at least about 92% identical to the amino acid sequence of SEQ ID NO: 1.

[0115] In some embodiments, a first set of amino acid substitutions for the uricase variant corresponds to the amino acid substitutions of SEQ ID NO: 1 described in group (a) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K. In some embodiments, a first set of amino acid substitutions for the uricase variant corresponds to the amino acid substitutions of SEQ ID NO: 1 described in group (b) V22L, Q26T, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

[0116] In some embodiments, the second set of amino acid substitutions for the uricase variant is a set of about 6 to about 10 amino acid substitutions selected from the following amino acid substitutions of SEQ ID NO: 1, one of K30E and K30F, one of L52T, H53D, T83E, V86N, S94D, K97D, N117G and N117D, F172W, T174N, R192T, G196D, R197D, Y225H, S228G, L239G, L279T, and N281Y. In one embodiment, the second set of amino acid substitutions for the uricase variant is a set of about 6 to about 7 amino acid substitutions. In another embodiment, the second set of amino acid substitutions for the uricase variant is a set of about 8 to about 10 amino acid substitutions.

[0117] In some embodiments, the second set of amino acid substitutions for the uricase variant is a set of about 6 to about 10 amino acid substitutions selected from the following amino acid substitutions of SEQ ID NO: 1, one of K30E and K30F, one of L52T, T83E, V86N, S94D, K97D, N117G and N117D, F172W, R192T, G196D, R197D, Y225H, S228G, L279T, and N281Y. In one embodiment, the second set of amino acid substitutions for the uricase variant is a set of about 6 to about 7 amino acid substitutions. In another embodiment, the second set of amino acid substitutions for the uricase variant is a set of about 8 to about 10 amino acid substitutions.

[0118] In some embodiments, the second set of amino acid substitutions for the uricase variant is a set of about 6 to about 10 amino acid substitutions selected from the following amino acid substitutions of SEQ ID NO: K30E, L52T, T83E, S94D, N117G and N117D, F172W, R192T, G196D, R197D, Y225H, S228G, L279T, and N281Y. In one embodiment, the second set of amino acid substitutions for the uricase variant is a set of about 6 to about 7 amino acid substitutions. In another embodiment, the second set of amino acid substitutions for the uricase variant is a set of about 8 to about 10 amino acid substitutions.

[0119] In some embodiments, the second set of amino acid substitutions for the uricase variant is a set of about 6 to about 10 amino acid substitutions selected from the following amino acid substitutions of SEQ ID NO: K30E, L52T, T83E, N117G and N117D, F172W, R192T, G196D, R197D, S228G, L279T, and N281Y. In one embodiment, the second set of amino acid substitutions for the uricase variant is a set of about 6 to about 7 amino acid substitutions. In another embodiment, the second set of amino acid substitutions for the uricase variant is a set of about 8 to about 10 amino acid substitutions.

[0120] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of about 6 to about 9 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: K30E, L52T, T83E, N117G and N117D, F172W, G196D, R197D, S228G, L279T, and N281Y.

[0121] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of about 6 to about 8 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: K30E, L52T, T83E, N117G, F172W, G196D, S228G, L279T, and N281Y.

[0122] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of about 6 to about 8 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: K30E, L52T, T83E, N117D, F172W, G196D, S228G, L279T, and N281Y.

[0123] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of about 6 to about 8 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: K30E, L52T, T83E, F172W, G196D, S228G, L279T, and N281Y.

[0124] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of about 6 to about 7 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: K30E, L52T, T83E, F172W, G196D, S228G, L279T, and N281Y.

[0125] In some embodiments, the uricase variants of the present disclosure induce about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less than 10% of the immune response induced by the non-deimmunized counterpart uricase on which the uricase variant is based (as measured, for example, by anti-uricase antibody titer). In further embodiments, the uricase variant and the counterpart uricase are homotetramers.

[0126] In some embodiments, the uricase variants of the present disclosure have at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, and at least about 90% (e.g., about 95%, about 97%, about 99%, or more) of the enzymatic activity of the non-immunized counterpart uricase on which the uricase variant is based. In further embodiments, the uricase variant and the counterpart uricase are homotetramers.

[0127] In this disclosure, if the N-terminal methionine residue is absent in the uricase variant, alternative embodiments include uricase variants of the same sequence in which the N-terminal methionine residue is present. Similarly, if the N-terminal methionine residue is present in the uricase variant, alternative embodiments include uricase variants of the same sequence in which the N-terminal methionine residue is absent. In some embodiments described herein, where methionine is absent at the N-terminus (position 1) of the uricase variant and instead another amino acid, such as threonine, is present, the uricase variant has had the N-terminal methionine residue removed by post-translational modification. In one embodiment, the N-terminal methionine residue is removed by endogenous bacterial methionine aminopeptidase after the uricase variant has been produced in a bacterium, such as E coli.

[0128] In one embodiment of the uricase variants disclosed herein, the uricase variant is isolated. In another embodiment, the uricase variant is purified. In some embodiments, the uricase variant includes a purification tag at the C-terminus, N-terminus, or both N and C-terminus to enable purification by affinity chromatography. In one embodiment, the purification tag is a polyhistidine tag (also referred to as the "his tag"). In one embodiment, the his tag contains six histidine residues. In another embodiment, the purification tag is a his-glu tag (HQ tag). In a further embodiment, the HQ tag has the amino acid sequence HQHQHQ (SEQ ID NO: 6). In another embodiment, the purification tag is a his-asp tag (HN tag). In a further embodiment, the HN tag has the amino acid sequence HNHNHNHNHNHN (SEQ ID NO: 7). In another embodiment, the purification tag is a histidine affinity tag (HAT). In further embodiments, HAT has the amino acid sequence KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8). In one embodiment of the uricase variant provided herein, the uricase variant is isolated and purified.

[0129] As discussed above, the uricase variants disclosed herein may be provided as monomeric proteins or as polymeric proteins, such as homodimers, homotetratomers, heterodimers, or heterotetramers. In one embodiment, the uricase variant disclosed herein is a monomer. In one embodiment, the uricase variant is a homodimer. In one embodiment, the uricase variant is a homotrimer. In a preferred embodiment, the uricase variant is a homotetramer. In another embodiment, the uricase variant is a heterodimer, a heterotrimer, or a heterotetramer.

[0130] In another aspect of this disclosure, uricase monomers are provided, including uricase variants disclosed herein.

[0131] In yet another aspect of this disclosure, a uricase dimer is provided comprising a first uricase monomer and a second uricase monomer, wherein the first uricase monomer is one of the uricase variants disclosed herein. In one embodiment, the second uricase monomer is one of the uricase variants disclosed herein and forms either a homodimer or a heterodimer with the first uricase monomer. In a preferred embodiment, the uricase dimer is a homodimer comprising two identical uricase variant monomers disclosed herein.

[0132] In yet another aspect of this disclosure, a uricase tetramer comprising first, second, third, and fourth uricase monomers is provided, the first uricase monomer being one of the uricase variants disclosed herein. In one embodiment, the second uricase monomer is one of the uricase variants disclosed herein. In a further embodiment, the third uricase monomer is one of the uricase variants disclosed herein. In yet another embodiment, the fourth uricase monomer is one of the uricase variants disclosed herein. In one embodiment, the uricase tetramer is a heterotetramer. In a preferred embodiment, the uricase tetramer is a homotetramer comprising four identical uricase variant monomers disclosed herein.

[0133] In one embodiment of the uricase variants disclosed herein, the uricase variant is not PEGylated. In another embodiment, the uricase variant is PEGylated, i.e., the uricase variant is covalently conjugated to polyethylene glycol (PEG) at, for example, one or more lysine residues. In a further embodiment, each of the uricase monomers of a multimeric uricase variant, such as a uricase homotetramer, is PEGylated. In one embodiment, the uricase variant is covalently conjugated to one or more molecules of monomethoxypoly(ethylene glycol) (mPEG). In a further embodiment, each mPEG molecule has a molecular weight of about 10 kDa. The uricase variant monomer can be conjugated to any desired number, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 PEG or mPEG molecules. In one embodiment, the uricase variant monomer is conjugated to about 8 to about 11 PEG or mPEG molecules, about 9 PEG or mPEG molecules, or about 10 PEG or mPEG molecules. In one embodiment, the average total molecular weight of the PEG portion of the PEGylated monomer uricase variant is about 10 kDa to about 150 kDa, about 30 kDa to 120 kDa, or about 50 kDa to about 100 kDa.

[0134] In one embodiment, the uricase variant disclosed herein is covalently bonded to PEG, e.g., mPEG, via a biocompatible linking group, the linkage being carried out using methods known in the art, e.g., Park et al, Anticancer Res., 1981, 1:373-376, and Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, JM Harris, ed., 1992, Plenum Press, New York, Chapter 21, the disclosures of each of these documents being incorporated herein by reference in their entirety. The biocompatible linking group is non-toxic and can be used in vitro or in vivo without causing injury, illness, disease, or death. In some embodiments, PEG can be bonded to the linking group via, for example, an ether linkage, an ester linkage, a thiol linkage, or an amide linkage. Suitable biocompatible linking groups include, for example, ester groups, amide groups, imide groups, carbamate groups, carboxyl groups, hydroxyl groups, carbohydrates, succinimide groups, epoxide groups, oxycarbonylimidazole groups, nitrophenyl groups, trisilate groups, aldehyde groups, isocyanate groups, vinylsulfone groups, tyrosine groups, cysteine ​​groups, histidine groups, or primary amines.

[0135] In another embodiment, the uricase variants disclosed herein are conjugated to PEG, for example, mPEG, via an amino group, sulfhydral group, hydroxyl group, or carboxyl group, without having a linking group. In one embodiment, PEG is conjugated to one or more lysine residues in the uricase variant. In one embodiment, PEG is conjugated to one or more cysteine ​​residues in the uricase variant. In one embodiment, PEG is conjugated to one or more serine residues in the uricase variant. In yet another embodiment, PEG is conjugated to one or more lysine residues, one or more cysteine ​​residues, one or more serine residues, or any combination of the above.

[0136] In another aspect, the disclosure relates to a uricase conjugate comprising at least two domains, wherein (i) a first (uricase) domain comprising one of the uricase variants disclosed herein is conjugated to (ii) a second domain comprising a first random coil polypeptide domain.

[0137] As used herein, the term “domain” refers to any region / part of an amino acid sequence that can autonomously adopt a particular structure and / or function. Therefore, in the context of the present invention, “domain” may represent a functional domain or a structural domain. As described herein, embodiments of the uricase conjugate or its fusion protein comprise at least one uricase domain and at least one domain / part that forms a random coil conformation (e.g., a PAS polypeptide domain). The uricase conjugate of the present invention may also comprise three or more domains. For example, as provided herein, uricase variants may exist as homotetramers; therefore, in one embodiment, the uricase conjugate of the present invention comprises four uricase domains and four random coil polypeptide domains. Furthermore, the fusion protein of the present invention may include, for example, an additional linker structure between the two domains / parts as defined herein, or another domain / part, such as a protease-sensitive cleavage site, affinity tags, such as a polyhistidine tag or Strep tag, a signal peptide, a retention peptide, a target-directed peptide, such as a membrane-transfer peptide, or an additional effector domain, such as an antibody fragment for a tumor target related to an antitumor toxin or an enzyme for prodrug activation. In another embodiment of the uricase fusion protein, the first (uricase) domain comprises a monomer of a uricase variant disclosed herein, and the fusion protein further comprises a random coil polypeptide domain at the C-terminus relative to the uricase domain, and a random coil polypeptide domain further comprises at the N-terminus relative to the uricase domain. In a further embodiment, the random coil polypeptide domain comprises a PAS polypeptide.

[0138] As used herein, the terms “random coil” or “random coil polypeptide domain” refer to a conformation of a polymer molecule, including an amino acid polymer, in which the individual monomer elements forming the polymer structure are essentially randomly oriented relative to adjacent monomer elements, but are still chemically bonded to those adjacent monomer elements. In particular, polypeptides or amino acid polymers employing / having / forming a “random coil” conformation substantially lack the defined secondary and tertiary structures. The properties of polypeptide random coils and methods for their experimental identification are known to those skilled in the art.

[0139] A first (uricase) domain containing one of the uricase variants disclosed herein is conjugated to a second domain containing a first random coil polypeptide domain, the conjugation of which, in one preferred embodiment, is achieved at the DNA level by operably ligating a uricase variant DNA sequence to a DNA sequence encoding a random coil polypeptide such that the DNA sequences are located within a single open reading frame, and subsequently by recombinant expression of a contiguous DNA sequence encoding the first and second domains. The ligation of the two domains at the DNA level eliminates the need for in vitro coupling or modification steps to achieve conjugate synthesis, which in other embodiments described herein may require, for example, the coupling of polyethylene glycol (PEG) to the uricase variant.

[0140] In embodiments in which the uricase conjugate is produced via the recombinant expression of a single DNA sequence, the uricase conjugate is referred to herein as a uricase "fusion protein." Specifically, a "fusion protein" refers to a protein composed of multiple polypeptide components, which are typically not linked in their native state but whose respective N-terminuses and C-terminuses are linked via peptide bonds to form a single continuous polypeptide. A uricase fusion protein may be a combination of two, three, four, or more different proteins. A uricase fusion protein may also include fusions with heterologous and homologous leader sequences having or not having N-terminal methionine residues, as well as fusion proteins containing additional sequences (e.g., polyhistidine tags) for the purification of the fusion protein.

[0141] In one embodiment, the second domain of the uricase conjugate comprises a first random coil polypeptide domain containing at least about 100 amino acid residues, e.g., a PA polypeptide, a PAS polypeptide, or an XTEN polypeptide. Without wishing to be constrained by theory, the random coil conformation mediates increased in vivo and / or in vitro stability of the uricase variant enzyme. Also without wishing to be constrained by theory, since the random coil polypeptide domain is not thought to adopt a stable structure or function on its own, the biological activity of the uricase variant conjugated by the random coil polypeptide domain is essentially conserved.

[0142] In one embodiment of a random coil polypeptide, the random coil polypeptide of a uricase conjugate contains two amino acids, proline (Pro) and alanine (Ala). In a further embodiment, the random coil polypeptide consists of two amino acids, proline (Pro) and alanine (Ala). If all amino acid residues in the polypeptide are Pro and Ala, or substantially all amino acids in the polypeptide are Pro and Ala, such polypeptide is referred to herein as a "PA polypeptide". In one embodiment, the uricase fusion protein contains a PA polypeptide. In further embodiments, the PA polypeptide is at least about 100 amino acid lengths, at least about 150 amino acid lengths, at least about 200 amino acid lengths, at least about 250 amino acid lengths, at least about 300 amino acid lengths, at least about 350 amino acid lengths, at least about 400 amino acid lengths, at least about 450 amino acid lengths, at least about 500 amino acid lengths, or at least about 550 amino acid lengths. For example, the PA polypeptide is about 200 amino acid lengths, about 300 amino acid lengths, about 400 amino acid lengths, about 500 amino acid lengths, or about 600 amino acid lengths.

[0143] In one embodiment of a random coil polypeptide, the random coil polypeptide in a uricase conjugate contains three amino acids: proline (Pro), alanine (Ala), and serine (Ser). In a further embodiment, the random coil polypeptide consists of three amino acids: proline (Pro), alanine (Ala), and serine (Ser). If all amino acid residues in the polypeptide are Pro, Ala, and Ser, or substantially all amino acids in the polypeptide are Pro, Ala, and Ser, such a polypeptide is referred to herein as a "PAS polypeptide." A PAS polypeptide may be referred to as a PAS domain when present in a uricase conjugate. In one embodiment, the uricase fusion protein comprises a PAS polypeptide, the PAS polypeptide comprising about 10 to about 30 tandem copies of a PAS sequence containing Pro, Ala, and Ser, for example, PAS10 has 10 tandem copies and a total of about 200 amino acids, PAS20 has 20 tandem copies and a total of about 400 amino acids, and PAS30 has 30 tandem copies and a total of about 600 amino acids. Exemplary amino acid sequences of PAS10, PAS20, and PAS30 are described in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively. Other examples of PAS polypeptides convenient for use herein are provided in PCT Publication WO2008 / 155134, the disclosure of which is incorporated in whole by reference.

[0144] In another embodiment, the random coil polypeptide is an elongated recombinant (XTEN) polypeptide. The XTEN polypeptide contains six amino acids A, E, G, S, and T in varying percentages, thereby forming a long, unstructured, hydrophilic amino acid sequence (Pasut, Polymers 2014, 6, 160-178). In one embodiment, the XTEN polypeptide is one of the polypeptides disclosed in U.S. Patent Application Publication 2015 / 0037359, the contents of which are incorporated by reference in whole for all purposes. In one embodiment, the XTEN polypeptide is at least about 800 amino acids long and consists of six hydrophilic, chemically stable amino acids Ala, Asp, Gly, Pro, Ser, and Thr in a non-repeating manner. In one embodiment, the XTEN polypeptide is 864 residues long. In another embodiment, the XTEN polypeptide is an 864aa fragment of the XTEN polypeptide. In one embodiment, the XTEN polypeptide contains the amino acid sequence described in Sequence ID No. 12. The XTEN polypeptide may be conjugated via chemical conjugation to a first domain containing a uricase variant as described herein, or it may be produced as a fusion protein with the uricase variant.

[0145] In some embodiments, the second domain of the uricase conjugate comprises a PAS polypeptide (referred herein to as the PAS domain in some embodiments) and includes an amino acid sequence comprising 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 include 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 wishing to be constrained by theory, the random coil conformation mediates increased in vivo and / or in vitro stability of the uricase variant enzyme. Details regarding various types of PAS polypeptides and the nucleic acids encoding them for use in the present invention can be found in PCT Publication WO2008 / 155134, the contents of which are incorporated by reference in whole for all purposes.

[0146] Random coils of uricase conjugate are formed under physiological conditions. For example, in one embodiment, the physiological conditions are parameters that are typically effective for higher organisms, particularly mammals, most preferably humans. Thus, the physiological conditions may be conditions normally found in the body fluids of mammals. The physiological conditions may relate to corresponding parameters found in a healthy body and parameters found in a diseased mammal or human patient. For example, a diseased mammal or human patient may have higher but physiological temperature conditions if the mammal or human is suffering from a fever.

[0147] Several buffers in experimental settings (e.g., for use in determining protein structure, particularly circular dichroism (CD) measurements, and other methods for determining the structural properties of protein / amino acid stretches), solvents, and / or excipients for pharmaceutical compositions are considered to represent physiological solutions and / or physiological conditions in vitro. Examples of such buffers include, for example, phosphate-buffered saline, Tris buffer, acetate buffer, citrate buffer, or similar buffers. Generally, the pH of buffers representing physiological solution conditions is in the range of 6.5 to 8.5, e.g., 7.0 to 8.0, e.g., 7.2 to 7.7, and the osmotic pressure may be in the range of 10 to 1000 mmol / kg H2O, more specifically, in the range of 50 to 500 mmol / kg H2O, e.g., 200 to 350 mmol / kg H2O.

[0148] Methods for determining whether an amino acid polymer forms / adopts a random coil conformation are known in the art. Such methods include CD spectroscopy, which represents optical absorption spectroscopy that measures the difference in absorbance of right-circularly polarized and left-circularly polarized light by a substance. The secondary structure of a protein can be determined by CD spectroscopy using far-ultraviolet spectra with wavelengths of approximately 190–250 nm. At these wavelengths, different secondary structures commonly found in polypeptides can be analyzed, as α-helices, parallel and antiparallel β-sheets, and random coil conformations each result in characteristic shapes and sizes of CD spectra. 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, hydrodynamic volume measurement by size exclusion chromatography, analytical ultracentrifugation or dynamic / static light scattering, and measurement of friction coefficient or intrinsic viscosity.

[0149] In one embodiment, the random coil polypeptide 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 polypeptide domain contains up to about 1000 amino acid residues, up to about 900 amino acid residues, up to about 800 amino acid residues, up to about 700 amino acid residues, or up to about 600 amino acid residues. In one embodiment, the random coil polypeptide domain contains up to about 500 amino acid residues, or up to about 450 amino acid residues.

[0150] In one embodiment, the random coil polypeptide domain contains about 100 to about 3000 amino acid residues. In a further embodiment, the random coil polypeptide domain contains about 100 to about 1000 amino acid residues. In some embodiments, the random coil polypeptide domain contains about 100 to about 800, about 100 to about 700, about 100 to about 600, about 100 to about 500, about 100 to about 400, or about 100 to about 300 amino acid residues.

[0151] In one embodiment, the random coil polypeptide domain comprises an amino acid sequence in which proline residues account for approximately 4% to 40% of the random coil polypeptide domain. In further embodiments, alanine and serine residues constitute the remaining approximately 60% to 96% of the random coil polypeptide domain. In some embodiments, the random coil polypeptide domain contains additional amino acids different from Ala, Ser, and Pro as trace components. As used in this context, the term “trace components” means that up to 10% of the amino acids in the random coil polypeptide domain are different from alanine, serine, and proline; for example, up to 8% of the amino acids in the random coil polypeptide domain; for example, up to 6% of the amino acids in the random coil polypeptide domain; for example, up to 5% of the amino acids in the random coil polypeptide domain; for example, up to 4% of the amino acids in the random coil polypeptide domain; for example, up to 3% of the amino acids in the random coil polypeptide domain; for example, up to 2% of the amino acids in the random coil polypeptide domain; and up to 1% of the amino acids in the random coil polypeptide domain are different from Ala, Ser, and Pro. In one embodiment of the random coil polypeptide domain, the polypeptide comprises amino acids other than Ala, Ser, and Pro, the other amino acids being selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. In another embodiment, the other amino acids comprise one or more non-natural amino acids.

[0152] In another embodiment, the random coil polypeptide domain includes multiple “amino acid repeats,” i.e., the same amino acid sequence occurring two or more times in the domain, where the “amino acid repeat” consists of Ala, Ser, and Pro residues (indicated herein as “PAS” or “APS”). In a further embodiment, six or fewer consecutive amino acid residues are identical in the random coil polypeptide domain, and proline residues constitute more than about 4% and less than about 40% of the amino acids in the random coil polypeptide domain. Non-limiting examples of “amino acid repeats” consisting of Ala, Ser, and Pro residues are provided herein; see, for example, SEQ ID NOs. 13, SEQ ID NOs. 15, SEQ ID NOs. 17, SEQ ID NOs. 19, SEQ ID NOs. 21, and SEQ ID NOs. 23 (Table 1A). Fragments and / or polymers of these sequences are used in several embodiments. A “fragment” contains at least three amino acids, including at least one Ala, one Ser, and / or one Pro. [Table 1A]

[0153] The above repeating sequences can be encoded by nucleic acid molecules having the sequences described in SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, and / or SEQ ID NO: 24 (Table 1A).

[0154] In one embodiment, a nucleotide sequence encoding the amino acid repeat of SEQ ID NO: 13 is provided. In a further embodiment, the nucleotide sequence is selected from one of the nucleotide sequences listed in Table 1B, i.e., one of SEQ ID NOs: 155 to 185. [Table 1B] TIFF2026517791000008.tif127170

[0155] In one embodiment, the amino acid repeats used in the random coil PAS polypeptide domain comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, and the amino acid repeat comprises at least one Ala, Ser, and Pro residue. In one embodiment, the amino acid repeat does not comprise more than 100 amino acid residues. In one embodiment, the amino acid repeat comprises at least about 4%, at least about 5%, at least about 6%, at least about 10%, at least about 15%, or at least about 20% Pro residues. In further embodiments, the amino acid repeat comprises less than about 40%, for example, less than about 35% Pro residues.

[0156] In one embodiment, the random coil polypeptide domain includes five or fewer identical consecutive amino acid residues, for example, four or fewer identical consecutive amino acid residues, for example, three or fewer identical consecutive amino acid residues.

[0157] In one embodiment, the random coil polypeptide domain contains more than 4% to less than 50% Ala residues, for example, more than 10% to less than 50% Ala residues, for example, more than 20% to less than 50% Ala residues.

[0158] In another embodiment, the random coil polypeptide domain contains more than 4% to less than 50% Ser residues, for example, more than 10% to less than 50% Ser residues, for example, more than 20% to less than 50% Ser residues.

[0159] In one embodiment, the random coil polypeptide domain comprises approximately 35% Pro residues, approximately 50% Ala residues, and approximately 15% Ser residues. Alternatively, the random coil polypeptide domain comprises approximately 35% Pro residues, approximately 15% Ala residues, and approximately 50% Ser residues.

[0160] In one embodiment of the present invention, the uricase conjugate comprises a PAS polypeptide in a random coil polypeptide domain containing the amino acid sequence described in Sequence ID No. 9.

[0161] In one embodiment of the present invention, the uricase conjugate comprises a PAS polypeptide in a random coil polypeptide domain containing the amino acid sequence described in SEQ ID NO: 10.

[0162] In one embodiment of the present invention, the uricase conjugate comprises a PAS polypeptide in a random coil polypeptide domain containing the amino acid sequence described in SEQ ID NO: 11.

[0163] In one embodiment, the uricase conjugate of the present invention is a fusion protein. The fusion protein described herein comprises a multidomain polypeptide comprising at least one uricase domain (i.e., a first domain) and at least one random coil polypeptide domain. In alternative embodiments, the uricase domain is bound to the random coil polypeptide domain via a non-peptide bond. Non-peptide bonds useful for crosslinking proteins are known in the art and may include disulfide bonds, e.g., disulfide bonds between Cys side chains, thioether bonds, or non-peptide covalent bonds induced by chemical crosslinkers, e.g., disaxinimidyl substrates (DSS) or sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate (Sulfo-SMPB), and non-covalent protein-protein interactions.

[0164] With respect to embodiments of fusion proteins, the two domains can be arranged in an order selected by those skilled in the art. For example, in one embodiment of a uricase conjugate fusion protein, the uricase domain is located at the amino(N-) terminus of the fusion protein, and the random coil polypeptide domain is located at the carboxy(C-) terminus of the fusion protein. However, this order can be reversed, for example, in one embodiment, the uricase domain is located in / at the carboxy(C-) terminus of the fusion protein, and the random coil polypeptide domain is located in / at the amino(N-) terminus.

[0165] In another embodiment, the random coil polypeptide domain is located at both the C-terminus and N-terminus of the fusion protein, and the uricase domain is located between the two random coil polypeptide domains.

[0166] In one embodiment of the random coil polypeptide domain provided herein, if the random coil polypeptide domain is present at the N-terminus of the fusion protein, the random coil polypeptide domain includes an N-terminal Met residue. In another embodiment, the fusion protein does not include an N-terminal Met residue, for example, because it has been removed post-translation.

[0167] In one embodiment, the uricase fusion protein includes an N-terminal Met residue. In another embodiment, the uricase fusion protein does not include an N-terminal Met residue, for example, because it has been removed post-translation. Therefore, if an N-terminal Met residue is present in the sequence provided herein, the alternative embodiment includes a fusion protein of the same sequence that does not include an N-terminal Met residue. Similarly, if an N-terminal Met residue is not present in the fusion protein provided herein, the alternative embodiment includes a fusion protein of the same sequence that does include an N-terminal Met residue.

[0168] In one embodiment of a uricase fusion protein, an amino acid spacer or linker sequence is located between the uricase domain and the random coil polypeptide (e.g., PAS) domain. In one embodiment, the amino acid spacer sequence is 1 amino acid long, 2 amino acid long, 3 amino acid long, 4 amino acid long, or 5 amino acid long. In another embodiment, the amino acid spacer sequence is approximately 2 to 5 amino acid long. In yet another embodiment, the amino acid spacer sequence is approximately 2 to 4 amino acid long. In a further embodiment, the amino acid spacer sequence is 2 amino acid long. In a further embodiment, the spacer sequence is Gly-Ser.

[0169] In one embodiment of the uricase conjugate described herein, the uricase conjugate includes a purification tag at the C-terminus, N-terminus, or both the N and C-terminuses. The purification tag is used to facilitate the purification of the uricase conjugate (e.g., uricase fusion protein) from an in vitro expression system, for example, by the use of immobilized metal affinity chromatography (IMAC). In one embodiment, the purification tag is located at the C-terminus of the uricase fusion protein. In a further embodiment, the purification tag is a polyhistidine tag (also called a "his tag"). In one embodiment, the his tag includes six histidine residues.

[0170] Alternative purification tags may also be used herein. For example, in one embodiment, a his-glu tag (HQ tag) is present at the C-terminus of one of the uricase conjugates described herein. In a further embodiment, the HQ tag has the amino acid sequence HQHQHQ (SEQ ID NO: 6). In another embodiment, the uricase conjugate includes a his-asp tag (HN tag) at its C-terminus to enable the purification of the conjugate. In one embodiment, the HN tag has the amino acid sequence HNHNHNHNHNHN (SEQ ID NO: 7). In yet another embodiment, the uricase conjugate includes a histidine affinity tag (HAT) at its C-terminus to enable the purification of the conjugate. In one embodiment, the HAT has the amino acid sequence KDHLIHNVHKEEHAHAHNK (SEQ ID NO: 8).

[0171] Regarding the fusion protein embodiments of the uricase conjugates of this disclosure, an exemplary configuration of a recombinant uricase fusion protein comprising a uricase domain containing one of the uricase variants disclosed herein and one or two random coil PAS polypeptide or XTEN polypeptide domains is provided in Figure 1. In one embodiment, the uricase fusion protein comprises a uricase domain and one random coil PAS polypeptide or XTEN polypeptide domain. In the uricase fusion protein, the uricase domain may be at the C-terminal end of one random coil PAS polypeptide or XTEN polypeptide domain, or at the N-terminal end of one random coil PAS polypeptide or XTEN polypeptide domain. In another embodiment, the uricase fusion protein comprises a uricase domain and two random coil PAS polypeptide domains or two XTEN polypeptide domains. In a uricase fusion protein, one of two random coil PAS polypeptide or XTEN polypeptide domains is at the N-terminus and the other is at the C-terminus, with the uricase domain located between the two random coil PAS polypeptide or XTEN polypeptide domains. In some embodiments, the spacer sequence Gly-Ser(GS) between the uricase domain and the C-terminal random coil PAS polypeptide (e.g., PAS10, PAS20, or PAS30) or XTEN polypeptide domain, as shown in Figure 1, is optional and may be absent. In one embodiment, the uricase fusion protein has a polyhistidine tag (also called a "his tag") containing, for example, six histidine residues at the C-terminus, N-terminus, or both C-terminus and N-terminus, for the purification of the uricase fusion protein. In some embodiments, the his tag is separated from its adjacent domains by the spacer sequence Gly-Ser(GS).

[0172] PAS10, PAS20, and PAS30 represent random coil PAS polypeptide domains containing 10, 20, and 30 tandem copies of a PAS sequence containing Pro, Ala, and Ser, for example, the PAS sequence described in SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 23, respectively.

[0173] In one embodiment, PAS10, PAS20, and PAS30 represent random coil PAS polypeptide domains containing 10, 20, and 30 tandem copies of the PAS sequence described in SEQ ID NO: 13, and therefore have the amino acid sequences described in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0174] In one embodiment, the DNA sequences encoding PAS10 of SEQ ID NO: 9, PAS20 of SEQ ID NO: 10, and PAS30 of SEQ ID NO: 11, respectively, include a total of 10, 20, and 30 tandem copies of one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 14 and SEQ ID NOs: 155-185.

[0175] In one embodiment, the DNA sequences encoding PAS10 of SEQ ID NO: 9, PAS20 of SEQ ID NO: 10, and PAS30 of SEQ ID NO: 11, respectively, contain a total of 10, 20, and 30 tandem copies of the same (single) nucleotide sequence selected from the group consisting of SEQ ID NO: 14 and SEQ ID NOs: 155-185.

[0176] In one embodiment, the DNA sequences encoding PAS10 of SEQ ID NO: 9, PAS20 of SEQ ID NO: 10, and PAS30 of SEQ ID NO: 11, respectively, include a total of 10, 20, and 30 tandem copies of two or more nucleotide sequences selected from the group consisting of SEQ ID NO: 14 and SEQ ID NOs: 155-185.

[0177] In one embodiment, the DNA sequences encoding PAS10 of SEQ ID NO: 9, PAS20 of SEQ ID NO: 10, and PAS30 of SEQ ID NO: 11, respectively, comprise 10, 20, and 30 unique (i.e., different) nucleotide sequences selected from the group consisting of SEQ ID NO: 14 and SEQ ID NOs: 155-185, and the nucleotide sequences are linked together in tandem via 3',5'-phosphodiester bonds.

[0178] In an exemplary embodiment, the DNA sequence encoding PAS10 of SEQ ID NO: 9 includes each of SEQ ID NOs: 176-185, tandemly linked together in ascending order of ID numbers via a 3',5'-phosphodiester bond in the 5'-3' direction.

[0179] In an exemplary embodiment, the DNA sequence encoding PAS20 of SEQ ID NO: 10 includes each of SEQ ID NOs: 166-185, tandemly linked together in ascending order of ID numbers via a 3',5'-phosphodiester bond in the 5'-3' direction.

[0180] In an exemplary embodiment, the DNA sequence encoding PAS30 of SEQ ID NO: 11 includes each of SEQ ID NOs: 156-185, tandemly linked together in ascending order of ID numbers via a 3',5'-phosphodiester bond in the 5'-3' direction.

[0181] In another exemplary embodiment, the DNA sequence encoding PAS30 of SEQ ID NO: 11 includes SEQ ID NO: 155 and SEQ ID NOs: 157-185, respectively, in the 5'-3' direction, and SEQ ID NOs: 155 and SEQ ID NOs: 157-185 are linked together in tandem via 3',5'-phosphodiester bonds in ascending order of ID number.

[0182] In one embodiment, the XTEN polypeptide domain of the uricase fusion protein contains the amino acid sequence of SEQ ID NO: 12.

[0183] Exemplary PAS-modified uricase fusion proteins, including amino acid sequences of uricase variants disclosed herein for a uricase domain in combination with one or two PAS domains according to the configuration of Figure 1, include those containing amino acid sequences selected from SEQ ID NOs. 1568-2288. These PAS-modified uricase fusion proteins are described in detail in Example 5 of this application.

[0184] In one embodiment, the uricase conjugate disclosed herein is a monomer. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein. In a further embodiment, the recombinant uricase fusion protein comprises a random coil PA polypeptide domain, a random coil PAS polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the above random coil polypeptide domains.

[0185] In another embodiment, the uricase conjugate is present in a tetramer, preferably a homotetramer. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein. In a further embodiment, the recombinant uricase fusion protein includes a random coil PA polypeptide domain, a random coil PAS polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the above random coil polypeptide domains.

[0186] In another embodiment, the uricase conjugate exists as a homodimer, heterodimer, or heterotetramer. In yet another embodiment, the uricase conjugate is a recombinant uricase fusion protein. In yet another embodiment, the recombinant uricase fusion protein includes a random coil PA polypeptide domain, a random coil PAS polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the above random coil polypeptide domains.

[0187] In one embodiment of the uricase conjugate disclosed herein, the uricase conjugate is a homotetramer comprising four identical uricase conjugate monomers. In a further embodiment, the homotetramer is a homotetramer recombinant uricase fusion protein comprising four identical recombinant uricase fusion protein monomers. In a further embodiment, the recombinant uricase fusion protein monomer comprises a random coil PA polypeptide domain, a random coil PAS polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the above random coil polypeptide domains.

[0188] In one embodiment of the uricase conjugate disclosed herein, the uricase conjugate is a homodimer comprising two identical uricase conjugate monomers. In a further embodiment, the homodimer is a homodimeric recombinant uricase fusion protein comprising two identical recombinant uricase fusion protein monomers. In a further embodiment, the recombinant uricase fusion protein monomer comprises a random coil PA polypeptide domain, a random coil PAS polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the above random coil polypeptide domains.

[0189] In one embodiment of the uricase conjugate disclosed herein, the uricase variant is not PEGylated. In another embodiment, the uricase conjugate is PEGylated, i.e., the uricase conjugate is covalently conjugated to polyethylene glycol (PEG) at, for example, one or more lysine residues. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein disclosed herein. In a further embodiment, the recombinant uricase fusion protein includes a random coil PA polypeptide domain, a random coil PAS polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the above random coil polypeptide domains. In one embodiment, the uricase conjugate is a homodimer or homotetramer disclosed herein, and each of the constituent monomers is PEGylated. In the PEGylated uricase conjugate, amino acid residues in the uricase domain, the random coil peptide domain, or both of the above domains may be covalently conjugated to PEG, for example, depending on the amino acid composition in each domain and the conjugation conditions. In one embodiment, a uricase conjugate, for example, a recombinant uricase fusion protein, is covalently conjugated to one or more mPEG molecules. In a further embodiment, each mPEG molecule has a molecular weight of about 10 kDa. In one embodiment, the monomeric recombinant uricase fusion protein is conjugated to any desired number, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 PEG or mPEG molecules. In one embodiment, the monomeric recombinant uricase fusion protein is conjugated to about 8 to about 11 PEG or mPEG molecules, about 9 PEG or mPEG molecules, or about 10 PEG or mPEG molecules. In one embodiment, the average total molecular weight of the PEG portion of the PEGylated monomer recombinant uricase fusion protein is approximately 10 kDa to 150 kDa, approximately 30 kDa to 120 kDa, or approximately 50 kDa to 100 kDa.In one embodiment, the uricase conjugate is PEGylated by covalent bonding to PEG, such as mPEG, via a biocompatible linking group, similar to the PEGylated uricase variant described above. In another embodiment, the uricase conjugate is PEGylated by covalent bonding to PEG, such as mPEG, without having a linking group, similar to the PEGylated uricase variant described above.

[0190] In another embodiment, the Disclosure provides isolated nucleic acids encoding uricase variants of the Disclosure. In one embodiment, the uricase variant exists as a uricase conjugate comprising the uricase variant domain disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising the uricase variant domain disclosed herein. The isolated nucleic acids have been removed from their natural environment and, additionally, may be substantially pure, e.g., at least 90% pure, or homogeneous. The isolated nucleic acids may be, for example, synthetic DNA, non-spontaneous mRNA, or cDNA. Methods for producing the disclosed nucleic acids are well known to those skilled in the art. See, for example, Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, which is incorporated herein by reference in its entirety. As an addition or alternative, the disclosed nucleic acids may be produced by introducing one or more mutations into a related nucleic acid having a similar nucleic acid sequence, the introduction of which is carried out using site-directed mutagenesis techniques known in the art, such as extension of duplicate gene segments by PCR, which is disclosed in Heckman et al., Nat Protoc 2007, 2, 924-932, which is incorporated herein by reference in its entirety. The disclosed nucleic acids can be cleaved with restriction endonucleases at appropriate sites, subsequently subjected to further enzymatic modification if desired, isolated, and ligated in vitro.

[0191] In another embodiment, the disclosure provides a nucleic acid vector comprising a nucleic acid encoding a uricase variant as disclosed herein. In one embodiment, the uricase variant exists as a uricase conjugate comprising a uricase variant domain as disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising a uricase variant domain as disclosed herein. In one embodiment, the nucleic acid encoding the uricase variant or a recombinant uricase fusion protein comprising a uricase variant domain is inserted into a vector for nucleic acid proliferation, such as a plasmid. In another embodiment, the nucleic acid encoding the uricase variant or a recombinant uricase fusion protein comprising a uricase variant domain is inserted into a suitable expression vector, i.e., a vector containing elements necessary for the transcription and translation of the inserted uricase variant coding sequence, such as a promoter sequence, a terminator sequence, a polyadenylation sequence, and an enhancer sequence. The vector may be, for example, a plasmid, a phage, a phagemid, an adenovirus, an AAV, or a lentivirus. Various host vector systems can be utilized to express coding sequences for recombinant uricase fusion proteins containing uricase variants or uricase variant domains. Exemplary systems include mammalian cell lines infected with viruses (e.g., vaccinia virus, adenovirus, etc.); insect cell lines infected with viruses (e.g., baculovirus); and microorganisms, such as yeast containing a yeast vector, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. The expression elements of these vectors vary in their intensity and specificity. In one embodiment, a bacterial expression vector is used. In a further embodiment, the bacterial expression vector is for use with E. coli.In one embodiment, the promoter in the bacterial expression vector is a T5 promoter induced by isopropyl β-D-1 thiogalactopyranoside (IPTG) and repressed by glucose, a T7 promoter induced by IPTG and repressed by glucose, a rhamnose (rham) promoter induced by rhamnose and repressed by glucose, or an alkaline phosphatase (phoA) promoter induced by phosphate starvation and repressed by the presence of phosphate.

[0192] Any known method for inserting DNA fragments into a vector may be used to construct an expression vector containing nucleic acids comprising one or more regulatory elements, e.g., appropriate transcription / translation control signals, and coding sequences for uricase variants or recombinant uricase fusion proteins operably linked thereto. These methods may include in vitro recombinant DNA and synthetic techniques, as well as in vivo recombination (genetic recombination). The expression of nucleic acid sequences encoding a uricase variant or recombinant uricase fusion protein comprising a uricase variant domain may be regulated by a second nucleic acid sequence, thereby causing the uricase variant or recombinant uricase fusion protein to be expressed in a host transformed with a recombinant DNA molecule. For example, the expression of a uricase variant or recombinant uricase fusion protein comprising a uricase variant domain may be controlled by any promoter / enhancer element known in the art. In some embodiments, the nucleic acid comprises nucleic acid sequences encoding a uricase variant or recombinant uricase fusion protein operably linked to a heterologous promoter. Exemplary promoters that can be used to control the expression of uricase variants or recombinant uricase fusion proteins using mammalian expression vectors include the initial promoter region of monkey virus 40 (SV40), the promoter contained within the 3' long-term repeat of Rous sarcoma virus, the herpesthymidine kinase promoter, and the regulatory sequence of the metallothione gene. Exemplary promoters useful for prokaryotic expression vectors include the β-lactamase promoter, the tac promoter, and the osmotically regulated osmB promoter.

[0193] In another embodiment, the disclosure provides a host cell comprising a nucleic acid vector disclosed herein. In a further embodiment, the host cell is capable of producing the uricase variant of the present invention. In one embodiment, the uricase variant exists as a uricase conjugate comprising the uricase variant domain disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising the uricase variant domain disclosed herein. A suitable cell line or host system can be selected based on the desired expression level of the uricase variant or recombinant uricase fusion protein, as well as / or post-translational processing and modification (e.g., glycosylation, cleavage). Suitable host cells include bacteria (e.g., E. coli), mammalian cells, plant cells, insect cells, fungi, yeasts, and transgenic plants and animals. Exemplary mammalian cell lines available in the art for the expression of heterologous proteins, such as recombinant uricase fusion proteins containing the uricase variant or uricase variant domain disclosed herein, include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, mouse melanoma cells, rat myeloma cells, human embryonic kidney cells, and human embryonic retinal cells. Introducing the vector into host cells can be achieved using techniques well known in the art. For eukaryotic cells, preferred techniques may include, for example, calcium phosphate transfection, diethylaminoethyl (DEAE)-dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses. For bacterial cells, preferred techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophages. Following introduction, expression from nucleic acids may be induced or made possible, and expression is carried out, for example, by culturing host cells under conditions for the expression of nucleic acid sequences encoding uricase variants or recombinant uricase fusion proteins, resulting in the production of the respective proteins.In one embodiment, a nucleic acid encoding a uricase variant or recombinant uricase fusion protein is integrated into the genome of a host cell, e.g., into a chromosome. Integration can be facilitated by including sequences that promote recombination in the genome according to standard techniques.

[0194] In one embodiment, the host cell is a mammalian cell, for example, a Chinese hamster ovary (CHO) cell or a human embryonic kidney cell. In a further embodiment, the expression vector for use in mammalian cells comprises actin (e.g., chicken β-actin), cytomegalovirus (CMV), CMV enhancer / elongation factor (CEF), CMV early enhancer / chicken β-actin (CAG), hybrid CMV enhancer / chicken β-actin (CBh), elongation factor 1α (EF1alpha), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or a monkey virus 40 (SV40) promoter.

[0195] In another embodiment, the host cell is a yeast cell. In a further embodiment, the expression vector for use in yeast cells comprises an alcohol oxidase (AOX), glyceraldehyde-3-phosphate dehydrogenase (GAP), alcohol dehydrogenase (ADH), or galactokinase 1 (GAL1) promoter.

[0196] In another embodiment, the host cell is an insect cell infected with a baculovirus. In a further embodiment, the expression vector for use with the baculovirus includes a polyhedrin gene promoter.

[0197] In another embodiment, the host cell is a bacterium, for example, E. coli. In a further embodiment, the expression vector for use in bacteria comprises an osmB promoter, a T7-lac promoter (see Shilling et al., Commun Biol 3, 214 (2020)), a pBAD promoter, a Tac promoter, a tet-inducible promoter, a cold shock protein A (cspA) promoter, or an inducible promoter of the alkaline phosphatase gene (phoA) derived from E. coli.

[0198] In another aspect, the Disclosure provides a method for recombinantly producing a uricase conjugate (e.g., recombinant uricase fusion protein) comprising a uricase variant or uricase variant domain disclosed herein. The method comprises (i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding a uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) disclosed herein, wherein the nucleic acid sequence is operably linked to a heterologous promoter under conditions that enable the host cell to express the nucleic acid sequence encoding the uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) and to recombinantly produce the uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein); and (ii) isolating the recombinantly produced uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein).

[0199] Uricase variants or uricase conjugates (e.g., recombinant uricase fusion proteins) can be produced in any suitable cell culture system, which may include prokaryotic cells, e.g., E. coli, BL21, or JM83, or eukaryotic cells, e.g., Pichia pastoris yeast strain X-33, or CHO cells. Further suitable cell lines known in the art are available from cell line depositories, such as the American Type Culture Collection (ATCC). Uricase variants or uricase conjugates (e.g., recombinant uricase fusion proteins) can be isolated from growth medium, cell lysates, or cell membrane fractions. The isolation and purification of the recombinant uricase variant or uricase conjugate produced by the present invention (e.g., recombinant uricase fusion protein) may be carried out by any of the conventional means, including ammonium sulfate precipitation, affinity columns, column chromatography, and gel electrophoresis, and may include the use of monoclonal or polyclonal antibodies, for example, monoclonal or polyclonal antibodies directed against a tag fused to the biologically active protein of the present invention. For example, the protein can be purified via Strep-tag II using streptavidin affinity chromatography (Skerra and Schmidt (2000). Methods Enzymol 326, pp. 271-304).

[0200] In one embodiment, the vector is under the control of an osmotically sensitive promoter. The osmotically sensitive promoter initiates transcription as a result of increased osmotic pressure sensed by the cell. In one embodiment, the host cell is E. coli, and the promoter is the osmB promoter, T7-lac promoter, pBAD promoter, Tac promoter, tet-inducible promoter, cold shock protein A (cspA) promoter, or an inducible promoter of the alkaline phosphatase gene (phoA) derived from E. coli.

[0201] In one embodiment, a uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) is isolated using a cationic surfactant, such as cetylpyridinium chloride (CPC). In one embodiment, the method further comprises purifying the recombinantly produced uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) using, for example, affinity chromatography, ammonium sulfate fractionation, or sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis. For example, a uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) can be prepared according to the method described in International Patent Application Publication No. 2000 / 008196, which is incorporated herein by reference in its entirety. In one embodiment, a uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) is produced in bacteria, isolated, and then subjected to endotoxin reduction using methods known in the art, such as those disclosed in the examples of this application.

[0202] In other embodiments, the disclosure provides a pharmaceutical composition comprising a uricase variant. In one embodiment, the uricase variant exists as or is contained within a uricase tetramer comprising a uricase variant monomer. In a further embodiment, the uricase tetramer is a homotetramer comprising four identical uricase variant monomers disclosed herein. In another embodiment, the uricase variant exists as a uricase conjugate comprising a uricase variant domain disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising a uricase variant domain disclosed herein. The pharmaceutical composition may include pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art for formulating protein therapeutics. Such materials are non-toxic and do not interfere with the potency of the uricase variants described herein. Such substances may include, for example, solvents, dispersion media, antimicrobial and antifungal agents, isotonic agents and absorption retarders. Some examples of pharmaceutically acceptable carriers are water, physiological saline, phosphate-buffered physiological saline, dextrose, glycerol, and ethanol, as well as combinations thereof. In one embodiment, the pharmaceutical composition includes an isotonic agent, such as a sugar and / or a polyhydric alcohol such as mannitol or sorbitol, or sodium chloride. Further examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifiers, preservatives, or buffers, which increase shelf life or effectiveness.

[0203] In one embodiment, a pharmaceutical composition containing a uricase variant may be formulated in liquid, semi-solid, or solid form, for example, in liquid solutions (e.g., injectable and injectable solutions), dispersions or suspensions, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration, therapeutic use, physicochemical properties of the uricase variant, and delivery route. The formulation may contain excipients, or combinations of excipients, such as sugars, amino acids, and surfactants. Liquid formulations may encompass a wide range of uricase variant concentrations and pH. Solid formulations may be produced, for example, by freeze-drying, spray-drying, or drying using supercritical fluid technology.

[0204] For intravenous or pain site injection, the active ingredient may be in a parenterally acceptable aqueous solution that is pyrogenic and has a suitable pK, isotonicity, and stability. Those skilled in the art can readily prepare suitable solutions using isotonic vehicles such as sodium chloride solution, Ringer's solution, and lactated Ringer's solution. Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included.

[0205] In some embodiments, the pharmaceutical composition is formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable for containing a high concentration of uricase variant. Sterile injectable solutions can be prepared by incorporating the uricase variant into a suitable solvent having one or a combination thereof of the components listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the uricase variant into a sterile vehicle containing a dispersion medium and other components from those listed above. For sterile powders for the preparation of sterile injectable solutions, the preparation method includes vacuum drying and lyophilization, from a pre-sterile filtered solution to obtain a powder of the active ingredient and any additional desired components. Appropriate fluidity of the solution can be maintained, for example, by using a coating such as lecithin, by maintaining the particle size of the dispersion, or by using a surfactant. Sustained absorption of the injectable composition can be brought about by including absorption-delaying agents, such as monostearate and gelatin, in the composition.

[0206] In some embodiments, the pharmaceutical composition may be prepared with a carrier that protects the uricase variant from rapid release, such as a controlled-release formulation, which includes implantable tablets, transdermal patches, and microencapsulated delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used.

[0207] In one embodiment, the pharmaceutical composition is a solution of the uricase variant disclosed herein, for example, a phosphate-buffered saline solution containing the uricase variant. In further embodiments, the solution is sterile and suitable for injection, for example, intravenous or subcutaneous injection.

[0208] In another embodiment, the disclosure provides a method for reducing elevated uric acid levels in a subject requiring treatment. The method comprises administering to the subject an effective amount of a pharmaceutical composition comprising a uricase variant disclosed herein. In one embodiment, the uricase variant exists as or is contained within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant exists as a uricase conjugate comprising a uricase variant domain disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising a uricase variant domain disclosed herein.

[0209] As used herein, “treatment,” “to treat,” or “to induce remission,” and their variations thereto, are interchangeable. These terms refer to an approach to obtain beneficial or desired outcomes, including but not limited to therapeutic and / or preventive benefits. Therapeutic benefit means any therapeutically related improvement in or effect on one or more diseases, conditions, or symptoms under treatment. In one embodiment, the term “to treat” includes (1) preventing or delaying the onset of clinical symptoms of a condition, disorder, or condition in a patient who is susceptible to or predisposed to a condition, disorder, or condition but has not yet experienced or shown any clinical or asymptomatic symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition (e.g., stopping, reducing, or delaying the onset of the disease, or in the case of maintenance treatment, its recurrence, or the onset of at least one clinical or asymptomatic symptom thereof); and (3) alleviating the condition (e.g., causing regression of the condition, disorder, or condition, or at least one of its clinical or asymptomatic symptoms, or reducing its severity).

[0210] "Effective dose" means an amount of a pharmaceutical composition containing the uricase variant of this disclosure that is sufficient to produce a desired therapeutic response. In one embodiment, the uricase variant exists as or is contained within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant exists as a uricase conjugate containing the uricase variant domain disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein containing the uricase variant domain disclosed herein. The therapeutically effective dose may vary depending on one or more of the following: the subject and disease state being treated, the subject's weight and age, the severity of the disease state, and the mode of administration.

[0211] The terms “subject,” “individual,” and “patient” are used interchangeably herein and refer to vertebrates, e.g., mammals. Mammals may be, for example, mice, rats, rabbits, cats, dogs, pigs, sheep, horses, non-human primates (e.g., cynomolgus macaques, chimpanzees), or humans. It also encompasses tissues, cells, or derivatives thereof of subjects obtained in vivo or cultured in vitro. Human subjects may be adults, teenagers, children (2 to 14 years), infants (1 to 24 months), or neonates (up to 1 month). In some embodiments, adults are elderly, approximately 65 years of age or older, or approximately 60 years of age or older. In some embodiments, subjects are pregnant women or women intending to become pregnant.

[0212] In one embodiment of the method, the subject is a human patient. In a further embodiment, the human patient is an adult human patient.

[0213] In one embodiment of the method, the uric acid level in the plasma or blood of the subject is reduced. In one embodiment, a preferred indicator for evaluating the effectiveness of the method includes normalization or reduction of plasma uric acid levels (PUA) in human patients, for example, a reduction or maintenance of PUA to 6.8 mg / dL or less or 6 mg / dL or less.

[0214] In some embodiments of the method, administration of the pharmaceutical compositions disclosed herein is carried out parenterally, for example, by intramuscular, intrathecal, subcutaneous, or intravenous administration. In one embodiment, the administration is intravenous (IV) administration. In another embodiment, the administration is subcutaneous (SC) administration. In yet another embodiment, the administration is rectal, topical, or pulmonary administration.

[0215] In one embodiment, the subject is a patient with gout, for example, a human patient with gout. In one embodiment, the gout is recurrent gout. In another embodiment, the gout is advanced gout, accompanied by the deposition of uric acid crystals that form nodules called tophi under the skin. In human patients, tophi may develop in several areas, for example, the fingers, hands, feet, elbows, or Achilles tendons along the outside of the ankles. In another embodiment, the patient with gout has kidney stones, which are uric acid crystals collected in the urinary tract.

[0216] In one embodiment, the subjects are patients with refractory gout, i.e., patients who have been resistant to different previous treatments. Previous treatments include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, corticosteroids, allopurinol, febuxostat, probenecid, KRYSTEXXA (pegroticase), and combinations thereof. In one embodiment, refractory gout is a chronic condition characterized by high serum uric acid levels, recurrent gout flare, chronic arthritis, and progressive nodular deposition. In another embodiment, refractory gout is associated with a high rate of cardiovascular and renal comorbidities.

[0217] In one embodiment, the subject is diagnosed with tumor lysis syndrome. In some embodiments, the subject diagnosed with tumor lysis syndrome has lymphoma (e.g., Burkitt lymphoma, non-Hodgkin lymphoma), acute lymphoblastic leukemia, or acute myeloid leukemia. In one embodiment, the subject diagnosed with tumor lysis syndrome is a human patient with a plasma uric acid concentration greater than 8 mg / dL. In another embodiment, the subject diagnosed with tumor lysis syndrome is a human patient with a plasma uric acid concentration greater than 15 mg / dL (hyperuricemia).

[0218] In another embodiment, the disclosure provides a method for treating gout in a subject requiring treatment. The method comprises administering to the subject an effective amount of a pharmaceutical composition comprising a uricase variant disclosed herein. In one embodiment, the uricase variant exists as or is contained within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant exists as a uricase conjugate comprising a uricase variant domain disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising a uricase variant domain disclosed herein. In one embodiment, the gout to be treated is the refractory gout described above. In one embodiment, the gout to be treated is recurrent gout. In another embodiment, the gout to be treated is progressive gout, characterized by the deposition of uric acid crystals formed subcutaneously in small nodules, which are referred to as tophi. In another embodiment, the subject having gout has kidney stones. In one embodiment, the subject is a human patient. In a further embodiment, the human patient is an adult human patient.

[0219] In some embodiments of the method, administration of the pharmaceutical compositions disclosed herein is carried out parenterally, for example, via intramuscular, intrathecal, scalculatory, or intravenous administration. In one embodiment, the administration is intravenous. In another embodiment, the administration is scalculatory. In yet another embodiment, the administration is rectal, topical, or pulmonary.

[0220] In another embodiment, the disclosure provides a method for treating tumor lysis syndrome in a subject requiring treatment. The method comprises administering to the subject an effective amount of a pharmaceutical composition comprising a uricase variant disclosed herein. In one embodiment, the uricase variant exists as or is contained within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant exists as a uricase conjugate comprising a uricase variant domain disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising a uricase variant domain disclosed herein. In one embodiment of the method, the subject is a human patient. In a further embodiment, the human patient is an adult human patient.

[0221] In some embodiments of the method, administration of the pharmaceutical composition is carried out parenterally, for example, via intramuscular, intrathecal, SC, or IV administration. In one preferred embodiment, the administration is IV administration. In another embodiment, the administration is SC administration. In yet another embodiment, the administration is rectal, topical, or pulmonary administration. In some embodiments, subjects having tumor lysis syndrome have lymphoma (e.g., Burkitt lymphoma, non-Hodgkin lymphoma), acute lymphoblastic leukemia, or acute myeloid leukemia. In one embodiment, subjects having tumor lysis syndrome are human patients with a plasma uric acid concentration greater than 8 mg / dL. In another embodiment, subjects having tumor lysis syndrome are human patients with a plasma uric acid concentration greater than 15 mg / dL (hyperuricemia).

[0222] In another embodiment, the disclosure provides a method for metabolizing uric acid using a uricase variant disclosed herein. In one embodiment, the uricase variant exists as or is contained within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant exists as a uricase conjugate comprising a uricase variant domain disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising a uricase variant domain disclosed herein.

[0223] In another embodiment, the disclosure provides the use of a composition comprising a uricase variant disclosed herein for reducing uric acid levels in a biological fluid. In one embodiment, the uricase variant exists or resides within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant exists as a uricase conjugate comprising a uricase variant domain disclosed herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising a uricase variant domain disclosed herein. In one embodiment, the biological fluid is from a patient with gout or refractory gout. In another embodiment, the biological fluid is from a patient diagnosed with tumor lysis syndrome. In a further embodiment, the biological fluid comprises blood. [Examples]

[0224] The present invention is further illustrated by reference to the following embodiments. However, it should be noted that these embodiments, like the embodiments described above, are illustrative and should not be construed as limiting the scope of the present invention in any way.

[0225] Example 1 - Preparation and Characterization of Deimmunized Non-PEGylated and PEGylated Chimeric Pig-Baby Melon Case Variants This example describes the development of highly engineered immunized, non-PEGylated, and PEGylated chimeric porcine-baboon melon case variants by utilizing a computer-driven protein manipulation platform. Compared to the wild-type chimeric porcine-baboon melon case of SEQ ID NO: 1, which constitutes pegroticase, the lead variant candidates exhibited better overall yield, activity, and thermal stability, while having reduced immunogenic performance due to mutagenic silencing (depletion) of the constituent CD4+ T cell epitopes.

[0226] method 1. Soluble enzyme activity assay Small-volume cultures of E. coli transformed with the expression vector pET-26b(+), which contains a cDNA sequence encoding wild-type chimeric pig-baboon uricase or a variant thereof, were grown and induced in deep 96-well plates. The expression vector pET-26b(+) encodes a His tag containing six histidine residues at the C-terminus of the inserted uricase gene. The induced E. coli cells were pelletized by centrifugation. The cell pellet was resuspended in BUGBUSTER® HT Protein Extraction Reagent (MilliporeSigma, MA, USA), pelletized by centrifugation, and the insoluble pellet was resolubilized in alkaline carbonic acid / bicarbonate buffer (pH 10.5). The resolubilized sample was combined with a uric acid substrate solution (0.1 M boric acid, 0.12 mM uric acid, pH 9.0) in a UV-transparent 96-well plate, and the absorbance at 293 nm was measured as a function of time. Uricase activity was measured as the slope of the absorbance-time curve at 293 nm. A steeper negative slope indicates higher activity. Therefore, the slope was quantified as a measure of vitality at arbitrary units.

[0227] 2. Expression assay The expression assay is based on the measurement of total activity in the soluble supernatant after lysis of the E. coli cell pellet in the alkaline carbonate / bicarbonate buffer (pH 10.5) described above, and a separate measurement (described below) of the activity of the immobilized uricase enzyme from the soluble supernatant (in a 96-well Ni-NTA HisSorb plate (Qiagen)), because the wild-type chimeric pig-baboon uricase or its variant produced in E. coli contains a His tag at the C-terminus. Theoretically, the HisSorb immobilized surface is saturated with the enzyme, and therefore the relative expression level is obtained by dividing the total activity by this saturated immobilized activity.

[0228] To measure the activity of immobilized uricase enzyme from the soluble supernatant, small volume cultures of E. coli transformed with the expression vector pET-26b(+) containing a cDNA sequence encoding wild-type chimeric pig-baboon uricase or a variant thereof were grown and induced in deep 96-well plates. The induced E. coli cells were pelletized by centrifugation. The cell pellet was resuspended in BUGBUSTER® HT Protein Extraction Reagent and pelletized by centrifugation, and the insoluble pellet was resolubilized in alkaline carbonate / bicarbonate buffer (pH 10.5). The resolubilized sample was collected and transferred to a 96-well Ni-NTA HisSorb plate (Qiagen) with immobilized metal ion affinity ligands at the bottom of the wells. The HisSorb plate was incubated for 1 hour to bind the His-tagged uricase enzyme to the wells, and then the supernatant was removed from the wells and the wells were washed five times with alkaline carbonate / bicarbonate buffer (pH 10.5). Subsequently, a uric acid substrate solution (0.1 M boric acid, 0.12 mM uric acid, pH 9.0) was added to a HisSorb plate, and the absorbance at 293 nm was measured as a function of time. Uricase activity was measured as the slope of the absorbance-time curve at 293 nm, with a steeper negative slope indicating higher activity. The relative expression level was calculated by dividing the activity of the total soluble enzyme fraction by the activity of the HisSorb-binding enzyme fraction.

[0229] 3. Differential Scanning Fluorescence (DSF) The melting temperatures of wild-type chimeric porcine-baboon melon case and its variants were determined using a C1000 thermal cycler with a CFX96 real-time system (Bio-Rad), performed according to Niesen et al., Nat Protoc. 2007, 2(9):2212-21, which is incorporated herein by reference in its entirety. Proteins were diluted to a final concentration of 66 μg / mL in PBS buffer containing SYPRO orange dye at a 5× final concentration. Triad samples of each protein were exposed to a temperature gradient from 40°C to 99°C. Melting temperatures were calculated using fluorescence acquisition and fluorescence loss of SYPRO orange with PRISM software.

[0230] 4.1 Step-by-step purification and determination of the percentage of soluble tetramer by size exclusion chromatography (SEC). 4.1. Purification of Inclusion Body E. coli cells were lysed in 50 ml of B-PER bacterial protein extraction reagent (ThermoFisher Scientific) per liter of culture supplemented with 1000 U / L of BENZONASE® endonuclease (MilliporeSigma). Specifically, the cell pellet was resuspended in B-PER and subsequently incubated at room temperature for 1 hour. The suspension was centrifuged at 25,000 rpm for 30 minutes at 4°C. The supernatant was removed and discarded, and the pellet was resuspended in 50 ml / L load buffer containing 100 mM sodium bicarbonate (pH 10.5) and 10 mM imidazole. After incubation at room temperature for 1 hour, the suspension was centrifuged at 25,000 rpm for 30 minutes at 4°C, the supernatant was removed, and the supernatant was saved for subsequent chromatography.

[0231] 4.2 Chromatographic Purification The preserved supernatant from Section 4.1 above was loaded onto a HISPREP FF 16 / 10 column at 5 ml / min. The column was then washed with 2 column volumes (cv) of load buffer containing 1% TRITON-114, followed by washing with 10 cv of load buffer, and subsequently eluted with 5 cv of 100 mM sodium bicarbonate (pH 10.5) and 500 mM imidazole. The eluted proteins were buffer-replaced with 100 mM sodium bicarbonate (pH 10.5), and the percentage of soluble tetramers was quantified by the HPLC-SEC analysis method described below.

[0232] 4.3. Determination of soluble tetramer percentage using SEC The size and concentration of uricase enzyme were determined by assessing the percentage of soluble tetramers in naked uricase enzyme samples using the HPLC-SEC analysis method. The HPLC-SEC analysis method was performed using a Waters Arc HPLC system with adjustable UV (TUV) and refractive index (RI) detectors, along with a Waters UPLC Premier protein SEC column (Thermo Scientific, catalog no. 186005225). The method measured hydrodynamic volume as a surrogate for molecular weight or size, using a Waters protein standard (Thermo Scientific, catalog no. 186006518) for molecular weight approximation. Specifically, a 12 μl solution of naked uricase enzyme containing either a Waters protein standard or 2 mg / mL of uricase protein in 100 mM sodium bicarbonate (pH 10.5) was injected into the Waters UPLC Premier protein SEC column of the Waters Arc HPLC system. Data acquisition and analysis were performed using a Waters TUV set to 280 nm. In the chromatogram of the naked uricase enzyme sample, one small peak with a shorter retention time representing aggregates and one large peak with a longer retention time representing soluble tetramers were observed. The soluble uricase tetramers had an approximate molecular weight of 100 kDa based on the retention time of the Waters protein standard.

[0233] 5. Determination of reaction rates for wild-type chimeric pig-baboon uricase and its variants. The reaction rates of wild-type chimeric pig-baboon uricase and its variants were determined by the following procedure. (1) Dilute the uricase protein sample to 0.2 mg / mL in a pH 10.5 buffer containing 0.1 M carbonic acid-bicarbonate. (2) Ali-coat 10 μL of diluted protein sample per well in a 96-well plate. (3) Add 190 μL of uric acid substrate solution containing 0.125 mM uric acid to each well. (4) Load the 96-well plate onto the plate reader. (5) Using a plate reader, measure the absorbance at 293 nm every 30 seconds for 20 minutes under a controlled temperature of 25°C. (6)V max The value should be calculated using BIOTEK Gen5 software.

[0234] 6. Determination of Michaelismenten kinetics for wild-type chimeric pig-baboon uricase and its variants. The kinetics of Michaelis-Menten in wild-type chimeric pig-hihiuricase and its variants were determined by the following procedure. (1) Dilute the uricase protein sample to 0.2 mg / mL in a pH 10.5 buffer containing 0.1 M carbonic acid-bicarbonate. (2) Ali-coat 10 μL of diluted protein sample per well into multiple wells of a 96-well plate. (3) Add 40 μL each of uric acid substrate solutions containing uric acid at the following concentrations: 2000 μM, 1700 μM, 1500 μM, 1200 μM, 1000 μM, 750 μM, 500 μM, 375 μM, 250 μM, 187 μM, 125 μM, and 93 μM to each well containing the diluted protein sample. (4) Load the 96-well plate onto the plate reader. (5) Using a plate reader, measure the absorbance at 293 nm every 30 seconds for 20 minutes under a controlled temperature of 25 °C. (6) V max values are calculated using BIOTEK Gen5 (trademark) software. (7) Using the Michaelis–Menten function of PRISM software, analyze the V max values as a function of uric acid concentration to derive Michaelis–Menten kinetic parameters.

[0235] 7. Purification Wild-type chimeric pig–rhesus uricase and its variants expressed in E. coli were subjected to multi-step purification by various means detailed below.

[0236] 7.1. Inclusion body purification E. coli cells were lysed with 50 ml of B-PER Bacterial Protein Extraction Reagent (ThermoFisher Scientific) per liter of culture supplemented with 1000 U / L of BENZONASE (registered trademark) endonuclease (MilliporeSigma). Specifically, the cell pellet was resuspended in B-PER and subsequently incubated at room temperature for 1 hour. The suspension was centrifuged at 25,000 rpm for 30 minutes at 4 °C. The supernatant was removed and discarded, and the pellet was resuspended in 50 ml / L of loading buffer containing 100 mM sodium bicarbonate (pH 10.5) and 10 mM imidazole. After incubation at room temperature for 1 hour, the suspension was centrifuged at 25,000 rpm for 30 minutes at 4 °C, the supernatant was removed, and stored for subsequent chromatography.

[0237] 7.2. Chromatographic purification The saved supernatant from section 7.1 above was loaded onto a HISPREP FF 16 / 10 column at 5 ml / min. The column was then washed with 2 column volumes (cv) of loading buffer containing 1% TRITON-114, followed by washing with 10 cv of loading buffer, and then eluted with 5 cv of 100 mM sodium bicarbonate (pH 10.5) and 500 mM imidazole. The eluted protein was analyzed by SDS-PAGE, activity, DSF, liquid chromatography-mass spectrometry (LC / MS), and dynamic light scattering (DLS). Thereafter, the eluate was concentrated to 15 ml using a stirred cell with a 50 kDa filter to a concentration of 3 mg / ml or less. The concentrated eluate was loaded onto a SUPERDEX 200 HILOAD 26 / 600 column and then eluted with 100 mM sodium bicarbonate (pH 10.5). Fractions containing tetrameric uricase identified as monodisperse by DLS were pooled, and the pooled tetrameric uricase fractions were analyzed by SDS-PAGE and DLS. The pooled tetrameric uricase fractions were then concentrated to 3 mg / ml using a stirred cell with a 50 kDa filter.

[0238] 7.3. Endotoxin reduction Endotoxin in preparations of wild-type chimeric pig-ape uricase and its variants was removed by the following procedure. (1) Add 1% TRITON-114 to the preparation, followed by incubation at 4 °C for 30 minutes and then at 37 °C for 30 minutes. (2) Centrifuge the mixture at 37 °C for 30 minutes in a pyrogen-free tube. (3) Decant the top layer into a new pyrogen-free tube. (4) Repeat steps (1)-(3) as necessary to reduce the endotoxin level to less than 0.5 EU / ml. (5) Repeat steps (2) and (3) one last time to remove residual TRITON-114. (6) Endotoxin should be tested using the horseshoe crab mebocyte lysate (LAL) assay (Charles River) by diluting the test sample 1:20 in endotoxin-free water and applying 25 μl to each sample well.

[0239] 8. PEGylation of wild-type chimeric pig-baboon uricase and its variants. PEGylation of wild-type chimeric pig-baboon uricase or its variants was performed using the following procedure. (1) Add methoxy-PEG-(CH2)5COO-NHS (chemical name: poly(oxy-1,2-ethanediyl), α-methyl-ω-{2-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyloxy}; NOF America Corporation, catalog number SUNBRIGHT ME-100HS) in a 50-fold molar excess of a molecular weight of 10 kDa to purified uricase protein on ice and incubate with stirring for 1 hour. (2) Verify the radius using DLS (radius of 14-15 nm = wild type), (3) Concentrate the PEGylated protein to 15 ml using a stirring cell with a 50 kDa filter. (4) Load the concentrated PEGylated protein onto a SUPERDEX 200 HILOAD 26 / 600 column and elute with PBS. (5) Pooling the fractions identified by DLS as simple variances, (6) Concentrate the pooled fraction to 5 mg / ml using a stirring cell with a 50 kDa filter.

[0240] 9. Determining the titer of anti-drug antibodies in mouse plasma using ELISA. High-binding 96-well ELISA plates (Costar No. 2592) were coated overnight at 4°C with a 5 μg / mL solution containing either one of the top six PEGylated chimeric porcine-baby melon case variants (i.e., HF0536, HF0554, HF0585, HF0668, HF0752, and HF0835) or PEGylated wild-type chimeric porcine-baby melon case in bicarbonate buffer. The coating solution was then removed, and the plates were washed three times with phosphate buffer containing 0.1% Tween 20 surfactant. The washed plates were blocked at 37°C for 15 minutes with 1x superblock buffer (ThermoFisher Scientific No. 37515), followed by removal of the superblock buffer. Mouse plasma in serial dilutions of 1:20 to 1:12,207 in LOWCROSS buffer (ThermoFisher Scientific, No. NC9831360) was added to the wells, and the plates were incubated at 37°C for 60 minutes. The plasma dilutions were removed, and the plates were washed three times with phosphate buffer containing 0.1% Tween 20 surfactant. The plates were then incubated with a 1:10,000 dilution of anti-mouse secondary antibody conjugated with horseradish peroxidase at 37°C for 30 minutes, and diluted in LOWCROSS buffer. The secondary antibody was then removed, and the plates were washed four times with phosphate buffer containing 0.1% Tween 20 surfactant. The anti-drug antibodies were then quantified by detection with TNB (3,3',5,5'-tetramethylbenzidine) substrate (Bethyl, No. E102), measurement of absorbance at 450 nm, and subtraction of background absorbance at a reference wavelength of 620 nm.

[0241] 10. Immunogenicity analysis of wild-type chimeric pig-baboon uricase and its variants using ex vivo cell immunoassay with human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy human donors were grown for 13 days with IL-2 in the presence of wild-type chimeric pig-baboon melon case protein or one of its variant proteins, and then restimulated with the same protein. Surface and intracellular staining for flow cytometry were then used to evaluate T cell activation markers and cytokine production. In short, PBMCs were placed in 48-well plates, 2 × 10⁶ per well. 6 Cells were seeded in 200 μl of RPMI medium with 5% human serum. Antigen stimulation or vehicle control was added at plating. Proteins were added at a final concentration of 10 μg / mL. IL-2 was added at a final concentration of 10 U / mL on day 4, and the medium was maintained thereafter with alternating daily changes. After 13 days of growth, the cells were re-stimulated for 10 hours under the same conditions in the presence of brefeldin A (to block cytokine secretion). Flow cytometry staining was then performed as follows: Cells were washed twice with PBS and then stained with Zombie Aqua® viable stain at a 1:1000 dilution at room temperature for 30 minutes. Cells were then washed with FACS buffer (1% FBS in 1×PBS) and subsequently incubated with Fc Block® at room temperature for 10 minutes. The cells were washed twice more with FACS buffer and then stained with the following antibodies diluted in BD Brilliant® staining buffer: CD3 BB515, CD4 SB600, CD8 BV650, CD25 APC Fire750, and CD69 BV750. The samples were incubated with surface staining in the dark at 4°C for 30 minutes in a final volume of 100 μl. The cells were then washed three times with FACS buffer, followed by intracellular staining. Intracellular staining was performed using the BD Cytofix / Cytoperm® Fixation / Permeabilization staining kit according to the manufacturer's instructions, with Ki67 AF647, IFN-γ BB700, TNF-α BV421, and IL-2 PE. After the final step, the samples were resuspended in FACS buffer and acquired using a Cytek® Northern Lights® full-spectrum flow cytometer.

[0242] The immunogenicity of wild-type chimeric pig-baboon uricase and its variants was measured by the stimulation index for each donor against each test uricase protein. The stimulation index was calculated as a factorial activation relative to the background, and in factorial activation, the percentage of cells positive for each combination of activating markers was divided by the percentage of cells positive for that combination of markers in the vehicle control group. The sum of the factorial increases in signal for each combination of markers was then reported as the stimulation index. Combinations of activating markers included (1) proliferation markers (i.e., Ki67), (2) activation markers (i.e., CD25 and CD69), and (3) functional markers (i.e., IL-2, INF-γ, and TNF-α).

[0243] result The monomeric wild-type chimeric pig-hihiuricase contains the amino acid sequence of SEQ ID NO: 1, where threonine is residue #1. The specified mutation in the chimeric pig-hihiuricase variant of this example is represented with threonine as residue #1, as in SEQ ID NO: 1. However, in this example, the wild-type chimeric pig-hihiuricase and its variant produced in E. coli have methionine (preceding threonine) as residue number 1 and threonine as residue number 2, which is due to the presence of an Ndel restriction site encoding methionine in-frame with the wild-type and variant chimeric pig-hihiuricase genes in the expression vector pET-26b(+) (MilliporeSigma, MA, USA). The expression vector pET-26b(+) encodes a His tag containing six histidine residues at the C-terminus of the inserted uricase gene.

[0244] 1. Design of a deimmunized library Deimmunization library design was performed to computationally optimize the uricase variant library and select one library for experimental construction. As illustrated in Figure 2A, a proprietary design algorithm was used to select mutations (both site- and site-specific substitutions) against wild-type chimeric pig-baboon uricase that defined a combinatorial library predicted to be enriched in clones with high function and low immunogenicity. In library design, the algorithm models the entire protein and evaluates the interrelationship effects of mutation combinations on both epitope content and protein stability and function. The algorithm generated hundreds of Pareto optimal and near-optimal designs with significantly different levels of epitope deletion versus function maintenance. The designs explored different population sizes (approximately 20,000–1 million members), different numbers of mutation target sites, and different numbers of substitutions per site (1–4). Two iterative rounds of library design and construction were performed, each round including filtering and testing of epitopes and functional scores, using the most deimmunized functional variants from the first round as a starting point for the second round. Specifically, in the first round, a library design with approximately 400,000 theoretical variants having combinatorial amino acid substitutions from 18 target sites of SEQ ID NO: 1 was selected. In the second round, a refined library design with approximately 18,000 theoretical variants having combinatorial amino acid substitutions from 12 additional target sites of SEQ ID NO: 1 (i.e., from a total of 30 target sites) was selected, as summarized in Table 2. In Table 2, the positions of amino acids from the 30 target sites present in the chimeric pig-baboon melon case variant following the second round library design, referred herein as “designed amino acids,” are denoted as follows, with threonine as residue number 1, as in SEQ ID NO: 1. The library members exhibited reduced MHC-II binding ability compared to wild-type chimeric pig-baboon melon case.Lead deimmunized and functional variant candidates from the second round of library design primarily contained 10–20 combinatorial amino acid substitutions out of 30 target sites. [Table 2]

[0245] In selecting this library design, functional chimeric pig-baboon melon case variants were identified via high-throughput screening (Figure 2B), and most or all immunodominant epitopes in the protein sequence were silenced as described below (Figure 2C).

[0246] 2. Library screening and functional evaluation Library screening and functional evaluation were performed to experimentally identify and characterize a diverse set of high-performance uricase variant candidates by (i) selecting active variants using a high-throughput halo-based assay and (ii) purifying them and analyzing the yield, activity, and stability of the top candidates in detail.

[0247] Primary screening of the combinatorial deimmunized uricase variant library was performed by the uricase halo assay (Figure 3A). Specifically, E. coli transformed with the expression vector pET-26b(+) containing a cDNA sequence encoding wild-type chimeric pig-baboon uricase or a variant thereof was seeded onto LB agar plates containing uric acid. Bacterial colonies expressing active uricase converted the suspended uric acid into water-soluble allantoin. Thus, a clear zone (halo) around the target bacteria indicates the production of the active enzyme. The diameter of each clear zone was recorded as a semi-quantitative measure of enzyme expression and activity. Variants selected based on halo size were seeded, grown, induced in deep 96-well plates, and the cell pellets were resuspended in lysis buffer. Uricase enzyme activity was quantified (i) in alkali-soluble lysate (carbonic acid / bicarbonate buffer at pH 10.5) and (ii) in enzyme immobilized on Ni-NTA HisSorb plates (Qiagen) from alkali-soluble lysate. A pseudo-estimate of expression levels was calculated as the quotient of total lysate activity versus immobilized lysate activity, as described in the "Methods" section above. Solution-phase kinetic analysis of whole-cell lysates allowed for precise selection of clones exhibiting the fastest kinetics (Figure 3B). Table 3 shows the 130 functional chimeric pig-baboon uricase variants identified. In the genes of the sequenced variants, Table 3 shows the sequence numbers for each variant and the mutations relative to wild-type chimeric pig-baboon uricase at sequence number 1. [Table 3] TIFF2026517791000011.tif254170TIFF2026517791000012.tif254170TIFF20265177910 00013.tif249170TIFF2026517791000014.tif249170TIFF2026517791000015.tif136170

[0248] The characteristics including the selection group of 21 non-PEGylated variants and the yield, Michaelis-Menten kinetic parameters, and melting temperature measured by DSF of non-PEGylated wild-type chimeric pig-rhesus uricase (WT) are summarized in Table 4B. The sequence numbers of the selected variants, the mutations relative to wild-type chimeric pig-rhesus uricase of SEQ ID NO: 1, and the amino acid sequences are shown in Table 4A.

Table 4A

Table 4B

[0249] Based on the data in Table 4B, the yields, activities, and T m of 21 non-PEGylated candidate variants and non-PEGylated WT comparator were scored as summarized in Table 4C.

Table 4C

[0250] Based on the total score data in Table 4C, the top six variant candidates were selected as shown in Table 4D. Each of these top six variant candidates has a high total score of 9 - 11 compared to the total score of 5 for WT.

Table 4D

[0251] 3. PEGylation of the top six chimeric pig-baboon melon case variant candidates and characterization of the PEGylated variants. As described above, the top six variant candidates produced in E. coli and wild-type chimeric pig-baboon uricase were purified and conjugated to polyethylene glycol (PEG). The successful PEGylation of wild-type chimeric pig-baboon uricase and the variants was confirmed by SDS-PAGE (Figure 4A). In SDS-PAGE, the band at approximately 34 kDa MW represents the non-PEGylated uricase species, and the band at approximately 291 kDa MW represents the PEGylated uricase species. The PEGylated uricase species is estimated to have approximately 10 PEG molecules per monomer by size exclusion chromatography-multi-angle static light scattering (SEC-MALS) analysis.

[0252] To confirm that each of the top six variant candidates was capable of forming a homotetramer, the unPEGylated (naked) wild-type chimeric porcine-baboon melon case and each of the top six unPEGylated (naked) variant candidates were subjected to one-step purification as described in the "Methods" section, followed by determination of the percentage of soluble tetramers using size exclusion chromatography (SEC). Figure 4B shows that each of the unPEGylated variants had a higher percentage of soluble tetramers than its unPEGylated wild-type counterpart, indicating that the top six variant candidates had reduced aggregation compared to wild-type chimeric porcine-baboon melon case.

[0253] The specific activity, Michaelis-Menten kinetics, and melting temperatures (measured by differential scanning fluorescence quantification) of PEGylated wild-type chimeric porcine-baboon uricase and the top six PEGylated variant candidates were determined. The data are shown in Figures 4C, 4D, 4E, and 4F. The results from these studies indicate that the specific activity of the PEGylated variants ranged from 90% to 133% of that of the PEGylated wild-type counterparts, and that the thermal stability of the PEGylated variants was either equivalent to or higher than that of the PEGylated wild-type counterparts. In summary, the top six PEGylated variant candidates exhibit good overall performance in vitro.

[0254] 4. Immunogenicity analysis The immunogenicity of the top six non-PEGylated chimeric pig-baby melon case variant candidates against non-PEGylated wild-type chimeric pig-baby melon case (WT) was evaluated by an ex vivo cell immunoassay using human peripheral blood mononuclear cells (PBMCs). Figure 5A is a schematic diagram of the ex vivo cell immunoassay measuring T cell activation; details of the assay are described in the "Methods" section. In the assay, immune cells from each individual human donor were incubated with either non-PEGylated wild-type chimeric pig-baby melon case (WT) or non-PEGylated deimmunized uricase variant (dURC) for a 14-day proliferation period. The T cells were then evaluated by flow cytometry for markers of proliferation (i.e., Ki67), activity (i.e., CD25 and CD69), and functionality (i.e., IL-2, INF-γ, and TNF-α). The sum of these activation markers was used to calculate the "stimulation index" for each donor against each test protein. The assay results are presented in Figure 5B, where the stimulation index (multiplicative activation relative to background) is shown for each of the top six non-PEGylated variant candidates or for each of the 20 individual human donors loaded with the non-PEGylated wild-type comparator, with each symbol representing a single donor. The bars in Figure 5B represent the median with a 95% confidence interval for each population. The assay results demonstrate a significant reduction in the T cell response with the non-PEGylated deimmunized uricase variant candidates HF0668, HF0752, and HF0835.

[0255] The immunogenicity of the top six candidate PEGylated chimeric porcine-porcine-porcine variants against PEGylated wild-type chimeric porcine-

[0256] Humanized DR4 HLA transgenic mice were subjected to primary immunization via intraperitoneal administration of 0.25 μg / kg of one of the PEGylated uricase variants or PEGylated wild-type chimeric pig-baboon uricase on day 1. Subsequently, they were subjected to additional immunization via intraperitoneal administration of 2.5 μg / kg of the same PEGylated uricase variant and PEGylated wild-type chimeric pig-baboon uricase on days 7, 14, 21, 28, and 35. Ten mice were used in each test protein group. Mice were administered at approximately 1 / 500 of the human equivalent dose using allometric scaling to minimize drug interference in the measurement of anti-drug antibodies. Mouse plasma was collected on day 42 and analyzed for anti-drug antibodies by ELISA (Figure 6A).

[0257] Figure 6B shows a curve representing the relationship between anti-drug antibody titer, determined by ELISA absorbance, and plasma dilution on a logarithmic scale, using 42-day plasma collected from humanized DR4 HLA transgenic mice immunized with PEGylated uricase variants or PEGylated wild-type counterparts. Higher ELISA absorbance at larger plasma dilutions indicates a stronger anti-drug antibody response. One summary measure of the overall response is the area under the plasma dose-response curve (AUC), where a larger AUC indicates a stronger anti-drug immune response. Figure 6C shows the anti-drug antibody titer AUC derived from Figure 6B. The results demonstrate that each of the top six PEGylated chimeric pig-uricase variant candidates was less immunogenic in DR4 mice than PEGylated wild-type chimeric pig-uricase, despite the low drug dose levels required by the study design.

[0258] 5. Efficacy Analysis The efficacy of weekly repeated dosing of three of the top six candidate PEGylated chimeric porcine-uricase variants against PEGylated wild-type chimeric porcine-uricase—HF0536, HF0554, and HF0752—was evaluated in humanized DR4 HLA transgenic mice. Specifically, as shown in Figure 7A, humanized DR4 HLA transgenic mice in groups of nine mice were either left untreated (i.e., untreated healthy mouse group) or administered an induction dose via intraperitoneal administration of 4.2 mg / kg of PEGylated uricase variant HF0536, HF0554, or HF0752, or PEGylated wild-type chimeric porcine-uricase (WT) on day 1, followed by maintenance doses via intraperitoneal administration of 1.4 mg / kg of each PEGylated test protein on days 7, 14, 21, and 28. The induction dose of PEGylated uricase at 4.2 mg / kg was 3 times the human equivalent dose by allometric scaling, and the maintenance dose of PEGylated uricase at 1.4 mg / kg was 1 times the human equivalent dose by allometric scaling. Mouse plasma was collected on days 5, 12, 19, 26, and 33, and uric acid analysis was performed. Figure 7B is a graph showing serum uric acid levels in each treated mouse group or untreated (no Rx) healthy mouse group at various weeks with uricase therapy. Figure 7C is a graph showing the percentage of mice in each group with a serum uric acid level of 0 at various weeks with or without uricase therapy (treated group) or untreated (no Rx) healthy mouse group. The PEGylated variants HF0752 and HF0554 maintained low mean serum uric acid levels close to 0 across seven doses. The PEGylated variant HF0752 maintained serum uric acid levels at 0 across seven doses in eight out of nine mice. Taken together, the data demonstrate that the PEGylated uricase variants HF0536, HF0554, and HF0752 exhibited superior efficacy compared to their PEGylated wild-type counterparts. The enhanced efficacy during repeated dosing is presumed to be due to a blunting of the anti-drug immune response, as designed. However, the enhanced efficacy may also play a role.

[0259] Example 2 - Identification and mitigation of undesirable post-translational modifications of the non-PEGylated deimmunized uricase variant HF0752 This example describes mass spectrometry peptide mapping and post-translational modification (PTM) analysis of the non-PEGylated deimmunized uricase variant HF0752, which contains the amino acid sequence of SEQ ID NO: 129 described in Example 1, and the non-PEGylated wild-type chimeric pig-baboon uricase (WT), which contains the amino acid sequence of SEQ ID NO: 1. PTM analysis revealed deamination of asparagine residues N5, N56, N117, N142, and N271 of both proteins to L-aspartic acid and L-isoaspartic acid, with the highest level of deamination found at N117. To mitigate potential risks to the safety and efficacy of the deimmunized uricase variant HF0752, subvariants of HF0752, each having an additional amino acid substitution at N117, i.e., N117S, N117H, N117G, N117D, or N117Q, were prepared to avoid deamination and tested for yield, thermal stability, and activity.

[0260] method Mass spectrometry peptide mapping and post-translational modification (PTM) analysis The uricase protein sample was first denatured by combining it with 8M urea in 100mM Tris-HCl buffer (pH=8.5). The sample was then subjected to reduction and alkylation via incubation with Tris(2-carboxyethyl)phosphine (TCEP) and chloroacetamide, respectively. The urea concentration was then reduced to 2M by diluting the sample with 100mM Tris-HCl buffer, and then trypsin was added to initiate protein digestion. The sample was then incubated at 37°C for 4 hours in a thermomixer. Formic acid was introduced into the sample to quench the digestion reaction. The digested peptide was injected into an LC-MS and data was acquired. The LC-MS system was an Orbitrap Eclipse® mass spectrometer coupled with a Vanquish® Neo nanoflow liquid chromatography system. Peptide separation was achieved using an Easy-Spray® PepMap Neo column. The obtained data was processed using Biopharma Finder® 5.0 software, which facilitated peptide identification and calculation of post-translational modification (PTM) abundances. Manual re-examination of the calculated PTM levels was performed to eliminate false positives and ensure the use of appropriate peptides for accurate calculations.

[0261] result Mass spectrometry peptide mapping and post-translational modification (PTM) analysis of the non-PEGylated deimmunized uricase variant HF0752 containing the amino acid sequence of SEQ ID NO: 129, and the non-PEGylated wild-type chimeric pig-baboon uricase (WT) containing the amino acid sequence of SEQ ID NO: 1, revealed deamination of asparagine residues N5, N56, N117, N142, and N271 of both proteins to L-aspartic acid and L-isoaspartic acid, with the highest level of deamination found at N117 (Table 5). [Table 5] High levels of deamination at N117 of HF0752 may pose a potential risk to its safety and efficacy as a therapeutic protein. Therefore, deamination was avoided by generating five HF0752 subvariants in which N117 was mutated to aspartic acid (HF0752-N117D), serine (HF0752-N117S), glycine (HF0752-N117G), glutamine (HF0752-N117Q), or histidine (HF0752-N117H) by site-directed mutagenesis of the HF0752 expression vector described in Example 1. The HF0752 subvariants were then expressed in parallel with the parent HF0752 variant, and deamination was analyzed using mass spectrometry peptide mapping. Yield, thermal stability, and activity were tested using the same methods as described in Example 1. Table 6 shows the amino acid sequences (each containing threonine as residue number 1) and gene sequences (each including the start codon ATG and stop codon TAA) of HF0752 and its five subvariants as presented in the sequence listing. [Table 6]

[0262] Table 7 shows the deamidation levels of HF0752 and its subvariants. The data in Table 7 indicate that mutations of Asn117 to any of the alternative residues Ser, His, Gly, or Asp completely eliminated deamidation at position Asn117. In addition, none of the mutations at Asn117 substantially increased the level of deamidation observed at other possible asparagine deamidation sites in the protein sequence. [Table 7]

[0263] Table 8 shows the yield of HF0752 and HF0752 subvariants, and the melting temperature (T) as determined by differential scanning fluorescence quantification.m The thermal stability measured by ( ) and the activity measured by the normalized 2 μg uricase reaction rate are shown. The data in Table 8 show that all HF0752 subvariants with mutations at position 117 performed relatively well compared to the HF0752 variant. The aspartic acid mutation present in HF0752-N117D appeared with the least effect on activity and thermal stability, which is consistent with aspartic acid being a product of asparagine deamidation. HF0752-N117G also showed thermal stability and activity similar to the parent HF0752 variant. [Table 8]

[0264] Example 3 - Immunogenicity analysis and mapping of T cell epitopes in the top six non-PEGylated deimmunized chimeric pig-bahiuricase variant candidates and non-PEGylated wild-type chimeric pig-bahiuricase (WT) using ex vivo cell immunoassay and MHC-related peptide proteomics (MAPP) assays with human peripheral blood mononuclear cells (PBMCs). This example describes the immunogenicity analysis and mapping of T cell epitopes in the top six non-PEGylated deimmunized chimeric porcine-baboon melon case variant candidates described in Example 1, namely HF0536, HF0554, HF0585, HF0668, HF0752, and HF0835, as well as non-PEGylated wild-type (WT) chimeric porcine-baboon melon case (WT), using ex vivo cell immunoassays and MHC-related peptide proteomics (MAPP) assays with human peripheral blood mononuclear cells (PBMCs). Note that the variant code names HF0536, HF0554, HF0585, HF0668, HF0752, and HF0835 used in Example 1 will be abbreviated as HF536, HF554, HF585, HF668, HF752, and HF835, respectively, in this example and subsequent examples, and in the figures described in these examples. Accordingly, the variant code names HF0536, HF0554, HF0585, HF0668, HF0752, and HF0835, and their respective abbreviated code name counterparts, will be used interchangeably in the examples section of this application. Consistent with the data presented in Example 1, the results of this example demonstrate that all of the top six non-PEGylated deimmunized chimeric pig-baboon uricase variants showed reductions in T cell response, as well as reductions in peptide presentation and immunogenicity regions with presented peptides, compared to WT uricase, with variants HF536 and HF752 showing the greatest overall reduction.

[0265] method 1. Immunogenicity analysis of wild-type chimeric pig-baboon uricase and its variants using ex vivo cell immunoassay with human peripheral blood mononuclear cells (PBMCs). PBMCs from healthy human donors were grown for 13 days with IL-2 in the presence of one of the following: non-PEGylated wild-type porcine-baboon melon case protein or its variant protein, and subsequently restimulated with a peptide corresponding to the protein used for initial stimulation. CD4 T cell cytokine production was then evaluated using surface and intracellular staining for flow cytometry.

[0266] In short, PBMC, 6.25 × 10 6 Cells were seeded in appropriate culture vessels in mL of RPMI medium containing 5% human serum per cell. Antigen stimulation or vehicle control was added at plating. IL-2 was added on day 4, and the culture was maintained thereafter with medium changes every 2-3 days. After 13 days of growth, cells from each stimulation condition were counted and redistributed into 96-well plates. Restimulation with peptides or peptide pools was performed at a final concentration of 2 μM, and all samples were treated overnight with brefeldin A (to block cytokine secretion). Flow cytometry staining was then performed with the following surface stains, Zombie Aqua®, and anti-CD3, anti-CD4, and anti-CD8 antibodies. Intracellular staining for IFN-γ, TNF-α, and IL-2 was performed using the BD Cytofix / Cytoperm® Fixation / Permeabilization staining kit according to the manufacturer's instructions. After the final step, the sample was resuspended in PBS and acquired using a Cytek® Northern Lights® full-spectrum flow cytometer.

[0267] Table 9 lists the peptides used to map CD4+ T cell epitopes in the wild-type chimeric pig-baboon melon case protein, which was used to restimulate cells grown in the presence of this protein. The peptides were 18 amino acids (aa) long with an offset of 2–4aa. For epitope mapping, the peptides used during restimulation were in a pool of two consecutive peptides spanning the length of the protein shown in Table 9. [Table 9] TIFF2026517791000030.tif254170TIFF2026517791000031.tif65170

[0268] To compare the CD4 T cell response to different epitopes in deimmunized uricase (dURC) variants with that in wild-type chimeric pig-baboon uricase protein, a subset of six regions was selected and evaluated. The regions to be evaluated were selected based on predicted immunogenicity, prioritizing regions that contained highly predicted immunogenic epitopes in the dURC variant and covered the mutation sites. Twelve peptides were selected to cover these regions, specifically the core epitopes within the six regions. During restimulation, each peptide corresponding to the protein sequence used for proliferation was used, as shown in Table 10. [Table 10]

[0269] The immunogenicity of wild-type chimeric pig-baboon melon case and its variants was reported as the sum of the percentage of positive CD4+ T cells for each cytokine (IFN-γ, TNF-α, and IL-2) for each donor (total cytokine). For graphing the heatmap, the maximum total cytokine value of any peptide covering this amino acid (AA) is shown. For Figure 13, the total cytokine value for each individual donor is further summed for each evaluated peptide across all donors. Background signals were subtracted for each condition as shown in the figure.

[0270] 2. Immunogenicity analysis of wild-type chimeric pig-baboon uricase and its variants using ex vivo cell immunoassay with MHC-related peptide proteomics (MAPP) assay. To evaluate peptides presented by antigen-presenting cells (APCs) derived from either wild-type chimeric pig-baboon melon case protein or one of its variant proteins, we implemented a MAPP assay approach. Briefly, monocytes were isolated from healthy human donors, differentiated into mature dendritic cells (DCs), and pulsed with the target protein. The pulsed cells were then collected and lysed, and the peptide MHC (pMHC) complex was pulled down using antibody-conjugated beads. The peptide was then eluted from the pMHC complex and analyzed by mass spectrometry (MS). The following is an outline of the MAPP assay procedure.

[0271] 2.1 Monocyte Isolation and Culture Monocytes were isolated from leukopak from healthy human donors. Leukopak was first processed to isolate PBMCs, and then monocytes were isolated using STEMCELL's EasySep® Human Monocyte Enrichment Kit without CD16 depletion according to the manufacturer's instructions. For each protein evaluated, 58 × 10⁴ 6 Monocytes were seeded in a T225 flask and cultured using the ImmunoCult® Dendritic Cell Culture Kit from STEMCELL according to the instructions. The target protein was added on day 4 of differentiation. On day 7, mature antigen-pulsed dendritic cells were harvested, washed with PBS, counted, and the pellet was frozen at -80°C.

[0272] 2.2 Peptide Isolation Next, a frozen cell pellet from mature dendritic cells was lysed using Pierce® IP Lysis Buffer. The cell fragments were then pelletized, the clear supernatant was collected, and added to CNBR beads pre-conjugated with pan HLA II monoclonal antibody clone IVA12. This mixture was then incubated overnight at 4°C with rotation. The bead supernatant mixture was then applied to a Poly-Prep® chromatography column and sequentially washed with the following buffers: Buffer A (150 mM NaCl, 20 mM Tris, pH 8.0), Buffer B (400 mM NaCl, 20 mM Tris, pH 8.0), Buffer A, and Buffer C (20 mM Tris, pH 8.0). Next, the pMHC complex was eluted from the beads in 1% TFA in water. The peptide was then eluted from the pMHC complex using a C18 column. The eluted peptide was dried using a vacuum system.

[0273] 2.3 Mass spectrometry analysis Samples from Section 2.2 were reconstituted using 95% H2O + 5% acetonitrile (ACN), and then injected into an LC-MS system. The LC-MS system was an Orbitrap Eclipse® mass spectrometer (Thermo Scientific, San Jose, CA) coupled with a Vanquish® Neo nanoflow liquid chromatography system (Thermo Scientific, San Jose, CA). Trap and elution injection modes were performed, in which the samples were first loaded onto a trap column (PepMap® Neo, 5 μm, C18, 300 μm × 5 mm trap cartridge) and then separated on an Easy-Spray® PepMap® Neo column (2 μm, C18, 75 μm × 150 mm). The solvent gradient started at 5% solvent B (ACN, 0.1% formic acid) for the first 5 minutes, then increased to 25% solvent B over the next 75 minutes, reaching 35% solvent B between 80 and 105 minutes. During the column washing phase, the gradient surged to 90% solvent B within 1 minute and maintained this level for a further 10 minutes. Data-dependent acquisition mode was performed. The system operated in data-dependent acquisition mode, and the ORBITRAP mass spectrometer performed a survey scan over the 375–1500 m / z range at a resolution of 120K. A normalized automatic gain control (AGC) target of 250% and a maximum injection time of 50 milliseconds were set for the survey scan. MS2 scanning was performed by the ion trap mass spectrometer. Precursor ions were fragmented using 30% high-energy collision dissociation (HCD), with a normalized AGC target of 100% and a maximum injection time of 35 milliseconds. A dynamic exclusion period of 60 seconds and a mass tolerance of 10 ppm were set.

[0274] Raw data was processed using PROTEOME DISCOVERER 2.5. The SEQUEST search engine was used to search the data against UNIPROT human protein sequences, in addition to uricase sequences that lacked enzyme specificity. Search parameters included peptide length (7–30 amino acids) and dynamic oxidation modifications. Furthermore, INFERYS® Rescoring and Percolator nodes were integrated into the post-SEQUEST search workflow to further refine the search results.

[0275] result The immunogenicity of the top six candidate non-PEGylated deimmunized chimeric pig-baby melon case variants HF536, HF554, HF585, HF668, HF752, and HF835 against non-PEGylated wild-type chimeric pig-baby melon case (WT) was evaluated by an ex vivo cell immunoassay using human peripheral blood mononuclear cells (PBMCs), also referred to in this example as the "PBMC assay." Figure 8 is a schematic diagram of the PBMC assay for measuring T cell activation; details of the assay are described in the "Methods" section. In the PBMC assay, immune cells from each of 12 individual human donors were incubated with either non-PEGylated wild-type chimeric pig-baby melon case (WT) or a non-PEGylated deimmunized uricase variant for a 14-day proliferation period. T cells were then evaluated by flow cytometry for cytokine production (IL-2, IFN-γ, and TNF-α) as an indicator of activation. The sum of %CD4+ T cells positive for each cytokine is referred to as the “total cytokine” value. The 12 donors used to evaluate the T cell response represented all seven HLA II supertypes, covering 25 of the 27 alleles contributing to the supertypes (see Greenbaum et al., Immunogenetics. 2011 Jun;63(6):325-35, which is incorporated herein by reference in its entirety). Thus, these donors represented the possible response well. The HLA II alleles covered for each supertype are shown in Table 11. [Table 11] TIFF2026517791000034.tif101170

[0276] In addition to evaluating T cell responses to WT and variant touricase proteins using PBMC assays, MAPP analysis was performed for each protein to identify differences in peptides presented in HLA II by monocyte-derived dendritic cells from healthy human donors. Figure 9 shows a schematic diagram of the MAPP assay; details of the assay are described in the "Methods" section.

[0277] Monocytes were differentiated into mature dendritic cells (DCs) and pulsed with the target protein. The pulsed cells were then collected, lysed, and the peptide MHC (pMHC) complex was pulled down using antibody-conjugated beads. The peptide was then eluted from the pMHC complex and analyzed by mass spectrometry (MS). A total of eight donors were used for MAPP analysis of WT proteins, and five donors were used for comparison between WT and variants. Table 11 shows the coverage of HLA II supertype alleles used for the MAPP assay.

[0278] 1. Immunogenicity analysis of wild-type chimeric pig-baboon melon case protein Figure 10 shows the CD4+ T cell epitope mapping results for wild-type (WT) chimeric pig-baboon uricase protein using a PBMC assay. In the PBMC assay, healthy human peripheral blood mononuclear cells (PBMCs) from each donor were grown for 14 days in the presence of WT uricase protein, restimulated with synthetic peptides covering the length of the protein, and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry analysis. For each donor evaluated, the total cytokine values ​​(%IL-2+, INF-γ+, and TNF-α+CD4+ T cell populations) for each test peptide with background subtraction were plotted as a heatmap. The cytokine value for each amino acid position in the protein was the highest cytokine value of the overlapping peptide covering the position. In Figure 10, the top bar labeled "Total" is the maximum total cytokine value across all 12 donors used. The grayscale indicates the total cytokine value represented by a range of gray tones. Values ​​exceeding the indicated range are shown in black (maximum cytokine value = 26.3).

[0279] Furthermore, wild-type chimeric pig-baboon melon case protein was subjected to MAPP analysis. In the MAPP analysis, monocytes from eight healthy human donors were differentiated into mature dendritic cells (DCs) and pulsed with the target protein. The pulsed cells were then collected and lysed, and the peptide MHC (pMHC) complex was pulled down using antibody-conjugated beads. The peptide was then eluted from the pMHC complex and analyzed by mass spectrometry (MS). Figure 11 shows the MAPP analysis results, and the heatmap in the MAPP analysis results shows the number of unique peptides overlapping at each amino acid position for each donor, as identified by the MAPP assay. The "Summary" row at the top of Figure 11 shows the maximum count of unique peptides from any one donor at each amino acid position. The grayscale shows the range of gray tones representing different count values.

[0280] Together, these data identify the most highly immunogenic regions in the WT chimeric pig-baboon melon case protein that needed to be addressed in the deimmunization candidates.

[0281] 2. Comparison of immunogenicity between wild-type chimeric pig-baboon uricase protein and deimmunized uricase variants. Figure 12 shows the results of CD4+ T cell epitope mapping of immunized uricase variants compared to wild-type chimeric pig-baboon uricase protein using a PBMC assay. In the PBMC assay, healthy human PBMCs from each of 12 donors were grown for 14 days in the presence of WT or variant uricase protein and restimulated with synthetic peptides corresponding to the proteins used for initial stimulation (see Table 10), covering six regions indicated by black bars on the heatmap. T cell activation was then evaluated as measured by intracellular cytokine staining and flow cytometry analysis. For each evaluated donor, the maximum total cytokine values ​​(%CD4+ T cells positive for IL-2, INF-γ, and TNF-α) for each test peptide with background subtraction are plotted as a heatmap. The cytokine value for each position in the protein is the highest cytokine value of the duplicate peptide covering that position. For comparison, the complete epitope mapping of WT uricase shown in Figure 10 is presented at the top of Figure 12. In Figure 12, the asterisk (*) symbol indicates the location of the mutation in each deimmunized uricase variant protein. The grayscale key shows various total cytokine values ​​represented by a range of gray tones. Values ​​exceeding the indicated range are shown in black (maximum cytokine value = 26.3). The data in Figure 12 shows that the most highly immunogenic regions of the WT protein are covered by six regions selected for evaluation of deimmunization candidates. The data also shows the impact of variant mutations on the T cell response in these regions.

[0282] Figure 13 shows the results of a further analysis comparing CD4+ T cell responses between WT and deimmunized variants, as determined by the PBMC assay. In the analysis, healthy human PBMCs from each of 12 donors were grown for 14 days in either WT or in the presence of variant touricase proteins, restimulated with synthetic peptides corresponding to the proteins used for initial stimulation (see Table 10), and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry analysis. For each evaluated donor, the total cytokine values ​​for each test peptide with background subtraction (%CD4+ T cells positive for IL-2, INF-γ, and TNF-α) were calculated. The total cytokine values ​​for each individual donor for each peptide were further summed across all evaluated donors, and the resulting values ​​were plotted for each tested peptide (represented by circles in Figure 13). The mean and standard deviation are also shown for each set of peptide responses for each evaluated protein. The mean values ​​of all peptide responses for each deimmunization variant were compared to the WT response using two-way ANOVA and Dunnett's multiple comparison test for single-pool variance. *p≦0.05, **p≦0.01, ***p≦0.001. The data in Figure 13 show that all deimmunization variants had a statistically significant reduction in T cell response compared to WT, with variants HF536 and HF752 having the largest reductions in T cell response overall.

[0283] Figure 14 shows the results of MAPP analysis comparing peptides presented in HLA II derived from either wild-type (WT) or deimmunized uricase protein. In the analysis, monocytes were differentiated into mature dendritic cells (DCs) and pulsed with the protein of interest. The pulsed cells were then harvested and lysed, and the peptide MHC (pMHC) complex was pulled down using antibody-conjugated beads. The peptide was then eluted from the pMHC complex and analyzed by mass spectrometry. In Figure 14, the value for each amino acid position in the heatmap is the maximum count of the unique peptide overlapping this position from either donor. For comparison, the MAPP analysis of WT uricase from all eight healthy donors is shown at the top, and all other rows show aggregated data from a subset of five of the eight donors commonly selected for MAPP studies of both wild-type and uricase variant proteins, namely donors 3, 6, 7, 13, and 14. The five donors included donors 3, 6, and 7, which were also used in the PBMC assay for the T cell epitope mapping experiment shown in Table 11. *The symbol indicates the location of the mutation in each deimmunized uricase variant protein. The grayscale key shows the range of gray tones corresponding to different count values. The data in Figure 14 show that all variants had a reduction in peptide presentation identified by both MAPP and PBMC assays, particularly in the most immunogenic region (amino acid positions 60-90). In addition, variants HF554 and HF752 had the least amount of peptide presentation overall.

[0284] Example 4 - Additional manipulation of lead deimmunized uricase variant candidates HF554 and HF752 for the preparation and screening of HF554 and HF752 uricase subvariants with further reduced immunogenicity The studies in Examples 1 and 3 identified HF554 and HF752 as candidate lead deimmunized uricase variants. However, the PBMC assay and MAPP analysis results in Example 3 revealed that regions in HF554 and HF752 carried residual immunogenicity risk, as evidenced by the identification of immunogenicity hotspots in HF554 and HF752 in their CD4+ T cell epitope mapping (see Figure 12) and their MAPP analysis (see Figure 14). This example describes additional manipulation of HF554 and HF752 by iterative design, construction, expression, and screening of libraries of HF554 and HF752 uricase subvariants having additional and / or alternative amino acid substitutions in these residual immunogenicity risk regions, compared to the parental HF554 and HF752 uricase variants, respectively. After each round of manipulation, functionally deimmunized HF554 and HF752 uricase subvariants were obtained, which, compared to the parental HF554 and HF752 uricase subvariants, exhibited further reduced immunogenicity while maintaining activity and stability. During each round of manipulation, functional HF554 and HF752 uricase subvariants were screened using a plate-based screening strategy.

[0285] method 1. Construction of expression plasmids for HF554 and HF752 uricase subvariants, and plate-based screening for functional HF554 and HF752 uricase subvariants. Repeat construction of expression plasmid libraries encoding HF554 and HF752 uricase subvariants, and plate-based screening for functional HF554 and HF752 uricase subvariants, were performed according to the following procedure. As described in Example 1, each expression plasmid encoding an HF554 or HF752 uricase subvariant encoded a His tag containing six histidine residues at the C-terminus of the HF554 or HF752 uricase subvariant gene. (1) Gene blocks of 450-1000 bp encoding 1-9 deimmunized amino acid substitutions for HF0752 or HF0554 were synthesized in 96-well plates. (2) A linear plasmid backbone was designed to have sufficient overlap with gene blocks of 24 or more base pairs and synthesized by PCR. (3) Gene blocks and linear plasmid backbone were assembled using the NEBuilder® HiFi DNA Assembly master mix. (4) Next, the HiFi reaction product was transformed into Invitrogen OneShot™ BL21 Star™ cells and grown overnight. (5) The cultures were subcultured overnight in MagicMedia (trademark) at a 1:50 ratio, grown for 20 hours, and then the cell pellets were harvested and frozen. (6) The cell pellet was dissolved using B-PER (trademark) Complete Bacterial Protein Extraction Reagent, the insoluble fraction was separated, and the fraction was resuspended in bicarbonate buffer (pH 10.5). (7) The protein was purified from the insoluble fraction using a HisPur® cobalt spin plate, and then desalted using a Zeba® spin desalting plate. (8) Next, the purified protein was quantified by BCA and normalized to 0.2 mg / mL. (9) Concentration-normalized proteins were subjected to activity, stability, and purity assays. The activity assay is described below. A stability assay, measuring the melting temperature of the protein, was performed using differential scanning fluorescence (DSF) as described in Example 1, with a thermal gradient of 30°C to 100°C. A purity assay was performed using dynamic light scattering (DLS). (10) Next, using the results from these assays from each round of screening, libraries of HF554 and HF752 uricase subvariants were designed in the following round.

[0286] 2. Activity assays performed in plate-based screening 10 μL of diluted protein (2 μg) was added to a UV-transparent 96-well plate. 190 μL of 0.125 mM uric acid in boric acid (pH 9) was added to each well, and the absorbance at 293 nm was monitored for 10 minutes using a plate reader. Uricase activity was measured as the slope of the absorbance-time curve at 293 nm, with a steeper negative slope indicating higher activity. Therefore, the slope was quantified as a measure of vitality in arbitrary units. This assay was repeated four times for each plate.

[0287] result 1. Additional manipulation of lead deimmunized uricase variant candidate HF554 for the creation and screening of functional HF554 uricase subvariants with further reduced immunogenicity. Table 12 lists seven regions (referred to as regions A-G) of the amino acid sequence of Sequence ID No. 53 for lead deimmunized uricase variant candidate HF554 that carry residual immunogenicity risk. These regions were identified based on the PBMC assay and MAPP analysis results for HF554 in Example 3. [Table 12]

[0288] HF554 uricase subvariants were constructed by designing single, double, and single triple mutation sets in the parental HF554 uricase variant amino acid sequence, targeting the residual immunogenicity risk region described above. HF554 subvariant expression plasmids were constructed, and HF554 subvariant proteins were produced using the methods described in the "Methods" section and screened for expression yield, melting temperature, and specific activity.

[0289] Table 13 shows 113 exemplary functional HF554 uricase subvariants identified from screening that possess at least 10% of the activity of the parental HF554 uricase variant. With the sequenced subvariant genes, Table 13 shows the sequence number for each HF554 uricase subvariant, the amino acid sequence of each HF554 uricase subvariant relative to the parental HF554 uricase variant amino acid sequence of Sequence ID No. 53, the mutation of each HF554 uricase subvariant relative to the wild-type chimeric pig-baboon uricase of Sequence ID No. 1, and the mean yield, melting temperature, and activity of each HF554 uricase subvariant. Data for the parental HF554 uricase variant are also included in Table 13 for comparison. [Table 13] TIFF2026517791000037.tif254170TIFF2026517791000038.tif254170TIFF20265177910 00039.tif254170TIFF2026517791000040.tif254170TIFF2026517791000041.tif254170 TIFF2026517791000042.tif254170TIFF2026517791000043.tif254170TIFF20265177910 00044.tif254170TIFF2026517791000045.tif254170TIFF2026517791000046.tif134170

[0290] 2. Additional manipulation of lead deimmunized uricase variant candidate HF752 for the generation and screening of functional HF752 uricase subvariants with further reduced immunogenicity. Table 14 lists seven regions (referred to as regions A-G) of the amino acid sequence of Sequence ID No. 129 for lead deimmunized uricase variant candidate HF752 that carry residual immunogenicity risk. These regions were identified based on the PBMC assay and MAPP analysis results for HF752 in Example 3. [Table 14]

[0291] We addressed the aforementioned residual immunogenicity risk area by performing five iterative rounds of HF752 uricase subvariant library design, construction, expression, and screening.

[0292] In the first round, we designed single, double, one triple, and one quadruple mutation sets in the parental HF752 uricase variant amino acid sequence, each independently targeting one of the residual immunogenicity risk regions A, B, C, D, and E of HF752. Expression plasmids of the designed HF752 subvariants were constructed, and the HF752 subvariant proteins were produced using the methods described in the "Methods" section and screened for expression yield, melting temperature, and specific activity.

[0293] Table 15 shows 139 exemplary functional HF752 uricase subvariants identified from the first round of screening, possessing at least 10% of the activity of the parental HF752 uricase variant, including the sequence number for each HF752 uricase subvariant's amino acid sequence, mutations of each HF752 uricase subvariant relative to the parental HF752 uricase variant amino acid sequence of Sequence ID No. 129, and mutations of each HF752 uricase subvariant relative to the wild-type chimeric pig-baboon uricase of Sequence ID No. 1, as well as the mean yield, melting temperature, and activity of each HF752 uricase subvariant. Data for the parental HF752 uricase variant are also included in Table 15 for comparison. [Table 15] TIFF2026517791000049.tif246170TIFF2026517791000050.tif248170TIFF2026517791000051.tif250170TIFF2026517791000052.t if254170TIFF2026517791000053.tif254170TIFF2026517791000054.tif254170TIFF2026517791000055.tif254170TIFF20265177910 00056.tif254170TIFF2026517791000057.tif254170TIFF2026517791000058.tif254170TIFF2026517791000059.tif254170TIFF202 6517791000060.tif254170TIFF2026517791000061.tif254170TIFF2026517791000062.tif254170TIFF2026517791000063.tif181170

[0294] Based on activity, stability, and yield data from the first round of screening for HF752 uricase subvariants, the following high-performance single and double mutants were selected and used in the next (i.e., second) round of library design. Regions A-R26T, R26F, K30E, and K30F, Regions B-K74Q, K79S, T83E, V86N, S94D, and K97D, Area C-F172W, F172L, F172W+T174N, and F172W+E177Q, Regions D-Y225H, S228G, and L239G, Regions E-L279T, N281F, and N281Y.

[0295] In the second round of library design, the functionally validated mutations described above from distinct regions A, B, C, D, and E were combined in pairs or triplicate to create HF752 uricase subvariants, each containing a set of duplex, triplicate, and quadruplicate mutations. These sets of mutations were designed to mitigate immunogenicity risk in two or three of the five target regions A, B, C, D, and E while maintaining a high level of functional fitness.

[0296] As shown in Table 16, after a second round of HF752 uricase subvariant library design, construction, expression, and screening, 173 exemplary functional HF752 uricase subvariants possessing at least 10% of the parental HF752 uricase variant activity were identified from screening. Table 16 lists the sequence number for each HF752 uricase subvariant, the mutation of each HF752 uricase subvariant relative to the parental HF752 variant amino acid sequence of Sequence ID No. 129, and the mutation of each HF752 uricase subvariant relative to the wild-type chimeric pig-baboon uricase of Sequence ID No. 1, as well as the mean yield, melting temperature, and activity of each HF752 uricase subvariant. Data for the parental HF752 variant are also included in Table 16 for comparison.

Table 16

[0297] Based on activity, stability, and yield data from the second round of screening for HF752 uricase subvariants, a high-performance set of mutations targeting two or three of the five residual immunogenicity regions A, B, C, D, and E of the parental HF752 variant was selected and used in the next (i.e., third) round of library design. The high-performance mutations included the L279T mutation associated with high activity, and the N281Y, N281F, or K97D mutations associated with high stability. In the third round of library design, 221 and 84 HF752 uricase subvariants were created by combining functionally validated sets of the above mutations targeting two or three of the five residual immunogenic regions A, B, C, D, and E. Each HF752 uricase subvariant has a set of mutations, each designed to reduce immunogenicity risk in four of the five target regions A, B, C, D, and E, as well as all of them, while maintaining a high level of functional fitness. Also in the third round of library design, a group of 24 L279T-containing HF752 uricase subvariants was included, each containing K97D, N281Y, or both additional mutations intended to increase thermal stability. These HF752 uricase subvariants with additive mutations were designed to target the residual immunogenicity region(s) of B, AB, BC, CE, AE, BE, ABE, CDE, or BCDE.

[0298] Additionally, in the third round of library design, we created 28 HF752 uricase subvariants with single point mutations in the parental HF752 variant, designed to reduce immunogenicity risk in new regions F and G while maintaining a high level of functional fitness.

[0299] As shown in Table 17, after a third round of HF752 uricase subvariant library design, construction, expression, and screening, 349 exemplary functional HF752 uricase subvariants possessing at least 10% of the parental HF752 uricase variant activity were identified from screening. Table 17 lists the sequence number for each HF752 uricase subvariant, the mutation of each HF752 uricase subvariant relative to the parental HF752 uricase variant amino acid sequence of Sequence ID No. 129, and the mutation of each HF752 uricase subvariant relative to the wild-type chimeric pig-baboon uricase of Sequence ID No. 1, as well as the mean yield, melting temperature, and activity of each HF752 uricase subvariant. Data for the parental HF752 uricase variant are also included in Table 17 for comparison. [Table 17] TIFF2026517791000085.tif197127TIFF2026517791000086.tif254170TIFF2026517791000087.tif254170TIFF2026517791000088.tif254170TIFF2026517791000089.tif254170TIFF2026517791000090.tif254170TIFF2026517791000091.tif254170TIFF2026517791000092.tif254170TIFF2026517791000093.tif254170TIFF2026517791000094.tif254170TIFF2026517791000095.tif254170TIFF2026517791000096.tif254170TIFF2026517791000097.tif254170TIFF2026517791000098.tif254170TIFF2026517791000099.tif254170TIFF2026517791000100.tif254170TIFF2026517791000101.tif254170TIFF2026517791000102.tif254170TIFF2026517791000103.tif254170TIFF2026517791000104.tif254170TIFF2026517791000105.tif254170TIFF2026517791000106.tif254170TIFF2026517791000107.tif254170TIFF2026517791000108.tif254170TIFF2026517791000109.tif254170TIFF2026517791000110.tif254170TIFF2026517791000111.tif254170TIFF2026517791000112.tif254170TIFF2026517791000113.tif254170TIFF2026517791000114.tif254170TIFF2026517791000115.tif254170TIFF2026517791000116.tif254170TIFF2026517791000117.tif254170TIFF2026517791000118.tif254170TIFF2026517791000119.tif254170TIFF2026517791000120.tif254170TIFF202651779100 0121.tif254170TIFF2026517791000122.tif254170TIFF2026517791000123.tif254170TIFF2026517791000124.tif254170TIFF2026517 791000125.tif254170TIFF2026517791000126.tif254170TIFF2026517791000127.tif254170TIFF2026517791000128.tif254170TIFF20 26517791000129.tif254170TIFF2026517791000130.tif254170TIFF2026517791000131.tif254170TIFF2026517791000132.tif160170.

[0300] Based on activity, stability, and yield data from the third round of screening for HF752 uricase subvariants, we identified 23 high-performance HF752 uricase subvariants with sets of mutations targeting all five of the residual immunogenic regions A, B, C, D, and E of the parental HF752 variant (Table 18). In addition, we also identified high-performance mutations in region F (L52T, H53D, or H53S) and region G (R192T or G196D). [Table 18] TIFF2026517791000134.tif254170TIFF2026517791000135.tif254170

[0301] In the next (i.e., fourth) round of library design, each of the 23 high-performance HF752 subvariants described above was used with the base design, and then combinatorially muted to incorporate high-performance mutations in region F (L52T, H53D, or H53S), region G (R192T, G196D, or R197D), or both regions F and G, to create new HF752 uricase subvariants. These new HF752 uricase subvariants were designed to target six residual immunogenicity risk regions of the HF752 uricase variant, i.e., regions A, B, C, D, E, E plus F or G, or seven residual immunogenicity risk regions, i.e., regions A, B, C, D, E, E plus regions F and G. Furthermore, when the underlying design was based on HF752 uricase subvariant numbers 600 (sequence number 622), 622 (sequence number 644), or 653 (sequence number 675), the underlying design was modified to include an additional N281Y stabilizing mutation.

[0302] As shown in Table 19, after the fourth round of HF752 uricase subvariant library design, construction, expression, and screening, 359 exemplary functional HF752 uricase subvariants possessing at least 10% of the parental HF752 uricase variant activity were identified from screening. Table 19 lists the sequence number for each HF752 uricase subvariant, the mutation of each HF752 uricase subvariant relative to the parental HF752 uricase variant amino acid sequence of Sequence ID No. 129, and the mutation of each HF752 uricase subvariant relative to the wild-type chimeric pig-baboon uricase of Sequence ID No. 1, as well as the mean yield, melting temperature, and activity of each HF752 uricase subvariant. Data for the parental HF752 uricase variant are also included in Table 19 for comparison. [Table 19-1] TIFF2026517791000137.tif231170TIFF2026517791000138.tif254170TIFF2026517791000139.tif254170TIFF2026517791000140.tif254170TIFF2026517791000141.tif254170TIFF2026517791000142.tif254170TIFF2026517791000143.tif254170TIFF2026517791000144.tif254170TIFF2026517791000145.tif254170TIFF2026517791000146.tif254170TIFF2026517791000147.tif254170TIFF2026517791000148.tif254170TIFF2026517791000149.tif254170TIFF2026517791000150.tif254170TIFF2026517791000151.tif254170TIFF2026517791000152.tif254170TIFF2026517791000153.tif254170TIFF2026517791000154.tif254170TIFF2026517791000155.tif254170TIFF2026517791000156.tif254170TIFF2026517791000157.tif254170TIFF2026517791000158.tif254170TIFF2026517791000159.tif254170TIFF2026517791000160.tif254170TIFF2026517791000161.tif254170TIFF2026517791000162.tif254170TIFF2026517791000163.tif254170TIFF2026517791000164.tif254170TIFF2026517791000165.tif254170TIFF2026517791000166.tif254170TIFF2026517791000167.tif254170TIFF2026517791000168.tif254170TIFF2026517791000169.tif254170TIFF2026517791000170.tif254170TIFF2026517791000171.tif25417 0TIFF2026517791000172.tif254170TIFF2026517791000173.tif254170TIFF2026 517791000174.tif254170TIFF2026517791000175.tif254170TIFF202651779100 0176.tif254170TIFF2026517791000177.tif254170TIFF2026517791000178.tif2 54170TIFF2026517791000179.tif196127TIFF2026517791000180.tif254170TIF F2026517791000181.tif254170TIFF2026517791000182.tif196127TIFF20265177 91000183.tif254170TIFF2026517791000184.tif254170TIFF2026517791000185. tif254170TIFF2026517791000186.tif254170TIFF2026517791000187.tif71170. [Table 19-2] TIFF2026517791000189.tif254170TIFF2026517791000190.tif254170TIFF20265177910 00191.tif254170TIFF2026517791000192.tif254170TIFF2026517791000193.tif214170

[0303] Based on activity, stability, and yield data from the fourth round of screening for HF752 uricase subvariants, we identified 35 high-performance HF752 uricase subvariants that possess a set of mutations targeting all seven of the residual immunogenicity regions A, B, C, D, E, F, G of the parent HF752 uricase variant (Table 20). [Table 20] TIFF2026517791000195.tif229170TIFF2026517791000196.tif227170TIFF2026517791000197.tif254170 TIFF2026517791000198.tif254170TIFF2026517791000199.tif254170TIFF2026517791000200.tif254170

[0304] In the next (i.e., fifth) round of library design, each of the 35 high-performance HF752 subvariants mentioned above was combined with either N117D or N117G, using the base design. When the base design was derived from high-performance HF752 subvariants lacking the L279T mutation (excluding HF752 subvariant numbers 1074 (sequence ID 1045), 1073 (sequence ID 1044), and 1106 (sequence ID 1077)), the mutation present in each of the base HF752 subvariants was combined with L279T in addition to N117D or N117G. As a result, the HF752 subvariants designed in the fifth round were N Mutations were found at 117, L279, or both, and in all seven residual immunogenicity risk regions A, B, C, D, E, F, and G. As shown in Example 2, N117D or N117G mutations completely eliminated deamidation at position Asn117 of the HF752 uricase variant, while having the least effect on activity and thermal stability. Additionally, as shown by the second round of screening results, the L279T mutation was associated with high activity of the HF752 uricase subvariant.

[0305] As shown in Table 21, after the fifth round of HF752 uricase subvariant library design, construction, expression, and screening, 145 exemplary functional HF752 uricase subvariants possessing at least 10% of the parental HF752 uricase variant activity were identified from screening. Table 21 lists the SEQ ID NO: for each HF752 uricase subvariant, its SEQ ID NO: for the amino acid sequence, the mutation of each HF752 uricase subvariant relative to the parental HF752 uricase variant amino acid sequence of SEQ ID NO: 129, and the mutation of each HF752 uricase subvariant relative to the wild-type chimeric pig-baboon uricase of SEQ ID NO: 1, as well as the mean yield, melting temperature, and activity of each HF752 uricase subvariant. Data for 35 high-performance baseline HF752 uricase subvariants and parental HF752 uricase variants, all tested in parallel under the same conditions, are also included in Table 21 for direct comparison. [Table 21] TIFF2026517791000202.tif254170TIFF2026517791000203.tif254170TIFF2026517791000204.tif254170TIFF2026517791000205.tif254170TIFF2026517791000206.tif254170TIFF2026517791000207.tif254170TIFF2026517791000208.tif254170TIFF2026517791000209.tif254170TIFF2026517791000210.tif254170TIFF2026517791000211.tif254170TIFF2026517791000212.tif254170TIFF2026517791000213.tif254170TIFF2026517791000214.tif254170TIFF2026517791000215.tif254170TIFF2026517791000216.tif254170TIFF2026517791000217.tif254170TIFF2026517791000218.tif254170TIFF2026517791000219.tif254170TIFF2026517791000220.tif254170TIFF2026517791000221.tif254170TIFF2026517791000222.tif254170TIFF2026517791000223.tif254170TIFF2026517791000224.tif254170TIFF2026517791000225.tif254170TIFF2026517791000226.tif254170TIFF2026517791000227.tif254170TIFF2026517791000228.tif254170TIFF2026517791000229.tif254170TIFF2026517791000230.tif254170TIFF2026517791000231.tif254170TIFF2026517791000232.tif254170TIFF2026517791000233.tif254170TIFF2026517791000234.tif254170TIFF2026517791000235.tif254170TIFF2026517791000236.tif254170.

[0306] Of the 145 exemplary functional HF752 uricase subvariants from the fifth round of screening, listed in Table 21, and the 35 high-performance baseline HF752 uricase subvariants identified from the previous (fourth) round of screening (for a total of 180 HF752 uricase subvariants), 161 HF752 uricase subvariants showed more than 70% of the parent HF752 variant activity. Of the 161 high-performance HF752 uricase subvariants, 145 showed more than 80% of the parent HF752 uricase variant activity, 140 showed more than 90% of the parent HF752 uricase variant activity, and 120 showed more than 100% of the parent HF752 uricase variant activity. According to the HF752 uricase subvariant design, each of the 161 high-performance HF752 uricase subvariants had two sets of amino acid substitutions. In aggregation, the first set of amino acid substitutions for the 161 high-performance HF752 uricase subvariants included the 13 amino acid substitutions present in the parent HF752 variant, namely V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K. The second set of amino acid substitutions for the 161 high-performance HF752 uricase subvariants included additional amino acid substitutions and / or one alternative amino acid substitution targeting all seven residual immunogenicity risk regions ABCDEFG, as well as N117D and N117G. The number of additional amino acid substitutions ranged from 6 to 10. One alternative amino acid substitution present in some of the high-performance HF752 subvariants is Q26T at position 26 of the wild-type chimeric pig-baboon uricase in SEQ ID NO: 1, where Q26T replaces Q26R in the parent HF752 uricase variant. Table 22 summarizes a second set of identified amino acid substitutions from 161 high-performance HF752 uricase subvariants. Table 22 shows the residual immunogenicity risk regions where amino acid substitutions from the second set are present.Additionally, the individual amino acid substitutions in the second set are ranked in descending order of frequency of occurrence among the 161 high-performance HF752 uricase subvariants (see the "Frequency Ranking" column in Table 22, where a rank of 1 indicates the highest frequency). [Table 22]

[0307] From 145 exemplary functional HF752 uricase subvariants from the fifth round of screening, 12 leading high-performance HF752 uricase subvariants listed in Table 23 were selected for further analysis, including immunogenicity analysis using the method described in Example 3. [Table 23] TIFF2026517791000239.tif254170TIFF2026517791000240.tif84170

[0308] Using the mutations present in the 12 lead high-performance HF752 uricase subvariants described above as templates, additional HF752 uricase subvariants were designed, and in these additional HF752 uricase subvariants, the mutation set in the template subvariants was modified, for example, by removing amino acid substitutions, incorporating additional amino acid substitutions, or both (Table 24). The newly designed HF752 uricase subvariants were constructed and studied for functional fitness and immunogenicity. [Table 24] TIFF2026517791000242.tif254170TIFF2026517791000243.tif254170TIFF2026517791000244.t if254170TIFF2026517791000245.tif254170TIFF2026517791000246.tif254170TIFF2026517791 000247.tif254170TIFF2026517791000248.tif254170TIFF2026517791000249.tif254170TIFF20 26517791000250.tif254170TIFF2026517791000251.tif254170TIFF2026517791000252.tif69170

[0309] Example 5 - Preparation and characterization of PAS-modified uricase fusion proteins containing an HF752 uricase variant or HF752 uricase subvariant for the uricase domain in combination with a PAS polypeptide domain. This example describes the preparation and characterization of PAS-modified HF752 variants and HF752 subvariant touricase fusion proteins in parallel with a PAS-modified wild-type chimeric pig-baboon melon case fusion protein as a control fusion protein. Table 25 shows the code names, sequence numbers, and domain compositions of the PAS-modified HF752 variant touricase fusion proteins and PAS-modified wild-type chimeric pig-baboon melon case fusion proteins prepared, produced, and studied for their pharmacokinetic (PK) and pharmacodynamic (PD) properties in this example. [Table 25]

[0310] As shown in Table 25, each monomer WT-CPAS20h and HF752-CPAS20h contained a single C-terminal PAS polypeptide domain having the amino acid sequence of PAS20. Since the amino acid sequence of wild-type chimeric pig-baboon uricase (i.e., Sequence ID No. 1) is present in each protein monomer of the PEGylated homotetramer KRYSTEXXA® (pegroticase), KRYSTEXXA® (pegroticase) was used as a comparative in the PK and PD studies described in this example. For brevity, KRYSTEXXA® (pegroticase) will be referred to simply as "pegroticase" in this example. The PEGylated HF752 uricase variant described in Example 1 was used as another comparative. In light of the favorable PK / PD results obtained with HF752-CPAS20h, we designed and tested PAS-modified HF752 subvariant uricase fusion proteins for functional fitness, PK / PD, and immunogenicity.

[0311] method 1. Production and expression of PAS-modified uricase fusion protein DNA encoding wild-type chimeric pig-baboon uricase or HF752 uricase variants for the uricase domain of the fusion proteins listed in Table 25, and DNA encoding the PAS20 polypeptide domain with a GS spacer were synthesized. The resulting DNA fragments encoding the uricase domain and PAS20 domain were cloned into the NdeI and BamHI digested expression vector pET-26b(+) (MilliporeSigma, MA, USA) using the NEBuilder® HiFi DNA Assembly Kit (New England Biolabs, Ipswich, MA, USA) to assemble the full-length coding sequences for each uricase fusion protein in the vector. The expression vector pET-26b(+) encodes a his tag containing six histidine residues at the C-terminus of the assembled full-length uricase fusion protein coding sequence. Expression of the uricase fusion protein was achieved by growing E. coli cultures transformed with the expression vector pET-26b(+) containing the full-length uricase fusion protein coding sequence, and by inducing protein expression with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0312] 2. Purification The PAS-modified uricase fusion protein expressed in E. coli was purified using the following procedure to obtain a soluble (non-aggregated) tetramer. (1) Pelletize 0.5 L of IPTG-inducible E. coli culture by centrifuging at 5,000 × g for 10 minutes. (2) Resuspend the E. coli pellet in 150 mL of 20 mM sodium borate buffer (pH 9.5). (3) The resuspended pellets are subjected to ultrasonic treatment. (4) Centrifuge the sonicated suspension at 17,000 × g for 20 minutes to decant the soluble fraction. (5) Dilute the soluble fraction with 20 mM sodium borate buffer (pH 9.5) to 150 mL and stir at 4°C. (6) While stirring, slowly add 19.5 g of solid ammonium sulfate. (7) Incubate the sample from step (6) at 4°C for 2 hours. (8) The sample from step (7) is pelletized by centrifuging at 17,000 × g for 20 minutes and decanting the soluble fraction. (9) Resuspend the protein pellet from step (8) in 40 mL of 20 mM sodium borate buffer (pH 9.5). (10) Desalt the suspension from step (9) using a Zeba® spin desalting column (ThermoFisher Scientific). (11) The desalted sample from step (10) is loaded onto a 70 mL Toyopearl® NH2-750F anion exchange column (Tosoh Bioscience), and the purified sample is eluted with 140 mL of 20 mM sodium borate buffer (pH 9.5) to further purify it. (12) The eluate from step (11) is replaced with 1×PBS buffer using a centrifugal spin filter. (13) Load the buffer-exchanged sample from step (12) onto a Superose® 6-size exclusion chromatography (SEC) column (Cytiva) to separate aggregates from the soluble tetramer (target) protein.

[0313] 3. In vivo PK and PD (potency) analysis of PAS-modified uricase fusion protein, as well as the comparative pegroticase and PEGylated HF752 uricase variant. Purified soluble tetrameric PAS-modified uricase fusion proteins prepared as described in Section 2 above, as well as pegroticase and PEGylated HF752 uricase variants serving as comparators, were subjected to in vivo PK and PD analysis in Wistar rats.

[0314] 3.1 Administration of PAS-modified uricase fusion protein, pegroticase, or PEGylated HF752 uricase variant to Wistar rats, and collection of rat blood samples. Female Wistar rats, each weighing approximately 220g, were purchased from Charles River. In the PK / PD studies described herein, rats were either left untreated or treated via intravenous administration of a single dose of 1 mg of pegroticase (core enzyme mass without considering PEG mass) / kg body weight, a single dose of 1 mg of PEGylated HF752 uricase variant (core enzyme mass without considering PEG mass) / kg body weight, or a single equimolar dose of PAS-modified uricase fusion protein WT-CPAS20h or HF752-CPAS20h (equivalent to 1 mg / kg body weight of pegroticase) (n=3 rats per group). 120 μL of blood was collected from each rat at various time points before and after administration. Table 26 shows the single doses (mg / kg of rat body weight) of pegroticase, PEGylated HF752 uricase variant, or PASized uricase fusion protein administered to Wistar rats, and the blood sample collection points selected in the PK / PD studies. [Table 26]

[0315] 3.2. Determination of uric acid concentration in rat plasma Rat blood samples collected as described in Section 3.1 above were processed to obtain rat plasma, and the plasma uric acid concentration at each time point was determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS). Details of the quantification of uric acid concentration in rat plasma are as follows.

[0316] 3.2.1 Preparation of standard uric acid samples using artificial human plasma to establish artificial human plasma and standard curves Since endogenous uric acid is present in rat plasma, artificial human plasma was used as a surrogate matrix for the preparation of standard uric acid samples to establish a standard curve. Artificial human plasma was prepared by dissolving 2 g of human serum albumin (Sigma - Aldrich) in 50 mL of PBS, followed by adjusting the pH to 7.4 with 1 M sodium hydroxide or 1 M phosphoric acid. Standard uric acid samples containing 0.1, 0.2, 1, 5, 10, 60, 80, and 100 μg / mL of uric acid in artificial human plasma were prepared and subjected to further processing and LC - MS MS / MS / MS analysis in parallel with the test rat plasma samples as described below.

[0317] 3.2.2. Processing of Standard Uric Acid Samples and Rat Plasma Samples for LC - MS / MS The standard uric acid samples and test rat plasma samples in Section 3.2.1 were processed according to the following procedure. (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 the standard uric acid sample or rat plasma sample. (2) Mix the samples from step (1) and centrifuge at 15,000 rpm for 15 minutes at 4 °C using an Eppendorf 5424R centrifuge. (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. (4) Mix the samples obtained in step (3) for the following LC - MS / MS analysis.

[0318] 3.2.3. LC - MS / MS Analysis of Standard Uric Acid Samples and Rat Plasma Samples for Quantifying Uric Acid Concentration Each 10 μl standard uric acid sample or rat plasma sample, processed as described in Section 3.2.2, was injected into an Acquity UPLC® BEH amide column on a Shimadzu Nexera® LC (Sciex), followed by gradient elution. Uric acid quantification was performed using a triple-quad API4500 mass spectrometer (Sciex) with ESI operating in negative mode.

[0319] LC-MS / MS data were collected using data acquisition software Analyst® 1.7 and MultiQuant® 3.0 (Sciex) or equivalents. Standard curves were created using data from standard uric acid samples. Calibration curves were constructed by plotting the peak area ratio of the reference standard and the internal standard against the nominal concentration of the existing reference standard. The calibration curves were fitted using least-squares regression analysis to obtain information on the slope of the calibration curve, the y-intercept, the correlation coefficient, and the inversely calculated calibration standard concentration.

[0320] Calibration curves obtained using standard uric acid samples prepared with artificial human plasma were shown using the standard addition method to accurately measure uric acid concentrations in rat plasma.

[0321] 3.3. Determination of rat plasma concentrations of PAS-modified uricase fusion protein, pegroticase, and PEGylated HF752 uricase variant. Rat blood samples collected as described in Section 3.1 above were processed to obtain rat plasma, and subsequently, the plasma concentrations of PAS-mediated uricase fusion protein, pegroticase, or PEGylated HF752 uricase variant at each time point were determined using the Amplex® Red Uric Acid / Uricase Assay Kit (ThermoFisher Scientific, catalog no. A22181). The assay kit provides a two-step fluorescence quantification method for quantifying PAS-mediated uricase fusion protein, pegroticase, or PEGylated HF752 uricase variant based on uricase enzyme activity. Firstly, the uricase enzyme activity of PAS-mediated uricase fusion protein, pegroticase, or PEGylated HF752 uricase variant converts uric acid to allantoin, hydrogen peroxide (H2O2), and carbon dioxide. Secondly, in the presence of horseradish peroxide (HRP), H2O2 reacts stoichiometrically with Amplex® Red reagent to produce resorphine, a red fluorescent oxidation product. In this example, rat plasma concentrations of PAS-modified uricase fusion protein, pegroticase, or PEGylated HF752 uricase variant were indirectly quantified by measuring the fluorescence of resorphine captured by a SpectraMax® miniplate reader (Molecular Devices) in kinetic mode for 30 minutes. Standard curves for quantifying PAS-modified uricase fusion protein, pegroticase, or PEGylated HF752 uricase variant were constructed using rat plasma from the same strain. Standard curve regressions were processed in an equilibrium model using the Excel add-in software XLift® by IDBS to back-calculate the concentrations of uricase fusion protein, pegroticase, and PEGylated HF752 uricase variant in rat plasma samples.

[0322] Rat plasma contained endogenous uricase enzymes, which also produced a fluorescent signal in the uricase assay. Furthermore, endogenous rat plasma uricase levels varied between rats and over time within a single rat, making it impossible to determine the true background uricase for a given sample. Therefore, the determination of plasma concentrations of exogenously administered PAS-mediated uricase fusion protein, pegroticase, or PEGylated HF752 uricase variants in study samples was approximated by subtracting the mean pre-dose background (endogenous) plasma uricase concentration for each PAS-mediated uricase fusion protein, pegroticase, or PEGylated HF752 uricase variant-treated rat group. The corresponding plasma concentrations of endogenous uricase in the untreated control rat group at pre-dose (time 0) and post-dose time points were similarly adjusted by subtracting the mean pre-dose endogenous plasma uricase concentration of the control rat group.

[0323] result 1. In vivo PK and PD analysis of PAS-modified wild-type chimeric pig-baboon uricase fusion protein (WT-CPAS20h) and PAS-modified HF752 variant uricase fusion protein (HF752-CPAS20h) compared to pegroticase and PEGylated HF752 uricase variant. In the PK / PD study, female Wistar rats in each group of three rats were either untreated or treated via intravenous administration of a single dose of 1 mg / kg body weight pegroticase, 1.99 mg / kg body weight WT-CPAS20h, 1.99 mg / kg body weight HF752-CPAS20h, or 1 mg / kg body weight PEGylated HF752 uricase variant (Table 26). Rat blood samples were collected before administration and at 0.5, 2, 6, 24, 72, and 96 hours after administration. Plasma concentrations of endogenous uricase in untreated control rats, or plasma concentrations of pegroticase, PAS-mediated uricase fusion protein, and PEGylated HF752 uricase variant, as well as plasma uric acid in treated rats, were determined as described in "Methods". The results are shown in Figures 15A to 15E, where the endogenous uricase or exogenously administered pegroticase, PAS-modified uricase fusion protein, and PEGylated HF752 uricase variant in rat plasma are generally referred to as "plasma uricase."

[0324] Figure 15A shows the plasma concentrations of endogenous uricase and uric acid in untreated control rats at the corresponding pre-drug time point (hour 0) and various corresponding post-drug time points up to 96 hours. The plasma concentration of endogenous uricase at hour 0 was adjusted to 0 μg / mL by subtracting the mean pre-drug endogenous plasma uricase concentration as described in the "Methods" section. At the corresponding post-drug time points, similarly adjusted plasma concentrations of endogenous uricase varied to below or above 0 μg / mL as described in the "Methods" section. Plasma concentrations of uric acid in untreated control rats also varied and were greater than 0 μg / mL at all time points.

[0325] Figure 15B shows the plasma concentrations of pegroticase and uric acid in pegroticase-treated rats at various time points, from pre-administration (hour 0) to up to 96 hours post-administration. Plasma concentrations of pegroticase plateaued between 0.5 and 6 hours post-administration and remained above detectable levels until at least 96 hours post-administration, while plasma concentrations of uric acid decreased to 0 μg / ml at all time points tested post-administration, i.e., from 0.5 to 96 hours post-administration, and remained at 0 μg / ml.

[0326] Figure 15C shows the plasma concentrations of WT-CPAS20h and uric acid in WT-CPAS20h-treated rats at various time points from pre-administration (hour 0) to up to 96 hours post-administration. Plasma concentrations of WT-CPAS20h peaked by 0.5 hours post-administration, remained at high levels for 24 hours post-administration, and then decreased to below the detection limit by 72 hours post-administration. Plasma concentrations of uric acid decreased to 0 μg / ml from 0.5 hours to 24 hours post-administration, remained at 0 μg / ml, and then rebounded to values ​​above 0 μg / ml between 24 hours and 72 hours post-administration.

[0327] Figure 15D shows the plasma concentrations of HF752-CPAS20h and uric acid in HF752-CPAS20h-treated rats at various time points from pre-administration (hour 0) to up to 96 hours post-administration. The changes in plasma concentrations of HF752-CPAS20h and uric acid after administration were similar to those in WT-CPAS20h-treated rats. Specifically, plasma concentrations of HF752-CPAS20h peaked by 0.5 hours post-administration, remained at high or detectable levels for 24 hours post-administration, and then decreased to below the detection limit by 72 hours post-administration. Plasma concentrations of uric acid decreased to 0 μg / ml from 0.5 hours to 24 hours post-administration, were maintained at 0 μg / ml, and then rebounded to values ​​above 0 μg / ml between 24 hours and 72 hours post-administration.

[0328] Figure 15E shows the plasma concentrations of PEGylated HF752 uricase variant and uric acid at various time points before administration (hour 0) and up to 96 hours after administration in rats treated with the PEGylated HF752 uricase variant. The changes in plasma concentrations of PEGylated HF752 uricase variant and uric acid after administration were similar to those in pegroticase-treated rats. Specifically, the plasma concentration of PEGylated HF752 uricase variant plateaued between 0.5 and 72 hours post-administration and remained at a detectable level for 96 hours post-administration, while the plasma concentration of uric acid decreased to 0 μg / ml and was maintained at 0 μg / ml at all time points tested post-administration, i.e., from 0.5 to 96 hours post-administration.

[0329] In summary, the results from the above PK / PD studies indicate that both PAS-modified HF752 uricase variant fusion protein and PEG-modified HF752 uricase variant were useful and effective in in vivo therapy. The results also show that plasma uric acid concentrations in untreated control rats varied from rat to rat and at different points in the study. Therefore, interpreting precise plasma uric acid concentration values ​​after treatment with PAS-modified uricase fusion protein, pegroticase, or PEG-modified HF752 uricase variant was not informative. However, the study results demonstrate a binary correlation between plasma uric acid concentrations at 0 μg / ml versus >0 μg / ml and the presence versus clearance of circulating exogenous uricase enzymes (i.e., PAS-modified uricase fusion protein, pegroticase, or PEG-modified HF752 uricase variant). Specifically, plasma uric acid concentrations of 0 μg / ml correlated with or indicated detectable levels of circulating exogenous uricase enzymes, while plasma uric acid concentrations rebounding above 0 μg / ml correlated with or indicated the elimination of exogenously administered uricase enzymes.

[0330] In light of the sufficient PK / PD performance of the PAS-modified HF752 uricase variant fusion protein HF752-CPAS20h in vivo, four sets of PAS-modified HF752 subvariant uricase fusion proteins were designed, constructed, expressed, and tested for functional fitness, PK / PD, and immunogenicity. Each set contained 180 PAS-modified HF752 subvariant uricase fusion proteins, and the uricase domains of the fusion proteins corresponded to 145 exemplary functional HF752 uricase subvariants from the fifth round of screening and 35 high-performance HF752 uricase subvariants identified from the fourth round of screening, as listed in Table 21 of Example 4. Although sharing the same group of HF752 subvariants for the uricase domain, the four sets differed in the type of PAS domain used and the composition of the PAS domain.

[0331] In the first set of PAS-modified HF752 subvariant uricase fusion proteins, their monomeric forms contained amino acid sequences selected from SEQ ID NOs. 1569-1748, and the PAS domains were configured in an "N10 / C10" format. In this format, each PAS-modified HF752 subvariant uricase fusion protein in the set had one PAS10 domain of SEQ ID NO. 9 at the N-terminus, another PAS10 domain of SEQ ID NO. 9 at the C-terminus, and one uricase domain between the two PAS10 domains containing the amino acid sequence of the HF752 subvariant.

[0332] In the second set of PAS-modified HF752 subvariant uricase fusion proteins, their monomeric forms contained amino acid sequences selected from SEQ ID NOs. 1749-1928, and the PAS domains were configured in the "N20 / C10" format. In this format, each PAS-modified HF752 subvariant uricase fusion protein in the set had one PAS20 domain from SEQ ID NO. 10 at the N-terminus, one PAS10 domain from SEQ ID NO. 9 at the C-terminus, and one uricase domain between the two PAS domains containing the amino acid sequence of the HF752 subvariant.

[0333] In a third set of PAS-modified HF752 subvariant uricase fusion proteins, their monomeric forms contained amino acid sequences selected from SEQ ID NOs. 1929-2108, and the PAS domains were configured in an "N10 / C20" format. In this format, each PAS-modified HF752 subvariant uricase fusion protein in the set had one PAS10 domain from SEQ ID NO. 9 at the N-terminus, one PAS20 domain from SEQ ID NO. 10 at the C-terminus, and one uricase domain between the two PAS domains containing the amino acid sequence of the HF752 subvariant.

[0334] In the fourth set of PAS-modified HF752 subvariant uricase fusion proteins, their monomeric forms contained amino acid sequences selected from SEQ ID NOs. 2109-2288, and the PAS domains were configured in an "N20 / C20" format. In this format, each PAS-modified HF752 subvariant uricase fusion protein in the set had one PAS20 domain of SEQ ID NO. 10 at the N-terminus, one PAS20 domain of SEQ ID NO. 10 at the C-terminus, and one uricase domain between the two PAS20 domains containing the amino acid sequence of the HF752 subvariant. * * * * * * * * *

[0335] While the described invention is described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. Furthermore, many modifications may be made to adopt specific circumstances, materials, substance compositions, processes, process steps, or steps to suit the spirit and scope of the described invention. All such modifications are intended to fall within the claims appended herein.

[0336] The patents, patent applications, published patent applications, journal articles, and protocols referenced herein are incorporated by reference in their entirety for all purposes.

Claims

1. A uricase variant comprising one or more amino acid mutations relative to the corresponding uricase protein, wherein the one or more amino acid mutations are located at one or more amino acid positions corresponding to one or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257 of SEQ ID NO: 1, and comprising an amino acid sequence that is at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO:

1.

2. The uricase variant according to claim 1, wherein the uricase variant comprises two or more amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to two or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

3. The uricase variant according to claim 1, wherein the uricase variant comprises three or more amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to three or more of the following amino acid residues of SEQ ID NO: 1, D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

4. The uricase variant according to claim 1, wherein the uricase variant comprises four or more amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to four or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

5. The uricase variant according to claim 1, wherein the uricase variant comprises five or more amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to five or more of the following amino acid residues of SEQ ID NO: 1, D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

6. The uricase variant according to claim 1, wherein the uricase variant comprises seven or more amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to seven or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257 of SEQ ID NO:

1.

7. The uricase variant according to claim 1, wherein the uricase variant contains 10 or more amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to 10 or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257 of SEQ ID NO:

1.

8. The uricase variant according to claim 1, wherein the uricase variant contains 15 or more amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to 15 or more of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257 of SEQ ID NO:

1.

9. The uricase variant according to claim 1, wherein the uricase variant contains about 5 to about 25 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 5 to about 25 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

10. The uricase variant according to claim 1, wherein the uricase variant contains about 5 to about 20 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 5 to about 20 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257 of SEQ ID NO:

1.

11. The uricase variant according to claim 1, wherein the uricase variant comprises about 10 to about 20 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 10 to about 20 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257 of SEQ ID NO:

1.

12. The uricase variant according to claim 1, wherein the uricase variant comprises about 10 to about 18 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 10 to about 18 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

13. The uricase variant according to claim 1, wherein the uricase variant comprises about 10 to about 16 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 10 to about 16 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

14. The uricase variant according to claim 1, wherein the uricase variant comprises about 10 to about 14 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 10 to about 14 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

15. The uricase variant according to claim 1, wherein the uricase variant comprises about 10 to about 13 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 10 to about 13 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

16. The uricase variant according to claim 1, wherein the uricase variant comprises about 10 to about 12 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 10 to about 12 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

17. The uricase variant according to claim 1, wherein the uricase variant comprises about 11 to about 20 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 11 to about 20 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

18. The uricase variant according to claim 1, wherein the uricase variant comprises about 11 to about 13 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 11 to about 13 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

19. The uricase variant according to claim 1, wherein the uricase variant comprises about 12 to about 14 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 12 to about 14 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

20. The uricase variant according to claim 1, wherein the uricase variant comprises about 13 to about 20 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 13 to about 20 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

21. The uricase variant according to claim 1, wherein the uricase variant comprises about 14 to about 20 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 14 to about 20 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

22. The uricase variant according to claim 1, wherein the uricase variant comprises about 15 to about 20 amino acid mutations relative to the corresponding uricase protein, and the amino acid mutations are located at amino acid positions corresponding to about 15 to about 20 of the following amino acid residues of SEQ ID NO: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66, N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

23. The uricase variant according to any one of claims 1 to 22, wherein the amino acid mutation is an amino acid substitution.

24. The uricase variant according to claim 23, wherein the amino acid substitution corresponds to one or more of the amino acid substitutions of Sequence ID No. 1 listed in Table A. Table 1

25. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to three or more of the following amino acid substitutions in SEQ ID NO: V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K.

26. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to five or more of the following amino acid substitutions in SEQ ID NO: V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K.

27. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to 10 or more of the following amino acid substitutions in SEQ ID NO: 1, V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K.

28. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to three or more of the following amino acid substitutions in SEQ ID NO: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K.

29. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to five or more of the following amino acid substitutions in SEQ ID NO: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K.

30. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to 10 or more of the following amino acid substitutions of SEQ ID NO: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K of SEQ ID NO:

1.

31. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to three or more of the following amino acid substitutions in SEQ ID NO: V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K.

32. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to five or more of the following amino acid substitutions in SEQ ID NO: V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K.

33. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to 10 or more of the following amino acid substitutions in SEQ ID NO: V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K.

34. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to three or more of the following amino acid substitutions in SEQ ID NO: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K.

35. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to five or more of the following amino acid substitutions in SEQ ID NO: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K.

36. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to 10 or more of the following amino acid substitutions of SEQ ID NO: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K of SEQ ID NO:

1.

37. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to three or more of the following amino acid substitutions in SEQ ID NO: V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

38. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to five or more of the following amino acid substitutions of Sequence ID No. 1: V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

39. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to 10 or more of the following amino acid substitutions of Sequence ID No. 1: V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

40. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to three or more of the following amino acid substitutions in SEQ ID NO: V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K.

41. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to five or more of the following amino acid substitutions in SEQ ID NO: V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K.

42. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to 10 or more of the following amino acid substitutions in SEQ ID NO: V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K.

43. The uricase variant according to claim 24, wherein the amino acid substitution corresponds to the amino acid substitution of SEQ ID NO: 1, which includes V22L, F75L, and Y127H.

44. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, Y127H, and K155G.

45. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, Y127H, and I257K.

46. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of Sequence ID No. 1, which include V22L, N70L, F75L, and Y127H.

47. The uricase variant according to claim 24, wherein the amino acid substitution corresponds to the amino acid substitution of Sequence ID No. 1, which includes V22L, F75L, A102V, and Y127H.

48. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, Y127H, and R209F.

49. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, and Y127H.

50. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, and Y127H.

51. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, and Y127H.

52. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K116M, and Y127H.

53. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K79G, and Y127H.

54. The uricase variant according to claim 24, wherein the amino acid substitution corresponds to the amino acid substitution of Sequence ID No. 1, which includes V22L, F75L, V106M, and Y127H.

55. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Y31H, F75L, and Y127H.

56. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70H, F75L, and Y127H.

57. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, A102L, and Y127H.

58. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, Y127H, and R209W.

59. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, D28H, F75L, and Y127H.

60. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K112S, and Y127H.

61. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, D28N, F75L, and Y127H.

62. The uricase variant according to claim 24, wherein the amino acid substitution corresponds to the amino acid substitution of Sequence ID No. 1, which includes V22L, F75L, V119T, and Y127H.

63. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, F114L, and Y127H.

64. The uricase variant according to claim 24, wherein the amino acid substitution corresponds to the amino acid substitution of SEQ ID NO: 1, which includes V22L, F75L, K116L, and Y127H.

65. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, Y127H, K155G, and I257K.

66. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, Y127H, and K155G.

67. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, A102V, Y127H, and K155G.

68. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, Y127H, K155G, and R209F.

69. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, Y127H, and K155G.

70. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of Sequence ID No. 1, which include V22L, Q26R, F75L, Y127H, and K155G.

71. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, Y127H, and K155G.

72. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K116M, Y127H, and K155G.

73. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K79G, Y127H, and K155G.

74. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, Y127H, and I257K.

75. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, A102V, Y127H, and I257K.

76. The uricase variant according to claim 24, wherein the amino acid substitution corresponds to the amino acid substitution of SEQ ID NO: 1, which includes V22L, F75L, Y127H, R209F, and I257K.

77. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, Y127H, and I257K.

78. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, Y127H, and I257K.

79. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, Y127H, and I257K.

80. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K116M, Y127H, and I257K.

81. The uricase variant according to claim 24, wherein the amino acid substitution corresponds to the amino acid substitution of SEQ ID NO: 1, which includes V22L, F75L, K79G, Y127H, and I257K.

82. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of Sequence ID No. 1, which include V22L, N70L, F75L, A102V, and Y127H.

83. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, Y127H, and R209F.

84. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, N70L, F75L, and Y127H.

85. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, N70L, F75L, and Y127H.

86. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, K76S, and Y127H.

87. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, K116M, and Y127H.

88. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, K79G, and Y127H.

89. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, A102V, Y127H, and R209F.

90. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, A102V, and Y127H.

91. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of Sequence ID No. 1, which include V22L, Q26R, F75L, A102V, and Y127H.

92. The uricase variant according to claim 24, wherein the amino acid substitution corresponds to the amino acid substitution of SEQ ID NO: 1, which includes V22L, F75L, K76S, A102V, and Y127H.

93. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, A102V, K116M, and Y127H.

94. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K79G, A102V, and Y127H.

95. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, Y127H, and R209F.

96. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, Y127H, and R209F.

97. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, Y127H, and R209F.

98. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K116M, Y127H, and R209F.

99. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K79G, Y127H, and R209F.

100. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, and Y127H.

101. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, K76S, and Y127H.

102. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, K116M, and Y127H.

103. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, K79G, and Y127H.

104. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, K76S, and Y127H.

105. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, K116M, and Y127H.

106. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, K79G, and Y127H.

107. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, K116M, and Y127H.

108. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, K79G, and Y127H.

109. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K79G, K116M, and Y127H.

110. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, A102V, Y127H, K155G, and I257K.

111. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, Y127H, K155G, R209F, and I257K.

112. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, N70L, F75L, Y127H, K155G, and I257K.

113. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, N70L, F75L, Y127H, K155G, and I257K.

114. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, K76S, Y127H, K155G, and I257K.

115. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, K116M, Y127H, K155G, and I257K.

116. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, K79G, Y127H, K155G, and I257K.

117. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, A102V, Y127H, K155G, I257K, and R209F.

118. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, A102V, Y127H, K155G, and I257K.

119. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, A102V, Y127H, K155G, and I257K.

120. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, A102V, Y127H, K155G, and I257K.

121. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, A102V, K116M, Y127H, K155G, and I257K.

122. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K79G, A102V, Y127H, K155G, and I257K.

123. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, Y127H, K155G, R209F, and I257K.

124. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, Y127H, K155G, R209F, and I257K.

125. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, Y127H, K155G, R209F, and I257K.

126. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K116M, Y127H, K155G, R209F, and I257K.

127. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K79G, Y127H, K155G, R209F, and I257K.

128. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, Y127H, K155G, and I257K.

129. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, K76S, Y127H, K155G, and I257K.

130. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, K116M, Y127H, K155G, and I257K.

131. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, K79G, Y127H, K155G, and I257K.

132. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, K76S, Y127H, K155G, and I257K.

133. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, K116M, Y127H, K155G, and I257K.

134. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, K79G, Y127H, K155G, and I257K.

135. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, K116M, Y127H, K155G, and I257K.

136. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, K79G, Y127H, K155G, and I257K.

137. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K79G, K116M, Y127H, K155G, and I257K.

138. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, N70L, F75L, A102V, Y127H, and R209F.

139. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, N70L, F75L, Y127H, and R209F.

140. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, N70L, F75L, K76S, and Y127H.

141. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, N70L, F75L, K76S, K116M, and Y127H.

142. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, N70L, F75L, K76S, K79G, K116M, and Y127H.

143. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, A102V, Y127H, and R209F.

144. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, A102V, and Y127H.

145. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, K76S, A102V, K116M, and Y127H.

146. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, K79G, A102V, K116M, and Y127H.

147. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, Y127H, and R209F.

148. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, K76S, K116M, Y127H, and R209F.

149. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, F75L, K76S, K79G, K116M, Y127H, and R209F.

150. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, K116M, and Y127H.

151. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, F75L, K76S, K79G, K116M, and Y127H.

152. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, F75L, K76S, K79G, K116M, and Y127H.

153. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, N70L, F75L, A102V, Y127H, K155G, R209F, and I257K.

154. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, N70L, F75L, K76S, A102V, Y127H, K155G, R209F, and I257K.

155. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, N70L, F75L, A102V, K116M, Y127H, K155G, R209F, and I257K.

156. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, S33D, N70L, F75L, K79G, A102V, Y127H, K155G, R209F, and I257K.

157. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, A102V, Y127H, K155G, R209F, and I257K.

158. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, K116M, Y127H, K155G, R209F, and I257K.

159. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, K79G, K116M, Y127H, K155G, and I257K.

160. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, N70L, F75L, K76S, A102V, Y127H, R209F, and I257K.

161. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, N70L, F75L, A102V, K116M, Y127H, R209F, and I257K.

162. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, N70L, F75L, K79G, A102V, Y127H, R209F, and I257K.

163. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, A102V, K116M, Y127H, R209F, and I257K.

164. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, A102V, K116M, Y127H, R209F, and I257K.

165. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, K79G, A102V, Y127H, R209F, and I257K.

166. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K79G, A102V, K116M, Y127H, R209F, and I257K.

167. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, K79G, K116M, Y127H, R209F, and I257K.

168. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, N70L, F75L, K76S, A102V, K116M, Y127H, and R209F.

169. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, N70L, F75L, K76S, K79G, A102V, Y127H, and R209F.

170. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of Sequence ID No. 1, which include V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K116M, Y127H, and R209F.

171. The uricase variant according to claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO: 1, which include V22L, Q26R, S33D, F75L, K76S, K79G, A102V, K116M, Y127H, and R209F.

172. A uricase variant according to any one of claims 25 to 171, further comprising an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:

1.

173. A uricase variant according to any one of claims 25 to 171, further comprising the amino acid substitution corresponding to N117G of SEQ ID NO:

1.

174. A uricase variant according to any one of claims 25 to 171, further comprising the amino acid substitution corresponding to N117D of SEQ ID NO:

1.

175. The uricase variant contains multiple amino acid mutations relative to the corresponding uricase protein, and the multiple amino acid mutations are the following combinations of amino acid residues of SEQ ID NO: 1, (1a) V22, D28, S33, N70, F75, K79, A102, Y127, K155, R209, and I257, (2a) V22, D28, S33, N70, F75, K79, A102, F114, V119, Y127, K155, R209, and I257, (3a) V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (4a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257, (5a) V22, Q26, Y31, N70, F75, K79, A102, Y127, K155, R209, and I257, (6a) V22, D28, Y31, N70, F75, K79, A102, K112, Y127, K155, R209, and I257, (7a) V22, Q26, S33, N70, F75, K79, A102, K112, Y127, K155, R209, and I257, (8a) V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (9a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (10a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (11a) V22, Q26, S33, N70, F75, K79, A102, K112, Y127, K155, R209, and I257, (12a) V22, D28, Y31, N70, F75, K79, A102, V106, K116, Y127, K155, R209, and I257, (13a) V22, Q26, Y31, N70, F75, K76, A102, Y127, K155, R209, and I257, (14a) V22, Q26, S33, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257, (15a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257, (16a) V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257, (17a) V22, Q26, S33, N70, F75, K76, A102, K112, Y127, K155, R209, and I257, (18a) V22, D28, Y31, N70, F75, K79, A102, V106, K116, Y127, K155, R209, and I257, (19a) V22, D28, Y31, N70, F75, K79, A102, K116, Y127, K155, R209, and I257, (20a) V22, Q26, S33, N70, F75, K76, A102, K116, V119, Y127, K155, R209, and I257, and (21a) V22, Q26, S33, N70, F75, K79, A102, F114, V119, Y127, K155, R209, and I257 The uricase variant according to claim 1, which is located at an amino acid position corresponding to one of the following.

176. The uricase variant according to claim 175, wherein the plurality of amino acid mutations are located at amino acid positions corresponding to the following combinations of amino acid residues of SEQ ID NO: V22, D28, S33, N70, F75, K79, A102, F114, V119, Y127, K155, R209, and I257 of the amino acid residues of SEQ ID NO:

1.

177. The uricase variant according to claim 175, wherein the plurality of amino acid mutations are located at amino acid positions corresponding to the following combinations of amino acid residues of SEQ ID NO: V22, Q26, Y31, N70, F75, K79, A102, Y127, K155, R209, and I257 of the amino acid residues of SEQ ID NO:

1.

178. The uricase variant according to claim 175, wherein the plurality of amino acid mutations are located at amino acid positions corresponding to the following combinations of amino acid residues of SEQ ID NO: V22, D28, Y31, N70, F75, K79, A102, K112, Y127, K155, R209, and I257 of the amino acid residues of SEQ ID NO:

1.

179. The uricase variant according to claim 175, wherein the plurality of amino acid mutations are located at amino acid positions corresponding to the following combinations of amino acid residues of SEQ ID NO: V22, Q26, S33, N70, F75, K79, A102, K112, Y127, K155, R209, and I257 of the amino acid residues of SEQ ID NO:

1.

180. The uricase variant according to claim 175, wherein the plurality of amino acid mutations are located at amino acid positions corresponding to the following combinations of amino acid residues of SEQ ID NO: V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257 of the amino acid residues of SEQ ID NO:

1.

181. The uricase variant according to claim 175, wherein the plurality of amino acid mutations are located at amino acid positions corresponding to the following combinations of amino acid residues of SEQ ID NO: V22, D28, Y31, N70, F75, K79, A102, K116, Y127, K155, R209, and I257 of the amino acid residues of SEQ ID NO:

1.

182. The uricase variant according to claim 175, wherein the plurality of amino acid mutations are amino acid substitutions.

183. The above amino acid substitution is the following combination of amino acid substitutions in SEQ ID NO: 1, (1b) V22L, D28H, S33D, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K, (2b) V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K, (3b) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209W, and I257T, (4b) V22L, D28T, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257T, (5b) V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K, (6b) V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K, (7b) V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K, (8b) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209F, and I257T, (9b) V22L, D28H, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209F, and I257T, (10b) V22L, D28T, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209F, and I257T, (11b) V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K, (12b) V22L, D28N, Y31H, N70L, F75L, K79G, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K, (13b) V22L, Q26R, Y31H, N70L, F75L, K76S, A102L, Y127H, K155G, R209F, and I257K, (14b) V22L, Q26R, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209W, and I257T, (15b) V22L, D28T, S33D, N70L, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209F, and I257K, (16b) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K, (17b) V22L, Q26R, S33D, N70H, F75L, K76S, A102V, K112S, Y127H, K155G, R209F, and I257K, (18b) V22L, D28N, Y31H, N70L, F75L, K79G, A102L, V106M, K116M, Y127H, K155G, R209F, and I257K, (19b) V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K. (20b) V22L, Q26R, S33D, N70L, F75L, K76S, A102L, K116M, V119T, Y127H, K155G, R209W, and I257K, and (21b) V22L, Q26R, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209F, and I257K A uricase variant according to claim 182, corresponding to one of the above.

184. The uricase variant according to claim 183, wherein the amino acid substitution corresponds to the following amino acid substitutions of SEQ ID NO: V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K.

185. The uricase variant according to claim 183, wherein the amino acid substitution corresponds to the following amino acid substitutions of SEQ ID NO: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K.

186. The uricase variant according to claim 183, wherein the amino acid substitution corresponds to the following amino acid substitutions of SEQ ID NO: V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K.

187. The uricase variant according to claim 183, wherein the amino acid substitutions correspond to the following amino acid substitutions of SEQ ID NO: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K of SEQ ID NO:

1.

188. The uricase variant according to claim 183, wherein the amino acid substitution corresponds to the following amino acid substitutions of Sequence ID No. 1: V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

189. The uricase variant according to claim 183, wherein the amino acid substitutions correspond to the following amino acid substitutions of SEQ ID NO: V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K.

190. A uricase variant according to any one of claims 183 to 189, further comprising an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:

1.

191. A uricase variant according to any one of claims 183 to 189, further comprising the amino acid substitution corresponding to N117G of SEQ ID NO:

1.

192. A uricase variant according to any one of claims 183 to 189, further comprising the amino acid substitution corresponding to N117D of SEQ ID NO:

1.

193. A uricase variant, wherein the uricase variant comprises a first set of amino acid substitutions and a second set of amino acid substitutions relative to the corresponding uricase protein. The first set of amino acid substitutions is group (a) or group (b), (a) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K, or (b) Corresponding to the amino acid substitutions of SEQ ID NO: 1 described in V22L, Q26T, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K, A uricase variant wherein the second set of amino acid substitutions is a set of about 6 to about 10 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1, one of K30E and K30F, one of L52T, H53D, K79S, T83E, V86N, S94D, K97D, one of N117G and N117D, one of F172W and F172L, T174N, E177Q, R192T, G196D, R197D, Y225H, S228G, L239G, L279T, and N281Y, wherein the uricase variant contains an amino acid sequence that is at least about 85%, at least about 90%, at least about 91%, or at least about 92% identical to SEQ ID NO:

1.

194. The uricase variant according to claim 193, wherein the first set of amino acid substitutions corresponds to the amino acid substitutions of SEQ ID NO: 1 described in group (a) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

195. The uricase variant according to claim 193, wherein the first set of amino acid substitutions corresponds to the amino acid substitutions of SEQ ID NO: 1 described in group (b) V22L, Q26T, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

196. The uricase variant according to any one of claims 193 to 195, wherein the second set of amino acid substitutions is a set of about 6 to about 10 amino acid substitutions selected from the following amino acid substitutions of Sequence ID No. 1, one of K30E and K30F, one of L52T, H53D, T83E, V86N, S94D, K97D, N117G and N117D, F172W, T174N, R192T, G196D, R197D, Y225H, S228G, L239G, L279T, and N281Y.

197. The uricase variant according to any one of claims 193 to 195, wherein the second set of amino acid substitutions is a set of about 6 to about 10 amino acid substitutions selected from the following amino acid substitutions of Sequence ID No. 1, one of K30E and K30F, one of L52T, T83E, V86N, S94D, K97D, N117G and N117D, F172W, R192T, G196D, R197D, Y225H, S228G, L279T, and N281Y.

198. The uricase variant according to any one of claims 193 to 195, wherein the second set of amino acid substitutions is a set of about 6 to about 10 amino acid substitutions selected from the following amino acid substitutions of Sequence ID No. 1: one of K30E, L52T, T83E, S94D, N117G and N117D, F172W, R192T, G196D, R197D, Y225H, S228G, L279T, and N281Y.

199. The uricase variant according to any one of claims 193 to 195, wherein the second set of amino acid substitutions is a set of about 6 to about 10 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of Sequence ID No. 1, one of K30E, L52T, T83E, N117G and N117D, F172W, R192T, G196D, R197D, S228G, L279T and N281Y.

200. The uricase variant according to any one of claims 193 to 199, wherein the second set of amino acid substitutions is a set of about 6 to about 7 amino acid substitutions.

201. The uricase variant according to any one of claims 193 to 199, wherein the second set of amino acid substitutions is a set of about 8 to about 10 amino acid substitutions.

202. The uricase variant according to any one of claims 193 to 195, wherein the second set of amino acid substitutions is a set of about 6 to about 9 amino acid substitutions selected from the following amino acid substitutions of Sequence ID No. 1, one of K30E, L52T, T83E, N117G and N117D, F172W, G196D, R197D, S228G, L279T and N281Y.

203. The uricase variant according to any one of claims 193 to 195, wherein the second set of amino acid substitutions is a set of about 6 to about 8 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of Sequence ID No. 1: K30E, L52T, T83E, N117G, F172W, G196D, S228G, L279T, and N281Y.

204. The uricase variant according to any one of claims 193 to 195, wherein the second set of amino acid substitutions is a set of about 6 to about 8 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of Sequence ID No. 1: K30E, L52T, T83E, N117D, F172W, G196D, S228G, L279T, and N281Y.

205. The uricase variant according to any one of claims 193 to 195, wherein the second set of amino acid substitutions is a set of about 6 to about 8 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of Sequence ID No. 1: K30E, L52T, T83E, F172W, G196D, S228G, L279T, and N281Y.

206. The uricase variant according to any one of claims 193 to 195, wherein the second set of amino acid substitutions is a set of about 6 to about 7 amino acid substitutions selected from the amino acid substitutions corresponding to the following amino acid substitutions of Sequence ID No. 1: K30E, L52T, T83E, F172W, G196D, S228G, L279T, and N281Y.

207. The uricase variant according to any one of claims 1 to 206, wherein the corresponding uricase protein comprises an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% identical to SEQ ID NO: 1, or at least 90% to about 100% identical to SEQ ID NO:

1.

208. The uricase variant according to any one of claims 1 to 207, wherein the corresponding uricase protein is a mammalian uricase.

209. The uricase variant according to claim 208, wherein the corresponding uricase protein is canine uricase, porcine uricase, bovine uricase, sheep uricase, or baboon uricase.

210. The uricase variant according to claim 207 or 208, wherein the corresponding uricase protein is a chimeric uricase containing two or more mammalian uricase domains.

211. The uricase variant according to claim 210, wherein the corresponding uricase protein is chimeric pig-baboon uricase.

212. The uricase variant according to claim 211, wherein the corresponding uricase protein is a chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO:

1.

213. The uricase variant according to claim 211, wherein the corresponding uricase protein is a chimeric pig-baboon uricase containing at least about 70% the same amino acid sequence as SEQ ID NO:

1.

214. The uricase variant according to claim 211, wherein the corresponding uricase protein is a chimeric pig-baboon uricase containing at least about 80% the same amino acid sequence as SEQ ID NO:

1.

215. The uricase variant according to claim 211, wherein the corresponding uricase protein is a chimeric pig-baboon uricase containing an amino acid sequence identical to that of SEQ ID NO: 1 by at least about 90%.

216. The uricase variant according to claim 211, wherein the corresponding uricase protein is a chimeric pig-baboon uricase containing an amino acid sequence that is approximately 90% to approximately 100% identical to that of SEQ ID NO:

1.

217. The uricase variant according to claim 211, wherein the corresponding uricase protein is a chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO:

4.

218. The uricase variant according to claim 211, wherein the corresponding uricase protein is a chimeric pig-baboon uricase containing the amino acid sequence of SEQ ID NO:

5.

219. The uricase variant according to any one of claims 1 to 218, wherein the corresponding uricase protein is further cleaved at its C-terminus.

220. The uricase variant according to claim 219, wherein the C-terminal cleavage comprises 4 to 13 amino acids.

221. The uricase variant according to any one of claims 1 to 220, wherein the corresponding uricase protein is further cleaved at its N-terminus.

222. The uricase variant according to claim 221, wherein the N-terminal cleavage comprises 4 to 13 amino acids.

223. A uricase variant according to any one of claims 1 to 222, comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO:

1.

224. A uricase variant according to any one of claims 1 to 222, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO:

1.

225. A uricase variant according to any one of claims 1 to 222, comprising an amino acid sequence that is at least about 92% identical to SEQ ID NO:

1.

226. The uricase variant according to any one of claims 1 to 225, wherein the uricase variant comprises an N-terminal methionine residue.

227. The uricase variant according to any one of claims 1 to 225, wherein the uricase variant does not have an N-terminal methionine residue.

228. The uricase variant according to any one of claims 1 to 225, wherein the uricase variant includes post-translational modification.

229. The uricase variant according to claim 228, wherein the post-translational modification is the removal of the N-terminal methionine residue of the uricase variant.

230. The uricase variant according to claim 1, comprising an amino acid sequence selected from SEQ ID NOs: 25 to 154.

231. The uricase variant according to claim 1, comprising an amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NOs. 25 to 154.

232. A uricase variant according to claim 1, comprising an amino acid sequence selected from SEQ ID NOs: 28, 45, 48, 49, 53, 66, 67, 101, 104, 112, 114, 117, 119, 123, 125, 129, 139, 142, 149, 150, and 152.

233. A uricase variant according to claim 1, comprising an amino acid sequence that is 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% identical to an amino acid sequence selected from SEQ ID NOs: 28, 45, 48, 49, 53, 66, 67, 101, 104, 112, 114, 117, 119, 123, 125, 129, 139, 142, 149, 150, and 152.

234. The uricase variant according to claim 1, comprising the amino acid sequence of SEQ ID NO:

45.

235. The uricase variant according to claim 1, comprising an amino acid sequence that is 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% identical to SEQ ID NO:

45.

236. The uricase variant according to claim 1, comprising the amino acid sequence of SEQ ID NO:

53.

237. The uricase variant according to claim 1, comprising an amino acid sequence that is 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% identical to SEQ ID NO:

53.

238. A uricase variant according to claim 237, comprising an amino acid sequence selected from SEQ ID NOs: 197 to 309.

239. The uricase variant according to claim 1, comprising the amino acid sequence of SEQ ID NO:

66.

240. A uricase variant according to claim 1, comprising an amino acid sequence that is 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% identical to sequence number 66.

241. The uricase variant according to claim 1, comprising the amino acid sequence of sequence number 114.

242. A uricase variant according to claim 1, comprising an amino acid sequence that is 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% identical to SEQ ID NO:

114.

243. The uricase variant according to claim 1, comprising the amino acid sequence of SEQ ID NO:

129.

244. A uricase variant according to claim 1, comprising an amino acid sequence that is 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% identical to SEQ ID NO:

129.

245. A uricase variant according to claim 244, comprising an amino acid sequence selected from SEQ ID NOs: 187, 189, 191, 193, and 195.

246. The uricase variant according to claim 244, comprising the amino acid sequence of SEQ ID NO:

187.

247. The uricase variant according to claim 244, comprising the amino acid sequence of SEQ ID NO:

191.

248. A uricase variant according to claim 244, comprising an amino acid sequence selected from SEQ ID NOs: 310 to 448.

249. A uricase variant according to claim 244, comprising an amino acid sequence selected from SEQ ID NOs: 449 to 621.

250. A uricase variant according to claim 244, comprising an amino acid sequence selected from SEQ ID NOs: 622 to 970.

251. A uricase variant according to claim 250, comprising an amino acid sequence selected from SEQ ID NOs: 622, 639, 640, 644, 646, 649, 657, 660, 668, 671, 673, 675, 676, 677, 678, 679, 680, 689, 694, 695, 704, 774, and 791.

252. A uricase variant according to claim 244, comprising an amino acid sequence selected from SEQ ID NOs: 971 to 1329.

253. A uricase variant according to claim 252, comprising an amino acid sequence selected from SEQ ID NOs: 971, 973, 976, 978, 979, 983, 985, 988, 991, 997, 1000, 1013, 1016, 1018, 1022, 1023, 1024, 1030, 1032, 1033, 1035, 1037, 1041, 1043, 1044, 1045, 1046, 1051, 1053, 1054, 1055, 1057, 1077, 1134, and 1142.

254. A uricase variant according to claim 244, comprising an amino acid sequence selected from sequence numbers 1330 to 1474.

255. A uricase variant according to claim 254, comprising an amino acid sequence selected from SEQ ID NOs: 1330, 1333, 1342, 1358, 1375, 1385, 1391, 1436, 1442, 1451, 1457, and 1465.

256. The uricase variant according to claim 244, comprising an amino acid sequence selected from sequence numbers 1475 to 1566.

257. The uricase variant according to claim 1, comprising the amino acid sequence of SEQ ID NO:

149.

258. The uricase variant according to claim 1, comprising an amino acid sequence that is 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% identical to SEQ ID NO:

149.

259. A uricase variant according to any one of claims 230 to 258, further comprising an N-terminal methionine residue.

260. The uricase variant according to any one of claims 1 to 259, wherein the uricase variant is not PEG-modified.

261. The uricase variant according to any one of claims 1 to 259, wherein the uricase variant is PEG-encapsulated.

262. The uricase variant according to any one of claims 1 to 261, wherein the uricase variant is isolated.

263. A uricase variant according to any one of claims 1 to 262, further comprising a purification tag at the C-terminus.

264. The uricase variant according to any one of claims 1 to 263, further comprising a purified tag at the N-terminus.

265. The uricase variant according to claim 263 or 264, wherein the purified tag is a polyhistidine tag.

266. The uricase variant according to any one of claims 1 to 265, wherein the uricase variant is a monomer.

267. The uricase variant according to any one of claims 1 to 265, wherein the uricase variant is a homodimer.

268. The uricase variant according to any one of claims 1 to 265, wherein the uricase variant is a homotetramer.

269. A uricase monomer comprising the uricase variant described in any one of claims 1 to 265.

270. A uricase dimer comprising a first uricase monomer and a second uricase monomer, wherein the first uricase monomer is a uricase variant according to any one of claims 1 to 265.

271. The uricase dimer according to claim 270, wherein the second uricase monomer is a uricase variant according to any one of claims 1 to 265.

272. The uricase dimer according to claim 271, wherein the uricase dimer is a homodimer.

273. The uricase dimer according to claim 271, wherein the uricase dimer is a heterodimer.

274. A uricase tetramer comprising a first, second, third, and fourth uricase monomer, wherein the first uricase monomer is a uricase variant according to any one of claims 1 to 265.

275. The uricase tetramer according to claim 274, wherein the second uricase monomer is a uricase variant according to any one of claims 1 to 265.

276. The uricase tetramer according to claim 275, wherein the third uricase monomer is a uricase variant according to any one of claims 1 to 265.

277. The uricase tetramer according to claim 276, wherein the fourth uricase monomer is a uricase variant according to any one of claims 1 to 265.

278. The uricase tetramer according to any one of claims 274 to 277, wherein the uricase tetramer is a homotetramer.

279. The uricase tetramer according to any one of claims 274 to 277, wherein the uricase tetramer is a heterotetramer.

280. A uricase conjugate comprising a first domain and a second domain, wherein the first domain comprises a uricase variant according to any one of claims 1 to 268, and the second domain comprises a first random coil polypeptide domain comprising at least about 100 amino acids.

281. The uricase conjugate according to claim 280, wherein the uricase conjugate is a fusion protein of the first domain and the second domain.

282. The uricase conjugate according to claim 281, wherein the first domain is located on the C-terminal side of the second domain.

283. The uricase conjugate according to claim 281, wherein the first domain is located on the N-terminal side of the second domain.

284. The uricase conjugate according to any one of claims 281 to 283, wherein an amino acid linker is present between the first domain and the second domain.

285. The uricase conjugate according to claim 284, wherein the amino acid linker is approximately 2 amino acid lengths to approximately 5 amino acid lengths.

286. The uricase conjugate according to claim 285, wherein the amino acid linker is 2 amino acid length.

287. The uricase conjugate according to claim 286, wherein the amino acid linker is Gly-Ser.

288. The uricase conjugate according to any one of claims 281 to 287, further comprising a third domain, wherein the third domain comprises a second random coil polypeptide domain comprising at least about 100 amino acids.

289. The uricase conjugate according to claim 288, wherein the first domain is located on the N-terminal side of the second domain and on the C-terminal side of the third domain.

290. The uricase conjugate according to claim 289, wherein an amino acid linker is present between the first domain and the second domain.

291. The uricase conjugate according to claim 289 or 290, wherein an amino acid linker is located between the first domain and the third domain.

292. The uricase conjugate according to claim 290 or 291, wherein the amino acid linker is approximately 2 amino acid lengths to approximately 5 amino acid lengths.

293. The uricase conjugate according to claim 292, wherein the amino acid linker is 2 amino acid length.

294. The uricase conjugate according to claim 293, wherein the amino acid linker is Gly-Ser.

295. The uricase conjugate according to any one of claims 280 to 294, wherein the second domain comprises a Pro-Ala-Ser (PAS) polypeptide.

296. The uricase conjugate according to any one of claims 288 to 295, wherein the third domain comprises a Pro-Ala-Ser (PAS) polypeptide.

297. The uricase conjugate according to claim 295 or 296, wherein the PAS polypeptide has the amino acid sequence described in SEQ ID NO:

9.

298. The uricase conjugate according to claim 295 or 296, wherein the PAS polypeptide has the amino acid sequence described in SEQ ID NO:

10.

299. The uricase conjugate according to claim 295 or 296, wherein the PAS polypeptide has the amino acid sequence described in SEQ ID NO:

11.

300. The uricase conjugate according to claim 295 or 296, wherein the PAS polypeptide comprises the amino acid sequence described in SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO:

23.

301. The uricase conjugate according to claim 300, wherein the PAS polypeptide comprises the amino acid sequence of Sequence ID No.

13.

302. The uricase conjugate according to claim 301, wherein the amino acid sequence of SEQ ID NO: 13 is encoded by a nucleotide sequence selected from SEQ ID NOs: 155 to 185.

303. The uricase conjugate according to any one of claims 280 to 302, wherein the second domain comprises an elongated recombinant (XTEN) polypeptide.

304. The uricase conjugate according to any one of claims 288 to 303, wherein the third domain comprises an elongated recombinant (XTEN) polypeptide.

305. The uricase conjugate according to claim 303 or 304, wherein the XTEN polypeptide has the amino acid sequence described in SEQ ID NO:

12.

306. The uricase conjugate according to any one of claims 280 to 305, wherein the second domain comprises a Pro-Ala (PA) polypeptide.

307. The uricase conjugate according to any one of claims 288 to 306, wherein the third domain comprises a Pro-Ala (PA) polypeptide.

308. The uricase conjugate according to any one of claims 280 to 307, wherein the first random coil polypeptide domain comprises about 100 to about 800 amino acids.

309. The uricase conjugate according to any one of claims 288 to 308, wherein the second random coil polypeptide domain comprises about 100 to about 800 amino acids.

310. The uricase conjugate according to any one of claims 280 to 307, wherein the first random coil polypeptide domain comprises about 100 to about 700 amino acids.

311. The uricase conjugate according to any one of claims 288 to 310, wherein the second random coil polypeptide domain comprises about 100 to about 700 amino acids.

312. The uricase conjugate according to any one of claims 280 to 307, wherein the first random coil polypeptide domain comprises about 100 to about 600 amino acids.

313. The uricase conjugate according to any one of claims 288 to 312, wherein the second random coil polypeptide domain comprises about 100 to about 600 amino acids.

314. The uricase conjugate according to any one of claims 280 to 307, wherein the first random coil polypeptide domain comprises about 100 to about 500 amino acids.

315. The uricase conjugate according to any one of claims 288 to 314, wherein the second random coil polypeptide domain comprises about 100 to about 500 amino acids.

316. The uricase conjugate according to any one of claims 280 to 307, wherein the first random coil polypeptide domain comprises about 100 to about 400 amino acids.

317. The uricase conjugate according to any one of claims 288 to 316, wherein the second random coil polypeptide domain comprises about 100 to about 400 amino acids.

318. The uricase conjugate according to any one of claims 280 to 307, wherein the first random coil polypeptide domain comprises about 100 to about 300 amino acids.

319. The uricase conjugate according to any one of claims 288 to 318, wherein the second random coil polypeptide domain comprises about 100 to about 300 amino acids.

320. The uricase conjugate according to any one of claims 281 to 319, wherein the first domain does not contain an N-terminal methionine residue.

321. The uricase conjugate according to any one of claims 281 to 320, wherein the first random coil polypeptide domain does not contain an N-terminal methionine residue.

322. The uricase conjugate according to any one of claims 288 to 321, wherein the second random coil polypeptide domain does not contain an N-terminal methionine residue.

323. The uricase conjugate according to any one of claims 281 to 322, further comprising a purified tag at the C-terminus.

324. The uricase conjugate according to any one of claims 281 to 323, further comprising a purified tag at the N-terminus.

325. The uricase conjugate according to claim 323 or 324, wherein the purified tag is a polyhistidine tag.

326. The uricase conjugate according to claim 281, wherein the monomeric form of the fusion protein comprises the amino acid sequence of SEQ ID NO: 1568.

327. The uricase conjugate according to claim 288, wherein the uricase conjugate is a fusion protein, and the monomeric form of the fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 1569 to 1748.

328. The uricase conjugate according to claim 288, wherein the uricase conjugate is a fusion protein, and the monomeric form of the fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 1749 to 1928.

329. The uricase conjugate according to claim 288, wherein the uricase conjugate is a fusion protein, and the monomeric form of the fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 1929 to 2108.

330. The uricase conjugate according to claim 288, wherein the uricase conjugate is a fusion protein, and the monomeric form of the fusion protein comprises an amino acid sequence selected from SEQ ID NOs: 2109 to 2288.

331. The uricase conjugate according to any one of claims 280 to 330, wherein the uricase conjugate is a monomer.

332. The uricase conjugate according to any one of claims 280 to 331, wherein the uricase conjugate is present in a homodimer.

333. The uricase conjugate according to any one of claims 280 to 331, wherein the uricase conjugate is present in a homotetramer.

334. The uricase conjugate according to any one of claims 280 to 330, wherein the uricase conjugate is a homotetramer.

335. The uricase conjugate according to any one of claims 280 to 330, wherein the uricase conjugate is a homodimer.

336. The uricase conjugate according to any one of claims 280 to 335, wherein the uricase variant is not PEG-conjugated.

337. The uricase conjugate according to any one of claims 280 to 335, wherein the uricase conjugate is PEG-conjugated.

338. An isolated nucleic acid encoding a uricase variant according to any one of claims 1 to 268 or a uricase conjugate according to any one of claims 280 to 337.

339. A nucleic acid vector comprising the nucleic acid described in claim 338.

340. A host cell comprising the nucleic acid vector according to claim 339.

341. A pharmaceutical composition comprising a uricase variant according to any one of claims 1 to 268, a uricase tetramer according to any one of claims 274 to 279, or a uricase conjugate according to any one of claims 280 to 337, and a pharmaceutically acceptable carrier.

342. A method for reducing elevated uric acid levels in a subject requiring such reduction, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 341.

343. The method according to claim 342, wherein the uric acid level is reduced in the plasma of the subject.

344. The method according to claim 342 or 343, wherein the subject is a patient with gout.

345. The method according to claim 344, wherein the subject is a patient with intractable gout.

346. The method according to any one of claims 342 to 345, wherein the subject is diagnosed with tumor lysis syndrome.

347. A method for treating gout in a subject requiring treatment, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 341.

348. The method according to claim 347, wherein the gout is intractable gout.

349. A method for treating tumor lysis syndrome in a subject requiring treatment, comprising administering to the subject an effective amount of the pharmaceutical composition according to claim 341.

350. The method according to any one of claims 342 to 349, wherein the subject is a human patient.

351. The method according to claim 350, wherein the human patient is an adult human patient.

352. The method according to any one of claims 342 to 351, wherein the pharmaceutical composition is administered parenterally.

353. The method according to claim 352, wherein the pharmaceutical composition is administered intravenously.

354. The method according to claim 352, wherein the pharmaceutical composition is administered subcutaneously.

355. A method for recombinantly producing a uricase variant according to any one of claims 1 to 268, (i) Culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding a uricase variant according to any one of claims 1 to 268, wherein the nucleic acid sequence is operably linked to a heterologous promoter under conditions that enable the host cell to express the nucleic acid sequence encoding the uricase variant and to recombinantly produce the uricase variant. (ii) A method comprising isolating the recombinantly produced uricase variant.

356. A method for recombinantly producing a uricase conjugate according to any one of claims 280 to 337, (i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding a uricase conjugate according to any one of claims 280 to 337, wherein the nucleic acid sequence is operably linked to a heterologous promoter under conditions that enable the expression of the nucleic acid sequence encoding the uricase conjugate and the recombinant production of the uricase conjugate by the host cell, (ii) A method comprising isolating the recombinantly produced uricase conjugate.

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