Uricase variants and methods of use thereof

Abstract

The present disclosure relates to uricase variants and their multimers, as well as uricase conjugates comprising a first domain comprising one of the uricase variants and conjugated to a second domain comprising a random coil polypeptide domain. Methods of using the same to treat gout, tumor lysis syndrome, and other diseases associated with elevated uric acid levels in body fluids, including blood, are provided. Methods of recombinantly producing the uricase variants or uricase conjugates disclosed herein are also provided.

Description

Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application URICASE VARIANTS AND METHODS OF USE THEREOF CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application Serial No. 63 / 500,541, filed May 5, 2023, the disclosure of which is incorporated by reference herein in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (INMD_191_01WO_SeqList_ST26.xml; Size: 3,082,460 bytes; and Date of Creation: April 30, 2024) are herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0003] In humans, uric acid is produced following breakdown of purines. Accumulation of uric acid in the blood (hyperuricemia) is manifested 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 accumulation of uric acid crystals in the joints, leading to inflammation of intense pain. Uric acid crystals are formed when high levels of uric acid are present in the blood.

[0005] Tumor lysis syndrome is a complication from the treatment of cancer, e.g., lymphomas, leukemias, including non-Hodgkin’s lymphoma, acute myeloid leukemia, and acute lymphoblastic leukemia. Tumor lysis syndrome occurs as large numbers of tumor cells are lysed, releasing their contents into the bloodstream. Tumor lysis syndrome is characterized by high blood uric acid (hyperuricemia), as well as high blood potassium (hyperkalemia), high blood phosphate (hyperphosphatemia), low blood calcium (hypocalcemia), and higher than normal levels of blood urea nitrogen (BUN). The metabolic abnormalities seen in tumor lysis syndrome can ultimately result in serious complications, such as acute uric acid nephropathy, acute kidney failure, seizures, cardiac arrhythmias, and death.

[0006] Uricases are enzymes catalyzing the oxidation of uric acid to a more soluble product, allantoin, a purine metabolite which is more readily excreted. Because humans do not produce enzymatically active uricase, due to several mutations in the gene for uricase acquired duringAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application the evolution of higher primates, exogenously administered uricase provides a therapy for diseases manifesting hyperuricemia, such as gout and tumor lysis syndrome.

[0007] The present invention addresses the need for deimmunized uricases for the treatment of gout, tumor lysis syndrome, and other diseases associated with elevated uric acid levels in body fluids, including blood. SUMMARY OF THE INVENTION

[0008] In one aspect, the present disclosure relates to a uricase variant comprising one or more amino acid mutations relative to a counterpart uricase protein, wherein the one or more amino acid mutations are at one or more amino acid positions 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, wherein 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 counterpart uricase protein is a chimeric pig-baboon uricase comprising the amino acid sequence of SEQ ID NO:1.

[0010] In another embodiment, the counterpart 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%, at least about 99%, or from about 90% to about 100% identical to SEQ ID NO:1. In a further embodiment, the counterpart uricase protein is a chimeric pig-baboon uricase comprising an amino acid sequence at least about 70%, at least about 80%, at least about 90%, or from about 90% to about 100% identical to SEQ ID NO:1.

[0011] In one embodiment, the uricase variant comprises two or more, three or more, four or more, five or more, seven or more, ten or more, fifteen or more, or from about 5 to about 25 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are selected from mutations at amino acid positions corresponding to 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 a further embodiment, each of the amino acid mutations is an amino acid substitution. In a further embodiment, 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 from about 5 to about 25 of the amino acid substitutions of SEQ ID NO:1 set forthAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application in Table A. In a further embodiment, the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising one of the exemplary groups of amino acid substitutions set forth in Table B. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution 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 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 an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of 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 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 comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of 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 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 comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of 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 of 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 comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a furtherAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of 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 of 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 comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of 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 of 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 an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of SEQ ID NO:1.

[0018] In one embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs:25-154. In a further embodiment, the uricase variant comprises 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 still a further embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs:45, 53, 66, 114, 129, and 149.

[0019] In one 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 an amino acid sequence selected from SEQ ID NOs: 25-154. In a further 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 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 even a further 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 an amino acid sequence selected from SEQ ID NOs:45, 53, 66, 114, 129, and 149.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0020] In one embodiment, the uricase variant comprises an amino acid sequence of SEQ ID NO:53. 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:53. Exemplary uricase variants include those comprising an amino acid sequence selected from SEQ ID NOs:197-309.

[0021] In one embodiment, the uricase variant comprises an 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 a uricase variant provided herein, the uricase variant comprises a first set of amino acid substitutions and a second set of amino acid substitutions relative to a counterpart uricase protein. The first set of amino acid substitutions corresponds to the amino acid substitutions of SEQ ID NO:1 set forth in group (a) or group (b) below: (a) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K; or (b) 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 from about 6 to about 10 (e.g., 6, 7, 8, 9, or 10) amino acid substitutions selected from amino acid substitutions correspondingAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application to the following amino acid substitutions of SEQ ID NO:1: one of K30E and K30F, 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. The uricase 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 a uricase variant disclosed herein, the uricase variant is non- PEGylated. In another embodiment, the uricase variant is PEGylated. In one embodiment, the uricase variant is PASylated. In another embodiment, the uricase variant is PASylated and PEGylated. In one embodiment, the uricase variant is XTENylated. In yet another embodiment, the uricase variant is XTENylated and PEGylated.

[0028] In one embodiment, a uricase variant disclosed herein is a monomer. In another embodiment, the uricase variant is a homodimer. In a preferred embodiment, the uricase variant is a homotetramer.

[0029] In another aspect of the present disclosure, a uricase dimer comprising a first uricase monomer and a second uricase monomer is provided, 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 the present disclosure, a uricase tetramer comprising a first, second, third and fourth uricase monomer is provided, 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. In a further embodiment, the third uricase monomer is one of the uricase variants disclosed herein. In a still further 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 aspect, the present disclosure relates to a uricase conjugate comprising at least two domains: (i) a first domain comprising one of the uricase variants disclosed herein and 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 domain and the second domain, referred to as “a uricase fusionAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application protein” or “a recombinant uricase fusion protein” in the present disclosure. In one embodiment, the uricase conjugate is present within a uricase tetramer, wherein the uricase tetramer comprises four uricase conjugate subunits.

[0032] In one embodiment, the random coil polypeptide comprises a PAS polypeptide. In one embodiment, the random coil polypeptide comprises an extended recombinant (XTEN) polypeptide. In 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 comprises 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 is present within 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 homotetrameric recombinant uricase fusion protein. In one embodiment of a uricase conjugate disclosed herein, the uricase conjugate is non-PEGylated. In another embodiment, the uricase conjugate is PEGylated.

[0035] In another aspect, the present disclosure provides an isolated nucleic acid which encodes a uricase variant or a uricase conjugate (e.g., a recombinant uricase fusion protein) disclosed herein.

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

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

[0038] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a uricase variant, a uricase tetramer, or a uricase conjugate, which in some embodiments is a recombinant uricase fusion protein, disclosed herein. In one embodiment, the uricase tetramer is a homotetramer comprising four identical uricase variant monomers disclosed herein.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0039] In another aspect, the present disclosure provides a method of reducing elevated uric acid levels in a subject in need of treatment. The method includes 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 uric acid levels are reduced in the plasma or blood of the subject. In one embodiment of the method, the administering is carried out via intravenous administration. In another embodiment, the administering is carried out via subcutaneous administration. In one embodiment of the method, the subject is a gout patient. In a further embodiment, the subject is a refractory gout patient. In one embodiment of the method, the subject has been diagnosed with tumor lysis syndrome.

[0040] In another aspect, the present disclosure provides a method of treating gout in a subject in need of treatment. The method includes administering to the subject an effective amount of the pharmaceutical composition disclosed herein. In one embodiment of the method, the gout 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 administering is carried out via intravenous administration. In another embodiment, the administering is carried out via subcutaneous administration.

[0041] In another aspect, the present disclosure provides a method of treating tumor lysis syndrome in a subject in need of treatment. The method includes 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 administering is carried out via intravenous administration. In another embodiment, the administering is carried out via subcutaneous administration.

[0042] In another aspect, the present disclosure provides a method of recombinantly producing a uricase variant or a uricase conjugate (e.g., a recombinant uricase fusion protein) disclosed herein. The method includes (i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding the uricase variant or the uricase conjugate (e.g., the recombinant uricase fusion protein) disclosed herein, wherein the nucleic acid sequence is operatively linked to a heterologous promoter under conditions to allow for expression of the nucleic acid sequence encoding the uricase variant or the uricase conjugate (e.g., the recombinant uricase fusion protein) and recombinant production of the uricase variant or the uricase conjugate (e.g., the uricase fusion protein) by the host cell; and (ii) isolating theAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application recombinantly produced uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein). BRIEF DESCRIPTION OF THE FIGURES

[0043] FIG. 1 is a schematic showing various configurations of uricase fusion proteins of the invention.

[0044] FIG. 2A shows the first stage in the uricase deimmunization workflow, in which immunodominant T cell epitopes in wild type chimeric pig-baboon uricase (WT) of which pegloticase is comprised (top) were targeted for mutational silencing using substitutions predicted to maintain protein structure and function (middle). Sets of mutations were computationally optimized to yield diverse combinatorial libraries that differentially balanced reduction in immunogenicity vs. retention of function (bottom).

[0045] FIG.2B shows the second stage in the uricase deimmunization workflow, in which one library design was selected and screened using a high throughput halo assay that reports uricase activity (top). Promising clones were picked into deep 96-well plates (middle), enabling medium throughput analysis of expression-normalized enzymatic activity in solution (bottom). The fastest enzymes were purified and analyzed in more detail.

[0046] FIG. 2C shows the third stage in the uricase deimmunization workflow, in which sensitive cellular immunoassays quantified antigen-specific proliferation of T cells among peripheral blood mononuclear cells (PBMCs) from a panel of genetically diverse donors. The immunoassays validated the computational prediction of reduced immunogenicity for each analyzed variant, and the data was used to guide lead candidate selection.

[0047] FIG. 3A is a picture showing growth of E. coli expressing the combinatorial deimmunized uricase library on agar containing uric acid, and halos created by active clones converting insoluble uric acid into soluble allantoin.

[0048] FIG.3B is a graphic representation showing absorbance at 293 nm over time of whole cell lysates from cultured colonies incubated with uric acid solution. Conversion of uric acid to allantoin results in decreasing absorbance, and specific activity is calculated from slopes.

[0049] FIG. 4A is an image of an SDS-PAGE gel showing bands at about MW 291 kDa representing PEGylated uricase species for the wild type chimeric pig-baboon uricase (WT) and top six chimeric pig-baboon uricase variant candidates.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0050] FIG. 4B is a graph showing the percentage of soluble tetramers using size exclusion chromatography (SEC) from one-step purification for non-PEGylated wild type chimeric pig- baboon uricase (WT) and non-PEGylated top six chimeric pig-baboon uricase variant candidates.

[0051] FIG. 4C is a graph showing specific activities measured by reaction velocity for PEGylated wild type chimeric pig-baboon uricase (WT) and PEGylated top six chimeric pig- baboon uricase variant candidates.

[0052] FIG. 4D is a graph showing Michaelis Menten kinetics parameter Vmax for PEGylated wild type chimeric pig-baboon uricase (WT) and PEGylated top six chimeric pig-baboon uricase variant candidates.

[0053] FIG. 4E is a graph showing apparent kcat / Kmfor PEGylated wild type chimeric pig- baboon uricase (WT) and PEGylated top six chimeric pig-baboon uricase variant candidates.

[0054] FIG. 4F is a graph showing melting temperatures (Tm) determined by differential scanning fluorimetry for PEGylated wild type chimeric pig-baboon uricase (WT) and PEGylated top six chimeric pig-baboon uricase variant candidates.

[0055] FIG.5A is a schematic of ex vivo cellular immunoassays to determine immunogenicity of non-PEGylated top six chimeric pig-baboon uricase variant candidates relative to non- PEGylated wild type chimeric pig-baboon uricase (WT).

[0056] FIG. 5B is a graph showing median CD4+ T cell stimulation index determined by the ex vivo cellular immunoassays for non-PEGylated top six chimeric pig-baboon uricase variant candidates and non-PEGylated wild type chimeric pig-baboon uricase (WT). The hashed line denotes the median stimulation index with the vehicle control.

[0057] FIG. 6A is a schematic of the study design for assessing the immunogenicity of PEGylated top six chimeric pig-baboon uricase variant candidates relative to PEGylated wild type chimeric pig-baboon uricase in humanized HLA transgenic mouse strain DR4.

[0058] FIG. 6B is a line graph showing the relationship between the antidrug antibody titer determined by ELISA absorbance and plasma dilution on a log scale, using day 42 plasma collected from humanized DR4 HLA transgenic mice immunized with the indicated PEGylated chimeric pig-baboon uricase variants or PEGylated wild type chimeric pig-baboon uricase (WT). There were 10 mice in each group, and each data point is presented as mean ± SEM. Abs = absorbance.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0059] FIG. 6C is a graph showing the areas under the plasma dose-response curves (AUCs) of FIG.6B as a summary measure of antidrug immune responses with the indicated PEGylated chimeric pig-baboon uricase variants or PEGylated wild type chimeric pig-baboon uricase (WT).

[0060] FIG. 7A is a schematic of the study design for assessing the efficacy of repeat dosing of PEGylated chimeric pig-baboon uricase variant candidates HF0536, HF0554, and HF0752 relative to PEGylated wild type chimeric pig-baboon uricase (WT) in humanized HLA transgenic mouse strain DR4.

[0061] FIG.7B is a graph showing serum uric acid levels in humanized DR4 HLA transgenic mice at various weeks of receiving either no treatment (No Rx), or receiving weekly repeat dose of one of PEGylated chimeric pig-baboon uricase variant candidates HF0536, HF0554, and HF0752, or PEGylated wild type chimeric pig-baboon uricase (WT). There were 9 mice in each group, and each data point is presented as mean ± SEM.

[0062] FIG. 7C is a graph showing the percentages of humanized DR4 HLA transgenic mice whose serum uric acid levels were 0 at various weeks of receiving either no treatment (No Rx) or receiving weekly repeat dose of one of PEGylated chimeric pig-baboon uricase variant candidates HF0536, HF0554, and HF0752, or PEGylated wild type chimeric pig-baboon uricase (WT). There were 9 mice in each group.

[0063] FIG. 8 is a schematic of the PBMC (peripheral blood mononuclear cell) assay used to measure CD4+ T-cell activation and epitopes for the non-PEGylated top six deimmunized chimeric pig-baboon uricase variant candidates as well as the non-PEGylated wild type chimeric pig-baboon uricase (WT).

[0064] FIG. 9 is a schematic of the workflow for the MHC-associated peptide proteomics (MAPPs) assay used to identify peptides derived from the non-PEGylated top six chimeric pig- baboon uricase variant candidates or the non-PEGylated wild type chimeric pig-baboon uricase (WT) and presented on HLA II monocyte derived dendritic cells.

[0065] FIG. 10 is a graph showing CD4+ T cell epitope mapping of the wild type chimeric pig-baboon uricase protein by the PBMC assay.

[0066] FIG. 11 is a graph showing MAPPs analysis results of the wild type chimeric pig- baboon uricase protein.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0067] FIG. 12 is a graph showing CD4+ T cell epitope mapping for the deimmunized uricase variants as compared to the wild type (WT) uricase by the PBMC assay.

[0068] FIG.13 is a graph showing CD4+ T cell responses to WT uricase compared with the deimmunized variants by the PBMC assay.

[0069] FIG.14 is graph showing the results of MAPPs analysis of the wild type uricase protein compared with the deimmunized variants.

[0070] FIG.15A is a graph showing plasma concentrations of the endogenous uricase as well as uric acid in untreated control Wistar rats (n = 3) at the corresponding pre-dose timepoint (at time 0) and at various corresponding post-dose timepoints up to 96 h in the pharmacokinetic (PK) study according to Example 5. Each data point is presented as mean ± SD.

[0071] FIG. 15B is a graph showing plasma concentrations of pegloticase and uric acid pre- dose (at time 0) and at various timepoints up to 96 h post-dose in Wistar rats (n = 3) intravenously administrated with a single dose of 1 mg / kg body weight pegloticase in the PK study according to Example 5. Each data point is presented as mean ± SD.

[0072] FIG.15C is a graph showing plasma concentrations of WT-CPAS20h and uric acid pre- dose (at time 0) and at various timepoints up to 96 h post-dose in Wistar rats (n = 3) intravenously administrated with a single dose of 1.99 mg / kg body weight WT-CPAS20h in the PK study according to Example 5. Each data point is presented as mean ± SD.

[0073] FIG.15D is a graph showing plasma concentrations of HF752-CPAS20h and uric acid pre-dose (at time 0) and at various timepoints up to 96 h post-dose in Wistar rats (n = 3) intravenously administrated with a single dose of 1.99 mg / kg body weight HF752-CPAS20h in the PK study according to Example 5. Each data point is presented as mean ± SD.

[0074] FIG. 15E is a graph showing plasma concentrations of PEGylated HF752 uricase variant and uric acid pre-dose (at time 0) and at various timepoints up to 96 h post-dose in Wistar rats (n = 3) intravenously administrated with a single dose of 1 mg / kg body weight PEGylated HF752 uricase variant in the PK study according to Example 5. Each data point is presented as mean ± SD. DETAILED DESCRIPTION OF THE INVENTION

[0075] Humans and higher primates lack uricase, the enzyme capable of oxidizing uric acid. As a result, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit, and can result in the crystallization of uric acid in joints. AcuteAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Nyborg et al., PLOS One, 2016, DOI:10.1371 / journal.pone.0167935, incorporated herein by reference in its entirety.

[0076] The U.S. Food and Drug Administration (FDA) has approved KRYSTEXXA® (pegloticase) for use against chronic refractory gout (Sundy et al., Jama, 2011, 306(7): p.711- 20). Pegloticase is a homotetrameric protein and each monomer is a single polypeptide chain comprised of the pig-baboon chimeric uricase having the amino acid sequence of SEQ ID NO:1. The homotetramer is hyper-PEGylated via random lysine conjugation to reduce immunogenicity and extend half-life. Because pegloticase is non-human in nature, it evokes strong immune responses, resulting in black box warnings, along with fairly rapid loss of therapeutic efficacy for many pegloticase patients due to the problems resulting from the development of antidrug 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 ADAs to pegloticase, highlighting a need for more effective uricase-based therapies. In general, the clinical complications associated with immunogenicity drastically reduce the utility of uricases.

[0077] Aspects of the present disclosure relate primarily to a recombinant uricase variant protein comprising one or more amino acid mutations in one or more T-cell epitopes such that the T-cell epitopes in the uricase variant protein are depleted, as compared to a counterpart uricase protein, e.g., a wild type (WT) uricase protein. Such uricase protein variants are referred to herein in some instances, as a “deimmunized uricase variant” or a “T cell epitope depleted” uricase variant. The use of such variants and methods of manufacturing the variants are also described herein.

[0078] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0079] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the present application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, representative methods and materials are herein described.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0080] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a carrier” includes mixtures of one or more carriers, two or more carriers, and the like and reference to “the method” includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0081] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the present application. Generally, the term “about,” as used herein in references to a measurable value such as an amount of weight, time, dose, etc. is meant to encompass values within an acceptable degree of variability in the art. In some embodiments, degree of variability is based on FDA guidelines.

[0082] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0083] The term “substantial identity” or “substantially identical,” as used in the context of protein sequences, refers to a sequence that has at least about 60% sequence identity to a reference sequence. Alternatively, percent identity can be any integer from about 60% to about 100%. Exemplary embodiments include 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% sequence identity, as compared to a reference sequence using the programs described herein; e.g., BLAST, using standard parameters, as described below.

[0084] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0085] Algorithms that are suitable 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 analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1997) Nucleic Acids Res.25: 3389-3402). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. For amino acid sequences, cumulative scores are calculated using a scoring matrix. Extension of the word hits in each direction is halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or 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 as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff. (1992). Proc. Natl. Acad. Sci. USA 89:10915-10919).

[0086] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.

[0087] As used herein, a “counterpart uricase protein” or “counterpart uricase” refers to a uricase protein that is compared to one of the uricase protein variants described herein, e.g., either through a primary sequence comparison or functional comparison. In one embodiment, a counterpart uricase protein does not comprise a mutation(s) in one or more T-cell epitopes. In some embodiments, the counterpart uricase protein is a WT uricase protein, for example, a naturally occurring mammalian uricase protein, such as a wild type dog, porcine, bovine, ovine or baboon uricase. In some embodiments, the counterpart uricase protein is a non-naturally occurring, or recombinant, uricase protein, such as a humanized uricase, e.g., a humanizedAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 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 the present disclosure, a chimeric pig-baboon uricase protein comprising the amino acid sequence of SEQ ID NO:1 serves as a counterpart enzyme for the exemplary uricase variants and amino acid mutations disclosed herein and, for ease of description, is referred to as the wild type (WT) chimeric pig-baboon uricase.

[0088] In one embodiment, the counterpart uricase protein is the WT chimeric pig-baboon uricase comprising the amino acid sequence of SEQ ID NO:1. In another embodiment, the counterpart uricase protein comprises an amino acid sequence that is substantially identical to SEQ ID NO:1. In accordance with the definition for “substantially identical” set forth herein, in one embodiment, the counterpart uricase protein comprises 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 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. In yet another embodiment, the counterpart uricase protein comprises an amino acid sequence that is from about 90% to about 100% identical to SEQ ID NO:1.

[0089] In a further embodiment, the counterpart uricase protein is a chimeric pig-baboon uricase comprising 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. One exemplary embodiment of such a counterpart uricase protein is a chimeric pig-baboon uricase comprising a different segment of the porcine uricase, a different segment of the baboon uricase, or both, as compared to the wild type chimeric pig-baboon uricase of SEQ ID NO:1. In a particular embodiment, the counterpart uricase protein is a chimeric pig-baboon uricase comprising the amino acid sequence of SEQ ID NO:4, which comprises amino acids (aa) 1–266 of the porcine uricase of SEQ ID NO:2 and aa 267–304 of the baboon uricase of SEQ ID NO:3. By comparison, the wild type chimeric pig-baboon uricase of SEQ ID NO:1 comprises aa 8-266 of the porcine uricase of SEQ ID NO:2 and aa 267–304 of the baboon uricase of SEQ ID NO:3. In another particular embodiment, the counterpart uricase protein is a chimeric pig-baboon uricase comprising the amino acid sequence of SEQ ID NO:5, which comprises aa 1–220 of the porcine uricase of SEQ ID NO:2 and aa 221–304 of the baboon uricase of SEQ ID NO:3.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0090] Other exemplary embodiments of a counterpart uricase protein comprising an amino acid sequence that is substantially identical to SEQ ID NO:1 include, but are not limited to, (1) mammalian uricases, e.g., a dog uricase whose amino acid sequence (with the NCBI reference number of NP_001011886.1, incorporated herein by reference in its entirety) is 93% identical to SEQ ID NO:1 determined by the protein-protein BLAST algorithm (BLASTP); a porcine uricase of SEQ ID NO:2 that is 99% identical to SEQ ID NO:1 determined by BLASTP; a bovine uricase whose amino acid sequence (with the NCBI reference number of NP_001069116.1, incorporated herein by reference in its entirety) is 91% identical to SEQ ID NO:1 determined by BLASTP; an ovine uricase whose amino acid sequence (with the NCBI reference number of XP_004002186.4, incorporated herein by reference in its entirety) is 90% identical to SEQ ID NO:1 determined by BLASTP; and a baboon uricase of SEQ ID NO:3 that is 91% identical to SEQ ID NO:1 determined by BLASTP; and (2) a chimeric uricase comprising two or more mammalian uricase domains, e.g., a chimeric pig-baboon uricase (described above), a chimeric dog-baboon uricase, and a chimeric pig-bovine uricase.

[0091] In one embodiment, the counterpart uricase protein is a truncated uricase, i.e., at either the C-terminus or N-terminus. In one embodiment, the counterpart uricase protein is a truncated uricase obtained by truncating a corresponding full-length uricase at either the C- terminus or N-terminus. In another embodiment, the counterpart uricase protein is a truncated uricase obtained by further truncating at either the C-terminus or N-terminus a corresponding uricase that has previously been truncated to a lesser extent. In a further embodiment, the truncated uricase is a truncated chimeric pig-baboon uricase. Accordingly, in one embodiment of a uricase variant of the present disclosure, the variant further comprises an amino acid truncation, as compared to a counterpart uricase protein without the corresponding amino acid truncation, in addition to the amino acid mutation(s) disclosed herein. The amino acid truncation, in one embodiment, is at or around the N- and / or C- terminus of the counterpart uricase protein. In one embodiment, the amino acid truncation is at the N-terminus of the counterpart uricase protein. In a further embodiment, the amino acid truncation begins at position 1, 2, 3, 4, 5 or 6 of the counterpart uricase protein. In another embodiment, the amino acid truncation comprises 4-13 amino acids at the N-terminus of the counterpart uricase protein. In another embodiment, the amino acid truncation comprises 4-13 amino acids at the C-terminus of the counterpart uricase protein. In another embodiment, the amino acid truncation comprises 4-13 amino acids at the C-terminus and 4-13 amino acids at the N- terminus of a counterpart uricase protein. In even another embodiment, the amino acidAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application truncation comprises 6 amino acids at the N-terminus of a counterpart uricase protein. In yet another embodiment, the amino acid truncation comprises 6 amino acids at the C-terminus of a counterpart uricase protein. In yet even another embodiment, the amino acid truncation comprises 6 amino acids at the C-terminus and 6 amino acids at the N-terminus of a counterpart uricase protein.

[0092] “Operably linked” refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. In the case of a promoter, a promoter that is operably linked to a coding sequence affects the expression of a coding sequence. The promoter or other control elements need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. For example, intervening untranslated yet transcribed sequences can be present 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 different domains of a uricase conjugate described herein, the domains can be operably linked in a single DNA sequence to allow for expression of the uricase fusion protein.

[0093] The term “pharmaceutically acceptable,” unless otherwise noted, is used to characterize a moiety (e.g., a salt, dosage form, or excipient) as being appropriate for use in accordance with sound medical judgment. In general, a pharmaceutically acceptable moiety has one or more benefits that outweigh any deleterious effect that the moiety may have. Deleterious effects may include, for example, excessive toxicity, irritation, allergic response, and other problems and complications.

[0094] “Recombinant protein,” as used herein, refers to any artificially produced protein and is distinguished from naturally produced proteins (i.e., proteins are produced in tissues of an animal that possesses only the natural gene for the specific protein of interest). As described herein, a recombinant uricase fusion protein comprises the first (uricase) domain bonded via a peptide bond (directly or through an amino acid linker) to one or more random coil polypeptide domains.

[0095] A “T-cell epitope” refers to a peptide epitope that binds to an MHCII molecule and subsequently forms a ternary complex with a cognate T-cell receptor (TCR). As used herein, one or more T-cell epitopes are “depleted” from a uricase variant if a peptide originating from the uricase variant is unable to bind an MHCII molecule, or if the peptide binds the MHCIIAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application molecule but subsequently does not bind a TCR, e.g., because of the lack of affinity to the MHCII molecule.

[0096] The terms “variant protein,” and “protein variant” are terms of the art, and used interchangeably herein. Each refers to a protein that is distinguished from a counterpart protein, such as the WT form of the protein, on the basis of the presence of one or more amino acid modifications, such as, for example, one or more amino acid substitutions, insertions, deletions, or a combination thereof. The term “mutant gene” is a term of the art and refers to a gene that is distinguished from the WT form of the gene on the basis of the presence of one or more nucleic acid modifications, such as, for example, one or more nucleic acid substitutions, insertions, deletions, or a combination thereof. In some embodiments, a mutant gene encodes a variant protein. However, a mutation in a gene may also be a silent mutation, i.e., a mutation that does not affect the amino acid sequence in the protein that is encoded by the gene. A mutant / variant may occur in nature or may be an engineered mutant / variant.

[0097] As used herein, the term “wild type” (abbreviated “WT”), unless otherwise specified, refers to the most prevalent form of an organism, strain, gene, protein, or characteristic as it occurs in nature and / or is distinguished from mutant or variant forms.

[0098] An amino acid modification may be an amino acid substitution, amino acid deletion and / or amino acid insertion. An amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. An amino acid substitution at a specific position on the protein sequence is denoted herein in the following manner: “one letter code of the WT amino acid residue -amino acid position- one letter code of the amino acid residue that replaces this WT residue”. For example, a uricase variant which has an amino acid substitution of W22L refers to a uricase variant protein in which the wild type residue at the 22ndposition (W or tryptophan) is replaced by L or leucine.

[0099] The designation of all amino acid positions of a uricase variant in the disclosure and the appended claims is with respect to the wild type chimeric pig-baboon uricase enzyme amino acid sequence of SEQ ID NO:1 (monomer). It will be understood by those skilled in the art that the corresponding amino acid positions in the amino acid sequences of other counterpart uricase proteins can be readily identified by aligning the amino acid sequence of one of the other counterpart uricase proteins with SEQ ID NO:1 using the BLAST, BLAST 2.0, or BLASTP algorithms described above, or according to other similar methods or algorithms known to those of skill in the art.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0100] The present disclosure provides in part, a uricase protein variant (e.g., a recombinant uricase protein variant), which is an engineered variant of a counterpart uricase protein, e.g., a WT uricase protein. The uricase variant, in embodiments described herein, is deimmunized compared to a counterpart uricase protein, e.g., because the variant is T-cell epitope depleted compared to a counterpart uricase protein. In one embodiment, the uricase variant is a variant of the 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 comprising an amino acid sequence that is substantially identical to SEQ ID NO:1 described above.

[0101] It is noted that the amino acid positions and mutations described herein are provided for uricase protein monomers. However, embodiments of the invention are also directed to multimeric, e.g., dimeric and tetrameric, forms of uricase. In the case of homodimers and homotetratmers, the amino acid mutations set forth for the monomeric protein will also be present in the remaining protein subunits. However, in embodiments where a heterodimeric and / or heterotetrameric uricase variant is provided, mutations in one subunit may or may not be present in the remaining uricase monomer subunits.

[0102] In one aspect of a uricase variant provided herein, a chimeric pig-baboon uricase variant is provided. Pegloticase is a homotetrameric protein and each monomer is a single polypeptide chain comprised of the pig-baboon chimeric uricase having the amino acid sequence of SEQ ID NO:1. The homotetramer is hyper-PEGylated via random lysine conjugation to reduce immunogenicity and extend half-life. As discussed in further detail herein, a uricase variant of the present invention in one embodiment, is PEGylated. In another embodiment, a uricase variant of the present invention is PASylated. In yet another embodiment, the uricase variant is PASylated and PEGylated. In yet another embodiment, a uricase variant of the present invention is XTENylated. In one embodiment, the chimeric pig- baboon uricase variant is T-cell epitope depleted, compared to a counterpart uricase (e.g., the WT chimeric-pig baboon uricase of SEQ ID NO:1 or a chimeric-pig baboon uricase comprising an amino acid sequence that is substantially identical to SEQ ID NO:1). In a further embodiment, the chimeric pig-baboon uricase variant has substantially the same stability and / or enzymatic activity as the counterpart uricase protein. The uricase variant provided herein, in one embodiment, is a monomer. In another embodiment, the uricase variant is provided as a homodimer or a homotetramer. In a preferred embodiment, the uricase variant is a homotetramer.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0103] In some embodiments of a uricase variant provided herein, the uricase variant comprises one or more amino acid mutations relative to a counterpart uricase protein, wherein the one or more amino acid mutations are at one or more amino acid positions 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 a further embodiment, the one or more amino acid mutations are amino acid substitutions. In a further embodiment, the amino acid substitutions correspond to one or more of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising the amino acid sequence of SEQ ID NO:1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins comprising an amino acid sequence that is substantially identical to SEQ ID NO:1 (nonlimiting examples provided above).

[0104] In one embodiment, the mutation(s) at one or more of the aforementioned amino acid positions eliminates one or more dominant immunogenic T cell epitopes present in the counterpart uricase protein such that the variant is T cell epitope depleted compared to the counterpart uricase protein. Mutations of the present disclosure include, but are not limited to, amino acid exchange(s), insertion(s), deletion(s), addition(s), substitution(s), inversion(s) and / or duplication(s). These mutations / modification(s) also include conservative and / or homologous amino acid exchange(s). In a preferred embodiment of the invention, the mutation(s) is an amino acid substitution.

[0105] In one embodiment, the uricase variant comprises 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, or about 20 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are selected from mutations at amino acid positions corresponding to 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, 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, or about 20 amino acid substitutions of SEQ ID NO:1 set forth in Table A.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0106] In another embodiment, the uricase variant comprises from about 5 to about 25 amino acid mutations, from about 5 to about 20 amino acid mutations, from about 10 to about 20 amino acid mutations, from about 10 to about 18 amino acid mutations, from about 10 to about 16 amino acid mutations, from about 10 to about 14 amino acid mutations, from about 10 to about 13 amino acid mutations, from about 10 to about 12 amino acid mutations, from about 11 to about 20 amino acid mutations, from about 11 to about 13 amino acid mutations, from about 12 to about 14 amino acid mutations, from about 13 to about 20 amino acid mutations, from about 14 to about 20 amino acid mutations, or from about 15 to about 20 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are selected from mutations at amino acid positions corresponding to 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the amino acid substitutions correspond to from about 5 to about 25, from about 5 to about 20, from about 10 to about 20, from about 10 to about 18, from about 10 to about 16, from about 10 to about 14, from about 10 to about 13, from about 10 to about 12, from about 11 to about 20, from about 11 to about 13, from about 12 to about 14, from about 13 to about 20, from about 14 to about 20, or from about 15 to about 20, amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0107] In one embodiment, the uricase variant comprises from about 10 to about 13 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 13 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 a further embodiment, each of the mutations isAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application an amino acid substitution. In a further embodiment, the from about 10 to about 13 amino acid substitutions correspond to from about 10 to about 13 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0108] In one embodiment, the uricase variant comprises from about 10 to about 14 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 14 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the from about 10 to about 14 amino acid substitutions correspond to from about 10 to about 14 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0109] In one embodiment, the uricase variant comprises from about 10 to about 15 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 15 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the from about 10 to about 15 amino acid substitutions correspond to from about 10 to about 15 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0110] In one embodiment, the uricase variant comprises from about 10 to about 16 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 16 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the from about 10 to about 16 amino acid substitutions correspond to from about 10 to about 16 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0111] In one embodiment, the uricase variant comprises from about 10 to about 17 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 17 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the from about 10 to about 17 amino acid substitutions correspond to from about 10 to about 17 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0112] In one embodiment, the uricase variant comprises from about 10 to about 18 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 18 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the from about 10 to about 18 amino acidAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application substitutions correspond to from about 10 to about 18 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0113] In one embodiment, the uricase variant comprises from about 10 to about 19 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 19 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the from about 10 to about 19 amino acid substitutions correspond to from about 10 to about 19 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0114] In one embodiment, the uricase variant comprises from about 10 to about 20 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 20 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the from about 10 to about 20 amino acid substitutions correspond to from about 10 to about 20 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0115] In one embodiment, the uricase variant comprises from about 11 to about 13 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 11 to about 13 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the from about 11 to about 13 amino acid substitutions correspond to from about 11 to about 13 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0116] In one embodiment, the uricase variant comprises from about 12 to about 14 amino acid mutations relative to a counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 12 to about 14 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 a further embodiment, each of the mutations is an amino acid substitution. In a further embodiment, the from about 12 to about 14 amino acid substitutions correspond to from about 12 to about 14 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to the amino acid sequence of SEQ ID NO:1.

[0117] In one embodiment, the uricase variant comprises one or more amino acid substitutions relative to a counterpart uricase protein, wherein the one or more amino acid substitutions correspond to one or more of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table A. Exemplary Amino Acid Substitutions of SEQ ID NO:1

[0118] In one embodiment, the uricase variant comprises amino acid substitutions relative to a counterpart uricase protein, wherein 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, from about 5 to about 25, from about 5 to about 20, from about 10 to about 20, from about 10 to about 18, from about 10 to about 16, from about 10 to about 14, from about 10 to about 13, from about 10 to about 12, from about 11 to about 20, from about 11 to about 13, from about 12 to about 14, from about 13 to about 20, from about 14 to about 20, or from about 15 to about 20 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to fromAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application about 10 to about 13 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to from about 10 to about 14 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to from about 10 to about 15 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to from about 10 to about 16 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to from about 10 to about 17 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to from about 10 to about 18 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to from about 10 to about 19 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to from about 10 to about 20 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to from about 11 to about 13 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In another embodiment, the amino acid substitutions correspond to from about 12 to about 14 of the amino acid substitutions of SEQ ID NO:1 set forth in Table A. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0119] In one embodiment, the uricase variant comprises amino acid substitutions relative to a counterpart uricase protein, wherein 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, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W and I257K. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution 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, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution 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, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F and I257K. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution 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, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W and I257K. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution 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 a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution 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 a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of SEQ ID NO:1. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0120] In some embodiments, the uricase variant comprises amino acid substitutions relative to a counterpart uricase protein, wherein the amino acid substitutions correspond to the aminoAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application acid substitutions of SEQ ID NO:1 comprising one of the exemplary groups of amino acid substitutions set forth in Table B. In one embodiment, the uricase variant comprises one amino acid substitution corresponding to one amino acid substitution selected from V22L, F75L, and Y127H set forth in an exemplary group of corresponding amino acid substitutions of SEQ ID NO:1 of Table B. In another embodiment, the uricase variant comprises two amino acid substitutions corresponding to a combination of two amino acid substitutions selected from V22L, F75L, and Y127H set forth in an exemplary group of corresponding amino acid substitutions of SEQ ID NO:1 of Table B. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of SEQ ID NO:1. T22.V22L, F75L, K116L, Y127HAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table B. Exemplary Groups of Amino Acid Substitutions of SEQ ID NO:156.V22L, F75L, K116M, Y127H, R209FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table B. Exemplary Groups of Amino Acid Substitutions of SEQ ID NO:190.V22L, Q26R, F75L, K76S, Y127H, K155G, I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table B. Exemplary Groups of Amino Acid Substitutions of SEQ ID NO:1124.V22L, Q26R, S33D, F75L, K79G, A102V, K116M, Y127H, R209F, I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table B. Exemplary Groups of Amino Acid Substitutions of SEQ ID NO:1

[0121] In one embodiment, the uricase variant comprises a plurality of amino acid mutations relative to a counterpart uricase protein, wherein the plurality of amino acid mutations are at amino acid positions corresponding to one of the following combinations of amino acid residues of SEQ ID NO:1:

[0122] (1a) V22, D28, S33, N70, F75, K79, A102, Y127, K155, R209, and I257;

[0123] (2a) V22, D28, S33, N70, F75, K79, A102, F114, V119, Y127, K155, R209, and I257;

[0124] (3a) V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257;

[0125] (4a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257;

[0126] (5a) V22, Q26, Y31, N70, F75, K79, A102, Y127, K155, R209, and I257;

[0127] (6a) V22, D28, Y31, N70, F75, K79, A102, K112, Y127, K155, R209, and I257;

[0128] (7a) V22, Q26, S33, N70, F75, K79, A102, K112, Y127, K155, R209, and I257;

[0129] (8a) V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257;

[0130] (9a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257;

[0131] (10a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257;

[0132] (11a) V22, Q26, S33, N70, F75, K79, A102, K112, Y127, K155, R209, and I257;Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0133] (12a) V22, D28, Y31, N70, F75, K79, A102, V106, K116, Y127, K155, R209, and I257;

[0134] (13a) V22, Q26, Y31, N70, F75, K76, A102, Y127, K155, R209, and I257;

[0135] (14a) V22, Q26, S33, N70, F75, K76, A102, V106, K116, Y127, L154, R209, and I257;

[0136] (15a) V22, D28, S33, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257;

[0137] (16a) V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257;

[0138] (17a) V22, Q26, S33, N70, F75, K76, A102, K112, Y127, K155, R209, and I257;

[0139] (18a) V22, D28, Y31, N70, F75, K79, A102, V106, K116, Y127, K155, R209, and I257;

[0140] (19a) V22, D28, Y31, N70, F75, K79, A102, K116, Y127, K155, R209, and I257;

[0141] (20a) V22, Q26, S33, N70, F75, K76, A102, K116, V119, Y127, K155, R209, and I257; and

[0142] (21a) V22, Q26, S33, N70, F75, K79, A102, F114, V119, Y127, K155, R209, and I257.

[0143] In one embodiment, the counterpart uricase protein is the wild type chimeric pig- baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1. In one embodiment, the uricase variant comprises an amino acid mutation at one amino acid position corresponding to one amino acid residue selected from V22, F75, and Y127 set forth in one of the combinations of corresponding amino acid residues of SEQ ID NO:1 of (1a) to (21a) above. In another embodiment, the uricase variant comprises two amino acid mutations at two amino acid positions corresponding to a combination of two amino acid residues selected from V22, F75, and Y127 set forth in one of the combinations of corresponding amino acid residues of SEQ ID NO:1 of (1a) to (21a) above.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0144] In one embodiment, each of the amino acid mutations is an amino acid substitution. In a further embodiment, the amino acid substitutions comprise / correspond to one of the following combinations of amino acid substitutions of SEQ ID NO:1:

[0145] (1b) V22L, D28H, S33D, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K;

[0146] (2b) V22L, D28H, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209W, and I257K;

[0147] (3b) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209W, and I257T;

[0148] (4b) V22L, D28T, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257T;

[0149] (5b) V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K;

[0150] (6b) V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K;

[0151] (7b) V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F, and I257K;

[0152] (8b) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209F, and I257T;

[0153] (9b) V22L, D28H, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209F, and I257T;

[0154] (10b) V22L, D28T, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209F, and I257T;

[0155] (11b) V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K;

[0156] (12b) V22L, D28N, Y31H, N70L, F75L, K79G, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K;

[0157] (13b) V22L, Q26R, Y31H, N70L, F75L, K76S, A102L, Y127H, K155G, R209F, and I257K;Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0158] (14b) V22L, Q26R, S33D, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, L154A, R209W, and I257T;

[0159] (15b) V22L, D28T, S33D, N70L, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209F, and I257K;

[0160] (16b) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K;

[0161] (17b) V22L, Q26R, S33D, N70H, F75L, K76S, A102V, K112S, Y127H, K155G, R209F, and I257K;

[0162] (18b) V22L, D28N, Y31H, N70L, F75L, K79G, A102L, V106M, K116M, Y127H, K155G, R209F, and I257K;

[0163] (19b) V22L, D28H, Y31H, N70L, F75L, K79G, A102L, K116L, Y127H, K155G, R209F, and I257K.

[0164] (20b) V22L, Q26R, S33D, N70L, F75L, K76S, A102L, K116M, V119T, Y127H, K155G, R209W, and I257K; and

[0165] (21b) V22L, Q26R, S33D, N70L, F75L, K79G, A102L, F114L, V119T, Y127H, K155G, R209F, and I257K.

[0166] In one embodiment, the uricase variant comprises one amino acid substitution corresponding to one amino acid substitution selected from V22L, F75L, and Y127H set forth in one of the combinations of corresponding amino acid substitutions of SEQ ID NO:1 of (1b) to (21b) above. In another embodiment, the uricase variant comprises two amino acid substitutions corresponding to a combination of two amino acid substitutions selected from V22L, F75L, and Y127H set forth in one of the combinations of corresponding amino acid substitutions of SEQ ID NO:1 of (1b) to (21b) above. In some embodiments, the uricase variants (1b) to (21b) may further comprise an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In other embodiments, the uricase variants (1b) to (21b) may further comprise an amino acid substitution corresponding to N117G or N117D of SEQ ID NO:1.

[0167] In one embodiment, the amino acid substitutions in a uricase variant of the present disclosure comprise / 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 comprises an amino acidAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, 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 a uricase variant of the present disclosure comprise / 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 comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, 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 a uricase variant of the present disclosure comprise / 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 comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, 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 a uricase variant of the present disclosure comprise / correspond to the following amino acid substitutions of 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 comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, 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 a uricase variant of the present disclosure comprise / 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 a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, 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 a uricase variant of the present disclosure comprise / 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 a further embodiment, the uricase variant further comprises an amino acid substitution corresponding to N117S, N117H, N117G, N117D, or N117Q of SEQ ID NO:1. In a further embodiment, theAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application uricase variant further comprises an amino acid substitution corresponding to N117G or N117D of SEQ ID NO:1. In one embodiment, the counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0168] In one embodiment, the uricase variant comprises one or more conservative amino acid mutations relative to a counterpart uricase protein, wherein the one or more conservative amino acid mutations are at one or more amino acid positions 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 another embodiment, the one or more conservative amino acid mutations are 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 the wild type chimeric pig-baboon uricase comprising 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 that comprises an amino acid sequence that is substantially identical to SEQ ID NO:1. In one embodiment, the one or more conservative amino acid mutations are conservative amino acid substitutions.

[0169] A conservative amino acid substitution is defined as a substitution of amino acids with generally similar properties (e.g., acidic, basic, aromatic, size, positively or negatively charged, polar, non-polar). Exemplary conservative substitutions that may be performed are among the groups of amino acids as follows:

[0170] (i) glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I);

[0171] (ii) aspartic acid (D) and glutamic acid (E);

[0172] (iii) alanine (A), serine (S) and threonine (T);

[0173] (iv) histidine (H), lysine (K) and arginine (R);

[0174] (v) asparagine (N) and glutamine (Q);

[0175] (vi) phenylalanine (F), tyrosine (Y) and tryptophan (W).Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0176] In embodiments described herein, the uricase variant having one or more conservative substitutions retains the structural stability of the counterpart uricase enzyme from which the variant is derived, i.e., a counterpart uricase variant or counterpart wild type uricase, and is enzymatically active even though its protein and DNA sequences are not the same as those of the counterpart uricase without the conservative substitutions.

[0177] In one embodiment, a uricase variant comprising at least one conservative mutation has the same uricase activity as does a uricase variant without such a mutation. In another embodiment, a uricase variant comprising at least one conservative mutation has substantially the same uricase activity, within 5% of the activity, within 10% of the activity, or within 30% of the activity of a uricase variant without such a mutation.

[0178] In one embodiment, the uricase variant of the disclosure is a humanized uricase variant. An exemplary method of generating humanized uricase enzymes is disclosed in U.S. Patent No. 8,586,535, the disclosure of which is incorporated by reference in its entirety. In one embodiment, the humanized uricase variant comprises one or more humanizing amino acid mutations relative to a non-human counterpart uricase protein, wherein the one or more humanizing amino acid mutations are at one or more amino acid positions 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 another embodiment, the one or more humanizing amino acid mutations are 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 the wild type chimeric pig-baboon uricase comprising the amino acid sequence of SEQ ID NO:1. In another embodiment, the counterpart uricase protein is one of the counterpart uricase proteins described above that comprise an amino acid sequence that is substantially identical to SEQ ID NO:1.

[0179] In one embodiment, the humanizing mutations are amino acid substitutions with the corresponding or homologous amino acid sequence in the non-active human uricase such that the variant retains uricase activity while improving the homology with the human uricase, thus reducing immunogenicity. In one embodiment, the humanized uricase variant comprises one or more humanizing amino acid mutations relative to a chimeric pig-baboon uricase, e.g., the wild type chimeric pig-baboon uricase comprising the amino acid sequence of SEQ ID NO:1,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application or one of the chimeric-pig baboon uricase described above that comprises an amino acid sequence that is substantially identical to SEQ ID NO:1. In the humanized uricase variant, a humanizing mutation can be a substitution(s) of the amino acid sequence in the porcine uricase segment, the baboon uricase segment, or both segments of the counterpart chimeric pig-baboon uricase protein.

[0180] In some embodiments of a uricase variant 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.

[0181] In one embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs:25-154. In a further embodiment, the uricase variant comprises 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 still a further embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs:45, 53, 66, 114, 129, and 149.

[0182] The present disclosure also provides a uricase variant 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:25-154. In a further 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 an amino acid sequence selected from 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 even a further 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 an amino acid sequence selected from SEQ ID NOs:45, 53, 66, 114, 129, and 149.

[0183] In one embodiment, the uricase variant comprises an amino acid sequence of SEQ ID NO:53. 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:53. Exemplary uricase variants include those comprising an amino acid sequence selected from SEQ ID NOs:197-309.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0184] In one embodiment, the uricase variant comprises an 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.

[0185] In one embodiment, the uricase variant comprises an amino acid sequence of SEQ ID NO:187.

[0186] In one embodiment, the uricase variant comprises an amino acid sequence of SEQ ID NO:191.

[0187] In a one embodiment, the uricase variant comprises 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.

[0188] 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.

[0189] 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.

[0190] In one embodiment, the uricase variant comprises an amino acid sequence selected from SEQ ID NOs:1475-1566.

[0191] In some embodiments of a uricase variant provided herein, the uricase variant comprises a first set of amino acid substitutions and a second set of amino acid substitutions relative to a counterpart uricase protein. The first set of amino acid substitutions corresponds to the amino acid substitutions of SEQ ID NO:1 set forth in group (a) or group (b) below:

[0192] (a) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K; or

[0193] (b) V22L, Q26T, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0194] The second set of amino acid substitutions is a set of from about 6 to about 10 (e.g., 6, 7, 8, 9, or 10) amino acid substitutions selected from 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, 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 counterpart uricase protein is the wild type chimeric pig-baboon uricase comprising 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, comprising an amino acid sequence that is substantially identical to SEQ ID NO:1. In some embodiments, the uricase variant further comprises one or more conservative amino acid substitutions and / or humanizing 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.

[0195] In some embodiments, the first set of amino acid substitutions of the uricase variant corresponds to the amino acid substitutions of SEQ ID NO:1 set forth in group (a): V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K. In some embodiments, the first set of amino acid substitutions of the uricase variant corresponds to the amino acid substitutions of SEQ ID NO:1 set forth in group (b): V22L, Q26T, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K.

[0196] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 10 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: one of K30E and K30F, L52T, H53D, T83E, V86N, S94D, K97D, one of N117G and N117D, F172W, T174N, R192T, G196D, R197D, Y225H, S228G, L239G, L279T, and N281Y. In one embodiment, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 7 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of the uricase variant is a set of from about 8 to about 10 amino acid substitutions.

[0197] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 10 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: one of K30E and K30F, L52T, T83E, V86N, S94D, K97D, one of N117G and N117D, F172W,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application R192T, G196D, R197D, Y225H, S228G, L279T, and N281Y. In one embodiment, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 7 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of the uricase variant is a set of from about 8 to about 10 amino acid substitutions.

[0198] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 10 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: K30E, L52T, T83E, S94D, one of N117G and N117D, F172W, R192T, G196D, R197D, Y225H, S228G, L279T, and N281Y. In one embodiment, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 7 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of the uricase variant is a set of from about 8 to about 10 amino acid substitutions.

[0199] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 10 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: K30E, L52T, T83E, one of N117G and N117D, F172W, R192T, G196D, R197D, S228G, L279T, and N281Y. In one embodiment, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 7 amino acid substitutions. In another embodiment, the second set of amino acid substitutions of the uricase variant is a set of from about 8 to about 10 amino acid substitutions.

[0200] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 9 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: K30E, L52T, T83E, one of N117G and N117D, F172W, G196D, R197D, S228G, L279T, and N281Y.

[0201] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 8 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: K30E, L52T, T83E, N117G, F172W, G196D, S228G, L279T, and N281Y.

[0202] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 8 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: K30E, L52T, T83E, N117D, F172W, G196D, S228G, L279T, and N281Y.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0203] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 8 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: K30E, L52T, T83E, F172W, G196D, S228G, L279T, and N281Y.

[0204] In some embodiments, the second set of amino acid substitutions of the uricase variant is a set of from about 6 to about 7 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: K30E, L52T, T83E, F172W, G196D, S228G, L279T, and N281Y.

[0205] In some embodiments, a uricase variant of the present disclosure elicits less than about 90%, less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% of the immune response (e.g., as measured by anti-uricase antibody titers) elicited by a non- deimmunized counterpart uricase on which the uricase variant is based. In a further embodiment, the uricase variant and the counterpart uricase are homotetramers.

[0206] In some embodiments, a uricase variant of the present disclosure possesses 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%, at least about 90% (e.g., about 95%, about 97%, about 99% or more) of the enzymatic activity of a non-deimmunized counterpart uricase on which the uricase variant is based. In a further embodiment, the uricase variant and the counterpart uricase are homotetramers.

[0207] In the present disclosure, if an N-terminal methionine residue is not present in a uricase variant, alternative embodiments include uricase variants of the same sequences that have the N-terminal methionine residue present. Similarly, if an N-terminal methionine residue is present in a uricase variant, alternative embodiments include uricase variants of the same sequences that do not include the N-terminal methionine residue. In some embodiments described herein where methionine is not at the N-terminus (position 1) of the uricase variant, and instead another amino acid is present, e.g., threonine, the uricase variant has had the N- terminal methionine residue removed by post-translational modification. The N-terminal methionine residue is removed, in one embodiment, by endogenous bacterial methionine aminopeptidase after the uricase variant is produced in bacteria, e.g., in E coli.

[0208] In one embodiment of a uricase variant disclosed herein, the uricase variant is isolated. In another embodiment, the uricase variant is purified. In some embodiments, theAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application uricase variant comprises a purification tag at the C-terminus, the N-terminus, or both the N- and C-terminus to allow for purification by affinity chromatography. In one embodiment, the purification tag is a polyhistidine tag (also referred to as a “his-tag”). The his-tag, in one embodiment, comprises six (6) 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 of 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 of HNHNHNHNHNHN (SEQ ID NO:7). In another embodiment, the purification tag is a histidine affinity tag (HAT). In a further embodiment, the HAT has the amino acid sequence of KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8). In one embodiment of a uricase variant provided herein, the uricase variant is isolated and purified.

[0209] As discussed above, the uricase variants disclosed herein may be provided as either a monomeric protein or a multimeric protein, e.g., a homodimer, a homotetratmer, a heterodimer or a heterotetramer. In one embodiment, a 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 one preferred embodiment, the uricase variant is a homotetramer. In another embodiment, the uricase variant is a heterodimer, a heterotrimer or a heterotetramer.

[0210] In another aspect of the present disclosure, a uricase monomer comprising a uricase variant disclosed herein is provided.

[0211] In still another aspect of the present disclosure, a uricase dimer comprising a first uricase monomer and a second uricase monomer is provided, 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.

[0212] In yet another aspect of the present disclosure, a uricase tetramer comprising a first, second, third and fourth uricase monomer is provided, 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. In a further embodiment, the third uricase monomer is one of the uricase variants disclosed herein. In a still further embodiment, the fourth uricase monomer is one of the uricase variants disclosed herein. In one embodiment,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application the uricase tetramer is a heterotetramer. In a preferred embodiment, the uricase tetramer is a homotetramer comprising four identical uricase variant monomers disclosed herein.

[0213] In one embodiment of a uricase variant disclosed herein, the uricase variant is non- PEGylated. In another embodiment, the uricase variant is PEGylated, i.e., the uricase variant is covalently conjugated to polyethylene glycol (PEG), e.g., at one or more lysine residues. In a further embodiment, each of the uricase monomers of a multimeric uricase variant, e.g., 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 is about 10 kDa in molecular weight. A uricase variant monomer may be conjugated to any desired number of PEG or mPEG molecules, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In one embodiment, a uricase variant monomer is conjugated to from 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 moiety of a PEGylated monomeric uricase variant is about 10 kDa to about 150 kDa, about 30 kDa to 120 kDa, or about 50 kDa to about 100 kDa.

[0214] In one embodiment, the uricase variant disclosed herein is covalently bonded to PEG, e.g., mPEG, via a biocompatible linking group, using methods known in the art, as described, for example, by Park et al, Anticancer Res., 1981, 1:373-376; and Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., 1992, Plenum Press, New York, Chapter 21, the disclosures of each of which are incorporated by reference herein in their entireties. A biocompatible linking group is non-toxic and can be used in vitro or in vivo without causing injury, sickness, disease, or death. In some embodiments, PEG can be bonded to the linking group, for example, via an ether bond, an ester bond, a thiol bond or an amide bond. Suitable biocompatible linking groups include, for example, an ester group, an amide group, an imide group, a carbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, a succinimide group, an epoxide group, an oxycarbonylimidazole group, a nitro phenyl group, a trysylate group, an aldehyde group, an isocyanate group, a vinylsulfone group, a tyrosine group, a cysteine group, a histidine group or a primary amine.

[0215] In another embodiment, the uricase variant disclosed herein is conjugated without a linking group to PEG, e.g., mPEG, through an amino group, a sulfhydral group, a hydroxyl group or a carboxyl group. In one embodiment, PEG is conjugated to one or more lysine residues on the uricase variant. In one embodiment, PEG is conjugated to one or more cysteineAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application residues on the uricase variant. In one embodiment, PEG is conjugated to one or more serine residues on 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 a combination of any of the foregoing.

[0216] In another aspect, the present disclosure relates to a uricase conjugate comprising at least two domains: (i) a first (uricase) domain comprising one of the uricase variants disclosed herein conjugated to (ii) a second domain comprising a first random coil polypeptide domain.

[0217] As used herein, the term “domain” relates to any region / part of an amino acid sequence that is capable of autonomously adopting a specific structure and / or function. In the context of the present invention, accordingly, a “domain” may represent a functional domain or a structural domain. As described herein, the uricase conjugate or its fusion protein embodiment of the present invention comprises at least one uricase domain and at least one domain / part forming random coil conformation (e.g., the PAS polypeptide domain). The uricase conjugate of the present invention also may include more than two domains. For example, as provided herein, because the uricase variant may exist as a homotetramer, in one embodiment, a uricase conjugate of the present invention comprises four uricase domains and four random coil polypeptide domains. Moreover, the fusion proteins of the present invention may comprise, e.g., an additional linker structure between the herein defined two domains / parts or another domain / part like, e.g. a protease sensitive cleavage site, an affinity tag such as the polyhistidine tag or the Strep-tag, a signal peptide, retention peptide, a targeting peptide like a membrane translocation peptide or additional effector domains like antibody fragments for tumor targeting associated with an anti-tumor toxin or an enzyme for prodrug-activation etc. In another embodiment of a 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 C-terminal to the uricase domain and a random coil polypeptide domain N-terminal to the uricase domain. In a further embodiment, the random coil polypeptide domains comprise PAS polypeptides.

[0218] As used herein, the term “random coil” or “random coil polypeptide domain” relates to a conformation of a polymeric molecule, including amino acid polymers, in which the individual monomeric elements that form the polymeric structure are essentially randomly oriented towards the adjacent monomeric elements while still being chemically bound to said adjacent monomeric elements. In particular, a polypeptide or amino acid polymer adopting / having / forming “random coil” conformation substantially lacks a defined secondaryAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application and tertiary structure. The nature of polypeptide random coils and their methods of experimental identification are known to the person skilled in the art.

[0219] The first (uricase) domain comprising one of the uricase variants disclosed herein is conjugated to the second domain comprising a first random coil polypeptide domain, in one preferred embodiment, at the DNA level via operably linking the uricase variant DNA sequence to a DNA sequence encoding the random coil polypeptide, such that the DNA sequences are present within one open reading frame, followed by recombinant expression of the continuous DNA sequence encoding the first and second domains. The linking of the two domains at the DNA level obviates the need for in vitro coupling or modification steps to achieve conjugate synthesis, which is needed, for example, for the coupling of polyethylene glycol (PEG) to a uricase variant, in other embodiments described herein.

[0220] In embodiments where the uricase conjugate is produced via 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 a plurality of polypeptide components, that while typically unjoined in their native state, are joined by their respective N-terminus and C-terminus through a peptide linkage to form a single continuous polypeptide. Uricase fusion proteins may be a combination of two, three or four or more different proteins. Uricase fusion proteins can also include fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; as well as fusion proteins that include additional sequences for purification of the fusion protein (e.g., a polyhistidine tag).

[0221] In one embodiment, the second domain of the uricase conjugate comprises the first random coil polypeptide domain comprising at least about 100 amino acid residues, e.g., a PA polypeptide, a PAS polypeptide, or an XTEN polypeptide. Without wishing to be bound by theory, the random coil conformation mediates an increased in vivo and / or in vitro stability of the uricase variant enzyme. Moreover, without wishing to be bound by theory, because the random coil polypeptide domain is thought to not adopt a stable structure or function by itself, the biological activity of the uricase variant to which it is conjugated is essentially preserved.

[0222] In one embodiment of a random coil polypeptide, the random coil polypeptide of a uricase conjugate comprises the two amino acids, proline (Pro) and alanine (Ala). In a further embodiment, the random coil polypeptide consists of the two amino acids, proline (Pro) and alanine (Ala). Where the amino acid residues in a polypeptide are all Pro and Ala, orAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application substantially all the amino acids are Pro and Ala in a polypeptide, such a polypeptide is referred to herein as a “PA polypeptide.” In one embodiment, a uricase fusion protein comprises a PA polypeptide. In a further embodiment, the PA polypeptide is at least about 100 amino acids long, at least about 150 amino acids long, at least about 200 amino acids long, at least about 250 amino acids long, at least about 300 amino acids long, at least about 350 amino acids long, at least about 400 amino acids long, at least about 450 amino acids long, at least about 500 amino acids long, or at least about 550 amino acids long, e.g., the PA polypeptide is about 200 amino acids long, about 300 amino acids long, about 400 amino acids long, about 500 amino acids long, or about 600 amino acids long.

[0223] In one embodiment of a random coil polypeptide, the random coil polypeptide of a uricase conjugate comprises the three amino acids, proline (Pro), alanine (Ala) and serine (Ser). In a further embodiment, the random coil polypeptide consists of the three amino acids, proline (Pro), alanine (Ala) and serine (Ser). Where the amino acid residues in a polypeptide are all Pro, Ala and Ser, or substantially all the amino acids are Pro, Ala and Ser in a polypeptide, such a polypeptide is referred to herein as a “PAS polypeptide.” A PAS polypeptide, when present in a uricase conjugate, may be referred to as a PAS domain. In one embodiment, a uricase fusion protein comprises a PAS polypeptide and the PAS polypeptide comprises from about 10 to about 30 tandem copies of a PAS sequence comprising Pro, Ala, and Ser, e.g., PAS10 with 10 tandem copies and a total of about 200 amino acids; PAS20 with 20 tandem copies and a total of about 400 amino acids; and PAS30 with 30 tandem copies and a total of about 600 amino acids. Exemplary amino acid sequences of PAS10, PAS20, and PAS30 are set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively. Other examples of PAS polypeptides amenable for use herein are provided in PCT Publication No. WO 2008 / 155134, the disclosure of which is incorporated by reference in its entirety.

[0224] In another embodiment, the random coil polypeptide is an extended recombinant (XTEN) polypeptide. An XTEN polypeptide includes six amino acids A, E, G, S and T at varying percentages so as to form a long unstructured hydrophilic amino acid sequence (Pasut, Polymers 2014, 6, 160-178). The XTEN polypeptide, in one embodiment, is one of the polypeptides disclosed in U.S. Patent Application Publication No. 2015 / 0037359, the content of which is incorporated by reference in its entirety for all purposes. The XTEN polypeptide, in one embodiment, is at least about 800 amino acids in length consisting of six hydrophilic chemically stable amino acids Ala, Asp, Gly, Pro, Ser, and Thr, in a nonrepetitive manner. In one embodiment, the XTEN polypeptide is 864 residues long. In another embodiment, theAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application XTEN polypeptide is a fragment of the 864 aa XTEN polypeptide. In one embodiment, the XTEN polypeptide comprises the amino acid sequence set forth in SEQ ID NO:12. XTEN polypeptides may be conjugated to the first domain comprising a uricase variant described herein via chemical conjugation or produced as a fusion protein with the uricase variant.

[0225] In some embodiments, the second domain of the uricase conjugate comprises a PAS polypeptide (referred to herein in some embodiments as a PAS domain) and comprises 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 forming the random coil comprise the amino acids proline (Pro), alanine (Ala) and serine (Ser). In a PAS domain, all or substantially all of the amino acids are Pro, Ala and Ser. Without wishing to be bound by theory, the random coil conformation mediates an increased in vivo and / or in vitro stability of the uricase variant enzyme. Details regarding various types of PAS polypeptides and nucleic acids encoding the same, for use in the present invention, can be found in PCT Publication No. WO 2008 / 155134, the content of which is incorporated by reference in its entirety for all purposes.

[0226] The random coil of a uricase conjugate is formed under physiological conditions. For example, in one embodiment, the physiological conditions are the parameters that are typically valid for higher forms of life, and in particular mammals, most preferably humans. As such, physiological conditions can be the conditions that are normally found in the body fluids of mammals. The physiological conditions may relate to the corresponding parameters found in the healthy body as well as the parameters as found in sick mammals or human patients. For example, a sick mammal or human patient may have a higher, yet physiological temperature condition when said mammal or said human suffers from fever.

[0227] Several buffers in experimental settings (e.g., for use in the determination of protein structures, in particular in circular dichroism (CD) measurements and other methods for determining the structural properties of a protein / amino acid stretch), solvents and / or excipients for pharmaceutical compositions, are considered to represent physiological solutions and / or physiological conditions in vitro. Examples of such buffers are, e.g., phosphate-buffered saline, Tris buffers, acetate buffers, citrate buffers or similar buffers. Generally, the pH of a buffer representing physiological solution conditions is in a range from 6.5 to 8.5, e.g., in a range from 7.0 to 8.0, e.g., in a range from 7.2 to 7.7 and the osmolarity may lie in a range from 10 to 1000 mmol / kg H2O, more particularly in a range from 50 to 500 mmol / kg H2O, e.g., in a range from 200 to 350 mmol / kg H2O.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0228] Methods for determining whether an amino acid polymer forms / adopts random coil conformation are known in the art. Such methods include CD spectroscopy, which represents a light absorption spectroscopy method in which the difference in absorbance of right- and left- circularly polarized light by a substance is measured. The secondary structure of a protein can be determined by CD spectroscopy using far-ultraviolet spectra with a wavelength between approximately 190 and 250 nm. At these wavelengths, the different secondary structures commonly found in polypeptides can be analyzed, since Į-helix, parallel and anti-parallel ȕ- sheet and random coil conformations each give rise to a characteristic shape and magnitude of the CD spectrum. Accordingly, by using CD spectrometry the skilled artisan is readily capable of determining whether an amino acid polymer forms / adopts random coil conformation at physiological conditions. Other established biophysical methods include nuclear magnetic resonance (NMR) spectroscopy, absorption spectrometry, infrared and Raman spectrometry, measurement of the hydrodynamic volume via size exclusion chromatography, analytical ultracentrifugation or dynamic / static light scattering as well as measurements of the frictional coefficient or intrinsic viscosity.

[0229] In one embodiment, a random coil polypeptide domain comprises 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, a random coil polypeptide domain comprises maximally about 1000 amino acid residues, maximally about 900 amino acid residues, maximally about 800 amino acid residues, maximally about 700 amino acid residues, or maximally about 600 amino acid residues. In one embodiment, a random coil polypeptide domain comprises maximally about 500 amino acid residues or of maximally about 450 amino acid residues.

[0230] In one embodiment, a random coil polypeptide domain comprises from about 100 to about 3000 amino acid residues. In a further embodiment, the random coil polypeptide domain comprises from about 100 to 1000 amino acid residues. In some embodiments, the random coil polypeptide domain comprises from about 100 to about 800, from about 100 to about 700, from about 100 to about 600, from about 100 to about 500, from about 100 to about 400, or from about 100 to about 300 amino acid residues.

[0231] In one embodiment, a random coil polypeptide domain comprises an amino acid sequence whereby proline residues represent about 4% to about 40% of the random coil polypeptide domain. In a further embodiment, alanine and serine residues comprise theAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application remaining about 60% to about 96% of the random coil polypeptide domain. In some embodiments, the random coil polypeptide domain comprises further amino acids differing from Ala, Ser and Pro as minor constituents. The term “minor constituent” as used in this context means that maximally 10% of the amino acids in a random coil polypeptide domain are different from alanine, serine and proline, e.g., maximally 8% of the amino acids in a random coil polypeptide domain, e.g., maximally 6% of the amino acids in a random coil polypeptide domain, e.g., maximally 5% of the amino acids in a random coil polypeptide domain, maximally 4% of the amino acids in a random coil polypeptide domain, maximally 3% of the amino acids in a random coil polypeptide domain, maximally 2% of the amino acids in a random coil polypeptide domain, maximally 1% of the amino acids in a random coil polypeptide domain are different from Ala, Ser and Pro. In one embodiment of a random coil polypeptide domain, the polypeptide comprises amino acids other than Ala, Ser and Pro, and the other amino acids are selected from the group consisting of Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val. In another embodiment, the other amino acids include one or more non-natural amino acids.

[0232] In another embodiment, a random coil polypeptide domain comprises a plurality of “amino acid repeats”, i.e., the same amino acid sequence occurring two or more times in the domain, wherein the “amino acid repeats” consist of Ala, Ser, and Pro residues (depicted herein as “PAS”, or as “APS”). In a further embodiment, no more than 6 consecutive amino acid residues are identical in the random coil polypeptide domain and the proline residues constitute more than about 4% and less than about 40% of the amino acids of the random coil polypeptide domain. Non-limiting examples of “amino acid repeats” consisting of Ala, Ser and Pro residues are provided herein; see, e.g., SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 and SEQ ID NO:23 (Table 1A). Fragments and / or multimers of these sequences are employed in some embodiments. A “fragment” comprises at least 3 amino acids and comprises at least one Ala, one Ser and / or one Pro. T a S18GCTCCGTCCTCCCCGTCCCCGTCCGCTCCGTCCTCCCCGTCCCCGGCTTCCCCGTCCTCCAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 1A – Exemplary PAS amino acid sequences for use in a random coil polypeptide domain, a S

[0233] The aforementioned repeat sequences may be encoded by nucleic acid molecules having the sequences set forth 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).

[0234] In one embodiment, a nucleotide sequence is provided that encodes the amino acid repeat of SEQ ID NO:13. In a further embodiment, the nucleotide sequence is selected from one of the nucleotide sequences set forth in Table 1B, i.e., one of SEQ ID NOs:155-185. T N S178GCTTCTCCTGCGGCCCCCGCGCCGGCTTCTCCGGCCGCTCCTGCGCCAAGTGCGCCGGCGAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 1B – Exemplary nucleotide sequences encoding the PAS amino acid repeat of SEQ ID N S

[0235] The amino acid repeat used in a random coil PAS polypeptide domain, in one embodiment, comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues, wherein the amino acid 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. The amino acid repeat, in one embodiment, 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 a further embodiment, the amino acid repeat comprises less than about 40%, e.g., less than about 35% Pro residues.

[0236] In one embodiment, a random coil polypeptide domain comprises no more than 5 identical consecutive amino acid residues, e.g., no more than 4 identical consecutive amino acid residues, e.g., no more than 3 identical consecutive amino acid residues.

[0237] In one embodiment, a random coil polypeptide domain comprises more than about 4% Ala residues but less than about 50% Ala residues, e.g., more than about 10% Ala residues but less than about 50% Ala residues, e.g., more than about 20% Ala residues but less than about 50% Ala residues.

[0238] In another embodiment, a random coil polypeptide domain comprises more than about 4% Ser residues but less than about 50% Ser residues, e.g., more than about 10% Ser residues but less than about 50% Ser residues, e.g., more than about 20% Ser residues but less than about 50% Ser residues.

[0239] In one embodiment, a random coil polypeptide domain comprises about 35% Pro residues, about 50% Ala residues and about 15% Ser residues. Alternatively, a random coil polypeptide domain comprises about 35% Pro residues, about 15% Ala residues and about 50% Ser residues.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0240] In one embodiment of the invention, the uricase conjugate comprises a PAS polypeptide in the random coil polypeptide domain comprising the amino acid sequence set forth in SEQ ID NO:9.

[0241] In one embodiment of the invention, the uricase conjugate comprises a PAS polypeptide in the random coil polypeptide domain comprising the amino acid sequence set forth in SEQ ID NO:10.

[0242] In one embodiment of the invention, the uricase conjugate comprises a PAS polypeptide in the random coil polypeptide domain comprising the amino acid sequence set forth in SEQ ID NO:11.

[0243] In one embodiment, the uricase conjugate of the present invention is a fusion protein. A fusion protein as described herein comprises at least one uricase domain (i.e., the first domain) and at least one random coil polypeptide domain in a multi-domain polypeptide. In an alternative embodiment, the uricase domain is bonded via a non-peptide bond to the random coil polypeptide domain. Non-peptide bonds that are useful for cross-linking proteins are known in the art and may include disulfide bonds, e.g., between Cys side chains, thioether bonds or non-peptide covalent bonds induced by chemical cross-linkers, such as disuccinimidyl suberate (DSS) or sulfosuccinimidyl 4-[pmaleimidophenyl] butyrate (Sulfo-SMPB), as well as non-covalent protein-protein interactions.

[0244] With respect to the fusion protein embodiments, the two domains can be arranged in an order selected by the ordinary skilled artisan. For example, in one embodiment of a uricase conjugate that is a fusion protein, the uricase domain is located at the amino (N-) terminus of the fusion protein and the random coil polypeptide domain is located at the carboxy (C-) terminus of the fusion protein. However, this order may also be reversed, e.g., in one embodiment, the uricase domain is located in / at the carboxy (C-) terminus and the random coil polypeptide domain is located in / at the amino (N-) terminus of the fusion protein.

[0245] In yet another embodiment, the random coil polypeptide domain is located at both the C-terminus and N-terminus of the fusion protein, and the uricase domain is in between both random coil polypeptide domains.

[0246] In one embodiment of a random coil polypeptide domain provided herein, where 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, theAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application fusion protein does not include an N-terminal Met residue, e.g., because it was removed after translation.

[0247] 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, e.g., because it was removed after translation. As such, for the sequences provided herein, if an N-terminal Met residue is present, alternative embodiments include fusion proteins of the same sequences that do not include the N-terminal Met residue. Similarly, if an N-terminal Met residue is not present in a fusion protein provided herein, alternative embodiments include fusion proteins of the same sequences that have the N-terminal Met residue present.

[0248] In one embodiment of a uricase fusion protein, an amino acid spacer or linker sequence is present between the uricase domain and the random coil polypeptide (e.g., PAS) domain. The amino acid spacer sequence in one embodiment, is one amino acid long, two amino acids long, three amino acids long, four amino acids long, or five amino acids long. In another embodiment, the amino acid spacer sequence is from about two amino acids long to about 5 amino acids long. In another embodiment, the amino acid spacer sequence is from about two amino acids long to about 4 amino acids long. In a further embodiment, the amino acid spacer sequence is two amino acids long. In a further embodiment, the spacer sequence is Gly-Ser.

[0249] In one embodiment of a uricase conjugate described herein, the uricase conjugate comprises a purification tag at the C-terminus, N-terminus or both the N- and C-terminus. The purification tag is employed to facilitate purification of the uricase conjugate (e.g., the uricase fusion protein) from an in vitro expression system, for example, via the use of immobilized metal affinity chromatography (IMAC). The purification tag, in one embodiment, is present at the C-terminus of the uricase fusion protein. In a further embodiment, the purification tag is a polyhistidine tag (also referred to as a “his-tag”). The his-tag, in one embodiment, comprises six (6) histidine residues.

[0250] Alternative purification tags can also be employed herein. For example, in one embodiment, a his-glu tag (HQ tag) is present at the C-terminus of one of the uricase conjugates described herein. In a further embodiment, the HQ tag has the amino acid sequence HQHQHQ (SEQ ID NO:6). In another embodiment, the uricase conjugate comprises a his-asp tag (HN tag) at the C-terminus, to allow for purification of the conjugate. The HN tag, in one embodiment, has the amino acid sequence: HNHNHNHNHNHN (SEQ ID NO:7). In yetAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application another embodiment, the uricase conjugate comprises a histidine affinity tag (HAT) at the C- terminus, to allow for purification of the conjugate. The HAT, in one embodiment, has the amino acid sequence: KDHLIHNVHKEEHAHAHNK (SEQ ID NO:8).

[0251] For the fusion protein embodiments of the uricase conjugate of the present disclosure, exemplary configurations of a recombinant uricase fusion protein comprising a uricase domain comprising one of the uricase variants disclosed herein, and one or two random coil PAS polypeptide or XTEN polypeptide domains are provided in FIG. 1. In one embodiment, the uricase fusion protein includes the uricase domain and one random coil PAS polypeptide or XTEN polypeptide domain. In the uricase fusion protein, the uricase domain may be C-terminal to the one random coil PAS polypeptide or XTEN polypeptide domain, or may be N-terminal to the one random coil PAS polypeptide or XTEN polypeptide domain. In another embodiment, the uricase fusion protein includes the uricase domain and two random coil PAS polypeptide domains or two XTEN polypeptide domains. In the uricase fusion protein, one of the two random coil PAS polypeptide or XTEN polypeptide domains is at the N-terminus and the other is at the C-terminus, and the uricase domain is 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 a C-terminal random coil PAS polypeptide (e.g., PAS10, PAS20, or PAS30) or XTEN polypeptide domain, as depicted in FIG. 1, is optional and can be absent. In one embodiment, the uricase fusion protein has a polyhistidine tag (also referred to as a “his-tag”) comprising, e.g., six histidine residues, at the C-terminus, the N-terminus, or both the C-terminus and the N-terminus for purification of the uricase fusion protein. In some embodiments, the his-tag is separated from its adjacent domain by the spacer sequence Gly-Ser (GS).

[0252] PAS10, PAS20, and PAS30 represent random coil PAS polypeptide domains respectively comprising 10, 20, and 30 tandem copies of a PAS sequence comprising Pro, Ala, and Ser, such as the PAS sequence set forth 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.

[0253] In one embodiment, PAS10, PAS20, and PAS30 represent random coil PAS polypeptide domains respectively comprising 10, 20, and 30 tandem copies of the PAS sequence of SEQ ID NO: 13, thereby having the amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0254] 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 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.

[0255] 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 a total of 10, 20, and 30 tandem copies of the same (one) nucleotide sequence selected from the group consisting of SEQ ID NO:14 and SEQ ID NOs:155-185.

[0256] 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 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.

[0257] 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 tandemly joined together via 3’,5’-phosphodiester bonds.

[0258] In an exemplary embodiment, the DNA sequence encoding PAS10 of SEQ ID NO:9 comprises, in the 5’ to 3’ direction, each of SEQ ID NOs:176-185 tandemly joined together in the ascending ID numerical order via 3’,5’-phosphodiester bonds.

[0259] In an exemplary embodiment, the DNA sequence encoding PAS20 of SEQ ID NO:10 comprises, in the 5’ to 3’ direction, each of SEQ ID NOs:166-185 tandemly joined together in the ascending ID numerical order via 3’,5’-phosphodiester bonds.

[0260] In an exemplary embodiment, the DNA sequence encoding PAS30 of SEQ ID NO:11 comprises, in the 5’ to 3’ direction, each of SEQ ID NOs:156-185 tandemly joined together in the ascending ID numerical order via 3’,5’-phosphodiester bonds.

[0261] In another exemplary embodiment, the DNA sequence encoding PAS30 of SEQ ID NO:11 comprises, in the 5’ to 3’ direction, each of SEQ ID NO:155 and SEQ ID NOs:157- 185 tandemly joined together in the ascending ID numerical order via 3’,5’-phosphodiester bonds.

[0262] In one embodiment, the XTEN polypeptide domain of the uricase fusion protein comprises the amino acid sequence of SEQ ID NO:12.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0263] Exemplary PASylated uricase fusion proteins comprising the amino acid sequence of a uricase variant disclosed herein for the uricase domain in combination with one or two PAS domains according to the configurations of FIG. 1 include those comprising an amino acid sequence selected from SEQ ID NOs:1568-2288. Those PASylated uricase fusion proteins are described in detail in Example 5 of the present application.

[0264] 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 still 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 foregoing random coil polypeptide domains.

[0265] In another embodiment, the uricase conjugate is present within a tetramer, preferably a homotetramer. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein. In a still 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 foregoing random coil polypeptide domains.

[0266] In another embodiment, the uricase conjugate is present within a homodimer, a heterodimer, or a heterotetramer. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein. In a still 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 foregoing random coil polypeptide domains.

[0267] In one embodiment of a uricase conjugate disclosed herein, the uricase conjugate is a homotetramer comprising four identical uricase conjugate monomers. In a further embodiment, the homotetramer is a homotetrameric recombinant uricase fusion protein comprising four identical recombinant uricase fusion protein monomers. In a still 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 foregoing random coil polypeptide domains.

[0268] In one embodiment of a 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 identicalAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application recombinant uricase fusion protein monomers. In a still 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 foregoing random coil polypeptide domains.

[0269] In one embodiment of a uricase conjugate disclosed herein, the uricase conjugate is non-PEGylated. In another embodiment, the uricase conjugate is PEGylated, i.e., the uricase conjugate is covalently conjugated to polyethylene glycol (PEG), e.g., at one or more lysine residues. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein disclosed herein. In a still 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 foregoing 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 a PEGylated uricase conjugate, amino acid residues in the uricase domain, in the random coil peptide domain, or in both of the foregoing domains may be covalently conjugated to PEG, depending on, e.g., the amino acid composition in the respective domains and conjugation conditions. In one embodiment, the uricase conjugate, e.g., a recombinant uricase fusion protein, is covalently conjugated to one or more molecules of mPEG. In a further embodiment, each mPEG molecule is about 10 kDa in molecular weight. In one embodiment, a monomeric recombinant uricase fusion protein is conjugated to any desired number of PEG or mPEG molecules, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. In one embodiment, a monomeric recombinant uricase fusion protein is conjugated to from 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 moiety of a PEGylated monomeric recombinant uricase fusion protein is about 10 kDa to about 150 kDa, about 30 kDa to 120 kDa, or about 50 kDa to about 100 kDa. In one embodiment, the uricase conjugate is PEGylated by being covalently bonded to PEG, e.g., mPEG, via a biocompatible linking group, analogous to the PEGylated uricase variant described previously. In another embodiment, the uricase conjugate is PEGylated by being covalently bonded to PEG, e.g., mPEG, without a linking group, also analogous to the PEGylated uricase variant described previously.

[0270] In another aspect, the present disclosure provides an isolated nucleic acid which encodes a uricase variant of the disclosure. In one embodiment, the uricase variant is presentAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 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. An isolated nucleic acid is removed from its natural environment, and may additionally be in substantially pure, e.g., at least 90% pure, or in homogeneous form. The isolated nucleic acid may be, for example, a synthetic DNA, a non- naturally occurring mRNA, or a cDNA. Methods of producing the disclosed nucleic acids are well known to the one of skill in the art. See, e.g., Maniatis, T., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., incorporated herein by reference in its entirety. Additionally or alternatively, the disclosed nucleic acids may be produced by introducing one or more mutations into a related nucleic acid with a similar nucleic acid sequence using site-directed mutagenesis techniques known in the art, such as extension of overlapping gene segments by PCR, disclosed in Heckman et al., Nat Protoc 2007, 2, 924–932, incorporated herein by reference in its entirety. The disclosed nucleic acids can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.

[0271] In another aspect, the present disclosure provides a nucleic acid vector comprising a nucleic acid encoding a uricase variant disclosed herein. In one embodiment, the uricase variant is present 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. In one embodiment, the nucleic acid coding for a uricase variant or a recombinant uricase fusion protein comprising a uricase variant domain is inserted into a vector, e.g., a plasmid, for multiplication of the nucleic acid. In another embodiment, the nucleic acid coding for a uricase variant or a recombinant uricase fusion protein comprising a uricase variant domain is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements, e.g., promoter sequences, terminator sequences, polyadenylation sequences, and enhancer sequences, for the transcription and translation of the inserted uricase variant-coding sequence. Vectors may be plasmids, phage, phagemids, adenoviral, AAV, lentiviral, for example. A variety of host-vector systems may be utilized to express the coding sequence of a uricase variant or a recombinant uricase fusion protein comprising a uricase variant domain. Exemplary systems include mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. The expressionAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application elements of these vectors vary in their strengths and specificities. 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 inducible by isopropyl ȕ-D-1 thiogalactopyranoside (IPTG) and repressible by glucose, a T7 promoter inducible by IPTG and repressible by glucose, a rhamnose (rham) promoter inducible by rhamnose and repressible by glucose, or an alkaline phosphatase (phoA) promoter inducible by phosphate starvation and repressible by the presence of phosphate.

[0272] Any of the methods known for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a nucleic acid comprising one or more regulatory elements such as appropriate transcriptional / translational control signals and a coding sequence for a uricase variant or a recombinant uricase fusion protein operably linked thereto. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination). Expression of nucleic acid sequence encoding a uricase variant or a recombinant uricase fusion protein comprising a uricase variant domain may be regulated by a second nucleic acid sequence so that the uricase variant or recombinant uricase fusion protein is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a uricase variant or a 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 a nucleic acid sequence encoding the uricase variant or the recombinant uricase fusion protein operatively linked to a heterologous promoter. Exemplary promoters which may be used to control uricase variant or recombinant uricase fusion protein expression using a mammalian expression vector include the simian virus 40 (SV40) early promoter region, the promoter contained in the 3’ long terminal repeat of Rous sarcoma virus, the herpes thymidine kinase promoter, and the regulatory sequences of the metallothionine gene. Exemplary promoters useful for prokaryotic expression vectors include the ȕ-lactamase promoter, the tac promoter, and the osmoregulated osmB promoter.

[0273] In another aspect, the present disclosure provides a host cell comprising a nucleic acid vector disclosed herein. In a further embodiment, the host cell is capable of producing a uricase variant of the invention. In one embodiment, the uricase variant is present 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. Appropriate cell lines or host systems can be chosen based on the desired expression level and / or post-translational processing and modification (e.g.,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application glycosylation, cleavage) of the uricase variant or recombinant uricase fusion protein. Suitable host cells include bacteria (e.g., E. coli), mammalian cells, plant cells, insect cells, fungi, yeast and transgenic plants and animals. Exemplary mammalian cell lines available in the art for expression of a heterologous protein, such as a uricase variant or a recombinant uricase fusion protein comprising a 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 retina cells. Introducing the vector into a host cell can be accomplished using techniques well known in the art. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, diethylaminoethyl (DEAE)- dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, for example. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage. The introduction may be followed by causing or allowing expression from the nucleic acid, e.g., by culturing host cells under conditions for expression of the nucleic acid sequence encoding the uricase variant or recombinant uricase fusion protein, leading to respective protein production. In one embodiment, the nucleic acid encoding the uricase variant or recombinant uricase fusion protein is integrated into the genome, e.g., chromosome, of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

[0274] In one embodiment, the host cell is a mammalian cell, e.g., a Chinese hamster ovary (CHO) cell, or a human embryonic kidney cell. In a further embodiment, the expression vector for use in the mammalian cell comprises the actin (e.g., chicken ȕ-actin), cytomegalovirus (CMV), CMV enhancer / elongation factor (CEF), CMV early enhancer / chicken ȕ-actin (CAG), hybrid CMV enhancer / chicken ȕ-actin (CBh), elongation factor-1Į (EF1alpha), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), or simian virus 40 (SV40) promoter.

[0275] In another embodiment, the host cell is a yeast cell. In a further embodiment, the expression vector for use in the yeast cell comprises the alcohol oxidase (AOX), glyceraldehyde-3-phosphate dehydrogenase (GAP), alcohol dehydrogenase (ADH), or galactokinase 1 (GAL1) promoter.

[0276] In another embodiment, the host cell is an insect cell infected with baculovirus. In a further embodiment, the expression vector for use with the baculovirus comprises the polyhedrin gene promoter.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0277] In another embodiment, the host cell is bacteria, e.g., E. coli. In a further embodiment, the expression vector for use in bacteria comprises the osmB promoter, the T7-lac promoter (see Shilling et al., Commun Biol 3, 214 (2020)), the pBAD promoter, the Tac promoter, a tet- inducible promoter, the cold-shock Protein A (cspA) promoter, or the inducible promoter of the alkaline phosphatase gene (phoA) derived from E. coli.

[0278] In another aspect, the present disclosure provides a method of recombinantly producing a uricase variant or a uricase conjugate (e.g., a recombinant uricase fusion protein) comprising a uricase variant domain disclosed herein. The method includes (i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding the uricase variant or the uricase conjugate (e.g., the recombinant uricase fusion protein) disclosed herein, wherein the nucleic acid sequence is operatively linked to a heterologous promoter under conditions to allow for expression of the nucleic acid sequence encoding the uricase variant or the uricase conjugate (e.g., the recombinant uricase fusion protein) and recombinant production of the uricase variant or the uricase conjugate (e.g., the recombinant uricase fusion protein) by the host cell; and (ii) isolating the recombinantly produced uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein).

[0279] The uricase variants or uricase conjugates (e.g., recombinant uricase fusion proteins) may be produced in any suitable cell-culture system including prokaryotic cells, e.g., E. coli, BL21 or JM83, or eukaryotic cells, e.g., Pichia pastoris yeast strain X-33 or CHO cells. Further suitable cell lines known in the art are obtainable from cell line depositories, such as the American Type Culture Collection (ATCC). The uricase variants or uricase conjugates (e.g., recombinant uricase fusion proteins) can be isolated from the growth medium, cellular lysates or cellular membrane fractions. The isolation and purification of the recombinantly produced uricase variants or uricase conjugates (e.g., recombinant uricase fusion proteins) of the invention may be performed by any conventional means, including ammonium sulphate precipitation, affinity columns, column chromatography, gel electrophoresis and the like and may involve the use of monoclonal or polyclonal antibodies directed, e.g., against a tag fused with the biologically active protein of the invention. For example, the protein can be purified via the Strep-tag II using streptavidin affinity chromatography (Skerra and Schmidt (2000). Methods Enzymol 326, pp.271-304).

[0280] In one embodiment, the vector is under the control of an osmotic pressure sensitive promoter. An osmotic pressure sensitive promoter initiates transcription as a result of increased osmotic pressure as sensed by the cell. In one embodiment, the host cell is E. coli, and theAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application promoter is the osmB promoter, the T7-lac promoter, the pBAD promoter, the Tac promoter, a tet-inducible promoter, the cold-shock Protein A (cspA) promoter, or the inducible promoter of the alkaline phosphatase gene (phoA) derived from E. coli.

[0281] In one embodiment, the uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) is isolated using a cationic surfactant, for example, cetyl pyridinium 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, the uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) can be made according to the methods described in International Patent Application Publication No. WO 2000 / 008196, incorporated herein by reference in its entirety. In one embodiment, the uricase variant or uricase conjugate (e.g., recombinant uricase fusion protein) is produced in bacteria and, following isolation, subjected to endotoxin mitigation using methods known in the art, such as one disclosed in the examples of the present application.

[0282] In another aspect, the present disclosure provides a pharmaceutical composition comprising a uricase variant. In one embodiment, the uricase variant is present as or within a uricase tetramer comprising uricase variant monomers. In a further embodiment, the uricase tetramer is a homotetramer comprising four identical uricase variant monomers disclosed herein. In another embodiment, the uricase variant is present 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 pharmaceutical composition may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabilizer 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 efficacy of the uricase variants described herein. Such materials may include, for example, solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, and ethanol, as well as combinations thereof. In one embodiment, the pharmaceutical composition includes an isotonic agent, for example, a sugar, and / or a polyalcohol, such as mannitol or sorbitol, or sodium chloride. Additional examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifying agents, preservatives or buffers, which increase the shelf life or effectiveness.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0283] In one embodiment, the pharmaceutical composition comprising the uricase variant may be formulated in liquid, semi-solid or solid forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration, therapeutic application, the physicochemical properties of the uricase variant and the route of delivery. Formulations may include excipients, or combinations of excipients, for example: sugars, amino acids and surfactants. Liquid formulations may include a wide range of uricase variant concentrations and pH. Solid formulations may be produced by lyophilization, spray drying, or drying by supercritical fluid technology, for example.

[0284] For intravenous injection, or injection at the site of affliction, the active ingredient may be in a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pK, isotonicity, and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride solution, Ringer’s solution, and a lactated Ringer’s solution. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included.

[0285] In some embodiments, the pharmaceutical composition is formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to contain a high uricase variant concentration. Sterile injectable solutions can be prepared by incorporating a uricase variant in an appropriate solvent with one or a combination of ingredients enumerated above, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the uricase variant into a sterile vehicle that contains a dispersion medium and other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by using a coating such as lecithin, by maintaining the particle size of a dispersion, or by using surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0286] In some embodiments, the pharmaceutical composition may be prepared with a carrier that protects the uricase variant against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

[0287] In one embodiment, the pharmaceutical composition is a solution of a uricase variant disclosed herein e.g., a phosphate buffered saline solution containing the uricase variant. In a further embodiment, the solution is sterile and suitable for injection, e.g., intravenous injection or subcutaneous injection.

[0288] In another aspect, the present disclosure provides a method of reducing elevated uric acid levels in a subject in need of treatment. The method includes administering to the subject an effective amount of the pharmaceutical composition comprising a uricase variant disclosed herein. In one embodiment, the uricase variant is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant is present 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.

[0289] As used herein, “treatment” or “treating,” or “ameliorating” and variations thereof are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. The term “treating” in one embodiment, includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in the patient that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (e.g., arresting, reducing or delaying the development of the disease, or a relapse thereof in case of maintenance treatment, of at least one clinical or subclinical symptom thereof); (3) relieving the condition (for example, by causing regression, or reducing the severity of the state, disorder or condition or at least one of its clinical or subclinical symptoms).

[0290] “Effective amount” means an amount of a pharmaceutical composition comprising a uricase variant of the present disclosure that is sufficient to result in the desired therapeutic response. In one embodiment, the uricase variant is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant is present as a uricase conjugate comprising the uricase variant domain, disclosedAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application herein. In a further embodiment, the uricase conjugate is a recombinant uricase fusion protein comprising the uricase variant domain, disclosed herein. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like.

[0291] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, such as a mammal. The mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., cynomolgus monkey, chimpanzee), or a human. A subject’s tissues, cells, or derivatives thereof, obtained in vivo or cultured in vitro are also encompassed. A human subject may be an adult, a teenager, a child (2 years to 14 years of age), an infant (1 month to 24 months), or a neonate (up to 1 month). In some embodiments, the adults are seniors about 65 years or older, or about 60 years or older. In some embodiments, the subject is a pregnant woman or a woman intending to become pregnant.

[0292] In one embodiment of the method, the subject is a human patient. In a further embodiment, the human patient is an adult human patient.

[0293] In one embodiment of the method, the uric acid levels are reduced in the plasma or blood of the subject. In one embodiment, suitable indicators for assessing effectiveness of the method include normalization or lowering of plasma uric acid levels (PUA), e.g., lowering or maintenance of PUA to 6.8 mg / dL or less, or 6 mg / dL or less in a human patient.

[0294] In some embodiments of the method, administration of the pharmaceutical composition disclosed herein is carried out parenterally, e.g., via 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 another embodiment, the administration is rectal, topical, or pulmonary administration.

[0295] In one embodiment, the subject is a gout patient, e.g., a gout human patient. In one embodiment, the gout is recurrent gout. In another embodiment, the gout is advanced gout, with deposits of uric acid crystals forming under the skin in nodules called tophi. In a human patient, tophi can develop in several areas, such as fingers, hands, feet, elbows or Achilles tendons along the backs of ankles. In another embodiment, the gout patient has kidney stones, which are uric acid crystals collected in the urinary tracts.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0296] In one embodiment, the subject is a refractory gout patient, i.e., refractory to a prior different treatment. The prior treatments include, but are not limited to, treatments with nonsteroidal anti-inflammatory drugs (NSAIDs), colchicine, corticosteroids, allopurinol, febuxostat, probenecid, KRYSTEXXA (pegloticase), and a combination of the foregoing. In one embodiment, refractory gout is a chronic condition characterized by high serum uric acid levels, recurrent gout flares, chronic arthritis, and progressive tophaceous deposition. In another embodiment, refractory gout is associated with high rates of cardiovascular and renal comorbidities.

[0297] In one embodiment, the subject has been diagnosed with tumor lysis syndrome. In some embodiments, the subject diagnosed with tumor lysis syndrome has a lymphoma (e.g., a Burkitt’s lymphoma, a non-Hodgkin’s 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 of > 8 mg / dL. In another embodiment, the subject diagnosed with tumor lysis syndrome is a human patient with a plasma uric acid concentration of > 15 mg / dL (hyperuricemia).

[0298] In another aspect, the present disclosure provides a method of treating gout in a subject in need of treatment. The method includes administering to the subject an effective amount of the pharmaceutical composition comprising a uricase variant disclosed herein. In one embodiment, the uricase variant is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant is present 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. In one embodiment, the gout treated is refractory gout, as described above. In one embodiment, the gout treated is recurrent gout. In another embodiment, the gout treated is advanced gout, characterized by deposits of uric acid crystals forming under the skin in nodules called tophi. In another embodiment, the subject with 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.

[0299] In some embodiments of the method, administration of the pharmaceutical composition disclosed herein is carried out parenterally, e.g., via intramuscular, intrathecal, SC, or IV administration. In one embodiment, the administration is IV administration. In another embodiment, the administration is SC administration. In another embodiment, the administration is rectal, topical, or pulmonary administration.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0300] In another aspect, the present disclosure provides a method of treating tumor lysis syndrome in a subject in need of treatment. The method includes administering to the subject an effective amount of the pharmaceutical composition comprising a uricase variant disclosed herein. In one embodiment, the uricase variant is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant is present 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. In one embodiment of the method, the subject is a human patient. In a further embodiment, the human patient is an adult human patient.

[0301] In some embodiments of the method, administration of the pharmaceutical composition is carried out parenterally, e.g., 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 another embodiment, the administration is rectal, topical, or pulmonary administration. In some embodiments, the subject with tumor lysis syndrome has a lymphoma (e.g., a Burkitt’s lymphoma, a non- Hodgkin’s lymphoma), acute lymphoblastic leukemia, or acute myeloid leukemia. In one embodiment, the subject with tumor lysis syndrome is a human patient with a plasma uric acid concentration of > 8 mg / dL. In another embodiment, the subject with tumor lysis syndrome is a human patient with a plasma uric acid concentration of > 15 mg / dL (hyperuricemia).

[0302] In another aspect, the present disclosure provides a method for metabolizing uric acid using a uricase variant disclosed herein. In one embodiment, the uricase variant is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant is present 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.

[0303] In another aspect, the present disclosure provides 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 is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant is present 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. In one embodiment, the biological fluid is from aAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application gout patient or a refractory gout patient. 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

[0304] The present invention is further illustrated by reference to the following Examples. However, it should be noted that these Examples, like the embodiments described above, are illustrative and are not to be construed as restricting the scope of the invention in any way. Example 1 – Creation and characterization of deimmunized non-PEGylated and PEGylated chimeric pig-baboon uricase variants.

[0305] This example describes the development of highly engineered, deimmunized non- PEGylated and PEGylated chimeric pig-baboon uricase variants by utilizing a computationally-driven protein engineering platform. Compared to the wild type chimeric pig- baboon uricase of SEQ ID NO:1, of which pegloticase is comprised, lead variant candidates exhibited better overall yield, activity and thermostability, while having reduced immunogenic potential due to mutagenic silencing (depletion) of constituent CD4+ T cell epitopes. Methods 1. Soluble Enzyme Activity Assay

[0306] Small volume cultures of E. coli transformed with the expression vector pET-26b(+) containing the cDNA sequence encoding the wild type chimeric pig-baboon uricase or its variant were grown and induced in deep 96-well plates. The expression vector pET-26b(+) encodes a His tag containing 6 histidine residues at the C-terminus of the inserted uricase gene. Induced E. coli cells were pelleted by centrifugation. Cell pellets were resuspended in BUGBUSTER® HT Protein Extraction Reagent (MilliporeSigma, MA, USA), pelleted by centrifugation, and the insoluble pellet was resolubilized in an alkaline carbonate / bicarbonate buffer (pH 10.5). The resolubilized sample was combined in a UV transparent 96-well plate with a uric acid substrate solution (0.1 M boric acid, 0.12 mM uric acid, pH 9.0), and absorbance at 293 nm was measured as a function of time. Uricase activity was measured as the slope of the absorbance at 293 nm vs time curve, with steeper negative slopes indicative of higher activity. As such, the slopes were quantified as raw activity measures in arbitrary units. 2. Expression AssayAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0307] The expression assay is based on measuring total activity in the soluble supernatant following lysis of E. coli cell pellets in the alkaline carbonate / bicarbonate buffer (pH 10.5), as described above, and separately measuring activity of immobilized uricase enzyme (on a 96- well Ni-NTA HisSorb plate (Qiagen)) from the soluble supernatant (described below), as the wild type chimeric pig-baboon uricase or its variant produced in E. coli contained a His tag at the C-terminus. In theory the HisSorb immobilized surface is saturated with enzyme, and thus dividing total activity by this saturated immobilized activity yields a relative expression level.

[0308] 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 the cDNA sequence encoding the wild type chimeric pig-baboon uricase or its variant were grown and induced in deep 96-well plates. The induced E. coli cells were pelleted by centrifugation. Cell pellets were resuspended in BUGBUSTER® HT Protein Extraction Reagent, pelleted by centrifugation, and the insoluble pellet was resolubilized in an alkaline carbonate / bicarbonate buffer (pH 10.5). The resolubilized sample was collected and transferred to a 96-well Ni-NTA HisSorb plate (Qiagen) bearing immobilized metal ion affinity ligands on the bottom of the wells. The HisSorb plate was incubated for one hour to bind the His-tagged uricase enzymes to the wells, followed by the removal of the supernatant from the wells and washing of the wells five times with the alkaline carbonate / bicarbonate buffer (pH 10.5). Thereafter, a uric acid substrate solution (0.1 M boric acid, 0.12 mM uric acid, pH 9.0) was added to the HisSorb plate, and absorbance at 293 nm was measured as a function of time. Uricase activity was measured as the slope of the absorbance at 293 nm vs time curve, with steeper negative slopes indicative of higher activity. Relative expression level was calculated as the activity of the total soluble enzyme fraction divided by the activity of the HisSorb-bound enzyme fraction. 3. Differential Scanning Fluorimetry (DSF)

[0309] DSF was performed according to Niesen et al., Nat Protoc. 2007, 2(9):2212-21, incorporated herein by reference in its entirety, to determine the melting temperature of the wild type chimeric pig-baboon uricase and its variants using a C1000 thermal cycler in conjunction with a CFX96 real-time system (Bio-Rad). Proteins were diluted to a final concentration of 66 μg / mL in PBS buffer containing SYPRO Orange dye at a final concentration of 5×. Triplicate samples of each protein were exposed to a temperature gradient from 40°C to 99°C. Melting temperatures were calculated using gain of fluorescence of SYPRO orange and loss of fluorescence using PRISM software.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 4. One-step purification and determination of the percentage of soluble tetramer by size exclusion chromatography SEC 4.1. Inclusion body purification

[0310] E. coli cells were lysed with 50 ml B-PER bacterial protein extraction reagent (ThermoFisher Scientific) per liter of culture with the addition of 1000 U / L BENZONASE® endonuclease (MilliporeSigma). Specifically, the cell pellet was resuspended in B-PER followed by incubation at room temperature for one hour. The suspension was centrifuged at 25,000 rpm for 30 min at 4 ^. 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 one hour, the suspension was centrifuged at 25,000 rpm for 30 min at 4 ^, and the supernatant was removed and reserved for subsequent chromatography. 4.2. Chromatographic Purification

[0311] The reserved 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 10 cv of load buffer, followed by elution with 5 cv of 100 mM sodium bicarbonate (pH 10.5) and 500 mM imidazole. Eluted proteins were buffer exchanged into 100 mM sodium bicarbonate (pH 10.5) and the percentage of soluble tetramer was quantified by an HPLC-SEC analytical method described below. 4.3. Determination of the percentage of soluble tetramer by SEC

[0312] The percentage of soluble tetramer in a naked uricase enzyme sample was determined using an HPLC-SEC analytical method to determine the size and concentration of the uricase enzyme. The HPLC-SEC analytical method used a Waters Arc HPLC system with a tunable UV (TUV) and refractive index (RI) detectors in conjunction with a Waters UPLC Premier Protein SEC Column (Thermo Scientific, Cat. No. 186005225). The method measured hydrodynamic volume as a surrogate assessment of molecular weight or size using the Waters protein standards (Thermo Scientific, Cat. No. 186006518) for molecular weight approximation. Specifically, the Waters protein standards or 12 μl of a naked uricase enzyme solution containing 2 mg / mL 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 was done by Waters TUV set to 280 nm. In the chromatogram of a naked uricase enzyme sample, one minor peak with a shorter retention time representingAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application aggregates and one major 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 times of the Waters protein standard. 5. Determination of reaction velocity for the wild type chimeric pig-baboon uricase and its variants

[0313] The reaction velocity for the wild type chimeric pig-baboon uricase and its variants was determined by the following procedure:

[0314] (1) diluting a uricase protein sample to 0.2 mg / mL in a buffer containing 0.1M carbonate-bicarbonate, pH 10.5;

[0315] (2) in a 96-well plate, aliquoting 10 μL of the diluted protein sample per well;

[0316] (3) adding 190 μL of a uric acid substrate solution containing 0.125 mM uric acid to each well;

[0317] (4) loading the 96-well plate onto a plate reader;

[0318] (5) measuring absorbance at 293 nm every 30 seconds for 20 minutes under the controlled temperature of 25oC on the plate reader;

[0319] (6) calculating Vmaxvalues using the BIOTEK Gen5 software. 6. Determination of Michaelis Menten kinetics for the wild type chimeric pig-baboon uricase and its variants

[0320] The Michaelis Menten kinetics for the wild type chimeric pig-baboon uricase and its variants were determined by the following procedure:

[0321] (1) diluting a uricase protein sample to 0.2 mg / mL in a buffer containing 0.1M carbonate-bicarbonate, pH 10.5;

[0322] (2) into a plurality of wells of a 96-well plate, aliquoting 10 μL of the diluted protein sample per well;

[0323] (3) adding to each well containing the diluted protein sample 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;

[0324] (4) loading the 96-well plate onto a plate reader;Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0325] (5) measuring absorbance at 293 nm every 30 seconds for 20 minutes under the controlled temperature of 25oC on the plate reader;

[0326] (6) calculating Vmax values using the BIOTEK Gen5TMsoftware;

[0327] (7) using the Michaelis-Menten function of the PRISM software, analyzing the Vmaxvalues as a function of the uric acid concentration to derive the Michaelis Menten kinetics parameters. 7. Purification

[0328] The wild type chimeric pig-baboon uricase and its variants expressed in E. coli were subject to multi-step purifications by various means, as detailed below. 7.1. Inclusion body purification

[0329] E. coli cells were lysed with 50 ml B-PER bacterial protein extraction reagent (ThermoFisher Scientific) per liter of culture with the addition of 1000 U / L BENZONASE® endonuclease (MilliporeSigma). Specifically, the cell pellet was resuspended in B-PER followed by incubation at room temperature for one hour. The suspension was centrifuged at 25,000 rpm for 30 min at 4 ^. 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 one hour, the suspension was centrifuged at 25,000 rpm for 30 min at 4 ^, and the supernatant was removed and reserved for subsequent chromatography. 7.2. Chromatographic Purification

[0330] The reserved 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 load buffer containing 1% TRITON-114 followed by 10 cv of load buffer, followed by elution 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). The elution was subsequently concentrated to 15 ml using stirred cell with 50 kDa filter to a concentration not exceeding 3 mg / ml. The concentrated elution was loaded onto a SUPERDEX 200 HILOAD 26 / 600 column, followed by elution with 100 mM sodium bicarbonate (pH 10.5). Fractions containing tetrameric uricase as identified by DLS as being monodisperse were pooled, and the pooled tetrameric uricase fractions wereAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application analyzed by SDS-PAGE and DLS. The pooled tetrameric uricase fractions were then concentrated using stirred cell with 50 kDa filter to 3 mg / ml. 7.3. Endotoxin Mitigation

[0331] Endotoxin in a preparation of the wild type chimeric pig-baboon uricase and its variant was removed by the following procedure:

[0332] (1) adding to the preparation 1% TRITON-114 followed by incubation for 30 minutes at 4^ and then 30 minutes at 37^;

[0333] (2) centrifuging the mixture in pyrogen free tubes for 30 minutes at 37^;

[0334] (3) decanting the top layer to fresh pyrogen free tubes;

[0335] (4) repeating steps (1)-(3) as needed to lower endotoxin level to < 0.5 EU / ml;

[0336] (5) repeating steps (2) and (3) one final time to remove residual TRITON-114;

[0337] (6) testing endotoxin using limulus amebocyte lysate (LAL) assay (Charles River) by diluting the test sample 1:20 in endotoxin free water and applying 25 μl to each sample well. 8. PEGylation of the wild type chimeric pig-baboon uricase and its variants

[0338] PEGylation of the wild type chimeric pig-baboon uricase or its variants was performed by the following procedure:

[0339] (1) adding 50 fold molar excess methoxy-PEG-(CH2)5COO-NHS of 10 kDa in molecular weight (Chemical Name: Poly(oxy-1,2-ethanediyl), Į-methyl-^- {2-[(2,5-dioxo-1- pyrrolidinyl)oxy]-6-oxohexyloxy}; NOF America Corporation, Cat# SUNBRIGHT ME- 100HS) to purified uricase protein on ice and incubating with stirring for one hour;

[0340] (2) verifying radius by DLS (14-15 nm radius ~= wild type);

[0341] (3) concentrating PEGylated protein using stirred cell with 50 kDa filter to 15 ml;

[0342] (4) loading the concentrated PEGylated protein onto a SUPERDEX 200 HILOAD 26 / 600 column and eluting with PBS;

[0343] (5) pooling fractions identified as being mono-disperse by DLS;

[0344] (6) concentrating the pooled fractions using a stirred cell with 50 kDa filter to 5 mg / ml. 9. Titer determination for antidrug antibody in mouse plasma using ELISAAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0345] A high-binding 96-well ELISA plate (Costar #2592) was coated with a 5 ^g / mL solution containing either one of PEGylated top six chimeric pig-baboon uricase variants (i.e., HF0536, HF0554, HF0585, HF0668, HF0752, and HF0835), or PEGylated wild type chimeric pig-baboon uricase, in bicarbonate buffer overnight at 4°C. The coating solution was then removed, and the plate was washed three times with phosphate buffer containing 0.1% Tween 20 detergent. The washed plate was blocked with 1x superblock buffer (ThermoFisher Scientific #37515) for 15 minutes at 37°C, followed by removal of the superblock buffer. Serial dilutions of 1:20 to 1:12,207 of mouse plasma in LOWCROSS buffer (ThermoFisher Scientific # NC9831360) were added to the wells, and the plate was incubated at 37oC for 60 minutes. Plasma dilutions were removed, and the plate was washed three times with phosphate buffer containing 0.1% Tween 20 detergent. The plate was then incubated for 30 minutes at 37^ with a 1:10,000 dilution of an anti-mouse secondary antibody conjugated with horseradish peroxidase, diluted in LOWCROSS buffer. The secondary antibody was then removed, and the plate was washed four times with phosphate buffer containing 0.1% Tween 20 detergent. Antidrug antibodies were then quantified by detection with TNB (3,3’,5,5’- tetramethylbenzidine) substrate (Bethyl #E102), measuring absorbance at 450 nm and subtracting background absorbance at a reference wavelength of 620 nm. 10. Immunogenicity analysis for the wild type chimeric pig-baboon uricase and its variants by ex vivo cellular immunoassays using human peripheral blood mononuclear cells (PBMCs)

[0346] PBMCs from healthy human donors were expanded with IL-2 in the presence of the wild type chimeric pig-baboon uricase protein or one of its variant proteins for 13 days, followed by restimulation with the same protein. Surface and intracellular staining for flow cytometry was then used to evaluate T cell activation markers and cytokine production. Briefly, PBMCs were seeded into 48-well plates at 2×106cell per well in a final volume of 200 μl RPMI media with 5% human serum. Antigen stimulation or vehicle control was added at the time of plating. Proteins were added at a final concentration of 10 μg / mL. IL-2 was added on day 4 at a final concentration of 10 U / mL and maintained thereafter with media changes every other day. After 13 days of expansion, cells were restimulated with the same conditions in the presence of brefeldin A (to block cytokine secretion) for 10 h. Flow cytometric staining was then performed as follows: Cells were washed with PBS twice and then stained with Zombie AquaTMlive dead stain at a 1:1000 dilution at room temperature for 30 minutes. Cells were then washed with FACS buffer (1% FBS in 1× PBS), followed by incubation with Fc BlockTMfor 10 minutes at room temperature. Cells were washed twice more with FACS buffer and thenAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application stained with the following antibodies diluted in BD BrilliantTMStain Buffer: CD3 BB515, CD4 SB600, CD8 BV650, CD25 APC Fire750, and CD69 BV750. Samples were incubated with surface stains for 30 min at 4oC in the dark in a final volume of 100 μl. Cells were then washed three times with FACS buffer before proceeding with intracellular stains. Intracellular staining was performed using BD Cytofix / CytopermTMFixation / Permeabilization staining kit as per the manufacturer's instructions with Ki67 AF647, IFN-Ȗ BB700, TNF-Į BV421, and IL-2 PE. Following the last step, samples were resuspended in FACS buffer and acquired on a Cytek® Northern LightsTMfull spectrum flow cytometer.

[0347] Immunogenicity of the wild type chimeric pig-baboon uricase and its variants was measured by a stimulation index for each donor against each test uricase protein. The stimulation index was calculated as a fold activation relative to background where the percentage of cells that were positive for each combination of activation markers were divided by the percentage of positive cells for that combination of markers in the vehicle control group. The sum of fold increase in signal for each combination of markers was then reported as the stimulation index. The combinations of activation markers included (1) a marker of proliferation (i.e., Ki67), (2) markers of activation (i.e., CD25 and CD69); and (3) markers of functionality (i.e., IL-2, INF-Ȗ, and TNF-Į). Results

[0348] The monomeric form of the wild type chimeric pig-baboon uricase comprises the amino acid sequence of SEQ ID NO:1, where threonine is residue #1. The specified mutations in the chimeric pig-baboon uricase variants of this example are indexed to threonine as residue #1, as in SEQ ID NO:1. However, in this example, the wild type chimeric pig-baboon uricase and its variants produced in E. coli possessed a methionine (preceding the threonine) as residue #1, with the threonine as residue #2, due to the presence of an Ndel restriction site encoding methionine in-frame with the wild type and the variant chimeric pig-baboon uricase genes in the expression vector pET-26b(+) (MilliporeSigma, MA, USA). The expression vector pET- 26b(+) encodes a His tag containing 6 histidine residues at the C-terminus of the inserted uricase gene. 1. Deimmunized library design

[0349] Deimmunized library design was carried out to computationally optimize uricase variant libraries and select one library for experimental construction. As illustrated in FIG.2A, using proprietary design algorithms, mutations (both positions and position-specificAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application substitutions) to the wild type chimeric pig-baboon uricase defining combinatorial libraries predicted to be enriched in clones with high function and low immunogenicity were selected. In designing a library, the algorithms model the entire protein and evaluate the interrelated effects of combinations of mutations on both epitope content and protein stability and function. The algorithms generated hundreds of Pareto optimal and near-optimal designs, striking different levels of aggressiveness in deleting epitopes vs. maintaining function. The designs explored different population sizes (about 20,000 to 1 million members), different numbers of mutational target sites, and different numbers of substitutions per site (1 to 4). Two iterative rounds of library design and construction were carried out, using the most deimmunized and functional variants from the first round as the starting points for the second round and involving filtering and inspection of epitope and function scores. Specifically, a library design with about 400,000 theoretical variants harboring combinatorial amino acid substitutions among 18 target sites of SEQ ID NO:1 was selected in the first round. In the second round, a refined library design was selected with about 18,000 theoretical variants harboring combinatorial amino acid substitutions among 12 additional target sites (i.e., among a total of 30 target sites) of SEQ ID NO:1, as summarized in Table 2. In Table 2, the positions of the amino acids among the 30 target sites present in the chimeric pig-baboon uricase variants according to the library design of the second round, referred to as “designed amino acids” herein, are indexed to threonine as residue #1, as in SEQ ID NO:1. The library members had reduced MHC-II binding potential as compared to the wild type chimeric pig-baboon uricase. Lead deimmunized and functional variant candidates from the second round of library design mostly had between 10 and 20 combinatorial amino acid substitutions among the 30 target sites.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 2. Designed amino acids in chimeric pig-baboon uricaseAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 2. Designed amino acids in chimeric pig-baboon uricase

[0350] Upon selection of this library design, functional chimeric pig-baboon uricase variants were identified via high throughput screening (FIG. 2B), with most or all immunodominant epitopes in the protein sequence silenced (FIG.2C), as described below. 2. Library screening and functional evaluation

[0351] Library screening and functional evaluation were performed to experimentally identify and characterize a diverse set of high-functioning uricase variant candidates by (i) employing a high-throughput halo-based assay to select active variants, and (ii) purification and detailed analysis of yield, activity, and stability for the top candidates.

[0352] Primary screening of the combinatorial deimmunized uricase variants library was conducted by uricase halo assay (FIG. 3A). Specifically, E. coli transformed with the expression vector pET-26b(+) containing the cDNA sequence encoding the wild type chimeric pig-baboon uricase or its variant was inoculated onto LB agar plates containing uric acid. Bacterial colonies expressing active uricase converted the suspended uric acid into water soluble allantoin, such that a clear zone (halo) around target bacteria indicates production of an active enzyme. The diameter of each clear zone was recorded as a semi-quantitative measure of enzyme expression and activity. The variants selected based on halo size were inoculated into deep 96-well plates, grown, induced, and cell pellets resuspended in lysis buffer. The uricase enzyme activity was quantified (i) in the alkaline-soluble lysate (pH 10.5 carbonate / bicarbonate buffer), and (ii) on enzyme immobilized from the alkaline-soluble lysate onto Ni-NTA HisSorb plates (Qiagen). A pseudo-estimate of expression level was calculated as the quotient of total lysate activity to immobilized lysate activity, as described in the “Methods” section above. Solution phase kinetic analysis on the whole cell lysates allowed for stringent selection of clones exhibiting the fastest kinetics (FIG. 3B). Table 3 shows 130 functional chimeric pig-baboon uricase variants identified. With the genes of the variantsAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application sequenced, Table 3 shows each variant’s SEQ ID NO and mutations relative to the wild type chimeric pig-baboon uricase of SEQ ID NO:1. Table 3. Exemplary functional chimeric pig-baboon uricase variants 17014K1751.155G / R209W / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 3. Exemplary functional chimeric pig-baboon uricase variants 511155201217 . 5G / R209F / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 3. Exemplary functional chimeric pig-baboon uricase variants 02020015.55G / R209W / I257TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 3. Exemplary functional chimeric pig-baboon uricase variants 015105591. 7KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 3. Exemplary functional chimeric pig-baboon uricase variants 11KK51170051712.57TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 3. Exemplary functional chimeric pig-baboon uricase variants 11052500K7 . 155G / R209F / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 3. Exemplary functional chimeric pig-baboon uricase variants 0K05RL

[0353] The characteristics, including the yield, Michaelis-Menten kinetics parameters, and melting temperatures measured by DSF, of a select group of 21 non-PEGylated variants, as well as non-PEGylated wild type chimeric pig-baboon uricase (WT), are summarized in Table 4B. The SEQ ID NOs, mutations to the wild type chimeric pig-baboon uricase of SEQ ID NO:1, and amino acid sequences of the select variants are shown in Table 4A.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 4A. SEQ ID NOs, mutations to wild type chimeric pig-baboon uricase of SEQ ID NO:1, aKHVIRLQVYVEEVPWKRFEKNGVKHVHAFIHTAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 4A. SEQ ID NOs, mutations to wild type chimeric pig-baboon uricase of SEQ ID NO:1, aYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 4A. SEQ ID NOs, mutations to wild type chimeric pig-baboon uricase of SEQ ID NO:1, aAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 4A. SEQ ID NOs, mutations to wild type chimeric pig-baboon uricase of SEQ ID NO:1, aKHVIRLQVYMEEVPWKRFEMNGVKHVHAFIHTAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 4A. SEQ ID NOs, mutations to wild type chimeric pig-baboon uricase of SEQ ID NO:1, aAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 4B. Yield, Michaelis-Menten kinetics parameters, and melting temperature of non- P P Tm

[0354] Based on the data of Table 4B, the yield, activity, and Tmof the 21 non-PEGylated candidate variants, as well as the non-PEGylated WT comparator, were scored, as summarized in Table 4C. T t vHF0536 4 3 3 10Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 4C. Scoring of yield, activity, and melting temperature of non-PEGylated wild t v

[0355] Based on the total score data of Table 4C, top six variant candidates were selected, as shown in Table 4D. Those top six variant candidates each have a high total score of between 9 and 11, as compared to the total score of 5 for the WT. T t sHF0554 3 4 3 10Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 4D. Scoring of yield, activity, and melting temperature of non-PEGylated wild t s3. PEGylation of the top six chimeric pig-baboon uricase variant candidates and characterization of the PEGylated variants

[0356] The top six variant candidates, as well as the wild type chimeric pig-baboon uricase, produced in E. coli as described above were purified and conjugated to polyethylene glycol (PEG). Successful PEGylation of the wild type chimeric pig-baboon uricase and the variants was confirmed by SDS-PAGE (FIG.4A). On the SDS-PAGE, the bands at about MW 34 kDa represent non-PEGylated uricase species, whereas the bands at about MW 291 kDa represent PEGylated uricase species estimated by size exclusion chromatography with multi-angle static light scattering (SEC-MALS) analysis to have about 10 PEG molecules per monomer.

[0357] To confirm that the top six variant candidates were each capable of forming a homotetramer, the non-PEGylated (naked) wild type chimeric pig-baboon uricase and each of the non-PEGylated (naked) top six variant candidates were subject to one-step purification, followed by determination of the percentage of soluble tetramers using size exclusion chromatography (SEC), as described in the “Methods” section. FIG.4B shows that each of the non-PEGylated variants had a higher proportion of soluble tetramers than the non-PEGylated wild type counterpart, indicating that the top six variant candidates had reduced aggregation as compared to the wild-type chimeric pig-baboon uricase.

[0358] The specific activities, Michaelis Menten kinetics, and the melting temperatures measured by differential scanning fluorimetry of the PEGylated wild type chimeric pig-baboon uricase and the PEGylated top six variant candidates were determined, with the data shown in FIGS. 4C, 4D, 4E, and 4F. The results from those studies indicate that the specific activities of the PEGylated variants ranged from 90% to 133% of the PEGylated wild-type counterpart,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application and that the thermostabilities of the PEGylated variants were either comparable to or higher than that of the PEGylated wild-type counterpart. Taken together, the PEGylated top six variant candidates display good overall performance in vitro. 4. Immunogenicity Analysis

[0359] An assessment of immunogenicity of the non-PEGylated top six chimeric pig-baboon uricase variant candidates relative to the non-PEGylated wild type chimeric pig-baboon uricase (WT) was made by ex vivo cellular immunoassays using human peripheral blood mononuclear cells (PBMCs). FIG.5A is a schematic of the ex vivo cellular immunoassays measuring T-cell activation, with the details of the assays described in the “Methods” section. In the assays, immune cells from each individual human donor were incubated with either non-PEGylated wild type chimeric pig-baboon uricase (WT) or a non-PEGylated deimmunized uricase variant (dURC) over a 14-day expansion period. T cells were then evaluated by flow cytometry for markers of proliferation (i.e., Ki67), activation (i.e., CD25 and CD69) and functionality (i.e., IL-2, INF-Ȗ, and TNF-Į). The sum of these activation markers was used to calculate a “stimulation index” for each donor against each test protein. The assay results are presented in FIG.5B, where stimulation index (fold activation relative to background) is shown for each of the 20 individual human donors challenged with each of the non-PEGylated top six variant candidates or the non-PEGylated wild type comparator, with each symbol representing a single donor. The bars in FIG.5B indicate the median value with 95% CI for each population. The assay results demonstrate a marked reduction in T cell responses with non-PEGylated deimmunized uricase variant candidates HF0668, HF0752 and HF0835.

[0360] The immunogenicity of the PEGylated top six chimeric pig-baboon uricase variant candidates relative to the PEGylated wild type chimeric pig-baboon uricase was assessed in humanized HLA transgenic mouse strain DR4. Humanized HLA transgenic mouse strain DR4 has functioning cellular and humoral immunity, but the murine class II MHC locus has been replaced with a functional form of human MHC II allele DRB1*0401, a common allele among human populations and a representative MHC II supertype. DR4 mice develop antidrug antibodies as a result of human DRB1*0401 restriction of T cell epitopes.

[0361] Humanized DR4 HLA transgenic mice were subject to initial immunization via intraperitoneal administration on Day 1 with 0.25 μg / kg of one of the PEGylated uricase variants or the PEGylated wild type chimeric pig-baboon uricase, followed by booster immunizations via intraperitoneal administration on Days 7, 14, 21, 28, and 35 with 2.5 μg / kgAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application of the same PEGylated uricase variant or the PEGylated wild type chimeric pig-baboon uricase, with 10 mice in each test protein group. Mice were dosed at about 1 / 500 of the human equivalent dose by allometric scaling to minimize drug interference in the measurement of antidrug antibodies. Mouse plasma was collected on Day 42 for antidrug antibody analysis by ELISA (FIG.6A).

[0362] FIG.6B shows the curves representing the relationship between the antidrug antibody titer determined by ELISA absorbance and plasma dilution on a log scale, using the Day 42 plasma collected from the humanized DR4 HLA transgenic mice immunized with the PEGylated uricase variants or the PEGylated wild type counterpart. Higher ELISA absorbance at larger plasma dilutions is indicative of a stronger antidrug antibody response. One summary measure of overall response is area under the plasma dose-response curve (AUC), where larger AUC indicates a stronger antidrug immune response. FIG. 6C shows the antidrug antibody titer AUC derived from FIG. 6B. The results demonstrate that each of the PEGylated top six chimeric pig-baboon uricase variant candidates was less immunogenic than the PEGylated wild type chimeric pig-baboon uricase in DR4 mice, despite low dosing levels required by the study design. 5. Efficacy Analysis

[0363] The efficacy of weekly repeat dosing of three of the PEGylated top six chimeric pig- baboon uricase variant candidates HF0536, HF0554, and HF0752 relative to the PEGylated wild type chimeric pig-baboon uricase was assessed in humanized DR4 HLA transgenic mice. Specifically, as shown in FIG.7A, humanized DR4 HLA transgenic mice in groups of 9 mice each were either left untreated (i.e., no treatment healthy mouse group), or subjected to an induction dose via intraperitoneal administration on Day 1 of 4.2 mg / kg of PEGylated uricase variant HF0536, HF0554, or HF0752, or the PEGylated wild type chimeric pig-baboon uricase (WT), followed by maintenance doses also via intraperitoneal administration on Days 7, 14, 21, and 28 of 1.4 mg / kg of the respective PEGylated test protein. The induction dose of 4.2 mg / kg PEGylated uricase was three times the human equivalent dose by allometric scaling, whereas the maintenance dose of 1.4 mg / kg PEGylated uricase was one time the human equivalent dose by allometric scaling. Mouse plasma was collected on Days 5, 12, 19, 26, and 33 for uric acid analysis. FIG. 7B is a graph showing the serum uric acid levels in each treatment mouse group at various weeks on uricase therapy, or in the no treatment (No Rx) healthy mouse group at the same time points. FIG. 7C is a graph showing the percentages of mice in each group whose serum uric acid levels were 0 at various weeks on uricase therapyAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application (in the treatment groups) or without therapy (in the no treatment (Rx) healthy mouse group). PEGylated variants HF0752 and HF0554 maintained low average serum uric acid levels near 0 through 7 doses. PEGylated variant HF0752 maintained serum uric acid levels at 0 in 8 of the 9 mice through 7 doses. Taken together, the data demonstrate that PEGylated uricase variants HF0536, HF0554, and HF0752 exhibited better efficacy than the PEGylated wild type counterpart. The enhanced efficacy during repeated dosing is presumably due to blunting of the antidrug immune response, as designed. However, enhanced potency may also play a role. Example 2 – Identification and mitigation of undesired post-translational modifications of non-PEGylated deimmunized uricase variant HF0752.

[0364] This example describes mass spectroscopy peptide mapping and post-translational modification (PTM) analyses of non-PEGylated deimmunized uricase variant HF0752 comprising the amino acid sequence of SEQ ID NO:129 described in Example 1, as well as non-PEGylated wild type chimeric pig-baboon uricase (WT) comprising the amino acid sequence of SEQ ID NO:1. The PTM analyses 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 levels of deamination found at N117. To mitigate potential risk to the safety and potency of the deimmunized uricase variant HF0752, subvariants of HF0752 each harboring an additional amino acid substitution at N117, i.e., N117S, N117H, N117G, N117D, or N117Q were made to avoid deamination and tested for yield, thermostability, and activity. Method Mass Spectroscopy Peptide Mapping and Post-translational Modification (PTM) Analysis

[0365] The uricase protein sample was initially denatured by combining it with 8 M urea in a 100 mM Tris-HCl buffer (pH = 8.5). Subsequently, the sample underwent reduction and alkylation through incubation with Tris (2-carboxyethyl) phosphine (TCEP) and chloroacetamide, respectively. The concentration of urea was then reduced to 2 M by diluting the sample with 100 mM Tris-HCl buffer, after which trypsin was added to initiate protein digestion. The sample was then incubated at 37°C for 4 hours in a thermomixer. To quench the digestion reaction, formic acid was introduced to the sample. The digested peptides were injected into LC-MS for data acquisition. The LC-MS system was an Orbitrap EclipseTMmass spectrometer coupled with a VanquishTMNeo nanoflow liquid chromatography system. Peptide separation was achieved using an Easy-SprayTMPepMap Neo column. The data obtained was processed using Biopharma FinderTM5.0 software, which facilitated theAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application identification of peptides and the calculation of post-translational modification (PTM) abundances. A manual review of the calculated PTM levels was performed to eliminate false positives and ensure the use of appropriate peptides for accurate calculations. Results

[0366] Mass spectroscopy peptide mapping and post-translational modification (PTM) analyses of non-PEGylated deimmunized uricase variant HF0752 comprising the amino acid sequence of SEQ ID NO:129 and non-PEGylated wild type chimeric pig-baboon uricase (WT) comprising 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 levels of deamination found at N117 (Table 5). T l L l f i i f i A i f - -Because the high levels of deamination at N117 of HF0752 may pose potential risk to the safety and potency for its use as a therapeutic protein, five HF0752 subvariants with N117 mutated to aspartic acid (HF0752-N117D), serine (HF0752-N117S), glycine (HF0752-N117G), glutamine (HF0752-N117Q), or histidine (HF0752-N117H) were made to avoid deamination by performing site-directed mutagenesis of the HF0752 expression vector described in Example 1. In parallel with the parent HF0752 variant, the HF0752 subvariants were then expressed and analyzed for deamination using mass spectroscopy peptide mapping, as well as tested for yield, thermostability, and activity using the methods similar to those described in Example 1. Table 6 shows the SEQ ID NOs of the amino acid sequences (each with threonine as residue #1) and gene sequences (each including the start codon ATG and stop codon TAA) of HF0752 and its five subvariants presented in the Sequence Listing.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 6. SEQ ID NOs of the amino acid sequences and gene sequences o

[0367] Table 7 shows the deamination levels of HF0752 and the HF0752 subvariants. The data of Table 7 indicates that mutation of Asn117 to any of the alternative residues Ser, His, Gly, or Asp completely eliminated deamidation at position Asn117. Additionally, none of the mutations at Asn117 substantially increased the level of deamidation observed at other possible asparagine deamidation sites in the protein sequence. T 5 a R D N. .

[0368] Table 8 shows the yield, thermostability measured by melting temperature (Tm) as determined by differential scanning fluorimetry, and activity measured by normalized 2 μg uricase reaction velocity of HF0752 and the HF0752 subvariants. The data of Table 8 indicates that, compared to the HF0752 variant, all of the HF0752 subvariants with mutations at the 117 position performed relatively well. The aspartic acid mutation present in HF0752-N117D appeared to have the least effect on activity and thermostability, consisting with aspartic acid being the product of asparagine deamidation. HF0752-N117G also exhibited the parent HF0752 variant-like thermostability and activity.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 8. Yield, melting temperature (Tm), and activity of HF0752 and HF0752 subvariants wExample 3 – Immunogenicity analyses of and mapping of T-cell epitopes in the non- PEGylated top six deimmunized chimeric pig-baboon uricase variant candidates and non-PEGylated wild type chimeric pig-baboon uricase by ex vivo cellular immunoassay using human peripheral blood mononuclearand by MHC- associated peptide proteomics (MAPPs) assay

[0369] This example describes immunogenicity analyses of and mapping of T-cell epitopes in the non-PEGylated top six deimmunized chimeric pig-baboon uricase variant candidates described in Example 1, i.e., HF0536, HF0554, HF0585, HF0668, HF0752, and HF0835, as well as non-PEGylated wild type (WT) chimeric pig-baboon uricase by ex vivo cellular immunoassay using human peripheral blood mononuclear cells (PBMCs) and by MHC- associated peptide proteomics (MAPPs) assay. It is noted that the variant code names HF0536, HF0554, HF0585, HF0668, HF0752, and HF0835 used in Example 1 are abbreviated as HF536, HF554, HF585, HF668, HF752, and HF835, respectively, for conciseness in this and subsequent examples as well as in the figures described in those examples. As such, the variant code names HF0536, HF0554, HF0585, HF0668, HF0752, and HF0835 and their respective abbreviated code name counterparts are used interchangeably in the Examples section of the present application. Consistent with the data presented in Example 1, the study results of this example demonstrate that all of the non-PEGylated top six deimmunized chimeric pig-baboon uricase variants had reductions in T cell responses, as well as reductions in the presentation of peptides and in immunogenic regions with presented peptides, as compared with the WT uricase, with the variants HF536 and HF752 exhibiting the greatest overall reductions. Methods 1. Immunogenicity analysis for the wild type chimeric pig-baboon uricase and its variants by ex vivo cellular immunoassay using human peripheral blood mononuclear cells (PBMCs)Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0370] PBMCs from healthy human donors were expanded with IL-2 in the presence of non- PEGylated wild type chimeric pig-baboon uricase protein or one of its variant proteins for 13 days, followed by restimulation with peptides corresponding to the protein used for initial stimulation. Surface and intracellular staining for flow cytometry was then used to evaluate CD4 T cell cytokine production.

[0371] Briefly, PBMCs were seeded at 6.25×106cell / mL RPMI media with 5% human serum into appropriate culture vessels. Antigen stimulation or vehicle control was added at the time of plating. IL-2 was added on day 4 and maintained thereafter with media changes every 2-3 days. After 13 days of expansion, cells from each stimulation condition were counted and redistributed into 96 well plates. Restimulation with peptides or pools of peptides was at a final concentration of 2 μM and all samples were treated with brefeldin A (to block cytokine secretion) overnight. Flow cytometric staining was then performed with the following surface stains: Zombie AquaTM, and anti-CD3, anti-CD4 and anti-CD8 antibodies. Intracellular staining for IFN-Ȗ, TNF-Į and IL-2 was performed using BD Cytofix / CytopermTMFixation / Permeabilization staining kit as per the manufacturer's instructions. Following the last step, samples were resuspended in PBS and acquired on a Cytek® Northern LightsTMfull spectrum flow cytometer.

[0372] Peptides used for restimulation of cells expanded in the presence of the wild type chimeric pig-baboon uricase protein for the purpose of mapping CD4+ T cell epitopes in this protein are listed in Table 9. The peptides were 18 amino acids (aa) in length with an offset of 2-4 aa. For epitope mapping, the peptides during restimulation were used in pools of two sequential peptides walking down the length of the protein as indicated in Table 9. T uYHSIKEVATTVQLTLSSK31 48 6 18 WTAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 9. Peptides used for CD4+ T cell epitope mapping of the wild type chimeric pig-baboon uTQSGFEGFIKDQFTTLPE160 17718 WTAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 9. Peptides used for CD4+ T cell epitope mapping of the wild type chimeric pig-baboon uDNPYGKITGTVKRKLSSR280 29718 WTAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0373] To compare CD4 T cell responses to the different epitopes in the deimmunized uricase (dURC) variants to those in the wild type chimeric pig-baboon uricase protein, a subset of 6 regions were selected to evaluate. The regions to evaluate were selected based on predicted immunogenicity with prioritization given to regions which contained epitopes of high predicted immunogenicity and covered the locations of mutations in the dURC variants. To cover these regions, 12 peptides were selected to cover the core epitopes within the 6 regions. During restimulation, each of the peptides corresponding to the sequence of the protein used for expansion was used as shown in Table 10. Table 10 Peptides used for CD4+ T cell epitope mapping of the wild type chimeric pig-baboon uricase p SS IVLQ IVLQ IVLQ IVLQ IVLQ IVLQ IVLQAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 10. Peptides used for CD4+ T cell epitope mapping of the wild type chimeric pig-baboon uricase p i i i i i S

[0374] Immunogenicity of the wild type chimeric pig-baboon uricase and its variants was reported as the sum of the % of CD4+ T cells positive for each cytokine (IFN-Ȗ, TNF-Į and IL-2) for each donor (summed cytokines). For graphing of the heat maps, the maximum summed cytokine value of any peptide covering that amino acid (AA) is shown. For FIG. 13, the summed cytokine value for each individual donor is further summed across all donors for each peptide evaluated. As indicated in the figures, background signal was subtracted for each condition. 2. Immunogenicity analysis for the wild type chimeric pig-baboon uricase and its variants by ex vivo cellular immunoassay using MHC-associated peptide proteomics (MAPPs) assay

[0375] To evaluate the peptides presented by antigen presenting cells (APCs) derived from either the wild type chimeric pig-baboon uricase protein or one of its variant proteins, a MAPPs assay approach was implemented. Briefly, monocytes were isolated from healthy human donors, differentiated to mature dendritic cells (DCs) and pulsed with the protein of interest. Pulsed cells were then harvested, lysed and peptide MHC (pMHC) complexes were pulled down using antibody conjugate beads. The peptides were then eluted from the pMHC complexes and analyzed by mass spectrometry (MS). The following is an outline of the procedures for the MAPPs assay. 2.1 Monocyte isolation and culturing

[0376] Monocytes were isolated from leukopaks from healthy human donors. Leukopaks were first processed to isolate PBMCs and then monocytes were isolated using the STEMCELL’s EasySep™ Human Monocyte Enrichment Kit without CD16 depletion as per the manufacturer’s instructions. For each protein evaluated, 58×106monocytes were seeded into a T225 flask and cultured using ImmunoCultTMDendritic Cell Culture Kit from STEMCELL according to the instructions. The protein of interest was added on day 4 ofAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application differentiation. On Day 7 the mature antigen pulsed dendritic cells were harvested, washed with PBS, counted, and pellets frozen at -80oC. 2.2 Peptide isolation

[0377] Frozen cell pellets from the matured dendritic cells were then lysed using PierceTMIP Lysis Buffer. Cell debris was then pelleted, the cleared supernatant was collected and added to CNBR beads pre-conjugated with the pan HLA II monoclonal antibody, clone IVA12. This mixture was then incubated overnight at 4oC with rotation. The bead supernatant mixture was then applied to a Poly-Prep®chromatography column and washed with each of the following buffers in sequence: 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 (Tris 20 mM, pH 8.0). The pMHC complexes were next eluted off the beads in 1% TFA in water. Peptides were then eluted from the pMHC complexes using C18 columns. The eluted peptides were dried down using a vacuum system. 2.3 Mass Spectrometry Analysis

[0378] The samples from Section 2.2 were reconstituted using 95% H2O + 5% acetonitrile (ACN) prior to their injections into an LC-MS system. The LC-MS system was an Orbitrap EclipseTMmass spectrometer (Thermo Scientific, San Jose, CA) coupled with a VanquishTMNeo nanoflow liquid chromatography system (Thermo Scientific, San Jose, CA). Trap and elute injection mode was performed where samples were first loaded on a trap column (PepMapTMNeo, 5 ^m, C18, 300 ^m × 5 mm Trap Cartridge) before separation on Easy- SprayTMPepMapTMNeo column (2 ^m, C18, 75 ^m × 150 mm). The solvent gradient started at 5% of solvent B (ACN, 0.1% formic acid) for the initial 5 minutes, then increased to 25% solvent B over the next 75 minutes, reaching 35% solvent B from 80 to 105 minutes. In the column wash stage, the gradient spiked to 90% solvent B within a minute and maintained this level for an additional 10 minutes. Data dependent acquisition mode was performed. The system operated in data-dependent acquisition mode, with the ORBITRAP mass analyzer conducting survey scans at a 120K resolution across an m / z range of 375 to 1500. A normalized automatic gain control (AGC) target of 250% and a maximum injection time of 50 ms were set for survey scan. MS2 scans were executed by the ion trap mass analyzer. Precursor ions were fragmented using 30% high-energy collisional dissociation (HCD) with a normalized AGC target of 100% and a maximum injection time of 35 ms. A dynamic exclusion period of 60 seconds and a mass tolerance of 10 ppm were set.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0379] Raw data were processed using PROTEOME DISCOVERER 2.5. Using the SEQUEST search engine, data were searched against the UNIPROT human protein sequences in addition to uricase sequences with no enzyme specificity. The search parameters included a peptide length of 7-30 amino acids and dynamic modification of oxidation. INFERYS®Rescoring and Percolator nodes were also integrated into the workflow following the SEQUEST search to further refine search results. Results

[0380] An assessment of immunogenicity of the non-PEGylated top six deimmunized chimeric pig-baboon uricase variant candidates HF536, HF554, HF585, HF668, HF752, and HF835 relative to the non-PEGylated wild type chimeric pig-baboon uricase (WT) was made by ex vivo cellular immunoassay using human peripheral blood mononuclear cells (PBMCs), also referred to as the “PBMC assay” in this example. FIG. 8 is a schematic of the PBMC assay measuring T-cell activation, with the details of the assay 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-baboon uricase (WT) or a non- PEGylated deimmunized uricase variant over a 14-day expansion period. T cells were then evaluated by flow cytometry for cytokine production as an indicator of activation (IL-2, IFN- Ȗ, and TNF-Į). The sum of the % CD4+ T cells positive for each of the cytokines is termed the “summed cytokine” value. The 12 donors used to evaluate T cell responses represented all 7 of the HLA II supertypes with coverage of 25 of the 27 alleles contributing to the supertypes (see Greenbaum et al., Immunogenetics. 2011 Jun;63(6):325-35, incorporated herein by reference in its entirety). As such, those donors were a good representation of possible responses. The HLA II alleles covered for each supertype are shown in Table 11. TA5P M M M D D D D D DDRB5*0101 X X X X X XAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 11. HLA II alleles and coverage of supertypes for donors used in immunogenicity studiesA5D D D D D D D D D D D M D D D D D D M D D D D D D D A D D D D D D D D D D D D D D D D DDQB1*0604 X X X XAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0381] In addition to evaluation of the T cell responses to the WT and variant uricase proteins by the PBMC assay, MAPPs analysis was also performed for each protein to identify differences in the peptides presented on HLA II by monocyte derived dendritic cells from healthy human donors. FIG.9 is a schematic of the MAPPs assay, with the details of the assays described in the “Methods” section.

[0382] The monocytes were differentiated to mature dendritic cells (DCs) and pulsed with the protein of interest. Pulsed cells were then harvested, lysed and peptide MHC (pMHC) complexes were pulled down using antibody conjugated beads. The peptides were then eluted from the pMHC complexes and analyzed by mass spectrometry (MS). A total of 8 donors were used for MAPPs analysis of the WT protein and 5 donors were used for the comparison between the WT and the variants. Coverage of the HLA II supertype alleles used for MAPPs assays are shown in Table 11. 1. Immunogenicity analysis of the wild type chimeric pig-baboon uricase protein

[0383] FIG. 10 shows the CD4+ T cell epitope mapping results of the wild type (WT) chimeric pig-baboon uricase protein using the PBMC assay, in which healthy human peripheral blood mononuclear cells (PBMCs) from each donor were expanded in the presence of the WT uricase protein for 14 days, 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 summed cytokine value (% IL-2+, INF-Ȗ+, and TNF-Į+ populations of CD4+ T cells) of each tested peptide with background subtraction was plotted as a heatmap. The cytokine value for each amino acid position in the protein was the highest cytokine value of the overlapping peptides covering the position. In FIG.10, the top bar, labeled “Aggregate,” is the max of the summed cytokine values across all 12 donors used. The gray scale shows the summed cytokine values represented by a range of gray shades. Values above the indicated range are presented as black (max cytokine value = 26.3).

[0384] The wild type chimeric pig-baboon uricase protein was also subjected to MAPPs analysis, where monocytes from 8 healthy human donors were differentiated to mature dendritic cells (DCs) and pulsed with the protein of interest. Pulsed cells were then harvested, lysed and peptide MHC (pMHC) complexes were pulled down using antibody conjugated beads. The peptides were then eluted from the pMHC complexes and analyzed by mass spectrometry (MS). FIG. 11 shows the MAPPs analysis results, with the heatmap thereinAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application showing the number of unique peptides identified by the MAPPs assay overlapping at each amino acid position for each donor. The “Aggregate” row at the top of FIG. 11 displays the max counts of unique peptides from any donor at each amino acid position. The gray scale shows a range of gray shades representing different count values.

[0385] Together these data identify the most highly immunogenic regions in the WT chimeric pig-baboon uricase protein that needed to be addressed in the deimmunized candidates. 2. Immunogenicity comparison of the wild type chimeric pig-baboon uricase protein and deimmunized uricase variants

[0386] FIG.12 shows the CD4+ T cell epitope mapping results of the deimmunized uricase variants as compared to the wild type chimeric pig-baboon uricase protein using the PBMC assay, in which healthy human PBMCs from each of 12 donors were expanded in the presence of the WT or variant uricase protein for 14 days, restimulated with synthetic peptide corresponding to the protein used for initial stimulation (see Table 10) covering the 6 regions indicated by black bars above the heatmaps and then evaluated for T cell activation as measured by intracellular cytokine staining and flow cytometry analysis. For each donor evaluated, the max summed cytokine value (% CD4+ T cells positive for IL-2, INF-Ȗ, and TNF-Į) of each tested peptide with background subtraction is plotted as a heatmap. The cytokine value for each position in the protein is the highest cytokine value of the overlapping peptides covering that position. For comparison, the full epitope mapping of the WT uricase shown in FIG. 10 is presented at the top of FIG.12. The * symbol in FIG.12 represents the positions of mutations in each deimmunized uricase variant protein. The gray scale key shows varying summed cytokine values represented by a range of gray shades. Values above the indicated range are presented as black (max cytokine value = 26.3). The data of FIG. 12 indicate that the most highly immunogenic regions of the WT protein are covered by the 6 regions selected for evaluation of the deimmunized candidates. The data also indicate the impact of the mutations in the variants on T cell responses in those regions.

[0387] FIG.13 shows the results of a further analysis comparing the CD4+ T cell responses between WT and the deimmunized variants determined by the PBMC assay. In the analysis, healthy human PBMCs from each of 12 donors were expanded in the presence of the WT or variant uricase protein for 14 days, restimulated with synthetic peptides corresponding to the protein used for initial stimulation (see Table 10) and then evaluated for T cell activation asAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application measured by intracellular cytokine staining and flow cytometry analysis. For each donor evaluated, the summed cytokine value (% CD4+ T cells positive for IL-2, INF-Ȗ, and TNF-Į) of each tested peptide with background subtraction was calculated. The individual donors’ summed cytokine values of each peptide were further summed across all donors evaluated, with the resultant value plotted for each of the tested peptides (represented by circles in FIG. 13). The mean and standard deviation are also shown for each set of peptide responses for each protein evaluated. The mean value of all peptide responses for each deimmunized variant was compared to the WT response by two-way ANOVA with Dunnett’s multiple comparison test for single pooled variance. * p ^ 0.05, **p ^ 0.01, ***p ^ 0.001. The data of FIG.13 indicates that all deimmunized variants had a statistically significant reduction in T cell responses when compared with WT and that variants HF536 and HF752 had the greatest reduction in T cell responses overall.

[0388] FIG. 14 shows the results of the MAPPs analysis comparing the peptides presented on HLA II derived from either the WT or deimmunized uricase proteins. In the analysis, monocytes were differentiated to mature dendritic cells (DCs) and pulsed with the protein of interest. Pulsed cells were then harvested, lysed and peptide MHC (pMHC) complexes were pulled down using antibody conjugated beads. The peptides were then eluted from the pMHC complexes and analyzed by mass spectrometry. In FIG. 14, the value for each amino acid position in the heatmaps is the max count of unique peptides overlapping that position from any donor. For comparison, the MAPPs analysis of WT uricase from all 8 healthy donors is shown at the top, while all other rows show aggregate data from a subset of 5 of the 8 donors, i.e., donors 3, 6, 7, 13, and 14, commonly selected for the MAPPs study of both the wild type and uricase variant proteins. The 5 donors included donors 3, 6 and 7 also used in the PBMC assay for the T cell epitope mapping experiments, as shown in Table 11. The * symbol represents the positions of mutations in each deimmunized uricase variant protein. The grayscale key shows a range of gray shades corresponding to different count values. The data of FIG. 14 shows that all variants had a reduction in the presentation of peptides, particularly in the most immunogenic region (amino acid positions 60-90) identified by both MAPPs and the PBMC assay. In addition, variants HF554 and HF752 had the fewest regions with presented peptides overall. Example 4 – Additional engineering of lead deimmunized uricase variant candidates HF554 and HF752 to create and screen for HF554 and HF752 uricase subvariants with further reduced immunogenicity.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0389] The studies of Examples 1 and 3 identified HF554 and HF752 as lead deimmunized uricase variant candidates. However, the PBMC assay and MAPPs analysis results of Example 3 revealed the regions in HF554 and HF752 with residual immunogenicity risk, evidenced by the immunogenic hotspots of HF554 and HF752 identified in their CD4+ T cell epitope mapping (see FIG. 12) and in their MAPPs analyses (see FIG. 14). This example describes additional engineering of HF554 and HF752 by iteratively designing, constructing, expressing, and screening libraries of HF554 and HF752 uricase subvariants that harbored additional and / or replacement amino acid substitutions in those residual immunogenicity risk regions, as compared to the parent HF554 and HF752 uricase variants, respectively. Following each round of engineering, functional deimmunized HF554 and HF752 uricase subvariants were obtained which had further reduced immunogenicity, while maintaining the activity and stability as compared to the parent HF554 and HF752 uricase variants, respectively. During each round of engineering, plate-based screening strategy was used to screen for functional HF554 and HF752 uricase subvariants. Methods 1. Construction of expression plasmids for HF554 and HF752 uricase subvariants and plate- based screening for functional HF554 and HF752 uricase subvariants

[0390] Iterative construction of libraries of expression plasmids 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 of the expression plasmids encoding an HF554 or HF752 uricase subvariant encoded a His tag containing 6 histidine residues at the C-terminus of the HF554 or HF752 uricase subvariant gene.

[0391] (1) Gene blocks between 450-1000 bp encoding 1-9 deimmunizing amino acid substitutions relative to HF0752 or HF0554 were synthesized in 96-well plates.

[0392] (2) Linear plasmid backbones were designed to have sufficient overlaps with the gene blocks of greater than or equal to 24 base pairs, and synthesized by PCR.

[0393] (3) The Gene blocks and linear plasmid backbones were assembled using the NEBuilder® HiFi DNA Assembly master mix.

[0394] (4) HiFi reactions products were then transformed into Invitrogen OneShot™ BL21 Star™ cells and grown overnight.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0395] (5) Overnight cultures were sub-cultured 1:50 into MagicMedia™ and grown for 20 hours before harvesting and freezing cell pellets.

[0396] (6) Cell pellets were lysed using B-PER™ Complete Bacterial Protein Extraction Reagent, and the insoluble fraction was separated and resuspended in bicarbonate buffer (pH 10.5).

[0397] (7) Proteins were purified from the insoluble fractions using HisPur™ Cobalt spin plates, and subsequently desalted using Zeba™ Spin desalting plates.

[0398] (8) Purified proteins were then quantified by BCA and normalized to 0.2 mg / mL.

[0399] (9) The concentration normalized proteins were subjected to activity, stability, and purity assays. The activity assay is described below. The stability assay measuring melting temperatures of the proteins was performed using differential scanning fluorimetry (DSF) as described in Example 1, with the thermal ramp of 30oC to 100oC. The purity assay was performed using dynamic light scattering (DLS).

[0400] (10) Results from these assays from each round of screening were then used to design the library of HF554 and HF752 uricase subvariants in the next round. 2. Activity assay performed in the plate-based screening

[0401] 10 uL of diluted proteins (2 μg) were added to a UV transparent 96-well plate. 190 uL of 0.125 mM uric acid in boric acid (pH 9) was added to each well, and the absorbance at 293 nm was monitored over 10 minutes in a plate reader. Uricase activity was measured as the slope of the absorbance at 293 nm vs time curve, with steeper negative slopes indicative of higher activity. As such, the slopes were quantified as raw activity measures in arbitrary units. This assay was repeated 4 times for each plate. Results 1. Additional engineering of lead deimmunized uricase variant candidate HF554 to create and screen for functional HF554 uricase subvariants with further reduced immunogenicity

[0402] Table 12 lists seven regions (designated as regions A to G) with residual immunogenicity risk of the amino acid sequence of SEQ ID NO:53 for lead deimmunized uricase variant candidate HF554. Those regions were identified based on the PBMC assay and MAPPs analysis results of Example 3 for HF554.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 12. Regions with residual immunogenicity risk in the amino acid sequence of SEQ ID NO:53 f R S 9 p a N

[0403] HF554 uricase subvariants were created by designing single, double, and one triple mutation sets on the parent HF554 uricase variant amino acid sequence targeting the above residual immunogenicity risk regions. The HF554 subvariant expression plasmids were constructed, and the HF554 subvariant proteins were produced and screened for expression yield, melting temperature, and specific activity by using the methods described in the “Methods” section.

[0404] Table 13 shows 113 exemplary functional HF554 uricase subvariants with at least 10% of the parent HF554 uricase variant activity identified from the screening. With the genes of the subvariants sequenced, Table 13 shows each HF554 uricase subvariant’s SEQ ID NO for its amino acid sequence, mutations relative to the parent HF554 uricase variant amino acid sequence of SEQ ID NO:53, mutations relative to the wild type chimeric pig-baboon uricase of SEQ ID NO:1, as well as the average yield, melting temperature, and activity of each HF554 uricase subvariant. Data for the parent HF554 uricase variant are also included in Table 13 for comparison. T H su d se v p H v 0 8H98D / A102L / Y127Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 3 0 5 2 9 5 7 1. . . . . . .7 N70L / F75L / K79G / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 1 2 3 2 7 0 2 7. . . . .8 N70L / F75L / K79G / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 7 0 1 7 5 3 8 9KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 4 2 5 7 7 2 9 0 2A102L / R113Q / Y12Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 5 5 1 0 5 6 4 0. . . . . . .2 N70L / F75L / K79G / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 8 9 0 0 5 7 3 1257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 2 4 5 2 8 0 6 3 9A102L / Y127H / K15Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 0 3 8 3 5 7 5 0. . . . . . .5 W N70L / F75L / K79G / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 5 0 3 0 6 2 9 8R209F / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 3 1 6 1 5 4 7 5 8A102L / Y127H / K15Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 9 6 0 1 2 5 1 1. . . . . . .9 W N70L / F75L / K79G / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 0 0 0 9 5 9 8 8209F / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 13. Exemplary functional HF554 uricase subvariants H su d se v p H v 1 2 6 2 3 8 9 H 1 v5G / R209F / I257K 2. Additional engineering of lead deimmunized uricase variant candidate HF752 to create and screen for functional HF752 uricase subvariants with further reduced immunogenicityAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0405] Table 14 lists seven regions (designated as regions A to G) with residual immunogenicity risk of the amino acid sequence of SEQ ID NO:129 for lead deimmunized uricase variant candidate HF752. Those regions were identified based on the PBMC assay and MAPPs analysis results of Example 3 for HF752. Table 14. Regions with residual immunogenicity risk in the amino acid sequence of SEQ ID NO:129 f R S 9 p a N

[0406] Five iterative rounds of HF752 uricase subvariant library design, construction, expression, and screening were carried out to address the above residual immunogenicity risk regions.

[0407] In the first round, single, double, one triple, and one quadruple mutation sets on the parent HF752 uricase variant amino acid sequence independently targeting each of the residual immunogenicity risk regions A, B, C, D, and E of HF752 were designed. The expression plasmids of the designed HF752 subvariants were constructed, and the HF752 subvariant proteins were produced and screened for expression yield, melting temperature, and specific activity by using the methods described in the “Methods” section.

[0408] Table 15 shows 139 exemplary functional HF752 uricase subvariants with at least 10% of the parent HF752 uricase variant activity identified from the first round of screening, including each HF752 uricase subvariant’s SEQ ID NO for its amino acid sequence, mutations relative to the parent HF752 uricase variant amino acid sequence of SEQ ID NO:129, and mutations relative to the wild type chimeric pig-baboon uricase of SEQ ID NO:1, as well as the average yield, melting temperature, and activity of each HF752 uricase subvariant. Data for the parent HF752 uricase variant are also included in Table 15 for comparison.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 3 8 5 9 7 8 4 8 56S / A102V / V106M / KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 9 9 1 4 9 1 3 2W / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 7 8 2 6 8 7 5 5 4S / A102V / V106M / K1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 0 8 9 7 0 6 9 7G / R209W / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 4 0 9 5 9 6 7 2. . . . . . .6 70H / F75L / K76S / V86Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 3 3 3 6 3 3 1 4R209W / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 8 4 1 0 9 2 4 7 2H / A102V / V106M / KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 5 2 9 4 1 9 4 2G / R209W / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 8 2 2 2 6 5 4 857KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 8 1 2 9 1 2 0 1Y127H / K155G / F172Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 5 2 2 3 1 2 1 92V / V106M / K116M / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 0 0 8 5 3 2 19RAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 2 8 4 2 4 1 6 3Y127H / K155G / R209Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 3 3 4 0 8 5 3 72V / V106M / K116M / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 3 4 4 3 6 6 3 02V / V106M / K116M / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 3 7 4 8 9 0 3. . . . . . .3 70H / F75L / K76S / A10Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 4 2 7 5 4 3 2W / I257K / L279TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 15. Exemplary functional HF752 uricase subvariants from the first round of library design, construction, ex s n 2 4

[0409] Based on the activity, stability, and yield data from the first round of screening of the HF752 uricase subvariants, the following high-performing single and double mutations were selected for use in the next (i.e., second) round of library design:

[0410] Region A – R26T, R26F, K30E, and K30F;

[0411] Region B – K74Q, K79S, T83E, V86N, S94D, and K97D;

[0412] Region C – F172W, F172L, F172W+T174N, and F172W+E177Q;

[0413] Region D – Y225H, S228G, and L239G;

[0414] Region E – L279T, N281F, and N281Y.

[0415] In the second round of library design, the above functionally validated mutations from the separate regions of A, B, C, D, and E were combined in pairs or triplets, creating HF752 uricase subvariants with sets of double, triple, and quadruple mutations designed to mitigate immunogenicity risk in two or three of the five target regions A, B, C, D, and E while maintaining high level functional fitness.

[0416] As shown in Table 16, following the second round of HF752 uricase subvariant library design, construction, expression, and screening, 173 exemplary functional HF752 uricase subvariants with at least 10% of the parent HF752 uricase variant activity wereAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application identified from the screening. Table 16 lists each HF752 uricase subvariant’s SEQ ID NO for its amino acid sequence, mutations relative to the parent HF752 variant amino acid sequence of SEQ ID NO:129, and mutations relative to the wild type chimeric pig-baboon uricase of SEQ ID NO:1, as well as the average yield, melting temperature, and activity of each HF752 uricase subvariant. Data for the parent HF752 variant are also included in Table 16 for comparison. Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design construction e s s v 6 8 5 0 56M / K116M / Y127Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 1 3 4 6 8 1Y127H / K155G / R2Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 6 2 9 8 8 37K / L279TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 2 6 9 6 5 8 0S94D / A102V / V10Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 5 9 8 3 8 2257K / L279TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 0 5 4 9 6 4. . . . . . .8 N70H / F75L / K76S / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 4 1 7 7 7 5Y127H / K155G / R2Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 0 4 8 8 2 3257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 8 5 1 4 5 5 0K76S / K97D / A102Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 4 8 7 0 5 0257K / N281FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 7 7 1 1 2 2. . . . . . .7 Y31H / N70H / F75L / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 0 4 3 4 8 8V / V106M / K116M / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 3 3 1 6 1 1257K / L279TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 3 9 8 3 4 2 6V / V106M / K116M / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 2 9 2 7 1 0116M / Y127H / K15Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 2 3 1 7 0 5116M / Y127H / K15Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 3 6 7 3 9 75G / F172W / R209WAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 8 8 6 6 5 4116M / Y127H / K15Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 7 0 8 3 3 65G / F172W / R209WAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 5 0 0 9 5 75G / F172W / E177Q / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 2 4 3 8 4 25G / F172W / T174N / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 3 8 9 7 4 85G / F172W / T174N / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 3 0 5 0 9 2V / V106M / K116M / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 6 9 3 7 5 95G / F172L / R209W / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 4 6 8 1 3 3M / K116M / Y127H / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 7 1 1 4 1 35G / F172W / E177Q / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 4 0 8 6 6 7116M / Y127H / K15Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 3 2 7 0 8 0K76S / T83E / A102Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 16. Exemplary functional HF752 uricase subvariants from the second round of library design, construction, ex s s v 0 3

[0417] Based on the activity, stability, and yield data from the second round of screening of the HF752 uricase subvariants, high-performing sets of mutations targeting two or three of the five residual immunogenicity regions A, B, C, D, and E of the parent HF752 variant were selected for use in the next (i.e., third) round of library design. The high performing mutations included the L279T mutation associated with high activity, and the mutation of N281Y, N281F, or K97D associated with high stability. In the third round of library design, the above- mentioned functionally validated sets of mutations targeting two or three of the five residual immunogenicity regions A, B, C, D, and E were combined, creating 221 and 84 HF752 uricase subvariants with sets of mutations designed to respectively mitigate immunogenicity risk in four and all of the five target regions A, B, C, D, and E while maintaining high level functional fitness. Also included in the third round of library design was a group of 24 L279T-containing HF752 uricase subvariants with an add-on mutation of K97D, N281Y, or both intended to increase thermostability. Those HF752 uricase subvariants with the add-on mutations were designed to target the residual immunogenicity region(s) of B, AB, BC, CE, AE, BE, ABE, CDE, or BCDE.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0418] Additionally, 28 HF752 uricase subvariants with single point mutations on the parent HF752 variant designed to mitigate immunogenicity risk in new regions F and G while maintaining high level functional fitness were created in the third round of library design.

[0419] As shown in Table 17, following the third round of HF752 uricase subvariant library design, construction, expression, and screening, 349 exemplary functional HF752 uricase subvariants with at least 10% of the parent HF752 uricase variant activity were identified from the screening. Table 17 lists each HF752 uricase subvariant’s SEQ ID NO for its amino acid sequence, mutations relative to the parent HF752 uricase variant amino acid sequence of SEQ ID NO:129, and mutations relative to the wild type chimeric pig-baboon uricase of SEQ ID NO:1, as well as the average yield, melting temperature, and activity of each HF752 uricase subvariant. Data for the parent HF752 uricase variant are also included in Table 17 for comparison.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, e H su se p H v 8 0K155G / F172W / R209Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 2 152K / N70H / F75L / K7Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 2 26M / K116M / Y127H / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 9 7 69TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 5 1 5G / N281F 6S / T83E / A102V / V10Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 7 1 79TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 2 3 0 5K155G / F172W / E177Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 4 8 39TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 6 5 9 5 781YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 1 9 2 9 1116M / Y127H / K155Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 3G / L279T 5L / K76S / A102V / V1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 5 6G / N281F D / A102V / V106M / KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 39TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 4G / F172W / E177Q / R2Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H vF172W / E177 31H / N70H / F75L / K7Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 3 0 1 3281Y 116M / Y127H / K155Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 0A / R209W / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 1 9 5 5N / S228G D / A102V / V106M / KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v5 L279T 70H / F75L / K76S / K97Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 42V / V106M / K116M / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 1 6 9 728G / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 7 6T 6S / K79S / K97D / A10Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 7 9G / R209W / I257KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 4 2 6G D / A102V / V106M / KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 5 979T / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 4 17KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 5 8 2G / F172W / R209W / YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v H 2 v (c fo HsubvariAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v a 6 7 ab116M / Y127H / K155Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v116M / Y127H / K155Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v57K / N281FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v57K / L279TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v57K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v7KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H vT 6S / K97D / A102V / V1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v5H 6S / S94D / A102V / V1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 806M / K116M / Y127H / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H vW / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H vK155G / F172W / E177Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 109W / I257K / L279TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 47K / N281FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 0 7W / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 957K / L279TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 728G / I257K / N281FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 4 07K / N281FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 028G / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v7K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 4N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 7G / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 5G / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H vG / I257K / L279TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 4 0W / T174N / L 70H / F75L / K76S / K97Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 6116M / Y127H / K155Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v116M / Y127H / K155Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v 3225H / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H vG / I257K / N281FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v28G / N281Y E / A102V / V106M / K1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 17. Exemplary functional HF752 uricase subvariants from the third round of library design, construction, ex H su se p H v H v (c H sabove)

[0420] Based on the activity, stability, and yield data from the third round of screening of the HF752 uricase subvariants, 23 high performing HF752 uricase subvariants with sets of mutationsAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application targeting all five of the residual immunogenicity regions ABCDE of the parent HF752 variant were identified (Table 18). Additionally, high-functioning mutations in region F (L52T, H53D, or H53S) and region G (R192T or G196D) were also identified. Table 18.23 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 0 3 4 157K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 18.23 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 2 3 4 4 64 / F172W / E1 / Y31H / N70H / F75Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 18.23 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 8 8 6 33 F172W / T17 / N70H / F75L / K76SAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 18.23 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 2 9 2 3YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 18.23 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 6 6 0Y

[0421] In the next (i.e., fourth) round of library design, each of the above-mentioned 23 high performing HF752 subvariants was used a basis design, which was then combinatorially mutated to incorporate high-functioning 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 designed toAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application target six residual immunogenicity risk regions, i.e., regions ABCDE plus F or G, or seven residual immunogenicity risk regions, i.e., regions ABCDE plus regions F and G, of the HF752 uricase variant. Further, when the basis design was from HF752 uricase subvariant #600 (SEQ ID NO:622) #622 (SEQ ID NO:644), or #653 (SEQ ID NO:675), the basis design was modified to include an additional N281Y stabilizing mutation.

[0422] As shown in Table 19, following the fourth round of HF752 uricase subvariant library design, construction, expression, and screening, 359 exemplary functional HF752 uricase subvariants with at least 10% of the parent HF752 uricase variant activity were identified from the screening. Table 19 lists each HF752 uricase subvariant’s SEQ ID NO for its amino acid sequence, mutations relative to the parent HF752 uricase variant amino acid sequence of SEQ ID NO:129, and mutations relative to the wild type chimeric pig-baboon uricase of SEQ ID NO:1, as well as the average yield, melting temperature, and activity of each HF752 uricase subvariant. Data for the parent HF752 uricase variant are also included in Table 19 for comparison. T ex H su se v p H v 1 3H / K155G / F172W / TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 5 0 1 1 0106M / K116M / Y127Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 8 9 9 2 628G / N281Y V106M / K116M / Y1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 6 1 8 5 8W / T174N / R 76S / S94D / A102V / VAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 9 9 1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 0 4 9 2 81F 172W / T174N / G196Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 6 3 9 3 / N281Y 27H / K155G / F172WAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 5 0 6 9 0A102V / V106M / K11Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 4 3 5 5 1A102V / V106M / K11Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 7 8 1 4 0W / R197D / S F75L / K76S / T83E / AAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 7 8 2 4279T / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 7 3 3 3 7 / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 4 6 4 725H / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 7 2 7 7 4279T / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 8 6 6 8 179T / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 1 7 8 9 2H / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 8 6 7 8 86D / R209W / L239G / IAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 1 9 2 2172W / E177Q / G196Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 7 8 9 127H / K155G / F172WAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 1 4 5 6F172L / G196D / R209Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 2 2 8 6172W / E177Q / R192Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 6 3 4 4 1F172W / G196D / R20Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 6 4 6 6 9172W / E177Q / R197Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 0 6 9 4 7 / T174N / R192T / R20Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 6 8 5 8172W / R197D / R209Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 4 8 3 3172L / R192T / R209Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 0 9 9 1 5 / N281Y 27H / K155G / F172WAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 1 1 5 9 / T174N / R192T / R20Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 1 8 1 527H / K155G / F172L / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 4 6 5 1 2F172W / R192T / R20Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 1 1 3 3Y 27H / K155G / F172WAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 7 1 1 8 5 / N281Y 27H / K155G / F172WAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 8 8 3 2 5M / Y127H / K155G / FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 9 3 8 5 / N281Y 27H / K155G / F172WAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 6 1 3 5 7N281Y 6M / Y127H / K155G / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 6 4 1 4 627H / K155G / F172L / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 6 1 0 9 3172W / G196D / R209Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v H 3 v (c H s 1 a 3 3 7 3A102V / V106M / K11Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 2 0 6 2M / Y127H / K155G / FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 4 3 5 0 3F172W / G196D / R20Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 6 8 7 5H / K155G / F172L / R2Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 8 8 9 2 9G / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 2 9 8 681YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 8 7 0 8 63 K79S / F172 Y31H / H53D / N70H / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 4 6 19 T83E / F172 H53S / N70H / F75L / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 5 0 6 2 528G / N281Y V106M / K116M / Y1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 7 2 2 0 6172W / E177Q / R197Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 8 9 9 57K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 5 5 4 8 281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 5 4 4 3 82 S94D / F172 H53D / N70H / F75L / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 8 1 5 8 3V106M / K116M / Y1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 2 4 8 8G / I257K / N281FAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 3 7 8 981YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 5 1 8 8281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 8 0 6 7 4 228G / N281Y V106M / K116M / Y1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 7 1 6 3 47K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 8 2 9 5 3YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 7 8 9 9 6K76S / T83E / A102V / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 4 2 2 5 739G / N281Y / K116M / Y127H / K1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 5 1 7 1281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 8 0 2 29 F172L / R197 N70H / F75L / K76S / TAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 2 0 .6 5281Y M / K116M / Y127H / Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 6 1 1 08G / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 9 2 5 8 5 581Y / V106M / K116M / Y1Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 8 4 1 6 / E177Q / R197D / R20Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 2 6 4 4 68G / I257K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 1 4 3 4 36 F172W / T174 N70H / F75L / K76S / SAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 1 7 8 8 0 / E177Q / R192T / R20Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 3 4 0 4 87K / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 6 6 6 6 73 F172W / T174 N70H / F75L / K76S / SAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 4 4 1 3 755G / F172W / R197DAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 19. Exemplary functional HF752 uricase subvariants from the fourth round of library design, construction, ex H su se v p H v 0 3 H 1 v (c H s 1 above)

[0423] Based on the activity, stability, and yield data from the fourth round of screening of the HF752 uricase subvariants, 35 high performing HF752 uricase subvariants with sets of mutations targeting all seven of the residual immunogenicity regions ABCDEFG of the parent HF752 uricase variant were identified (Table 20).Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 20.35 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 3 3 3 1 5Y K116M / Y127H / KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 20.35 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 3 0 5 3 5W / R192T / S H / F75L / K76S / S94Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 20.35 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 3 3 5 881YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 20.35 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 5 8 0 3 3155G / F172W / G19Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 20.35 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 5 8 0 5 002V / V106M / K116Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 20.35 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 8 5 0 53 T83E / F172 / L52T / N70H / F75LAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 20.35 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 3 3 5 3N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 20.35 high performing HF752 uricase subvariants with the best overall activity, stability, and y s y 3 5

[0424] In the next (i.e., fifth) round of library design, each of the above-mentioned 35 high performing HF752 subvariants was used a basis design, whose mutations were combined with N117D or N117G. When the basis design was from a high performing HF752 subvariant lacking the L279T mutation (excluding HF752 subvariants #1074 (SEQ ID NO:1045), #1073 (SEQ ID NO:1044), and #1106 (SEQ ID NO:1077), the mutations present in each of the basis HF752 subvariant were combined with L279T in addition to N117D or N117G. As a result, the designed HF752 subvariants in the fifth round bore mutations at N117, L279, or both, as well as mutations in all of the seven residual immunogenicity risk regions ABCDEFG of the HF752 uricase variant. As shown in Example 2, the N117D or N117G mutation completely eliminated deamidation at position Asn117 of the HF752 uricase variant while having the least effect on activity and thermostability. Additionally, as indicated by the second round of screening results, the L279T mutation was associated with high activity of the HF752 uricase subvariants.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application

[0425] As shown in Table 21, following the fifth round of HF752 uricase subvariant library design, construction, expression, and screening, 145 exemplary functional HF752 uricase subvariants with at least 10% of the parent HF752 uricase variant activity were identified from the screening. Table 21 lists each HF752 uricase subvariant’s SEQ ID NO for its amino acid sequence, mutations relative to the parent HF752 uricase variant amino acid sequence of SEQ ID NO:129, and mutations relative to the wild type chimeric pig-baboon uricase of SEQ ID NO:1, as well as the average yield, melting temperature, and activity of each HF752 uricase subvariant. Data for the 35 high performing basis HF752 uricase subvariants and the parent HF752 uricase variant, all of which were tested in parallel under the same conditions, are also included in Table 21 for direct comparison.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 2 1 0 1 07 1 32 1 0657K / L279T / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 06 1 70 1 9 1 9 1 281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 52 1 49 1 06 1 53 1 68 1. . . . . . .37 94D / N117D / F L52T / N70H / F75L / KAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 41 1 4 1 5 1 56 1 98102V / V106M / K116Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 09 1 00 1 01 1 92 1 89172W / G196D / R209Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 85 1 26 1 80 1 70 1 336D / R209W / S228G / IAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 99 1 63 1 49 1 57 1 3781YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 36 1 53 1 19 1 35 1 19 1 18172W / R192T / 76S / T83E / A102V / VAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 77 1 31 1 68 1 85 1 2927H / K155G / F172WAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 61 1 23 1 65 1 74 1 6479T / N281YAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, construction, ex s d s v v 1 64 1 30 1 40 1 59 1 02 1 62R197D / S228 F75L / K76S / T83E / AAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table 21. Exemplary functional HF752 uricase subvariants from the fifth round of library design, constructi...

Claims

Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application CLAIMS 1. A uricase variant comprising one or more amino acid mutations relative to a counterpart uricase protein, wherein the one or more amino acid mutations are at one or more amino acid positions 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, wherein 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.

2. The uricase variant of claim 1, comprising two or more amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to two 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.

3. The uricase variant of claim 1, comprising three or more amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are 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 of claim 1, comprising four or more amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to four 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.

5. The uricase variant of claim 1, comprising five or more amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are 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,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

6. The uricase variant of claim 1, comprising seven or more amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to seven 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.

7. The uricase variant of claim 1, comprising ten or more amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to ten 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.

8. The uricase variant of claim 1, comprising fifteen or more amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to fifteen 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.

9. The uricase variant of claim 1, comprising from about 5 to about 25 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 5 to about 25 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.

10. The uricase variant of claim 1, comprising from about 5 to about 20 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 5 to about 20 of the following amino acid residues of SEQ ID NO:1: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

11. The uricase variant of claim 1, comprising from about 10 to about 20 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 20 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.

12. The uricase variant of claim 1, comprising from about 10 to about 18 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 18 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.

13. The uricase variant of claim 1, comprising from about 10 to about 16 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 16 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.

14. The uricase variant of claim 1, comprising from about 10 to about 14 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 14 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.

15. The uricase variant of claim 1, comprising from about 10 to about 13 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 13 of the following amino acid residues of SEQ ID NO:1: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

16. The uricase variant of claim 1, comprising from about 10 to about 12 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 10 to about 12 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.

17. The uricase variant of claim 1, comprising from about 11 to about 20 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 11 to about 20 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.

18. The uricase variant of claim 1, comprising from about 11 to about 13 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 11 to about 13 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.

19. The uricase variant of claim 1, comprising from about 12 to about 14 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 12 to about 14 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.

20. The uricase variant of claim 1, comprising from about 13 to about 20 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 13 to about 20 of the following amino acid residues of SEQ ID NO:1: D18, I20, V22, H24, Q26, D28, Y31, H32, S33, A38, I65, K66,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application N70, F75, K76, K79, S80, A102, V106, K112, F114, K116, N117, V119, Y127, L154, K155, K158, R209, and I257.

21. The uricase variant of claim 1, comprising from about 14 to about 20 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 14 to about 20 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.

22. The uricase variant of claim 1, comprising from about 15 to about 20 amino acid mutations relative to the counterpart uricase protein, wherein the amino acid mutations are at amino acid positions corresponding to from about 15 to about 20 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.

23. The uricase variant of any one of claims 1-22, wherein the amino acid mutations are amino acid substitutions.

24. The uricase variant of claim 23, wherein the amino acid substitutions correspond to one or more of the amino acid substitutions of SEQ ID NO:1 set forth in Table A:N70L K155GAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application Table A.

25. The uricase variant of claim 24, wherein the amino acid substitutions correspond to three 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.

26. The uricase variant of claim 24, wherein the amino acid substitutions correspond to five 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.

27. The uricase variant of claim 24, wherein the amino acid substitutions correspond to 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.

28. The uricase variant of claim 24, wherein the amino acid substitutions correspond to three or more of the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K.

29. The uricase variant of claim 24, wherein the amino acid substitutions correspond to five or more of the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K.

30. The uricase variant of claim 24, wherein the amino acid substitutions correspond to ten or more of the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F and I257K.

31. The uricase variant of claim 24, wherein the amino acid substitutions correspond to three or more of the following amino acid substitutions of SEQ ID NO:1: V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F and I257K.

32. The uricase variant of claim 24, wherein the amino acid substitutions correspond to five or more of the following amino acid substitutions of SEQ ID NO:1: V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 33. The uricase variant of claim 24, wherein the amino acid substitutions correspond to ten or more of the following amino acid substitutions of SEQ ID NO:1: V22L, D28N, Y31H, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209F and I257K.

34. The uricase variant of claim 24, wherein the amino acid substitutions correspond to three or more of the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W and I257K.

35. The uricase variant of claim 24, wherein the amino acid substitutions correspond to five or more of the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W and I257K.

36. The uricase variant of claim 24, wherein the amino acid substitutions correspond to ten or more of the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W and I257K.

37. The uricase variant of claim 24, wherein the amino acid substitutions correspond to three 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.

38. The uricase variant of claim 24, wherein the amino acid substitutions correspond to five 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.

39. The uricase variant of claim 24, wherein the amino acid substitutions correspond to 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.

40. The uricase variant of claim 24, wherein the amino acid substitutions correspond to three 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.

41. The uricase variant of claim 24, wherein the amino acid substitutions correspond to five 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.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 42. The uricase variant of claim 24, wherein the amino acid substitutions correspond to 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.

43. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, and Y127H.

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

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

46. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, N70L, F75L, and Y127H.

47. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, A102V, and Y127H.

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

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

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

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

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

53. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, K79G, and Y127H.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 54. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, V106M, and Y127H.

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

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

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

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

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

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

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

62. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, V119T, and Y127H.

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

64. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, K116L, and Y127H.

65. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, Y127H, K155G, and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 66. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, N70L, F75L, Y127H, and K155G.

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

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

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

70. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, Q26R, F75L, Y127H, and K155G.

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

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

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

74. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, N70L, F75L, Y127H, and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 75. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, A102V, Y127H, and I257K.

76. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, Y127H, R209F, and I257K, 77. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, S33D, F75L, Y127H, and I257K.

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

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

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

81. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, K79G, Y127H, I257K.

82. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, N70L, F75L, A102V, and Y127H.

83. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, N70L, F75L, Y127H, and R209F.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 84. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, S33D, N70L, F75L, and Y127H.

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

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

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

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

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

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

91. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, Q26R, F75L, A102V, and Y127H.

92. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, K76S, A102V, and Y127H,Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 93. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, A102V, K116M, and Y127H.

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

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

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

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

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

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

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

101. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, S33D, F75L, K76S, and Y127H.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 102. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, S33D, F75L, K116M, and Y127H.

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

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

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

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

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

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

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

110. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, N70L, F75L, A102V, Y127H, K155G, and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 111. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, N70L, F75L, Y127H, K155G, R209F, and I257K.

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

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

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

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

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

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

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

119. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, Q26R, F75L, A102V, Y127H, K155G, and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 120. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, K76S, A102V, Y127H, K155G, and I257K.

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

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

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

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

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

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

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

128. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, Q26R, S33D, F75L, Y127H, K155G, and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 129. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, S33D, F75L, K76S, Y127H, K155G, and I257K.

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

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

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

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

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

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

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

137. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, K79G, K116M, Y127H, K155G, and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 138. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, S33D, N70L, F75L, A102V, Y127H, and R209F.

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

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

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

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

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

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

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

146. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, F75L, K76S, K79G, A102V, K116M, and Y127H.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 147. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, Q26R, S33D, F75L, K76S, Y127H, and R209F.

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

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

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

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

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

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

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

155. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, S33D, N70L, F75L, A102V, K116M, Y127H, K155G, R209F, and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 156. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, S33D, N70L, F75L, K79G, A102V, Y127H, K155G, R209F, and I257K.

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

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

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

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

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

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

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

164. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, Q26R, S33D, F75L, K76S, A102V, K116M, Y127H, R209F, and I257K.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 165. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, Q26R, S33D, F75L, K76S, K79G, A102V, Y127H, R209F, and I257K.

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

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

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

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

170. The uricase variant of claim 24, wherein the amino acid substitutions correspond to the amino acid substitutions of SEQ ID NO:1 comprising V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K116M, Y127H, and R209F.

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

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

1.

173. The uricase variant of any one of claims 25-171, further comprising an amino acid substitution corresponding to N117G of SEQ ID NO:

1.

174. The uricase variant of any one of claims 25-171, further comprising an amino acid substitution corresponding to N117D of SEQ ID NO:1.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 175. The uricase variant of claim 1, comprising a plurality of amino acid mutations relative to the counterpart uricase protein, wherein the plurality of amino acid mutations are at amino acid positions corresponding to one of 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.

176. The uricase variant of claim 175, wherein the plurality of amino acid mutations are at amino acid positions corresponding to the following combination of amino acid residues ofAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application SEQ ID NO:1: V22, D28, S33, N70, F75, K79, A102, F114, V119, Y127, K155, R209, and I257.

177. The uricase variant of claim 175, wherein the plurality of amino acid mutations are at amino acid positions corresponding to the following combination of amino acid residues of SEQ ID NO:1: V22, Q26, Y31, N70, F75, K79, A102, Y127, K155, R209, and I257.

178. The uricase variant of claim 175, wherein the plurality of amino acid mutations are at amino acid positions corresponding to the following combination of amino acid residues of SEQ ID NO:1: V22, D28, Y31, N70, F75, K79, A102, K112, Y127, K155, R209, and I257.

179. The uricase variant of claim 175, wherein the plurality of amino acid mutations are at amino acid positions corresponding to the following combination of amino acid residues of SEQ ID NO:1: V22, Q26, S33, N70, F75, K79, A102, K112, Y127, K155, R209, and I257.

180. The uricase variant of claim 175, wherein the plurality of amino acid mutations are at amino acid positions corresponding to the following combination of amino acid residues of SEQ ID NO:1: V22, Q26, Y31, N70, F75, K76, A102, V106, K116, Y127, K155, R209, and I257.

181. The uricase variant of claim 175, wherein the plurality of amino acid mutations are at amino acid positions corresponding to the following combination of amino acid residues of SEQ ID NO:1: V22, D28, Y31, N70, F75, K79, A102, K116, Y127, K155, R209, and I257.

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

183. The uricase variant of claim 182, wherein the amino acid substitutions correspond to one of the following combinations 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;Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application (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.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application (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.

184. The uricase variant of claim 183, wherein the amino acid substitutions 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.

185. The uricase variant of claim 183, wherein the amino acid substitutions correspond to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, Y31H, N70L, F75L, K79G, A102L, Y127H, K155G, R209F, and I257K.

186. The uricase variant of claim 183, wherein the amino acid substitutions 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.

187. The uricase variant of claim 183, wherein the amino acid substitutions correspond to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, S33D, N70L, F75L, K79G, A102V, K112S, Y127H, K155G, R209W, and I257K.

188. The uricase variant of claim 183, wherein the amino acid substitutions 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.

189. The uricase variant of claim 183, wherein the amino acid substitutions 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.

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

1.

191. The uricase variant of any one of claims 183-189, further comprising an amino acid substitution corresponding to N117G of SEQ ID NO:

1.

192. The uricase variant of any one of claims 183-189, further comprising an amino acid substitution corresponding to N117D of SEQ ID NO:1.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 193. A uricase variant comprising a first set of amino acid substitutions and a second set of amino acid substitutions relative to a counterpart uricase protein, wherein the first set of amino acid substitutions corresponds to the amino acid substitutions of SEQ ID NO:1 set forth in group (a) or group (b), (a) V22L, Q26R, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K; or (b) V22L, Q26T, Y31H, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, R209W, and I257K; wherein the second set of amino acid substitutions is a set of from about 6 to about 10 amino acid substitutions selected from 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, 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 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.

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

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

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

197. The uricase variant of any one of claims 193-195, wherein the second set of amino acid substitutions is a set of from about 6 to about 10 amino acid substitutions selected fromAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: one of K30E and K30F, L52T, T83E, V86N, S94D, K97D, one of N117G and N117D, F172W, R192T, G196D, R197D, Y225H, S228G, L279T, and N281Y.

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

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

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

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

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

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

204. The uricase variant of any one of claims 193-195, wherein the second set of amino acid substitutions is a set of from about 6 to about 8 amino acid substitutions selected fromAtty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: K30E, L52T, T83E, N117D, F172W, G196D, S228G, L279T, and N281Y.

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

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

207. The uricase variant of any one of claims 1-206, wherein the counterpart 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 from about 90% to about 100% identical to SEQ ID NO:

1.

208. The uricase variant of any one of claims 1-207, wherein the counterpart uricase protein is a mammalian uricase.

209. The uricase variant of claim 208, wherein the counterpart uricase protein is a dog uricase, a porcine uricase, a bovine uricase, an ovine uricase, or a baboon uricase.

210. The uricase variant of claim 207 or 208, wherein the counterpart uricase protein is a chimeric uricase comprising two or more mammalian uricase domains.

211. The uricase variant of claim 210, wherein the counterpart uricase protein is a chimeric pig-baboon uricase.

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

1.

213. The uricase variant of claim 211, wherein the counterpart uricase protein is a chimeric pig-baboon uricase comprising an amino acid sequence at least about 70% identical to SEQ ID NO:1.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 214. The uricase variant of claim 211, wherein the counterpart uricase protein is a chimeric pig-baboon uricase comprising an amino acid sequence at least about 80% identical to SEQ ID NO:

1.

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

1.

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

1.

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

4.

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

5.

219. The uricase variant of any one of claims 1-218, wherein the counterpart uricase protein is further truncated at the C-terminus.

220. The uricase variant of claim 219, wherein the C-terminus truncation comprises 4-13 amino acids.

221. The uricase variant of any one of claims 1-220, wherein the counterpart uricase protein is further truncated at the N-terminus.

222. The uricase variant of claim 221, wherein the N-terminus truncation comprises 4-13 amino acids.

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

1.

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

1.

225. The uricase variant of any one of claims 1-222, comprising an amino acid sequence that is at least about 92% identical to SEQ ID NO:1.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 226. The uricase variant of any one of claims 1-225, wherein the uricase variant comprises an N-terminal methionine residue.

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

228. The uricase variant of any one of claims 1-225, wherein the uricase variant comprises a post-translational modification.

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

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

231. The uricase variant of 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:25-154.

232. The uricase variant of 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. The uricase variant of 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 of claim 1, comprising an amino acid sequence of SEQ ID NO:

45.

235. The uricase variant of claim 1, comprising an amino acid sequence that 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 of claim 1, comprising an amino acid sequence of SEQ ID NO:53.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 237. The uricase variant of 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. The uricase variant of claim 237, comprising an amino acid sequence selected from SEQ ID NOs:197-309.

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

66.

240. The uricase variant of 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:

66.

241. The uricase variant of claim 1, comprising an amino acid sequence of SEQ ID NO:

114.

242. The uricase variant of 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 of claim 1, comprising an amino acid sequence of SEQ ID NO:

129.

244. The uricase variant of 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. The uricase variant of claim 244, comprising an amino acid sequence selected from SEQ ID NOs:187, 189, 191, 193, and 195.

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

187.

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

191.

248. The uricase variant of claim 244, comprising an amino acid sequence selected from SEQ ID NOs:310-448.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 249. The uricase variant of claim 244, comprising an amino acid sequence selected from SEQ ID NOs:449-621.

250. The uricase variant of claim 244, comprising an amino acid sequence selected from SEQ ID NOs:622-970.

251. The uricase variant of 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. The uricase variant of claim 244, comprising an amino acid sequence selected from SEQ ID NOs:971-1329.

253. The uricase variant of 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. The uricase variant of claim 244, comprising an amino acid sequence selected from SEQ ID NOs:1330-1474.

255. The uricase variant of 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 of claim 244, comprising an amino acid sequence selected from SEQ ID NOs:1475-1566.

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

149.

258. The uricase variant of claim 1, comprising an amino acid sequence that 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. The uricase variant of any one of claims 230-258, further comprising an N-terminal methionine residue.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 260. The uricase variant of any one of claims 1-259, wherein the uricase variant is non- PEGylated.

261. The uricase variant of any one of claims 1-259, wherein the uricase variant is PEGylated.

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

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

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

265. The uricase variant of claim 263 or 264, wherein the purification tag is a polyhistidine tag.

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

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

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

269. A uricase monomer comprising a uricase variant of any one of claims 1-265.

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

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

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

273. The uricase dimer of claim 271, wherein the uricase dimer is a heterodimer.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 274. A uricase tetramer comprising a first, second, third and fourth uricase monomer, wherein the first uricase monomer is a uricase variant of any one of claims 1-265.

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

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

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

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

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

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

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

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

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

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

285. The uricase conjugate of claim 284, wherein the amino acid linker is from about two amino acids long to about 5 amino acids long.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 286. The uricase conjugate of claim 285, wherein the amino acid linker is two amino acids long.

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

288. The uricase conjugate of any one of claims 281-287, 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 of claim 288, wherein the first domain is N-terminal to the second domain and C-terminal to the third domain.

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

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

292. The uricase conjugate of claim 290 or 291, wherein the amino acid linker is from about two amino acids long to about 5 amino acids long.

293. The uricase conjugate of claim 292, wherein the amino acid linker is two amino acids long.

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

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

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

297. The uricase conjugate of claim 295 or 296, wherein the PAS polypeptide has the amino acid sequence set forth in SEQ ID NO:

9.

298. The uricase conjugate of claim 295 or 296, wherein the PAS polypeptide has the amino acid sequence set forth in SEQ ID NO:10.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 299. The uricase conjugate of claim 295 or 296, wherein the PAS polypeptide has the amino acid sequence set forth in SEQ ID NO:

11.

300. The uricase conjugate of claim 295 or 296, wherein the PAS polypeptide comprises an amino acid sequence set forth 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 of claim 300, wherein the PAS polypeptide comprises the amino acid sequence of SEQ ID NO:

13.

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

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

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

305. The uricase conjugate of claim 303 or 304, wherein the XTEN polypeptide has the amino acid sequence set forth in SEQ ID NO:

12.

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

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

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

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

310. The uricase conjugate of any one of claims 280-307, wherein the first random coil polypeptide domain comprises from about 100 amino acids to about 700 amino acids.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 311. The uricase conjugate of any one of claims 288-310, wherein the second random coil polypeptide domain comprises from about 100 amino acids to about 700 amino acids.

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

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

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

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

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

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

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

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

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

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

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

323. The uricase conjugate of any one of claims 281-322, further comprising a purification tag at the C-terminus.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 324. The uricase conjugate of any one of claims 281-323, further comprising a purification tag at the N-terminus.

325. The uricase conjugate of claim 323 or 324, wherein the purification tag is a polyhistidine tag.

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

327. The uricase conjugate of 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:1569-1748.

328. The uricase conjugate of 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:1749-1928.

329. The uricase conjugate of 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:1929-2108.

330. The uricase conjugate of 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:2109-2288.

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

332. The uricase conjugate of any one of claims 280-331, wherein the uricase conjugate is present within a homodimer.

333. The uricase conjugate of any one of claims 280-331, wherein the uricase conjugate is present within a homotetramer.

334. The uricase conjugate of any one of claims 280-330, wherein the uricase conjugate is a homotetramer.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 335. The uricase conjugate of any one of claims 280-330, wherein the uricase conjugate is a homodimer.

336. The uricase conjugate of any one of claims 280-335, wherein the uricase conjugate is non-PEGylated.

337. The uricase conjugate of any one of claims 280-335, wherein the uricase conjugate is PEGylated.

338. An isolated nucleic acid which encodes the uricase variant of any one of claims 1- 268, or the uricase conjugate of any one of claims 280-337.

339. A nucleic acid vector comprising the nucleic acid of claim 338.

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

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

342. A method of reducing elevated uric acid levels in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 341.

343. The method of claim 342, wherein the uric acid levels are reduced in the plasma of the subject.

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

345. The method of claim 344, wherein the subject is a refractory gout patient.

346. The method of any one of claims 342-345, wherein the subject has been diagnosed with tumor lysis syndrome.

347. A method of treating gout in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 341.

348. The method of claim 347, wherein the gout is refractory gout.Atty. Docket No.: INMD-191 / 01WO 315953-4287 PCT Application 349. A method of treating tumor lysis syndrome in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 341.

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

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

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

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

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

355. A method of recombinantly producing the uricase variant of any one of claims 1-268, comprising: (i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding the uricase variant of any one of claims 1-268, wherein the nucleic acid sequence is operatively linked to a heterologous promoter under conditions to allow for expression of the nucleic acid sequence encoding the uricase variant and recombinant production of the uricase variant by the host cell; and (ii) isolating the recombinantly produced uricase variant.

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

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