Uricase variants and methods of use thereof

Abstract

The present disclosure relates to uricase variant polypeptides and their multimers, as well as uricase conjugates comprising (i) a first domain comprising one of the uricase variant polypeptides conjugated to (ii) a second domain comprising a random coil polypeptide domain. Methods of using the uricase variant polypeptides and uricase conjugates 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 variant polypeptides and uricase conjugates disclosed herein are also provided.

Description

Atty. Docket No.: INMD-220 / 01 WO PCT ApplicationURICASE VARIANTS AND METHODS OF USE THEREOFCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. Provisional Application Serial No. 63 / 717,188, filed November 6, 2024, 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_220_01WO_SeqList_ST26.xml; Size: 1,618,738 bytes; and Date of Creation: October 31, 2025) 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 duringAty. Docket No.: INMD-220 / 01 WO 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 provides a uricase variant polypeptide comprising (i) from 10 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R192T, R209W, Y225H, one of I257K and I257R, and N281Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119H, V119E, V119M and V119Q. The uricase variant polypeptide 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 uricase variant polypeptide is a variant of a counterpart uricase protein, and 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 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.

[0010] In one aspect, the present disclosure provides another uricase variant polypeptide comprising (i) from 12 to 20 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, one of I257K and I257R, L279T, and N281 Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationI100N and I1OOS, one of R101K and R101E, one of Y105A, Y105S, Y105G and Y105W, F114L, one of N117G and N117E, one of V119E, V119H and V119R, one of K120G and K120S, E138R, R192T, G196D, and R197D. The uricase variant polypeptide 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.

[0011] In one embodiment, the uricase variant polypeptide is a variant of a counterpart uricase protein, and 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 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.

[0012] In one embodiment, (i) the from 12 to 20 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T, and N281Y.

[0013] In another embodiment, (i) the from 12 to 20 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T, and N281Y.

[0014] In a further embodiment, the uricase variant polypeptide comprising (i) the from 12 to 20 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T, and N281Y, exhibits a higher melting temperature than a counterpart uricase variant polypeptide in which I257R is substituted with I257K. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 1°C to about 5°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 2°CAtty. Docket No.: INMD-220 / 01 WOPCT Application to about 4°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is about 3°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation.

[0015] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

[0016] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257R, L279T and N281Y.

[0017] In one aspect, the present disclosure provides yet another uricase variant polypeptide comprising (i) from 10 to 15 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, one of I257K and I257R, L279T and N281Y, and (ii) from one to 12 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, one of Q26T and Q26E, L72T, one of F75L and F75T, G77E, S80D, T83E, one of V86K, V86T and V86H, one of S95G and S95E, K97D, one of I100E, I100N and I100S, one of R101E and R101Q, one of Y105G, Y105A, Y105W and Y105S, one of V106M and V106G, Fl 14L, one ofNl 17G andNl 17E, one of VI 19E, VI 19H, VI 19R and VI 19N, K120S, E138R, R192T, G196D and R197D. The uricase variant polypeptide 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.

[0018] In one embodiment, the uricase variant polypeptide is a variant of a counterpart uricase protein, and 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 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 aboutAtty. Docket No.: INMD-220 / 01 WOPCT Application99%, 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.

[0019] In one embodiment, (i) the from 10 to 15 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T and N281Y.

[0020] In another embodiment, (i) the from 10 to 15 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y.

[0021] In a further embodiment, the uricase variant polypeptide comprising (i) the from 10 to 15 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y, exhibits a higher melting temperature than a counterpart uricase variant polypeptide in which I257R is substituted with I257K. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 1°C to about 5°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 2°C to about 4°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is about 3°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation.

[0022] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0023] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0024] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

[0025] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

[0026] In one aspect, the present disclosure provides yet another uricase variant polypeptide comprising (i) from 12 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, one of I257K and I257R, L279T and N281Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105W, Y105S, Y105G and Y105A, one of N117G and N117E, V119H, R192T and R197D. The uricase variant polypeptide 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.

[0027] In one embodiment, the uricase variant polypeptide is a variant of a counterpart uricase protein, and 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 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,Aty. Docket No.: INMD-220 / 01 WOPCT Application 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.

[0028] In one embodiment, (i) the from 12 to 18 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T and N281Y.

[0029] In another embodiment, (i) the from 12 to 18 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y.

[0030] In a further embodiment, the uricase variant polypeptide comprising (i) the from 12 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, KI 16M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y, exhibits a higher melting temperature than a counterpart uricase variant polypeptide in which I257R is substituted with I257K. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 1°C to about 5°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 2°C to about 4°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is about 3°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation.

[0031] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0032] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L,Aty. Docket No.: INMD-220 / 01 WO PCT ApplicationH24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0033] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

[0034] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

[0035] In one aspect, the present disclosure provides exemplary functional uricase variant polypeptides. In one embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 136-268. In a further embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 163, 165, 166, 169, 171, 172, 177, 183, 189, 190, 191, 204, 205, and 217.

[0036] In one embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 269-581, and 1408. In a further embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356, 360, 363, 366, 369, 371, 372, 375, 389, 390, 392, 417, 419, 422, 424, 426, 433, 460, 462, 524, 525, 563, 565, and 1408.

[0037] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 524.

[0038] In another embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 1408.

[0039] In one embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 582-1009, 1409, and 1410. In a further embodiment, the exemplary functional uricase variant polypeptide comprises an amino acidAtty. Docket No.: INMD-220 / 01 WO PCT Application sequence selected from the group consisting of SEQ ID NOs: 583, 588, 591, 592, 597, 599, 601, 605, 606, 624, 625, 626, 630, 632, 633, 634, 635, 637, 647, 652, 656, 922, 924, 928, 931, 939, 940, 946, 949, 958, 961, 964, 979, 982, 988, 1409, and 1410. In a further embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 922, 924, 928, 931, 939, 940, 946, 949, 958, 961, 964, 979, 982, 988, 1409, and 1410.

[0040] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 928.

[0041] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:949.

[0042] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 1409.

[0043] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 1410.

[0044] In one embodiment of a uricase variant polypeptide disclosed herein, the uricase variant polypeptide is non-PEGylated. In another embodiment, the uricase variant polypeptide is PEGylated.

[0045] In one embodiment of a uricase variant polypeptide disclosed herein, the uricase variant polypeptide is non-PEGylated and PATylated. In another embodiment, the uricase variant polypeptide is PEGylated and PATylated.

[0046] In one embodiment of a uricase variant polypeptide disclosed herein, the uricase variant polypeptide is non-PEGylated and XTENylated. In yet another embodiment, the uricase variant polypeptide is PEGylated and XTENylated.

[0047] In one embodiment of a uricase variant polypeptide disclosed herein, the uricase variant polypeptide is non-PEGylated and PASylated. In another embodiment, the uricase variant polypeptide is PEGylated and PASylated. In one embodiment, the PAS polypeptide of the PASylated uricase variant 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.

[0048] In one embodiment, a uricase variant polypeptide disclosed herein is a monomer. In another embodiment, the uricase variant polypeptide is a homodimer. In a preferred embodiment, the uricase variant polypeptide is a homotetramer.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0049] 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 variant polypeptides disclosed herein. In one embodiment, the second uricase monomer is one of the uricase variant polypeptides 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.

[0050] 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 variant polypeptides disclosed herein. In one embodiment, the second uricase monomer is one of the uricase variant polypeptides disclosed herein. In a further embodiment, the third uricase monomer is one of the uricase variant polypeptides disclosed herein. In a still further embodiment, the fourth uricase monomer is one of the uricase variant polypeptides 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.

[0051] 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 variant polypeptides disclosed herein and conjugated to (ii) a second domain comprising a first random coil polypeptide domain comprising at least about 50 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 fusion 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.

[0052] In one embodiment, the random coil polypeptide comprises a Pro-Ala-Thr (PAT) 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. In another embodiment, the random coil polypeptide comprises a Pro- Ala-Ser (PAS) polypeptide.

[0053] In some embodiments, the uricase conjugate is a PATylated uricase fusion protein whose monomeric form comprises an amino acid sequence selected from SEQ ID NOs: 1192- 1407.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0054] 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.

[0055] In another aspect, the present disclosure provides an isolated nucleic acid which encodes a uricase variant polypeptide or a uricase conjugate (e.g., a recombinant uricase fusion protein) disclosed herein.

[0056] In another aspect, the present disclosure provides a nucleic acid vector comprising the nucleic acid encoding a uricase variant polypeptide or a uricase conjugate (e.g., a recombinant uricase fusion protein) disclosed herein.

[0057] In another aspect, the present disclosure provides a host cell comprising a nucleic acid vector disclosed herein.

[0058] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a uricase variant polypeptide, 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.

[0059] 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.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0060] 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.

[0061] 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.

[0062] In another aspect, the present disclosure provides a method of recombinantly producing a uricase variant polypeptide 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 polypeptide 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 polypeptide or the uricase conjugate (e.g., the recombinant uricase fusion protein) and recombinant production of the uricase variant polypeptide or the uricase conjugate (e.g., the uricase fusion protein) by the host cell; and (ii) isolating the recombinantly produced uricase variant polypeptide or uricase conjugate (e.g., recombinant uricase fusion protein).BRIEF DESCRIPTION OF THE FIGURES

[0063] FIG. 1A is a schematic showing various configurations of uricase fusion proteins comprising PAS or XTEN polypeptide domain(s) of the invention.

[0064] FIG. IB is a schematic showing various configurations of uricase fusion proteins comprising PAT or PA polypeptide domain(s) of the invention.

[0065] FIG. 2 is a schematic of the PBMC (peripheral blood mononuclear cell) assay used to measure CD4+ T-cell activation and epitopes for wild type (WT) chimeric pig-baboonAty. Docket No.: INMD-220 / 01 WO PCT Application uricase as well as deimmunized HF752 uricase variant and HF752 uricase subvariants according to Example 1.

[0066] FIG. 3 is a graph showing full CD4+ T cell epitope mapping of wild type chimeric pig-baboon uricase (“WT Uricase”), HF752 uricase variant (“HF752”), and HF752 uricase subvariant #1428 (“Sv. #1428”) by the PBMC assay according to Example 1.

[0067] FIG. 4 is a graph showing targeted CD4+ T cell epitope mapping of 12 HF752 uricase subvariants (represented by their subvariant (Sv.) serial numbers) as compared to wild type chimeric pig-baboon uricase (“WT URC”) by the PBMC assay according to Example 1.

[0068] FIG. 5A 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 PK / PD study 1 according to Example 3. Each data point is presented as mean ± SD.

[0069] FIG. 5B 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 PK / PD study 1 according to Example 3. Each data point is presented as mean ± SD.

[0070] FIG. 5C is a graph showing plasma concentrations of WT-CXTENh and uric acid predose (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 2.16 mg / kg body weight WT-CXTENh in PK / PD study 1 according to Example 3. Each data point is presented as mean ± SD.

[0071] FIG. 6A is a graph showing plasma concentrations of the endogenous uricase as well as uric acid in untreated control Wistar rats (n = 6) at the corresponding pre-dose timepoint (at time 0) and at various corresponding post-dose timepoints up to 240 h in PK / PD study 2 according to Example 3. Each data point is presented as mean ± SD.

[0072] FIG. 6B is a graph showing plasma concentrations of pegloticase and uric acid pre-dose (at time 0) and at various timepoints up to 240 h post-dose in Wistar rats (n = 6) intravenously administrated with a single dose of 1 mg / kg body weight pegloticase in PK / PD study 2 according to Example 3. Each data point is presented as mean ± SD.

[0073] FIG. 6C is a graph showing plasma concentrations of XTEN144-WT-XTEN144 and uric acid pre-dose (at time 0) and at various timepoints up to 240 h post-dose in Wistar rats (n = 6) intravenously administrated with a single dose of 1.74 mg / kg body weight XTEN144-WT-Aty. Docket No.: INMD-220 / 01 WO PCT ApplicationXTEN144 in PK / PD study 2 according to Example 3. Each data point is presented as mean ± SD.

[0074] FIG. 6D is a graph showing plasma concentrations of XTEN214-WT-XTEN216 and uric acid pre-dose (at time 0) and at various timepoints up to 240 h post-dose in Wistar rats (n = 6) intravenously administrated with a single dose of 2.16 mg / kg body weight XTEN214-WT- XTEN216 in PK / PD study 2 according to Example 3. Each data point is presented as mean ± SD.

[0075] FIG. 7 is a graph showing the immunogenicity of subvariant (Sv.) #256R.1428, Sv. #347R.1445, and Sv. #368R.1445 as compared to WT chimeric pig-baboon uricase of SEQ ID NO: 1, determined by ex vivo cellular FluoroSpot assay using human peripheral blood mononuclear cells (PBMCs) (also referred to as “FluoroSpot PBMC assay”) according to Example 6.DETAILED DESCRIPTION OF THE INVENTION

[0076] 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. Acute 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 / joumal. pone.0167935, incorporated herein by reference in its entirety.

[0077] 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, highlightingAty. Docket No.: INMD-220 / 01 WO PCT Application a need for more effective uricase-based therapies. In general, the clinical complications associated with immunogenicity drastically reduce the utility of uricases.

[0078] Aspects of the present disclosure relate primarily to a recombinant uricase variant polypeptide comprising one or more amino acid mutations, e.g., one or more amino acid substitutions, in one or more T-cell epitopes such that the T-cell epitopes in the uricase variant polypeptide are depleted, as compared to a counterpart uricase protein, e.g., a wild type (WT) uricase protein. Such uricase variant polypeptides are referred to herein in some instances, as a “deimmunized uricase variant (polypeptide)” or a “T cell epitope depleted” uricase variant polypeptide. The use of such variants and methods of manufacturing the variants are also described herein.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0083] 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”).

[0084] 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.

[0085] 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.

[0086] 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 el 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 accumulationAtty. Docket No.: INMD-220 / 01 WOPCT Application 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).

[0087] 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.

[0088] One of ordinary skill in the art will appreciate that for each of the peptide and protein sequences provided herein, a DNA sequence suitable for cloning can be determined via the use of a codon table and appropriate start and stop codons.

[0089] As used herein, a “counterpart uricase protein” or “counterpart uricase” refers to a uricase protein that is compared to one of the uricase variant polypeptides 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 humanized porcine uricase, or a chimeric mammalian uricase, e.g., a chimeric pig-baboon uricase having the amino acid sequence of SEQ ID NO: 1. In 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.

[0090] 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 isAty. Docket No.: INMD-220 / 01 WOPCT Application 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.

[0091] 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.

[0092] 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%Aty. Docket No.: INMD-220 / 01 WOPCT Application 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.

[0093] 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 polypeptide of the present disclosure, the variant polypeptide 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 acid 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.

[0094] “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, interveningAtty. Docket No.: INMD-220 / 01 WOPCT Application 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.

[0095] 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.

[0096] “Recombinant protein,” or “recombinant polypeptide,” as used herein, refers to any artificially produced protein or polypeptide 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.

[0097] 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 polypeptide if a peptide originating from the uricase variant polypeptide is unable to bind an MHCII molecule, or if the peptide binds the MHCII molecule but subsequently does not bind a TCR, e.g., because of the lack of affinity to the MHCII molecule.

[0098] The terms “variant protein,” “variant polypeptide,” and “protein variant” are terms of the art, and used interchangeably herein. Each refers to a protein or polypeptide 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. In the present disclosure, the terms “variant protein,” “variant polypeptide,” and “protein variant” encompass a full-length form as well as a truncated form of a uricase variant protein or polypeptide, e.g., a catalytically active fragment of a uricase variant protein or polypeptide. 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 basisAtty. Docket No.: INMD-220 / 01 WOPCT Application 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 or polypeptide. 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.

[0099] 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.

[0100] 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 polypeptide which has an amino acid substitution of W22L refers to a uricase variant polypeptide in which the wild type residue at the 22ndposition (W or tryptophan) is replaced by L or leucine.

[0101] The designation of all amino acid positions of a uricase variant polypeptide 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.

[0102] The present disclosure provides in part, a uricase variant polypeptide (e.g., a recombinant uricase variant polypeptide), which is an engineered variant of a counterpart uricase protein, e.g., a WT uricase protein. The uricase variant polypeptide, in embodiments described herein, is deimmunized compared to a counterpart uricase protein, e.g., because the uricase variant polypeptide is T-cell epitope depleted compared to a counterpart uricase protein. In one embodiment, the uricase variant polypeptide is a variant of the WT chimeric pig-baboon uricase having the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricaseAty. Docket No.: INMD-220 / 01 WOPCT Application variant polypeptide 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.

[0103] 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 polypeptide is provided, mutations in one subunit may or may not be present in the remaining uricase monomer subunits.

[0104] In one aspect of a uricase variant polypeptide provided herein, a chimeric pigbaboon uricase variant polypeptide 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 polypeptide of the present invention in one embodiment, is PEGylated. In another embodiment, a uricase variant polypeptide of the present invention is PASylated. In yet another embodiment, the uricase variant polypeptide is PASylated and PEGylated. In yet another embodiment, a uricase variant polypeptide of the present invention is XTENylated. In yet another embodiment, a uricase variant polypeptide of the present invention is PATylated. In one embodiment, the chimeric pig-baboon uricase variant polypeptide 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 polypeptide has substantially the same stability and / or enzymatic activity as the counterpart uricase protein. The uricase variant polypeptide provided herein, in one embodiment, is a monomer. In another embodiment, the uricase variant polypeptide is provided as a homodimer or a homotetramer. In a preferred embodiment, the uricase variant polypeptide is a homotetramer.

[0105] In one aspect, the present disclosure provides a uricase variant polypeptide comprising (i) from 10 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R192T, R209W, Y225H, one of I257K and I257R, and N281Y, and (ii) from one to nine amino acid 1Atty. Docket No.: INMD-220 / 01 WOPCT Application substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119H, V119E, V119M and V119Q. The uricase variant polypeptide 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.

[0106] In one embodiment, the uricase variant polypeptide is a variant of a counterpart uricase protein, and 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 polypeptide further comprises one or more conservative amino acid substitutions and / or humanizing amino acid mutations disclosed herein. In one embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1.

[0107] In one embodiment, (i) the from 10 to 18 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K, and N281Y.

[0108] In another embodiment, (i) the from 10 to 18 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257R, and N281Y.

[0109] In a further embodiment, the uricase variant polypeptide comprising (i) the from 10 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257R, and N281Y, exhibits a higher melting temperature than a counterpart uricase variant polypeptide in which I257R is substituted with I257K. In still a further embodiment, theAtty. Docket No.: INMD-220 / 01 WOPCT Application melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 1°C to about 5°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 2°C to about 4°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is about 3°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation.

[0110] In some embodiments, (i) the from 10 to 18 amino acid substitutions are from 11 to 18, from 12 to 18, from 13 to 18, from 14 to 18, from 15 to 18, from 16 to 18, from 17 to 18, or 18 amino acid substitutions.

[0111] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, Fl 14L, N117D, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

[0112] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K and V86T, S95G, K97D, Y105W, Fl 14L, one of N117D and N117H, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

[0113] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, and V86E, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

[0114] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, one of N117D and N117H, and one of V119H, V119E, V119M and V119Q.

[0115] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, Fl 14L, one of N117D and N117H, and one of VI 19M and VI 19Q.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0116] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, Fl 14L, and one of N117D and N117H.

[0117] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86T, and V86E, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

[0118] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to six amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D and N117D.

[0119] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to four amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Y105W, F114L, one of N117D and N117H, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

[0120] In one embodiment, (ii) the from one to nine amino acid substitutions are from two to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, V86H and V86E; S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119M, V119Q, V119H and V119E.

[0121] In one embodiment, (ii) the from one to nine amino acid substitutions are from three to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, V86H and V86E; S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119M, V119Q, V119H and V119E.

[0122] In one embodiment, (ii) the from one to nine amino acid substitutions are from four to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, V86H and V86E; S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119M, V119Q, V119H and V119E.Atty. Docket No.: INMD-220 / 01 WO PCT Application

[0123] In some embodiments, (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to one or a combination of the exemplary groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table A.

[0124] In some embodiments, (ii) the from one to nine amino acid substitutions comprise from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 2 to 4, from 2 to 5, from 2 to 6, from 3 to 6, from 2 to 8, from 3 to 7, or from 3 to 4 amino acid substitutions.

[0125] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0126] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0127] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0128] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86H, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0129] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0130] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86E, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0131] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0132] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, F114L, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0133] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationQ26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117D, V119H, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0134] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117D, V119M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0135] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117D, V119Q, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0136] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106M, K116M, N117D, V119M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0137] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106M, K116M, N117D, V119Q, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0138] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117H, V119E, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0139] In one aspect, the present disclosure provides another uricase variant polypeptide comprising (i) from 12 to 20 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, one of I257K and I257R, L279T, and N281 Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, one of R101K and R101E, one of Y105A, Y105S, Y105G and Y105W, F114L, one of N117G and N117E, one of V119E, V119H and V119R, one of K120G and K120S, E138R, R192T, G196D, and R197D. The uricase variant polypeptide comprises anAtty. Docket No.: INMD-220 / 01 WOPCT Application amino acid sequence that is at least about 85%, at least about 90%, or at least about 95% identical to SEQ ID NO: 1.

[0140] In one embodiment, the uricase variant polypeptide is a variant of a counterpart uricase protein, and 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 polypeptide further comprises one or more conservative amino acid substitutions and / or humanizing amino acid mutations disclosed herein. In one embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1.

[0141] In one embodiment, (i) the from 12 to 20 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T, and N281Y.

[0142] In another embodiment, (i) the from 12 to 20 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T, and N281Y.

[0143] In a further embodiment, the uricase variant polypeptide comprising (i) the from 12 to 20 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T, and N281Y, exhibits a higher melting temperature than a counterpart uricase variant polypeptide in which I257R is substituted with I257K. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 1°C to about 5°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the meltingAtty. Docket No.: INMD-220 / 01 WOPCT Application temperature of the uricase variant polypeptide harboring the I257R mutation is from about 2°C to about 4°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is about 3°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation.

[0144] In some embodiments, (i) the from 12 to 20 amino acid substitutions are from 13 to 20, from 14 to 20, from 15 to 20, from 16 to 20, from 17 to 20, from 18 to 20, from 19 to 20, or 20 amino acid substitutions.

[0145] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, one of V119E and V119H, R192T, G196D, and R197D.

[0146] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, one of R101K and R101E, one of Y105A, Y105S, Y105G and Y105W, F114L, one of N117G and N117E, one of V119E, V119H and V119R, one of K120G and K120S, E138R, G196D, and R197D.

[0147] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, one of V119E and V119H, G196D, and R197D.

[0148] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, V119H, G196D, and R197D.

[0149] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K,Atty. Docket No.: INMD-220 / 01 WOPCT Application one of Y105A, Y105S, Y105G and Y105W, N117G, one of V119E and V119H, G196D, and R197D.

[0150] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, V119E, G196D, and R197D.

[0151] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, T83E, one of V86T and V86K, S95G, one of I100E and I100N, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, one of VI 19E and VI 19H, G196D, and R197D.

[0152] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, one of VI 19E and VI 19H, G196D, and R197D.

[0153] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, V86T, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, one of VI 19E and VI 19H, G196D, and R197D.

[0154] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to seven amino acid substitutions selected from substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, V86T, one of I100E and I100N, one of Y105A, Y105S, Y105G and Y105W, N117G, G196D, and R197D.

[0155] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from 2 to 6 amino acid substitutions selected from substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, one of I100E, I100N and I100S, one of Y105A, Y105S, Y105W and Y105G, N117G and G196D.Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0156] In some embodiments, (ii) the from one to nine amino acid substitutions comprise from 1 to 8, from 2 to 8, from 3 to 8, from 4 to 8, from 5 to 8, from 1 to 7, from 2 to 7, from 3 to 7, from 4 to 7, from 5 to 7, or from 3 to 6 amino acid substitutions.

[0157] In one embodiment, (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to one or a combination of the exemplary groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table B 1.

[0158] In some embodiments, (ii) the from one to nine amino acid substitutions are 4, 5, 6, or 7 amino acid substitutions, comprising amino acid substitutions corresponding to one of the exemplary groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table B2. In one embodiment, (ii) the from one to nine amino acid substitutions are 4 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 5 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 6 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 7 amino acid substitutions.

[0159] In some embodiments, (ii) the from one to nine amino acid substitutions are 5, 6, or 7 amino acid substitutions, comprising amino acid substitutions corresponding to one of the exemplary groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table B3. In one embodiment, (ii) the from one to nine amino acid substitutions are 5 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 6 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 7 amino acid substitutions.Aty. Docket No.: INMD-220 / 01 WO PCT ApplicationAty. Docket No.: INMD-220 / 01 WO PCT Application

[0160] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0161] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, F114L,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationK116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0162] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117G, V119E, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0163] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117G, K120G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0164] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0165] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0166] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0167] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, R101K, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0168] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0169] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117G, V119R, K120S, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0170] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, R101E, A102V, Y105S, V106M, K116M, N117G, Y127H, E138R, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0171] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0172] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, R101K, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0173] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0174] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L,Atty. Docket No.: INMD-220 / 01 WO PCT ApplicationQ26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, A102V, V106M, K116M, N117G, V119E, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0175] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0176] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0177] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0178] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0179] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

[0180] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G,Aty. Docket No.: INMD-220 / 01 WOPCT ApplicationV106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0181] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0182] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0183] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257R, L279T and N281Y.

[0184] In yet another aspect, the present disclosure provides a uricase variant polypeptide comprising (i) from 10 to 15 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, one of I257K and I257R, L279T and N281Y, and (ii) from one to 12 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: H24R, one of Q26T and Q26E, L72T, one of F75L and F75T, G77E, S80D, T83E, one of V86K, V86T and V86H, one of S95G and S95E, K97D, one of I100E, I100N and I100S, one of R101E and R101Q, one of Y105G, Y105A, Y105W and Y105S, one of V106M and V106G, Fl 14L, one ofNl 17G andNl 17E, one of VI 19E, VI 19H, VI 19R and VI 19N, K120S, E138R, R192T, G196D and R197D. In this aspect, the uricase variant polypeptide 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.

[0185] In one embodiment, the uricase variant polypeptide is a variant of a counterpart uricase protein, and the counterpart uricase protein is the wild type chimeric pig-baboon uricaseAtty. Docket No.: INMD-220 / 01 WOPCT Application 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 polypeptide further comprises one or more conservative amino acid substitutions and / or humanizing amino acid mutations disclosed herein. In one embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1.

[0186] In one embodiment, (i) the from 10 to 15 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T and N281Y.

[0187] In another embodiment, (i) the from 10 to 15 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y.

[0188] In a further embodiment, the uricase variant polypeptide comprising (i) the from 10 to 15 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y, exhibits a higher melting temperature than a counterpart uricase variant polypeptide in which I257R is substituted with I257K. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 1°C to about 5°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 2°C to about 4°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is about 3°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0189] In some embodiments, (i) the from 10 to 15 amino acid substitutions are from 11 to 15, from 12 to 15, from 13 to 15, from 14 to 15, or 15 amino acid substitutions.

[0190] In one embodiment, (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: H24R, Q26T, L72T, F75L, G77E, S80D, T83E, one of V86K andV86T, one of S95G and S95E, K97D, one of I100E, I100N and I100S, one of Y105G, Y105A, Y105W and Y105S, V106M, one of N117G and N117E, VI 19H, R192T, G196D and R197D.

[0191] In one embodiment, (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: H24R, one of Q26T and Q26E, L72T, one of F75L and F75T, G77E, S80D, T83E, one of V86K, V86T and V86H, S95G, K97D, one of I100E, I100N and I100S, one of R101E and R101Q, one of Y105G, Y105A, Y105W and Y105S, one of V106M and V106G, F114L, N117G, one of VI 19E, VI 19R and VI 19N, K120S, E138R, G196D and R197D.

[0192] In one embodiment, (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: H24R, Q26T, L72T, F75L, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100N and I100S, one of Y105G, Y105A, Y105W and Y105S, V106M, N117G, G196D and R197D.

[0193] In one embodiment, (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: Q26T, L72T, F75L, T83E, one of V86K and V86T, K97D, one of I100E and I100N, one of Y105G and Y105A, V106M, N117G, G196D and R197D.

[0194] In one embodiment, (ii) the from one to 12 amino acid substitutions comprise from one to 11 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Q26T, F75L, T83E, one of V86K and V86T, K97D, I100E, Y105G, V106M, N117G, G196D and R197D.

[0195] In one embodiment, (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: one of Q26T and Q26E, one of F75L and F75T, T83E, one of V86K, V86T and V86H, K97D, I100E, one of R101E and R101Q, one of Y105G, Y105W and Y105S, one of V106M and V106G, F114L, N117G, one of V119E, V119R and V119N, K120S, E138R, G196D and R197D.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0196] In some embodiments, (ii) the from one to 12 amino acid substitutions comprise from 2 to 12, from 3 to 12, from 4 to 12, from 5 to 12, from 6 to 12, from 7 to 12, from 8 to 12, from 9 to 12, from 1 to 11, from 2 to 11, from 3 to 11, from 4 to 11, from 5 to 11, from 6 to 11, from 1 to 10, from 2 to 10, from 3 to 10, from 4 to 10, from 5 to 10, or from 6 to 10 amino acid substitutions.

[0197] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0198] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, F114L, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0199] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117G, V119E, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0200] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117G, V119N, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0201] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0202] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0203] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationQ26T, Y31H, L52T, N70H, F75L, K76S, T83E, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0204] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, R101Q, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0205] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106G, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0206] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0207] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117G, V119R, K120S, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0208] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, R101E, A102V, Y105G, V106M, K116M, N117G, Y127H, E138R, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0209] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0210] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86H, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0211] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0212] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0213] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95E, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0214] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y

[0215] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26E, Y31H, L52T, N70H, F75T, K76S, T83E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0216] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0217] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0218] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationQ26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0219] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

[0220] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0221] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0222] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

[0223] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0224] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationV106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0225] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0226] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0227] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0228] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0229] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0230] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0231] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0232] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

[0233] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

[0234] In one aspect, the present disclosure provides yet another uricase variant polypeptide comprising (i) from 12 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, one of I257K and I257R, L279T and N281Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105W, Y105S, Y105G and Y105A, one of N117G and N117E, V119H, R192T and R197D. The uricase variant polypeptide 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.

[0235] In one embodiment, the uricase variant polypeptide is a variant of a counterpart uricase protein, and 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 polypeptide further comprises one or more conservative amino acidAtty. Docket No.: INMD-220 / 01 WOPCT Application substitutions and / or humanizing amino acid mutations disclosed herein. In one embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 85% identical to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1. In another embodiment, the uricase variant polypeptide has an amino acid sequence that is at least about 95% identical to the amino acid sequence of SEQ ID NO: 1.

[0236] In one embodiment, (i) the from 12 to 18 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T and N281Y.

[0237] In another embodiment, (i) the from 12 to 18 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y.

[0238] In a further embodiment, the uricase variant polypeptide comprising (i) the from 12 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, KI 16M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y, exhibits a higher melting temperature than a counterpart uricase variant polypeptide in which I257R is substituted with I257K. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 1°C to about 5°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is from about 2°C to about 4°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation. In still a further embodiment, the melting temperature of the uricase variant polypeptide harboring the I257R mutation is about 3°C higher than the melting temperature of the counterpart uricase variant polypeptide harboring the I257K mutation.

[0239] In some embodiments, (i) the from 12 to 18 amino acid substitutions are from 13 to 18, from 14 to 18, from 15 to 18, from 16 to 18, from 17 to 18, or 18 amino acid substitutions.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0240] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105S, Y105G and Y105A, N117G, R192T and R197D.

[0241] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E and I100N, one of Y105S, Y105G and Y105A, N117G, R192T and R197D.

[0242] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E and I100N, one of Y105S, Y105G and Y105A, N117G, and R197D.

[0243] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E and I100N, one of Y105S, Y105G and Y105A, N117G, and R197D.

[0244] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E and I100N, one of Y105G and Y105A, N117G, and R197D.

[0245] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, S80D, T83E, one of V86K and V86T, K97D, one of I100E and I100N, one of Y105G and Y105A, N117G, and R197D.

[0246] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, one of N117G and N117E, and R197D.

[0247] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding toAtty. Docket No.: INMD-220 / 01 WOPCT Application the following amino acid substitutions of SEQ ID NO: 1 : T83E, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

[0248] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, one of N117G and N117E, and R197D.

[0249] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

[0250] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and llOON, one of Y105G, Y105A, Y105W and Y105S, one of N117G and N117E, and R197D.

[0251] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: H24R, T83E, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

[0252] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, G77E, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, one of N117G and N117E, and R197D.

[0253] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, G77E, T83E, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

[0254] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, N117G, and R197D.Aty. Docket No.: INMD-220 / 01 WOPCT Application

[0255] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, T83E, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

[0256] In one embodiment, (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, T83E, V86T, S95G, K97D, I100E, Y105G, N117G, and R197D.

[0257] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to seven amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, one of V86K and V86T, one of S95G and K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, R192T, and R197D.

[0258] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K and V86T, one of S95G and K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, R192T, and R197D.

[0259] In one embodiment, (ii) the from one to nine amino acid substitutions comprise from one to seven amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, one of V86K and V86T, one of S95G and K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, R192T, and R197D.

[0260] In some embodiments, (ii) the from one to nine amino acid substitutions comprise from 2 to 9, from 3 to 9, from 4 to 9, from 5 to 9, from 6 to 9, from 7 to 9, from 6 to 8, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 2 to 9, from 3 to 8, from 4 to 7 amino acid substitutions.

[0261] In some embodiments, (ii) the from one to nine amino acid substitutions are 5, 6, 7, 8, or 9 amino acid substitutions, comprising amino acid substitutions corresponding to one of the exemplary groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table Cl. In one embodiment, (ii) the from one to nine amino acid substitutions are 5 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 6 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 7 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acidAty. Docket No.: INMD-220 / 01 WO PCT Application substitutions are 8 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 9 amino acid substitutions.

[0262] In some embodiments, (ii) the from one to nine amino acid substitutions are 6, 7, 8, or 9 amino acid substitutions, comprising amino acid substitutions corresponding to one of the exemplary groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table C2. In one embodiment, (ii) the from one to nine amino acid substitutions are 6 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 7 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 8 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 9 amino acid substitutions.Atty. Docket No.: INMD-220 / 01 WO PCT Application

[0263] In some embodiments, (ii) the from one to nine amino acid substitutions are 7, 8, or 9 amino acid substitutions, comprising amino acid substitutions corresponding to one of the exemplary groups of amino acid substitutions of SEQ ID NO:1 set forth in Table DI. In one embodiment, (ii) the from one to nine amino acid substitutions are 7 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 8 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 9 amino acid substitutions.

[0264] In some embodiments, (ii) the from one to nine amino acid substitutions are 8 or 9 amino acid substitutions, comprising amino acid substitutions corresponding to one of the exemplary groups of amino acid substitutions of SEQ ID NO:1 set forth in Table D2. In one embodiment, (ii) the from one to nine amino acid substitutions are 8 amino acid substitutions. In one embodiment, (ii) the from one to nine amino acid substitutions are 9 amino acid substitutions.Aty. Docket No.: INMD-220 / 01 WO PCT ApplicationAty. Docket No.: INMD-220 / 01 WO PCT Application

[0265] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0266] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

[0267] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0268] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0269] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V, Y105G,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationV106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

[0270] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0271] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0272] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0273] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0274] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0275] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0276] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0277] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0278] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0279] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

[0280] In one embodiment, the uricase variant polypeptide comprises amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

[0281] In one aspect, the present disclosure provides exemplary functional uricase variant polypeptides. In one embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from SEQ ID NOs:45-90.

[0282] In one embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from SEQ ID NOs:91-135.Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0283] In one embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 136-268. In a further embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 163, 165, 166, 169, 171, 172, 177, 183, 189, 190, 191, 204, 205, and 217.

[0284] In one embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 269-581 and 1408. In a further embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 356, 360, 363, 366, 369, 371, 372, 375, 389, 390, 392, 417, 419, 422, 424, 426, 433, 460, 462, 524, 525, 563, 565, and 1408.

[0285] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:417.

[0286] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:419.

[0287] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:422.

[0288] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:424.

[0289] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:426.

[0290] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:433.

[0291] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:460.

[0292] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:462.

[0293] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:524.

[0294] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:525.Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0295] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:563.

[0296] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:565.

[0297] In another embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 1408.

[0298] In one embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 582-1009, 1409, and 1410. In a further embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 583, 588, 591, 592, 597, 599, 601, 605, 606, 624, 625, 626, 630, 632, 633, 634, 635, 637, 647, 652, 656, 922, 924, 928, 931, 939, 940, 946, 949, 958, 961, 964, 979, 982, 988, 1409, and 1410. In a further embodiment, the exemplary functional uricase variant polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 922, 924, 928, 931, 939, 940, 946, 949, 958, 961, 964, 979, 982, 988, 1409, and 1410.

[0299] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:922.

[0300] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 928.

[0301] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 931.

[0302] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:939.

[0303] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:940.

[0304] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:949.

[0305] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 958.Atty. Docket No.: INMD-220 / 01 WO PCT Application

[0306] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO:961.

[0307] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 1409.

[0308] In one embodiment, the exemplary functional uricase variant polypeptide comprises the amino acid sequence of SEQ ID NO: 1410.

[0309] In one embodiment, a uricase variant polypeptide of the present disclosure comprises one or more conservative amino acid mutations relative to a counterpart uricase protein. 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 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.

[0310] In another embodiment, the present disclosure provides a uricase variant polypeptide comprising one or more conservative amino acid substitutions relative to a counterpart uricase variant polypeptide disclosed herein. In a further embodiment, the one or more conservative amino acid substitutions are at one or more amino acid positions where the counterpart uricase variant polypeptide harbor one or more of the disclosed amino acid substitutions relative to a counterpart wild type uricase. The counterpart wild type uricase, in one embodiment, is the wild type chimeric pig-baboon uricase comprising the amino acid sequence of SEQ ID NO: 1. In another embodiment, the counterpart wild type uricase is one of the counterpart uricase proteins provided herein that comprises an amino acid sequence that is substantially identical to SEQ ID NO: 1.

[0311] 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:

[0312] (i) glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I);

[0313] (ii) aspartic acid (D) and glutamic acid (E);

[0314] (iii) alanine (A), serine (S) and threonine (T);

[0315] (iv) histidine (H), lysine (K) and arginine (R);Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0316] (v) asparagine (N) and glutamine (Q);

[0317] (vi) phenylalanine (F), tyrosine (Y) and tryptophan (W).

[0318] In embodiments described herein, the uricase variant polypeptide 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.

[0319] In one embodiment, a uricase variant polypeptide 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 polypeptide 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.

[0320] In one embodiment, the uricase variant polypeptide of the present 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 polypeptide comprises one or more humanizing amino acid mutations relative to a non-human counterpart uricase protein. 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 herein that comprise an amino acid sequence that is substantially identical to SEQ ID NO: 1.

[0321] 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 uricase variant polypeptide retains uricase activity while improving the homology with the human uricase, thus reducing immunogenicity. In one embodiment, the humanized uricase variant polypeptide 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, or one of the chimeric-pig baboon uricase described herein that comprises an amino acid sequence that is substantially identical to SEQ ID NO: 1. In the humanized uricase variant polypeptide, a humanizing mutation can be a substitution(s)Aty. Docket No.: INMD-220 / 01 WOPCT Application 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.

[0322] In one embodiment, a uricase variant polypeptide of the present disclosure is T-cell epitope depleted compared to the counterpart uricase protein. In some embodiments, a uricase variant polypeptide 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 polypeptide is based. In a further embodiment, the uricase variant polypeptide and the counterpart uricase are homotetramers.

[0323] In some embodiments, a uricase variant polypeptide 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 polypeptide is based. In a further embodiment, the uricase variant polypeptide and the counterpart uricase are homotetramers.

[0324] In the present disclosure, if an N-terminal methionine residue is not present in a uricase variant polypeptide, alternative embodiments include uricase variant polypeptides 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 polypeptide, alternative embodiments include uricase variant polypeptides 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 polypeptide, and instead another amino acid is present, e.g., threonine, the uricase variant polypeptide 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 polypeptide is produced in bacteria, e.g., in E coli.

[0325] In one embodiment of a uricase variant polypeptide disclosed herein, the uricase variant polypeptide is isolated. In another embodiment, the uricase variant polypeptide is purified. In some embodiments, the uricase variant polypeptide 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 (alsoAtty. Docket No.: INMD-220 / 01 WOPCT Application 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 I<DHLIHNVHI<EEHAHAHNI< (SEQ ID NO:8). In one embodiment of a uricase variant polypeptide provided herein, the uricase variant polypeptide is isolated and purified.

[0326] As discussed above, the uricase variant polypeptides 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 polypeptide disclosed herein is a monomer. In one embodiment, the uricase variant polypeptide is a homodimer. In one embodiment, the uricase variant polypeptide is a homotrimer. In one preferred embodiment, the uricase variant polypeptide is a homotetramer. In another embodiment, the uricase variant polypeptide is a heterodimer, a heterotrimer or a heterotetramer.

[0327] In another aspect of the present disclosure, a uricase monomer comprising a uricase variant polypeptide disclosed herein is provided.

[0328] 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 variant polypeptides disclosed herein. In one embodiment, the second uricase monomer is one of the uricase variant polypeptides 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.

[0329] 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 variant polypeptides disclosed herein. In one embodiment, the second uricase monomer is one of the uricase variant polypeptides disclosed herein. In a further embodiment, the third uricase monomer is one of the uricase variant polypeptides disclosed herein. In a still further embodiment, the fourth uricase monomer is one of the uricase variantAty. Docket No.: INMD-220 / 01 WOPCT Application polypeptides 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.

[0330] In one embodiment of a uricase variant polypeptide disclosed herein, the uricase variant polypeptide is non-PEGylated. In another embodiment, the uricase variant polypeptide is PEGylated, i.e., the uricase variant polypeptide 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 polypeptide, e.g., a uricase homotetramer, is PEGylated. In one embodiment, the uricase variant polypeptide 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 polypeptide is about 10 kDa to about 150 kDa, about 30 kDa to 120 kDa, or about 50 kDa to about 100 kDa.

[0331] In one embodiment, the uricase variant polypeptide 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, Anti cancer 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.

[0332] In another embodiment, the uricase variant polypeptide disclosed herein is conjugated without a linking group to PEG, e.g., mPEG, through an amino group, a sulfhydral group, aAty. Docket No.: INMD-220 / 01 WOPCT Application hydroxyl group or a carboxyl group. In one embodiment, PEG is conjugated to one or more lysine residues on the uricase variant polypeptide. In one embodiment, PEG is conjugated to one or more cysteine residues on the uricase variant polypeptide. In one embodiment, PEG is conjugated to one or more serine residues on the uricase variant polypeptide. 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.

[0333] 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 variant polypeptides disclosed herein conjugated to (ii) a second domain comprising a first random coil polypeptide domain.

[0334] 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 polypeptide 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 polypeptide 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.

[0335] 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 randomlyAtty. Docket No.: INMD-220 / 01 WOPCT Application 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 secondary and tertiary structure. The nature of polypeptide random coils and their methods of experimental identification are known to the person skilled in the art.

[0336] The first (uricase) domain comprising one of the uricase variant polypeptides 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 DNA sequence of the uricase variant polypeptide 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.

[0337] 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).

[0338] In one embodiment, the second domain of the uricase conjugate comprises the first random coil polypeptide domain comprising, e.g., a PA polypeptide, a PAS polypeptide, a PAT polypeptide, or an XTEN polypeptide with at least about 30, at least about 50, at least about 70, at least about 100, at least about 150, or at least about 200 amino acid residues. 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 polypeptide. Moreover, without wishing to be bound by theory, because the random coil polypeptide domain is thought to not adopt a stableAty. Docket No.: INMD-220 / 01 WOPCT Application structure or function by itself, the biological activity of the uricase variant polypeptide to which it is conjugated is essentially preserved.

[0339] 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, or 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 30 amino acids long, at least about 50 amino acids long, at least about 70 amino acids long, 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 40 amino acids long, about 60 amino acids long, about 80 amino acids long, about 100 amino acids long, about 120 amino acids long, about 140 amino acids long, about 160 amino acids long, 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. In one embodiment, the PA polypeptide is 401 amino acids long (also referred to herein as PA401) and has the amino acid sequence of SEQ ID NO:25. Other examples of PA polypeptides and their coding nucleotide sequences amenable for use herein are provided in U.S. Pat. No. 10,844,094B2, the disclosure of which is incorporated by reference in its entirety.

[0340] 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, the PAS polypeptide is at least about 30 amino acids long, at least about 50 amino acids long, at least about 70 amino acids long, 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 acidsAtty. Docket No.: INMD-220 / 01 WOPCT Application 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 PAS polypeptide is about 40 amino acids long, about 60 amino acids long, about 80 amino acids long, about 100 amino acids long, about 120 amino acids long, about 140 amino acids long, about 160 amino acids long, 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. In one embodiment, a uricase fusion protein comprises a PAS polypeptide and the PAS polypeptide (i.e., the PAS domain) comprises from about 10 to about 30 tandem copies of a PAS sequence comprising Pro, Ala, and Ser, e.g., a PAS10 domain with 10 tandem copies of a PAS sequence, a PAS20 domain with 20 tandem copies of a PAS sequence, and / or a PAS30 domain with 30 tandem copies of a PAS sequence. In a further embodiment, the PAS sequence comprises the amino acid sequence of 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. In still a further embodiment, the PAS sequence comprises the amino acid sequence of SEQ ID NO: 13. In one embodiment, the PAS 10 domain has a total of about 200 amino acids; the P AS20 domain has a total of about 400 amino acids; and the PAS30 domain has a total of about 600 amino acids. An exemplary amino acid sequence of a PAS 10 domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 1414, and exemplary amino acid sequences of a PAS20 domain and a PAS30 domain comprise the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 11, respectively. In another embodiment, a uricase fusion protein comprises a PAS domain and the PAS domain comprises from about 2 to about 9 tandem copies of a PAS sequence comprising Pro, Ala, and Ser, e.g., a PAS3 domain with 3 tandem copies of a PAS sequence, a PAS5 domain with 5 tandem copies of a PAS sequence, and / or a PAS8 domain with 8 tandem copies of a PAS sequence. In a further embodiment, the PAS sequence comprises the amino acid sequence of 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. In still a further embodiment, the PAS sequence comprises the amino acid sequence of SEQ ID NO: 13. In one embodiment, the PAS3 domain has a total of about 60 amino acids; the PAS5 domain has a total of about 100 amino acids; and the PAS8 domain has a total of about 160 amino acids. Exemplary amino acid sequences of a PAS3 domain, a PAS5 domain, and a PAS8 domain comprise the amino acid sequences of SEQ ID NO: 1411, SEQ ID NO: 1412, and SEQ ID NO: 1413, respectively. Other examples of PAS polypeptides and their coding nucleotide sequences amenable for use herein are provided in PCT Publication Nos. WO 2008 / 155134 and WO 2024 / 124142, as well as in U.S. Pat. Nos.Aty. Docket No.: INMD-220 / 01 WOPCT Application11,548,931B2 and 8,563,521B2, the disclosure of each of which is incorporated by reference in its entirety.

[0341] 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 threonine (Thr). In a further embodiment, the random coil polypeptide consists of the three amino acids, proline (Pro), alanine (Ala) and threonine (Thr). Where the amino acid residues in a polypeptide are all Pro, Ala and Thr, or substantially all the amino acids are Pro, Ala and Thr in a polypeptide, such a polypeptide is referred to herein as a “PAT polypeptide.” A PAT polypeptide, when present in a uricase conjugate, may be referred to as a PAT domain. In one embodiment, a uricase fusion protein comprises a PAT polypeptide. In a further embodiment, the PAT 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 PAT 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. In one embodiment, the PAT polypeptide is 200 amino acids long (also referred to herein as PAT200) and has the amino acid sequence of SEQ ID NO:27. In a further embodiment, PAT200 is encoded by the nucleotide sequence of SEQ ID NO:28. In another embodiment, the PAT polypeptide is 300 amino acids long (also referred to herein as PAT300) and has the amino acid sequence of SEQ ID NO:29. In a further embodiment, PAT300 is encoded by the nucleotide sequence of SEQ ID NO:30. In another embodiment, the PAT polypeptide is 400 amino acids long (also referred to herein as PAT400) and has the amino acid sequence of SEQ ID NO:31. In a further embodiment, PAT400 is encoded by the nucleotide sequence of SEQ ID NO:32. PAT polypeptides amenable for use herein are provided in Shirian J et al., bioRxiv [Preprint], 2024 Jun 27:2024.06.27.600979, the disclosure of which is incorporated by reference in its entirety.

[0342] 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,Aty. Docket No.: INMD-220 / 01 WOPCT Application 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, the 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. In another embodiment, the XTEN polypeptide comprises part of the amino acid sequence of SEQ ID NO: 12. For example, the XTEN polypeptide, referred to herein as “XTEN216,” comprises amino acids 1-216 of SEQ ID NO: 12, as set forth in SEQ ID NO:42. As another example, the XTEN polypeptide, referred to herein as “XTEN214,” comprises amino acids 212-425 of SEQ ID NO: 12, as set forth in SEQ ID NO:43. In still another embodiment, the XTEN polypeptide comprises 144 amino acids with the amino acid sequence set forth in SEQ ID NO:44, which is referred herein as “XTEN144” and disclosed in Geething NC et al., PLoS ONE 5(4): el0175 (2010), incorporated herein by reference in its entirety. XTEN polypeptides may be conjugated to the first domain comprising a uricase variant polypeptide described herein via chemical conjugation or produced as a fusion protein with the uricase variant polypeptide.

[0343] 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 30 amino acid residues (e.g., about 40 amino acid residues), at least about 50 amino acid residues (e.g., about 60 amino acid residues), at least about 70 amino acid residues (e.g., about 80 amino acid residues), at least about 100 amino acid residues (e.g., about 100 amino acid residues), at least about 150 amino acid residues (e.g., about 160 amino acid residues), or at least about 200 amino acid residues (e.g., about 200 amino acid residues) that form a random coil conformation. The 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. In one embodiment, the second domain of the uricase conjugate comprises a PAS3 domain, a PAS5 domain, a PAS8 domain, a PAS10 domain, a PAS20 domain, or a PAS30 domain disclosed herein. In another embodiment, the second domain of the uricase conjugate comprises a PAS3 domain, a PAS5 domain, a PAS8 domain, or a PAS 10 domain disclosed herein. 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 polypeptide. Details regarding various types of PAS polypeptides and nucleic acids encoding the same, for use in the present invention, can be found in PCTAtty. Docket No.: INMD-220 / 01 WOPCT ApplicationPublication No. WO 2008 / 155134, the content of which is incorporated by reference in its entirety for all purposes.

[0344] 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.

[0345] 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.

[0346] 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 a-helix, parallel and anti-parallel P- 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, analyticalAty. Docket No.: INMD-220 / 01 WOPCT Application ultracentrifugation or dynamic / static light scattering as well as measurements of the frictional coefficient or intrinsic viscosity.

[0347] In one embodiment, a random coil polypeptide domain comprises at least about 30 amino acid residues, at least about 50 amino acid residues, at least about 60 amino acid residues, at least about 70 amino acid residues, 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.

[0348] In one embodiment, a random coil polypeptide domain comprises from about 30 to about 3000 amino acid residues. In a further embodiment, the random coil polypeptide domain comprises from about 60 to 1000 amino acid residues. In some embodiments, the random coil polypeptide domain comprises from about 60 to about 800, from about 60 to about 700, from about 60 to about 600, from about 60 to about 500, from about 60 to about 400, from about 60 to about 300, from about 60 to about 200, from about 60 to about 160, from about 60 to about 100, about 60, about 100, about 160, or about 200 amino acid residues. In a further embodiment, the random coil polypeptide domain comprises a PAS polypeptide, e.g., a PAS3 domain, a PAS5 domain, a PAS8 domain, or a PAS 10 domain.

[0349] 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 the 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, maximallyAtty. Docket No.: INMD-220 / 01 WO PCT Application3% 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, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Vai. In another embodiment, the other amino acids include one or more non-natural amino acids.

[0350] 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.Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0351] 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 1 A).

[0352] In one embodiment, a nucleotide sequence encoding the amino acid repeat of SEQ ID NO: 13 is one of the nucleotide sequences set forth in Table IB, i.e., one of SEQ ID NOs: 1415-1445. In a further embodiment, the nucleotide sequence is one of SEQ ID NOs: 1426-1445.

[0353] 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,Atty. Docket No.: INMD-220 / 01 WOPCT Application22, 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.

[0354] 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.

[0355] 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.

[0356] 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.

[0357] 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.

[0358] In one embodiment of the invention, the uricase conjugate comprises a PAS polypeptide in the random coil polypeptide domain, i.e., a PAS3 domain, comprising the amino acid sequence set forth in SEQ ID NO: 1411.

[0359] In one embodiment of the invention, the uricase conjugate comprises a PAS polypeptide in the random coil polypeptide domain, i .e., a PAS5 domain, comprising the amino acid sequence set forth in SEQ ID NO: 1412.

[0360] In one embodiment of the invention, the uricase conjugate comprises a PAS polypeptide in the random coil polypeptide domain, i.e., a PAS8 domain, comprising the amino acid sequence set forth in SEQ ID NO: 1413.Aty. Docket No.: INMD-220 / 01 WOPCT Application

[0361] In one embodiment of the invention, the uricase conjugate comprises a PAS polypeptide in the random coil polypeptide domain, i.e., a PAS10 domain, comprising the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO: 1414.

[0362] In one embodiment of the invention, the uricase conjugate comprises a PAS polypeptide in the random coil polypeptide domain, i.e., a PAS20 domain, comprising the amino acid sequence set forth in SEQ ID NO: 10.

[0363] In one embodiment of the invention, the uricase conjugate comprises a PAS polypeptide in the random coil polypeptide domain, i.e., a PAS30 domain, comprising the amino acid sequence set forth in SEQ ID NO: 11.

[0364] 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. In another embodiment, the uricase domain is chemically conjugated to the random coil polypeptide domain via one or more peptide bonds.

[0365] 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, a uricase domain amino acid sequence is located at the amino (N-) terminus of the fusion protein and an amino acid sequence of 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, an amino acid sequence of the uricase domain is located in / at the carboxy (C-) terminus and a random coil polypeptide domain amino acid sequence is located in / at the amino (N-) terminus of the fusion protein.

[0366] In yet another embodiment, an amino acid sequence of the random coil polypeptide domain is located at both the C-terminus and N-terminus of the fusion protein, and an amino acid sequence of the uricase domain is in between both random coil polypeptide domain amino acid sequences.Aty. Docket No.: INMD-220 / 01 WOPCT Application

[0367] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two XTEN polypeptide domain amino acid sequences.

[0368] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAT polypeptide domain amino acid sequences.

[0369] In some embodiments of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences. In one embodiment, the amino acid sequence of the PAS polypeptide domain at the N-terminus of the fusion protein and the amino acid sequence of the PAS polypeptide domain at the C-terminus of the fusion protein each independently comprise an amino acid sequence selected from the group consisting of an amino acid sequence of a PAS3 domain, an amino acid sequence of a PAS5 domain, an amino acid sequence of a PAS8 domain, an amino acid sequence of a PAS 10 domain, an amino acid sequence of a PAS20 domain, and an amino acid sequence of a PAS30 domain disclosed herein. In a further embodiment, the amino acid sequence of the PAS polypeptide domain at the N-terminus of the fusion protein and the amino acid sequence of the PAS polypeptide domain at the C-terminus of the fusion protein each independently comprise an amino acid sequence selected from the group consisting of an amino acid sequence of a PAS3 domain, an amino acid sequence of a PAS5 domain, an amino acid sequence of a PAS8 domain, and an amino acid sequence of a PAS 10 domain disclosed herein.

[0370] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein and the PAS polypeptide domain at the C-terminus of the fusion protein each being a PAS3 domain. In one embodiment, the PAS3 domain comprises the amino acid sequence of SEQ ID NO: 1411.

[0371] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS3 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS5 domain. In a further embodiment, the PAS3 domain comprises the amino acid sequence of SEQ ID NO: 1411, and the PAS5 domain comprises the amino acid sequence of SEQ ID NO: 1412.

[0372] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS3 domain and the PASAty. Docket No.: INMD-220 / 01 WOPCT Application polypeptide domain at the C-terminus of the fusion protein being a PAS8 domain. In a further embodiment, the PAS3 domain comprises the amino acid sequence of Sl'.Q ID NO: 1411, and the PAS8 domain comprises the amino acid sequence of SEQ ID NO: 1413.

[0373] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS3 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS 10 domain. In a further embodiment, the PAS3 domain comprises the amino acid sequence of SEQ ID NO: 1411, and the PAS10 domain comprises the amino acid sequence of SEQ ID NON or SEQ ID NO: 1414.

[0374] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS5 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS3 domain. In a further embodiment, the PAS5 domain comprises the amino acid sequence of SEQ ID NO: 1412, and the PAS3 domain comprises the amino acid sequence of SEQ ID NO: 1411.

[0375] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein and the PAS polypeptide domain at the C-terminus of the fusion protein each being a PAS5 domain. In one embodiment, the PAS5 domain comprises the amino acid sequence of SEQ ID NO: 1412.

[0376] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS5 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS8 domain. In a further embodiment, the PASS domain comprises the amino acid sequence of SEQ ID NO: 1412, and the PAS8 domain comprises the amino acid sequence of SEQ ID NO: 1413.

[0377] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PASS domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS 10 domain. In a further embodiment, the PASS domain comprises the amino acid sequence of SEQ ID NO: 1412, and the PAS10 domain comprises the amino acid sequence of SEQ ID NON or SEQ ID NO: 1414.Aty. Docket No.: INMD-220 / 01 WOPCT Application

[0378] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS8 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS3 domain. In a further embodiment, the PAS8 domain comprises the amino acid sequence of SEQ ID NO: 1413, and the PAS3 domain comprises the amino acid sequence of SEQ ID NO: 1411.

[0379] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS8 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS5 domain. In a further embodiment, the PAS8 domain comprises the amino acid sequence of SEQ ID NO: 1413, and the PAS5 domain comprises the amino acid sequence of SEQ ID NO: 1412.

[0380] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein and the PAS polypeptide domain at the C-terminus of the fusion protein each being a PAS8 domain. In one embodiment, the PAS8 domain comprises the amino acid sequence of SEQ ID NO: 1413.

[0381] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS8 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS 10 domain. In a further embodiment, the PASS domain comprises the amino acid sequence of SEQ ID NO: 1413, and the PAS10 domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 1414.

[0382] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS 10 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS3 domain. In a further embodiment, the PAS10 domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 1414, and the PAS3 domain comprises the amino acid sequence of SEQ ID NO: 1411.

[0383] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PASAtty. Docket No.: INMD-220 / 01 WOPCT Application polypeptide domain at the N-terminus of the fusion protein being a PAS 10 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS5 domain. In a further embodiment, the PAS10 domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 1414, and the PAS5 domain comprises the amino acid sequence of SEQ ID NO: 1412.

[0384] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein being a PAS 10 domain and the PAS polypeptide domain at the C-terminus of the fusion protein being a PAS8 domain. In a further embodiment, the PAS10 domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 1414, and the PAS8 domain comprises the amino acid sequence of SEQ ID NO:1413.

[0385] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS polypeptide domain amino acid sequences, with the PAS polypeptide domain at the N-terminus of the fusion protein and the PAS polypeptide domain at the C-terminus of the fusion protein each being a PAS 10 domain. In one embodiment, the PAS10 domain comprises the amino acid sequence of SEQ ID NO:9 or SEQ ID NO: 1414.

[0386] In one embodiment of the fusion protein, an amino acid sequence of the uricase domain is between two PAS 10 domain amino acid sequences (which, e.g., each comprise the amino acid sequence of SEQ ID NO: 9) located at the C-terminus and N-terminus of the fusion protein, respectively. In another embodiment of the fusion protein, an amino acid sequence of a PAS20 domain (which, e.g., comprises the amino acid sequence of SEQ ID NO: 10) is located at the N-terminus of the fusion protein, an amino acid sequence of a PAS 10 domain (which, e.g., comprises the amino acid sequence of SEQ ID NO:9) is located at the C-terminus of the fusion protein, and an amino acid sequence of the uricase domain is between the two PAS domain amino acid sequences.

[0387] 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, the fusion protein does not include an N-terminal Met residue, e.g., because it was removed after translation.Aty. Docket No.: INMD-220 / 01 WOPCT Application

[0388] 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.

[0389] 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 to about 5 amino acids long. In another embodiment, the amino acid spacer sequence is from about two amino acids 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.

[0390] 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.

[0391] 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 yet 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: I<DHLIHNVHI<EEHAHAHNI< (SEQ ID NO:8).Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0392] 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 variant polypeptides disclosed herein, and one or two random coil PAS polypeptide domains, XTEN polypeptide domains, PAT polypeptide domains, or PA polypeptide domains, are provided in FIGS. 1 A and IB. In one embodiment, the uricase fusion protein includes the uricase domain and one random coil domain selected from a PAS polypeptide domain, an XTEN polypeptide domain, a PAT polypeptide domain, or a PA polypeptide domain. In the uricase fusion protein, an amino acid sequence of the uricase domain may be C-terminal to an amino acid sequence of the one random coil PAS polypeptide domain, XTEN polypeptide domain, PAT polypeptide domain, or PA polypeptide domain, or may be N-terminal to an amino acid sequence of the one random coil PAS polypeptide domain, XTEN polypeptide domain, PAT polypeptide domain, or PA polypeptide domain. In another embodiment, the uricase fusion protein includes the uricase domain and two random coil PAS polypeptide domains, two random coil XTEN polypeptide domains, two random coil PAT polypeptide domains, or two random coil PA polypeptide domains. In the uricase fusion protein, an amino acid sequence of one of the two random coil PAS polypeptide, XTEN polypeptide, PAT polypeptide, or PA polypeptide domains is at the N-terminus and an amino acid sequence of the other is at the C-terminus, and an amino acid sequence of the uricase domain is between the amino acid sequences of the two random coil PAS polypeptide, XTEN polypeptide, PAT polypeptide, or PA polypeptide domains. In some embodiments, the spacer sequence Gly-Ser (GS) between the uricase domain and a C-terminal random coil domain, as depicted in FIGS. 1 A and IB, 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).

[0393] PAS3, PAS5, PAS8, PAS10, PAS20, and PAS30 represent random coil PAS polypeptide domains (i.e., PAS domains) respectively comprising 3, 5, 8, 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.

[0394] In one embodiment, PAS3, PAS5, PAS8, PAS10, PAS20, and PAS30 represent random coil PAS polypeptide domains (i.e., PAS domains) respectively comprising 3, 5, 8, 10,Aty. Docket No.: INMD-220 / 01 WOPCT Application20, and 30 tandem copies of the PAS sequence of SEQ ID NO: 13, thereby comprising the amino acid sequences as set forth in SEQ ID NO.T41 1, SEQ ID NO: 1412, SEQ ID NO: 1413, SEQ ID NO:9 or 1414, SEQ ID NO: 10, and SEQ ID NO: 11, respectively.

[0395] In one embodiment, the XTEN polypeptide domain of the uricase fusion protein comprises the amino acid sequence of SEQ ID NO: 12. In another embodiment, the XTEN polypeptide domain of the uricase fusion protein comprises XTEN216 having the amino acid sequence of SEQ ID NO:42. In another embodiment, the XTEN polypeptide domain of the uricase fusion protein comprises XTEN214 having the amino acid sequence of SEQ ID NO:43. In another embodiment, the XTEN polypeptide domain of the uricase fusion protein comprises XTEN144 having the amino acid sequence of SEQ ID NO:44.

[0396] PAT200, PAT300, and PAT400 represent random coil PAT polypeptide domains respectively comprising PAT polypeptides of 200, 400, and 600 amino acids long. Exemplary amino acid sequences of PAT200, PAT300, and PAT400 are set forth in SEQ ID NOs:27, 29, and 31, respectively. In a further embodiment, PAT200, PAT300, and PAT400 are encoded by the nucleotide sequences set forth in SEQ ID NOs:28, 30, and 32, respectively.

[0397] In one embodiment, the PA polypeptide domain of the uricase fusion protein comprises PA401 with the amino acid sequence of SEQ ID NO:25.

[0398] Exemplary PATylated uricase fusion proteins comprising the amino acid sequence of a uricase variant polypeptide disclosed herein for the uricase domain in combination with two PAT polypeptide domains according to the configurations of FIG. IB include those comprising an amino acid sequence selected from SEQ ID NOs: 1192-1407. Those PATylated uricase fusion proteins are described in detail in Example 4 of the present application.

[0399] 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 PAT polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the foregoing random coil polypeptide domains.

[0400] 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 coilAty. Docket No.: INMD-220 / 01 WOPCT ApplicationPAT polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the foregoing random coil polypeptide domains.

[0401] 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 PAT polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the foregoing random coil polypeptide domains.

[0402] 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 PAT polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the foregoing random coil polypeptide domains.

[0403] 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 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 PAT polypeptide domain, a random coil XTEN polypeptide domain, or a combination of the foregoing random coil polypeptide domains.

[0404] 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 PAT 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 constituentAtty. Docket No.: INMD-220 / 01 WOPCT Application 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 polypeptide 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 polypeptide described previously.

[0405] In another aspect, the present disclosure provides an isolated nucleic acid which encodes a uricase variant polypeptide of the disclosure. In one embodiment, the uricase variant polypeptide 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. 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., NatProtoc 2007, 2, 924-932, incorporated herein by reference in its entirety.Aty. Docket No.: INMD-220 / 01 WOPCT ApplicationThe 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.

[0406] In another aspect, the present disclosure provides a nucleic acid vector comprising a nucleic acid encoding a uricase variant polypeptide disclosed herein. In one embodiment, the uricase variant polypeptide 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 polypeptide 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 polypeptide 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 polypeptide-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 polypeptide 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 expression 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 P-D-l 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.

[0407] 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 polypeptide or a recombinant uricase fusion protein operably linked thereto. These methods may include in vitro recombinant DNA and syntheticAtty. Docket No.: INMD-220 / 01 WOPCT Application techniques and in vivo recombination (genetic recombination). Expression of nucleic acid sequence encoding a uricase variant polypeptide 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 polypeptide or recombinant uricase fusion protein is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a uricase variant polypeptide 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 polypeptide or the recombinant uricase fusion protein operatively linked to a heterologous promoter. Exemplary promoters which may be used to control uricase variant polypeptide 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 P-lactamase promoter, the tac promoter, and the osmoregulated osmB promoter.

[0408] 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 polypeptide of the invention. In one embodiment, the uricase variant polypeptide 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., glycosylation, cleavage) of the uricase variant polypeptide or recombinant uricase fusion protein. Suitable host cells include bacteria (e.g., E. colt), 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 polypeptide 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. ForAtty. Docket No.: INMD-220 / 01 WOPCT Application 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 polypeptide or recombinant uricase fusion protein, leading to respective protein production. In one embodiment, the nucleic acid encoding the uricase variant polypeptide 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.

[0409] 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 P-actin), cytomegalovirus (CMV), CMV enhancer / elongation factor (CEF), CMV early enhancer / chicken P-actin (CAG), hybrid CMV enhancer / chicken P-actin (CBh), elongation factor- la (EFl alpha), glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), or simian virus 40 (SV40) promoter.

[0410] 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 (GALI) promoter.

[0411] 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.

[0412] 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.

[0413] In another aspect, the present disclosure provides a method of recombinantly producing a uricase variant polypeptide 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 polypeptide or the uricase conjugate (e.g., the recombinant uricaseAty. Docket No.: INMD-220 / 01 WOPCT Application 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 polypeptide or the uricase conjugate (e.g., the recombinant uricase fusion protein) and recombinant production of the uricase variant polypeptide or the uricase conjugate (e.g., the recombinant uricase fusion protein) by the host cell; and (ii) isolating the recombinantly produced uricase variant polypeptide or uricase conjugate (e.g., recombinant uricase fusion protein).

[0414] The uricase variant polypeptides 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 variant polypeptides 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 variant polypeptides 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).

[0415] 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 the 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.

[0416] In one embodiment, the uricase variant polypeptide 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 polypeptide 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. ForAty. Docket No.: INMD-220 / 01 WOPCT Application example, the uricase variant polypeptide 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 polypeptide 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.

[0417] In another aspect, the present disclosure provides a pharmaceutical composition comprising a uricase variant polypeptide. In one embodiment, the uricase variant polypeptide 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 polypeptide 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.

[0418] In one embodiment, the pharmaceutical composition comprising the uricase variant polypeptide 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 polypeptide 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 uricaseAty. Docket No.: INMD-220 / 01 WOPCT Application variant polypeptide concentrations and pH. Solid formulations may be produced by lyophilization, spray drying, or drying by supercritical fluid technology, for example.

[0419] 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.

[0420] 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 polypeptide concentration. Sterile injectable solutions can be prepared by incorporating a uricase variant polypeptide 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 polypeptide 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.

[0421] In some embodiments, the pharmaceutical composition may be prepared with a carrier that protects the uricase variant polypeptide against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid.

[0422] In one embodiment, the pharmaceutical composition is a solution of a uricase variant polypeptide disclosed herein e.g., a phosphate buffered saline solution containing the uricase variant polypeptide. In a further embodiment, the solution is sterile and suitable for injection, e.g., intravenous injection or subcutaneous injection.Aty. Docket No.: INMD-220 / 01 WOPCT Application

[0423] 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 polypeptide disclosed herein. In one embodiment, the uricase variant polypeptide is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant polypeptide 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.

[0424] 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).

[0425] “Effective amount” means an amount of a pharmaceutical composition comprising a uricase variant polypeptide of the present disclosure that is sufficient to result in the desired therapeutic response. In one embodiment, the uricase variant polypeptide is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant polypeptide 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 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.

[0426] 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, aAtty. Docket No.: INMD-220 / 01 WOPCT Application 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.

[0427] In one embodiment of the method, the subject is a human patient. In a further embodiment, the human patient is an adult human patient.

[0428] 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.

[0429] 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.

[0430] 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.

[0431] 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. InAty. Docket No.: INMD-220 / 01 WOPCT Application another embodiment, refractory gout is associated with high rates of cardiovascular and renal comorbidities.

[0432] 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).

[0433] 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 polypeptide disclosed herein. In one embodiment, the uricase variant polypeptide is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant polypeptide 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.

[0434] 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.

[0435] 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 polypeptide disclosed herein. In one embodiment, the uricase variant polypeptide is present asAtty. Docket No.: INMD-220 / 01 WOPCT Application or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant polypeptide 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.

[0436] 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 nonHodgkin’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).

[0437] In another aspect, the present disclosure provides a method for metabolizing uric acid using a uricase variant polypeptide disclosed herein. In one embodiment, the uricase variant polypeptide is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant polypeptide 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.

[0438] In another aspect, the present disclosure provides use of a composition comprising a uricase variant polypeptide disclosed herein for reducing uric acid levels in a biological fluid. In one embodiment, the uricase variant polypeptide is present as or within a uricase tetramer, preferably a uricase homotetramer, as disclosed herein. In another embodiment, the uricase variant polypeptide 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 a 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.Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0439] Additional Embodiments

[0440] Embodiment 1. A uricase variant polypeptide, comprising (i) from 10 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119H, V119E, V119M and V119Q, wherein the uricase variant polypeptide 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.

[0441] Embodiment 2. The uricase variant polypeptide of embodiment 1, wherein (i) the from 10 to 18 amino acid substitutions are from 11 to 18 amino acid substitutions.

[0442] Embodiment 3. The uricase variant polypeptide of embodiment 1 , wherein (i) the from 10 to 18 amino acid substitutions are from 12 to 18 amino acid substitutions.

[0443] Embodiment 4. The uricase variant polypeptide of embodiment 1, wherein (i) the from 10 to 18 amino acid substitutions are from 13 to 18 amino acid substitutions.

[0444] Embodiment 5. The uricase variant polypeptide of embodiment 1 , wherein (i) the from 10 to 18 amino acid substitutions are from 14 to 18 amino acid substitutions.

[0445] Embodiment 6. The uricase variant polypeptide of embodiment 1, wherein (i) the from 10 to 18 amino acid substitutions are from 15 to 18 amino acid substitutions.

[0446] Embodiment 7. The uricase variant polypeptide of embodiment 1, wherein (i) the from 10 to 18 amino acid substitutions are from 16 to 18 amino acid substitutions.

[0447] Embodiment 8. The uricase variant polypeptide of embodiment 1, wherein (i) the from 10 to 18 amino acid substitutions are from 17 to 18 amino acid substitutions.

[0448] Embodiment 9. The uricase variant polypeptide of embodiment 1, wherein (i) the from 10 to 18 amino acid substitutions are 18 amino acid substitutions.

[0449] Embodiment 10. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationT83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, Fl 14L, N117D, and one ofV119H, V119E, V119M and V119Q.

[0450] Embodiment 11. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K and V86T, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

[0451] Embodiment 12. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, and V86E, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

[0452] Embodiment 13. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, one of N117D and N117H, and one of V119H, V119E, V119M and V119Q.

[0453] Embodiment 14. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of VI 19M and VI 19Q.

[0454] Embodiment 15. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, Fl 14L, and one of N117D and N117H.

[0455] Embodiment 16. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D,Aty. Docket No.: INMD-220 / 01 WO PCT ApplicationT83E, one of V86H, V86T, and V86E, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

[0456] Embodiment 17. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions comprise from one to six amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D and N117D.

[0457] Embodiment 18. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions comprise from one to four amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Y105W, F114L, one of N117D and N117H, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

[0458] Embodiment 19. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions are from two to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: ES80D, T83E, one of V86K, V86T, V86H and V86E; S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119M, V119Q, V119H and V119E.

[0459] Embodiment 20. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions are from three to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, V86H and V86E; S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119M, V119Q, V119H and V119E.

[0460] Embodiment 21. The uricase variant polypeptide of any one of embodiments 1-9, wherein (ii) the from one to nine amino acid substitutions are from four to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, V86H and V86E; S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119M, V119Q, V119H and V119E.Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0461] Embodiment 22. The uricase variant polypeptide of any one of embodiments 1-21, wherein (ii) the from one to nine amino acid substitutions comprise an amino acid substitution corresponding to N117D of SEQ ID NO: 1.

[0462] Embodiment 23. The uricase variant polypeptide of any one of embodiments 1-22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to S80D and K97D of SEQ ID NO: 1.

[0463] Embodiment 24. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to T83E and K97D of SEQ ID NO: 1.

[0464] Embodiment 25. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to T83E and S95G of SEQ ID NO: 1.

[0465] Embodiment 26. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D and a substitution selected from V86K, V86T, V86H or V86E.

[0466] Embodiment 27. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : K97D and a substitution selected from V86K, V86T, V86H or V86E.

[0467] Embodiment 28. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S95G and a substitution selected from V86K, V86T, V86H or V86E.

[0468] Embodiment 29. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86K and K97D.

[0469] Embodiment 30. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutionsAty. Docket No.: INMD-220 / 01 WO PCT Application corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, one of V86K, V86T, V86H and V86E, and K97D.

[0470] Embodiment 31. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, one of V86K, V86T, V86H and V86E, and K97D.

[0471] Embodiment 32. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Y105W and an amino acid substitution selected from VI 19M, VI 19Q or VI 19E.

[0472] Embodiment 33. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : N117H and an amino acid substitution selected from VI 19M, VI 19Q or VI 19E.

[0473] Embodiment 34. The uricase variant polypeptide of any one of embodiments 1- 22, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Y105W and one of VI 19M, VI 19Q, VI 19H and VI 19E.

[0474] Embodiment 35. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 8 amino acid substitutions.

[0475] Embodiment 36. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 7 amino acid substitutions.

[0476] Embodiment 37. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 6 amino acid substitutions.

[0477] Embodiment 38. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 5 amino acid substitutions.Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0478] Embodiment 39. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 4 amino acid substitutions.

[0479] Embodiment 40. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 4 amino acid substitutions.

[0480] Embodiment 41. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 5 amino acid substitutions.

[0481] Embodiment 42. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 6 amino acid substitutions.

[0482] Embodiment 43. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 6 amino acid substitutions.

[0483] Embodiment 44. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 8 amino acid substitutions.

[0484] Embodiment 45. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 7 amino acid substitutions.

[0485] Embodiment 46. The uricase variant polypeptide of any one of embodiments 1- 34, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 4 amino acid substitutions.

[0486] Embodiment 47. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0487] Embodiment 48. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ IDAtty. Docket No.: INMD-220 / 01 WOPCT ApplicationNO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0488] Embodiment 49. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0489] Embodiment 50. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86H, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0490] Embodiment 51. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0491] Embodiment 52. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86E, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0492] Embodiment 53. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0493] Embodiment 54. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, F114L, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0494] Embodiment 55. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117D, V119H, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0495] Embodiment 56. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117D, V119M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0496] Embodiment 57. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117D, V119Q, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0497] Embodiment 58. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106M, K116M, N117D, V119M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0498] Embodiment 59. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106M, K116M, N117D, V119Q, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0499] Embodiment 60. The uricase variant polypeptide of embodiment 1, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117H, V119E, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

[0500] Embodiment 61. A uricase variant polypeptide, comprising (i) from 12 to 20 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T, and N281Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, one of R101K and R101E, one of Y105A, Y105S, Y105G and Y105W, F114L, one of N117G and N117E, one of VI 19E, VI 19H and VI 19R, one of K120GandK120S, E138R, R192T, G196D, andR197D,Aty. Docket No.: INMD-220 / 01 WO PCT Application wherein the uricase variant polypeptide 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.

[0501] Embodiment 62. The uricase variant polypeptide of embodiment 61, wherein (i) the from 12 to 20 amino acid substitutions are from 13 to 20 amino acid substitutions.

[0502] Embodiment 63. The uricase variant polypeptide of embodiment 61, wherein (i) the from 12 to 20 amino acid substitutions are from 14 to 20 amino acid substitutions.

[0503] Embodiment 64. The uricase variant polypeptide of embodiment 61, wherein (i) the from 12 to 20 amino acid substitutions are from 15 to 20 amino acid substitutions.

[0504] Embodiment 65. The uricase variant polypeptide of embodiment 61, wherein (i) the from 12 to 20 amino acid substitutions are from 16 to 20 amino acid substitutions.

[0505] Embodiment 66. The uricase variant polypeptide of embodiment 61, wherein (i) the from 12 to 20 amino acid substitutions are from 17 to 20 amino acid substitutions.

[0506] Embodiment 67. The uricase variant polypeptide of embodiment 61, wherein (i) the from 12 to 20 amino acid substitutions are from 18 to 20 amino acid substitutions.

[0507] Embodiment 68. The uricase variant polypeptide of embodiment 61, wherein (i) the from 12 to 20 amino acid substitutions are from 19 to 20 amino acid substitutions.

[0508] Embodiment 69. The uricase variant polypeptide of embodiment 61, wherein (i) the from 12 to 20 amino acid substitutions are 20 amino acid substitutions.

[0509] Embodiment 70. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, one of V119E and V119H, R192T, G196D, and R197D.

[0510] Embodiment 71. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, one of R101K and R101E, one of Y105A, Y105S, Y105G and Y105W, F114L, one of N117G and N117E, one of V119E, V119H and V119R, one of K120G and K120S, E138R, G196D, and R197D.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0511] Embodiment 72. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, one of V119E and V119H, G196D, and R197D.

[0512] Embodiment 73. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, VI 19H, G196D, and R197D.

[0513] Embodiment 74. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, N117G, one of VI 19E and VI 19H, G196D, and R197D.

[0514] Embodiment 75. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, VI 19E, G196D, and R197D.

[0515] Embodiment 76. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E and I100N, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, one of V119E and V119H, G196D, and R197D.

[0516] Embodiment 77. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S,Aty. Docket No.: INMD-220 / 01 WO PCT ApplicationY105G and Y105W, one of N117G and N117E, one of V119E and V119H, G196D, and R197D.

[0517] Embodiment 78. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, V86T, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one ofN117G and N117E, one of VI 19E and VI 19H, G196D, and R197D.

[0518] Embodiment 79. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions comprise from one to seven amino acid substitutions selected from substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, T83E, V86T, one of I100E and I100N, one of Y105A, Y105S, Y105G and Y105W, N117G, G196D, and R197D.

[0519] Embodiment 80. The uricase variant polypeptide of any one of embodiments 61- 69, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 6 amino acid substitutions selected from substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, one of I100E, I100N and I100S, one of Y105A, Y105S, Y105W and Y105G, N117G and G196D.

[0520] Embodiment 81. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 8 amino acid substitutions.

[0521] Embodiment 82. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 8 amino acid substitutions.

[0522] Embodiment 83. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 8 amino acid substitutions.

[0523] Embodiment 84. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 4 to 8 amino acid substitutions.Aty. Docket No.: INMD-220 / 01 WOPCT Application

[0524] Embodiment 85. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 5 to 8 amino acid substitutions.

[0525] Embodiment 86. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 7 amino acid substitutions.

[0526] Embodiment 87. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 7 amino acid substitutions.

[0527] Embodiment 88. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 7 amino acid substitutions.

[0528] Embodiment 89. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 4 to 7 amino acid substitutions.

[0529] Embodiment 90. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 5 to 7 amino acid substitutions.

[0530] Embodiment 91. The uricase variant polypeptide of any one of embodiments 61- 80, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 6 amino acid substitutions.

[0531] Embodiment 92. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) I100E, I100N or I100S and (b) Y105G, Y105A, Y105S or Y105W.

[0532] Embodiment 93. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) R101K or R101E and (b) Y105G, Y105A, Y105S or Y105W.

[0533] Embodiment 94. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acidAtty. Docket No.: INMD-220 / 01 WO PCT Application substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) VI 19E, VI 19H or VI 19R and (b) K120G or K120S.

[0534] Embodiment 95. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) N117E or N117G and (b) VI 19E, VI 19H or VI 19R.

[0535] Embodiment 96. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) R101K or R101E, (b) Y105G, Y105A, Y105S or Y105W and (c) E138R.

[0536] Embodiment 97. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) S80D, (b) V86T or V86K, and (c) Y105A, Y105S, Y105G or Y105W.

[0537] Embodiment 98. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) S80D, (b) V86T or V86K, (c) I100E, I100N or I100S and (d) Y105A, Y105S, Y105G or Y105W.

[0538] Embodiment 99. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) S80D, (b) V86T or V86K, (c) R10 IK or R10 IE and (d) Y105A, Y105S, Y105G or Y105W.

[0539] Embodiment 100. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) S80D, (b) V86T or V86K, and (c) VI 19E, VI 19H or VI 19R.

[0540] Embodiment 101. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) V86T or V86K, (b) S95G, (c) N117G or N117E and (d) VI 19E, VI 19H or VI 19R.Atty. Docket No.: INMD-220 / 01 WO PCT Application

[0541] Embodiment 102. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) V86T or V86K, (b) I100E, I100N or I100S and (c) Y105A, Y105S or Y105G.

[0542] Embodiment 103. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) V86T or V86K, (b) S95G, (c) I100E, I100N or I100S and (d) Y105A, Y105S or Y105G.

[0543] Embodiment 104. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) S80D, (b) V86T or V86K, (c) I100E, I100N or I100S, (d) Y105A, Y105S or Y105G, and (e) R192T.

[0544] Embodiment 105. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) S80D, (b) V86T or V86K, (c) I100E, I100N or I100S, (d) Y105A, Y105S or Y105G, and (e) R197D.

[0545] Embodiment 106. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) V86T or V86K, (b) I100E, I100N or I100S, (c) Y105A, Y105S or Y105G, and (d) R197D.

[0546] Embodiment 107. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) S80D, (b) V86T or V86K, (c) Y105A, Y105S or Y105G, and (d) R197D.

[0547] Embodiment 108. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : 1100E and Y105G.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0548] Embodiment 109. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : 1100N and Y105 A.

[0549] Embodiment 110. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : I100S and Y105S.

[0550] Embodiment 111. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : R101K and Y105G.

[0551] Embodiment 112. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : N117E and V119H.

[0552] Embodiment 113. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : VI 19R and K120S.

[0553] Embodiment 114. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : R101E, Y105S and El 38R.

[0554] Embodiment 115. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86T and Y105W.

[0555] Embodiment 116. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acidAtty. Docket No.: INMD-220 / 01 WOPCT Application substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86T, R101K and Y105G.

[0556] Embodiment 117. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86T, IlOOE and Y105G.

[0557] Embodiment 118. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V86T, I100N and Y105A.

[0558] Embodiment 119. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V86T, I100S and Y105S.

[0559] Embodiment 120. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86T, I100E, Y105G and R192T.

[0560] Embodiment 121. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86T, I100E, Y105G and R197D.

[0561] Embodiment 122. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V86K, I100N, Y105A and R197D.

[0562] Embodiment 123. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86K, Y105W and R197D.Atty. Docket No.: INMD-220 / 01 WO PCT Application

[0563] Embodiment 124. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : N117G and G196D.

[0564] Embodiment 125. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, N117G and G196D.

[0565] Embodiment 126. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : N117E and G196D.

[0566] Embodiment 127. The uricase variant polypeptide of any one of embodiments 61- 91, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, N117E and G196D.

[0567] Embodiment 128. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0568] Embodiment 129. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, F114L, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0569] Embodiment 130. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117G, V119E, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.Aty. Docket No.: INMD-220 / 01 WOPCT Application

[0570] Embodiment 131. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117G, K120G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0571] Embodiment 132. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0572] Embodiment 133. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0573] Embodiment 134. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0574] Embodiment 135. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, R101K, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0575] Embodiment 136. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0576] Embodiment 137. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ IDAtty. Docket No.: INMD-220 / 01 WOPCT ApplicationNO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117G, V119R, K120S, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0577] Embodiment 138. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, R101E, A102V, Y105S, V106M, K116M, N117G, Y127H, E138R, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0578] Embodiment 139. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0579] Embodiment 140. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, R101K, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0580] Embodiment 141. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0581] Embodiment 142. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, A102V, V106M, K116M, N117G, V119E, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0582] Embodiment 143. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, K97D,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationA102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0583] Embodiment 144. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0584] Embodiment 145. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0585] Embodiment 146. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0586] Embodiment 147. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, KI 16M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

[0587] Embodiment 148. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0588] Embodiment 149. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0589] Embodiment 150. The uricase variant polypeptide of embodiment 61, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0590] Embodiment 151. A uricase variant polypeptide, comprising (i) from 10 to 15 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Y31H, L52T, N70H, K76S, A102V, KI 16M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T and N281Y, and (ii) from one to 12 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, one of Q26T and Q26E, L72T, one of F75L and F75T, G77E, S80D, T83E, one of V86K, V86T and V86H, one of S95G and S95E, K97D, one of I100E, I100N and I100S, one of R101E and R101Q, one of Y105G, Y105A, Y105W and Y105S, one of V106M and V106G, F114L, one of N117G and N117E, one of V119E, VI 19H, VI 19R and VI 19N, K120S, E138R, R192T, G196D and R197D, wherein the uricase variant polypeptide 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.

[0591] Embodiment 152. The uricase variant polypeptide of embodiment 151, wherein (i) the from 10 to 15 amino acid substitutions are from 11 to 15 amino acid substitutions.

[0592] Embodiment 153. The uricase variant polypeptide of embodiment 151, wherein (i) the from 10 to 15 amino acid substitutions are from 12 to 15 amino acid substitutions.

[0593] Embodiment 154. The uricase variant polypeptide of embodiment 151, wherein (i) the from 10 to 15 amino acid substitutions are from 13 to 15 amino acid substitutions.

[0594] Embodiment 155. The uricase variant polypeptide of embodiment 151, wherein (i) the from 10 to 15 amino acid substitutions are from 14 to 15 amino acid substitutions.

[0595] Embodiment 156. The uricase variant polypeptide of embodiment 151, wherein (i) the from 10 to 15 amino acid substitutions are 15 amino acid substitutions.

[0596] Embodiment 157. The uricase variant polypeptide of any one of embodiments 151- 156, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, Q26T, L72T, F75L, G77E, S80D, T83E, one of V86K andV86T, one of S95G and S95E,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationK97D, one of I100E, I100N and I100S, one of Y105G, Y105A, Y105W and Y105S, V106M, one of N117G and N117E, VI 19H, R192T, G196D and R197D.

[0597] Embodiment 158. The uricase variant polypeptide of any one of embodiments 151- 156, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, one of Q26T and Q26E, L72T, one of F75L and F75T, G77E, S80D, T83E, one of V86K, V86T and V86H, S95G, K97D, one of I100E, I100N and I100S, one of R101E and R101Q, one of Y105G, Y105A, Y105W and Y105S, one of V106M and V106G, F114L, N117G, one of VI 19E, VI 19R and VI 19N, K120S, E138R, G196D and R197D.

[0598] Embodiment 159. The uricase variant polypeptide of any one of embodiments 151- 156, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, Q26T, L72T, F75L, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100N and I100S, one of Y105G, Y105A, Y105W and Y105S, V106M, N117G, G196D and R197D.

[0599] Embodiment 160. The uricase variant polypeptide of any one of embodiments 151- 156, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Q26T, L72T, F75L, T83E, one of V86K and V86T, K97D, one of I100E and I100N, one of Y105G and Y105A, V106M, N117G, G196D and R197D.

[0600] Embodiment 161. The uricase variant polypeptide of any one of embodiments 151- 156, wherein (ii) the from one to 12 amino acid substitutions comprise from one to 11 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Q26T, F75L, T83E, one of V86K and V86T, K97D, I100E, Y105G, V106M, N117G, G196D and R197D.

[0601] Embodiment 162. The uricase variant polypeptide of any one of embodiments 151- 156, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : one of Q26T and Q26E, one of F75L and F75T, T83E, one of V86K, V86T and V86H, K97D, I100E, one of R101E and R101Q, one of Y105G, Y105W and Y105S, one of V106M and V106G, Fl 14L, N117G, one of VI 19E, VI 19R and VI 19N, K120S, E138R, G196D and R197D.Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0602] Embodiment 163. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 2 to 12 amino acid substitutions.

[0603] Embodiment 164. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 3 to 12 amino acid substitutions.

[0604] Embodiment 165. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 4 to 12 amino acid substitutions.

[0605] Embodiment 166. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 5 to 12 amino acid substitutions.

[0606] Embodiment 167. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 6 to 12 amino acid substitutions.

[0607] Embodiment 168. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 7 to 12 amino acid substitutions.

[0608] Embodiment 169. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 8 to 12 amino acid substitutions.

[0609] Embodiment 170. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 9 to 12 amino acid substitutions.

[0610] Embodiment 171. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 1 to 11 amino acid substitutions.

[0611] Embodiment 172. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 2 to 11 amino acid substitutions.Aty. Docket No.: INMD-220 / 01 WO PCT Application

[0612] Embodiment 173. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 3 to 11 amino acid substitutions.

[0613] Embodiment 174. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 4 to 11 amino acid substitutions.

[0614] Embodiment 175. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 5 to 11 amino acid substitutions.

[0615] Embodiment 176. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 6 to 11 amino acid substitutions.

[0616] Embodiment 177. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 1 to 10 amino acid substitutions.

[0617] Embodiment 178. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 2 to 10 amino acid substitutions.

[0618] Embodiment 179. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 3 to 10 amino acid substitutions.

[0619] Embodiment 180. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 4 to 10 amino acid substitutions.

[0620] Embodiment 181. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 5 to 10 amino acid substitutions.

[0621] Embodiment 182. The uricase variant polypeptide of any one of embodiments 151- 162, wherein (ii) the from one to 12 amino acid substitutions comprise from 6 to 10 amino acid substitutions.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0622] Embodiment 183. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0623] Embodiment 184. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, F114L, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0624] Embodiment 185. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117G, V119E, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0625] Embodiment 186. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117G, V119N, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0626] Embodiment 187. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0627] Embodiment 188. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0628] Embodiment 189. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ IDAtty. Docket No.: INMD-220 / 01 WO PCT ApplicationNO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0629] Embodiment 190. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, R101Q, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0630] Embodiment 191. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106G, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0631] Embodiment 192. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0632] Embodiment 193. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117G, V119R, K120S, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0633] Embodiment 194. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, R101E, A102V, Y105G, V106M, K116M, N117G, Y127H, E138R, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0634] Embodiment 195. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, A102V, V106M,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationK116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0635] Embodiment 196. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86H, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0636] Embodiment 197. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0637] Embodiment 198. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0638] Embodiment 199. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95E, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0639] Embodiment 200. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0640] Embodiment 201. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26E, Y31H, L52T, N70H, F75T, K76S, T83E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application

[0641] Embodiment 202. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0642] Embodiment 203. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, A102V, V106M, KI 16M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

[0643] Embodiment 204. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0644] Embodiment 205. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

[0645] Embodiment 206. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0646] Embodiment 207. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0647] Embodiment 208. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationY105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

[0648] Embodiment 209. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0649] Embodiment 210. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0650] Embodiment 211. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0651] Embodiment 212. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0652] Embodiment 213. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0653] Embodiment 214. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WO PCT Application

[0654] Embodiment 215. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0655] Embodiment 216. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0656] Embodiment 217. The uricase variant polypeptide of embodiment 151, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

[0657] Embodiment 218. A uricase variant polypeptide, comprising (i) from 12 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T and N281Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105W, Y105S, Y105G and Y105A, one of N117G and N117E, VI 19H, R192T and R197D, wherein the uricase variant polypeptide 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.

[0658] Embodiment 219. The uricase variant polypeptide of embodiment 218, wherein (i) the from 12 to 18 amino acid substitutions are from 13 to 18 amino acid substitutions.

[0659] Embodiment 220. The uricase variant polypeptide of embodiment 218, wherein (i) the from 12 to 18 amino acid substitutions are from 14 to 18 amino acid substitutions.

[0660] Embodiment 221. The uricase variant polypeptide of embodiment 218, wherein (i) the from 12 to 18 amino acid substitutions are from 15...

Claims

1. Atty. Docket No.: INMD-220 / 01 WO PCT ApplicationCLAIMS1. A uricase variant polypeptide, comprising (i) from 10 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, KI 16M, Y127H, K155G, F172W, R192T, R209W, Y225H, one of I257K and I257R, and N281Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of V119H, V119E, V119M and V119Q, wherein the uricase variant polypeptide 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 polypeptide of claim 1, comprising (i) from 10 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K, and N281Y.

3. The uricase variant polypeptide of claim 1, comprising (i) from 10 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257R, and N281Y.

4. The uricase variant polypeptide of any one of claims 1-3, wherein (i) the from 10 to 18 amino acid substitutions are from 11 to 18 amino acid substitutions.

5. The uricase variant polypeptide of any one of claims 1-3, wherein (i) the from 10 to 18 amino acid substitutions are from 12 to 18 amino acid substitutions.

6. The uricase variant polypeptide of any one of claims 1-3, wherein (i) the from 10 to 18 amino acid substitutions are from 13 to 18 amino acid substitutions.

7. The uricase variant polypeptide of any one of claims 1-3, wherein (i) the from 10 to 18 amino acid substitutions are from 14 to 18 amino acid substitutions.

8. The uricase variant polypeptide of any one of claims 1-3, wherein (i) the from 10 to 18 amino acid substitutions are from 15 to 18 amino acid substitutions.

9. The uricase variant polypeptide of any one of claims 1-3, wherein (i) the from 10 to 18 amino acid substitutions are from 16 to 18 amino acid substitutions.Atty. Docket No.: INMD-220 / 01 WOPCT Application10. The uricase variant polypeptide of any one of claims 1-3, wherein (i) the from 10 to 18 amino acid substitutions are from 17 to 18 amino acid substitutions.

11. The uricase variant polypeptide of any one of claims 1-3, wherein (i) the from 10 to 18 amino acid substitutions are 18 amino acid substitutions.

12. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, F114L, N117D, and one of V119H, V119E, V119M and V119Q.

13. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K and V86T, S95G, K97D, Y105W, F114L, one ofN117D and N117H, and one of V119H, V119E, V119M and V119Q.

14. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, and V86E, S95G, K97D, Y105W, F114L, one ofN117D and N117H, and one of V119H, V119E, V119M and V119Q.

15. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, one of N117D and N117H, and one of VI 19H, VI 19E, VI 19M and VI 19Q.

16. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, Fl 14L, one of N117D and N117H, and one of VI 19M and V119Q.

17. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selectedAtty. Docket No.: INMD-220 / 01 WO PCT Application from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D, Y105W, Fl 14L, and one of N117D and N117H.

18. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86T, and V86E, S95G, K97D, Y105W, F114L, one ofN117D and N117H, and one of V119H, V119E, V119M and V119Q.

19. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions comprise from one to six amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86H, V86K, V86T, and V86E, S95G, K97D and N117D.

20. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions comprise from one to four amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Y105W, F114L, one of N117D and N117H, and one of V119H, V119E, V119M and V119Q.

21. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are from two to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: ES80D, T83E, one of V86K, V86T, V86H and V86E; S95G, K97D, Y105W, Fl 14L, one of N117D and N117H, and one of VI 19M, VI 19Q, VI 19H and VI 19E.

22. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are from three to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, V86H and V86E; S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of VI 19M, VI 19Q, VI 19H and VI 19E.

23. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are from four to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86K, V86T, V86H and V86E; S95G, K97D, Y105W, F114L, one of N117D and N117H, and one of VI 19M, VI 19Q, VI 19H and VI 19E.Atty. Docket No.: INMD-220 / 01 WO PCT Application24. The uricase variant polypeptide of any one of claims 1-23, wherein (ii) the from one to nine amino acid substitutions comprise an amino acid substitution corresponding to N117D of SEQ ID NO: 1.

25. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to S80D and K97D of SEQ ID NO: 1.

26. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to T83E and K97D of SEQ ID NO: 1.

27. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to T83E and S95G of SEQ ID NO: 1.

28. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D and a substitution selected from V86K, V86T, V86H or V86E.

29. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : K97D and a substitution selected from V86K, V86T, V86H or V86E.

30. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S95G and a substitution selected from V86K, V86T, V86H or V86E.

31. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86K and K97D.

32. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, one of V86K, V86T, V86H and V86E, and K97D.Aty. Docket No.: INMD-220 / 01 WOPCT Application33. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, one of V86K, V86T, V86H and V86E, and K97D.

34. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Y105W and an amino acid substitution selected from VI 19M, VI 19Q or VI 19E.

35. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : N117H and an amino acid substitution selected from VI 19M, VI 19Q or VI 19E.

36. The uricase variant polypeptide of any one of claims 1-24, wherein (ii) the from one to nine amino acid substitutions comprise amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Y105W and one of V119M, V119Q, V119H and V119E.

37. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 8 amino acid substitutions.

38. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 7 amino acid substitutions.

39. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 6 amino acid substitutions.

40. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 5 amino acid substitutions.

41. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 4 amino acid substitutions.

42. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 4 amino acid substitutions.

43. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 5 amino acid substitutions.Atty. Docket No.: INMD-220 / 01 WOPCT Application44. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 6 amino acid substitutions.

45. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 6 amino acid substitutions.

46. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 8 amino acid substitutions.

47. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 7 amino acid substitutions.

48. The uricase variant polypeptide of any one of claims 1-36, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 4 amino acid substitutions.

49. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, K97D, and N117D.

50. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are 3 or 4 amino acid substitutions, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, K97D, and Ni l 7D.

51. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are 4, 5, 6, 7, 8, or 9 amino acid substitutions, comprising amino acid substitutions corresponding to one of the following groups of amino acid substitutions of SEQ ID NO:1: (a) S80D, T83E, K97D, and N117D; (b) T83E, V86T, K97D, and N117D; (c) T83E, V86K, K97D, and N117D; (d) T83E, K97D, Y105W, and N117D; (e) T83E, K97D, N117D, and VI 19M; (f) T83E, K97D, N117D, and VI 19Q.

52. The uricase variant polypeptide of any one of claims 1-11, wherein (ii) the from one to nine amino acid substitutions are 4 amino acid substitutions corresponding to one of the following groups of amino acid substitutions of SEQ ID NO:1: (a) S80D, T83E, K97D, and N117D; (b) T83E, V86T, K97D, and N117D; (c) T83E, V86K, K97D, and N117D; (d) T83E, K97D, Y105W, and N117D; (e) T83E, K97D, N117D, and VI 19M; (f) T83E, K97D, N117D, and V119Q.Atty. Docket No.: INMD-220 / 01 WOPCT Application53. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

54. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

55. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

56. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86H, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

57. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

58. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86E, K97D, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

59. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, A102V, V106M, K116M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

60. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H,Aty. Docket No.: INMD-220 / 01 WOPCT ApplicationL52T, N70H, F75L, K76S, T83E, A102V, V106M, Fl 14L, KI 16M, N117D, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

61. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, KI 16M, N117D, VI 19H, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

62. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, KI 16M, N117D, VI 19M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

63. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, KI 16M, N117D, VI 19Q, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

64. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106M, K116M, N117D, V119M, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

65. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106M, KI 16M, N117D, VI 19Q, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

66. The uricase variant polypeptide of claim 1 or 2, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, KI 16M, N117H, VI 19E, Y127H, K155G, F172W, R192T, R209W, Y225H, I257K and N281Y.

67. A uricase variant polypeptide, comprising (i) from 12 to 20 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO:1: V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, one of I257K and I257R, L279T, and N281Y, and (ii) from one to nine amino acid substitutions selected from amino acidAty. Docket No.: INMD-220 / 01 WO PCT Application substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, one of R101K and R101E, one of Y105A, Y105S, Y105G and Y105W, F114L, one of N117G and N117E, one of VI 19E, VI 19H and VI 19R, one of K120GandK120S, E138R, R192T, G196D, andR197D, wherein the uricase variant polypeptide 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.

68. The uricase variant polypeptide of claim 67, comprising (i) from 12 to 20 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T, and N281Y.

69. The uricase variant polypeptide of claim 67, comprising (i) from 12 to 20 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, K97D, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T, and N281Y.

70. The uricase variant polypeptide of any one of claims 67-69, wherein (i) the from 12 to 20 amino acid substitutions are from 13 to 20 amino acid substitutions.

71. The uricase variant polypeptide of any one of claims 67-69, wherein (i) the from 12 to 20 amino acid substitutions are from 14 to 20 amino acid substitutions.

72. The uricase variant polypeptide of any one of claims 67-69, wherein (i) the from 12 to 20 amino acid substitutions are from 15 to 20 amino acid substitutions.

73. The uricase variant polypeptide of any one of claims 67-69, wherein (i) the from 12 to 20 amino acid substitutions are from 16 to 20 amino acid substitutions.

74. The uricase variant polypeptide of any one of claims 67-69, wherein (i) the from 12 to 20 amino acid substitutions are from 17 to 20 amino acid substitutions.

75. The uricase variant polypeptide of any one of claims 67-69, wherein (i) the from 12 to 20 amino acid substitutions are from 18 to 20 amino acid substitutions.

76. The uricase variant polypeptide of any one of claims 67-69, wherein (i) the from 12 to 20 amino acid substitutions are from 19 to 20 amino acid substitutions.Aty. Docket No.: INMD-220 / 01 WO PCT Application77. The uricase variant polypeptide of any one of claims 67-69, wherein (i) the from 12 to 20 amino acid substitutions are 20 amino acid substitutions.

78. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one ofN117G and N117E, one of VI 19E and VI 19H, R192T, G196D, and R197D.

79. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, one of R101K and R101E, one of Y105A, Y105S, Y105G and Y105W, Fl 14L, one of N117G and N117E, one of VI 19E, VI 19H and VI 19R, one of K120G and K120S, E138R, G196D, and R197D.

80. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, one of Y105A, Y105S, Y105G and Y105W, one of N117G and Ni l 7E, one of VI 19E and VI 19H, G196D, and R197D.

81. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, VI 19H, G196D, and R197D.

82. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, N117G, one of VI 19E and VI 19H, G196D, and R197D.

83. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K,Atty. Docket No.: INMD-220 / 01 WO PCT ApplicationS95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and N117E, VI 19E, G196D, and R197D.

84. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, S95G, one of I100E and I100N, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and Ni l 7E, one of VI 19E and VI 19H, G196D, and R197D.

85. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and Ni l 7E, one of VI 19E and VI 19H, G196D, and R197D.

86. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, V86T, S95G, one of I100E, I100N and I100S, R101K, one of Y105A, Y105S, Y105G and Y105W, one of N117G and Ni l 7E, one of VI 19E and VI 19H, G196D, and R197D.

87. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions comprise from one to seven amino acid substitutions selected from substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, V86T, one of I100E and I100N, one of Y105A, Y105S, Y105G and Y105W, N117G, G196D, and R197D.

88. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 6 amino acid substitutions selected from substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, T83E, one of V86T and V86K, one of I100E, I100N and I100S, one of Y105A, Y105S, Y105W and Y105G, N117G and G196D.

89. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 8 amino acid substitutions.

90. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 8 amino acid substitutions.Aty. Docket No.: INMD-220 / 01 WOPCT Application91. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 8 amino acid substitutions.

92. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 4 to 8 amino acid substitutions.

93. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 5 to 8 amino acid substitutions.

94. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 7 amino acid substitutions.

95. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 7 amino acid substitutions.

96. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 7 amino acid substitutions.

97. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 4 to 7 amino acid substitutions.

98. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 5 to 7 amino acid substitutions.

99. The uricase variant polypeptide of any one of claims 67-88, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 6 amino acid substitutions.

100. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) I100E, I100N or I100S and (b) Y105G, Y105A, Y105S or Y105W.

101. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) R101K or R101E and (b) Y105G, Y105A, Y105S or Y105W.

102. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) V119E, V119H or V119R and (b) K120G or K120S.Atty. Docket No.: INMD-220 / 01 WO PCT Application103. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) N117E or N117G and (b) V119E, V119H or V119R.

104. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ IDNO:1: (a) R101K orRlOlE, (b) Y105G, Y105A, Y105S or Y105W and (c) E138R.

105. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) S80D, (b) V86T or V86K, and (c) Y105A, Y105S, Y105G or Y105W.

106. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) S80D, (b) V86T or V86K, (c) I100E, I100N or I100S and (d) Y105A, Y105S, Y105G or Y105W.

107. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) S80D, (b) V86T or V86K, (c) R101K or R101E and (d) Y105A, Y105S, Y105G or Y105W.

108. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) S80D, (b) V86T or V86K, and (c) V119E, V119H or V119R.

109. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) V86T or V86K, (b) S95G, (c) N117G or N117E and (d) VI 19E, VI 19H or VI 19R.

110. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to theAtty. Docket No.: INMD-220 / 01 WO PCT Application following amino acid substitutions of SEQ ID NO:1: (a) V86T or V86K, (b) I100E, I100N or I100S and (c) Y105A, Y105S or Y105G.

111. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) V86T or V86K, (b) S95G, (c) I100E, I100N or I100S and (d) Y105A, Y105S or Y105G.

112. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) S80D, (b) V86T or V86K, (c) I100E, I100N or I100S, (d) Y105A, Y105S or Y105G, and (e) R192T.

113. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) S80D, (b) V86T or V86K, (c) I100E, I100N or I100S, (d) Y105A, Y105S or Y105G, and (e) R197D.

114. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: (a) V86T or V86K, (b) I100E, I100N or I100S, (c) Y105A, Y105S or Y105G, and (d) R197D.

115. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : (a) S80D, (b) V86T or V86K, (c) Y105A, Y105S or Y105G, and (d) R197D.

116. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : 1100E and Y105G.

117. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: 1100N and Y105A.

118. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: 1100S and Y105S.Aty. Docket No.: INMD-220 / 01 WOPCT Application119. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: R101K and Y105G.

120. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : N117E and VI 19H.

121. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : VI 19R and K120S.

122. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: R101E, Y105S and E138R.

123. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, V86T and Y105W.

124. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, V86T, R101K and Y105G.

125. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, V86T, I100E and Y105G.

126. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V86T, I100N and Y105A.

127. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: V86T, I100S and Y105S.

128. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, V86T, I100E, Y105G and R192T.Atty. Docket No.: INMD-220 / 01 WO PCT Application129. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86T, I100E, Y105G and R197D.

130. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V86K, I100N, Y105A and R197D.

131. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86K, Y105W and R197D.

132. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : N117G and G196D.

133. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, N117G and G196D.

134. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : N117E and G196D.

135. The uricase variant polypeptide of any one of claims 67-99, wherein (ii) the from one to nine amino acid substitutions comprise the amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, N117E and G196D.

136. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are 4, 5, 6, or 7 amino acid substitutions, comprising amino acid substitutions corresponding to one of the groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table B2.

137. The uricase variant polypeptide of any one of claims 67-77, wherein (ii) the from one to nine amino acid substitutions are 5, 6, or 7 amino acid substitutions, comprising amino acid substitutions corresponding to one of the groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table B3.

138. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E,Atty. Docket No.: INMD-220 / 01 WO PCT ApplicationY31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

139. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, F114L, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

140. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117G, V119E, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

141. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117G, K120G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

142. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

143. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

144. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

145. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, R101K, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application146. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

147. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, A102V, V106M, K116M, N117G, V119R, K120S, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

148. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, K97D, R101E, A102V, Y105S, V106M, K116M, N117G, Y127H, E138R, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

149. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

150. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, R101K, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

151. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

152. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, A102V, V106M, K116M, N117G, V119E, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application153. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

154. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

155. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

156. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

157. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

158. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

159. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationY31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

160. The uricase variant polypeptide of claim 67 or 68, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

161. The uricase variant polypeptide of claim 67 or 69, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26R, K30E, Y31H, L52T, N70H, F75L, K76S, S80D, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257R, L279T and N281Y.

162. A uricase variant polypeptide, comprising (i) from 10 to 15 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, one of I257K and I257R, L279T and N281 Y, and (ii) from one to 12 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, one of Q26T and Q26E, L72T, one of F75L and F75T, G77E, S80D, T83E, one of V86K, V86T and V86H, one of S95G and S95E, K97D, one of I100E, I100N and I100S, one of R101E and R101Q, one of Y105G, Y105A, Y105W and Y105S, one of V106M and V106G, F114L, one of N117G and N117E, one of V119E, VI 19H, VI 19R and VI 19N, K120S, E138R, R192T, G196D and R197D, wherein the uricase variant polypeptide 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.

163. The uricase variant polypeptide of claim 162, comprising (i) from 10 to 15 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T and N281Y.

164. The uricase variant polypeptide of claim 162, comprising (i) from 10 to 15 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Y31H, L52T, N70H, K76S, A102V, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y.Aty. Docket No.: INMD-220 / 01 WOPCT Application165. The uricase variant polypeptide of any one of claims 162-164, wherein (i) the from 10 to 15 amino acid substitutions are from 11 to 15 amino acid substitutions.

166. The uricase variant polypeptide of any one of claims 162-164, wherein (i) the from 10 to 15 amino acid substitutions are from 12 to 15 amino acid substitutions.

167. The uricase variant polypeptide of any one of claims 162-164, wherein (i) the from 10 to 15 amino acid substitutions are from 13 to 15 amino acid substitutions.

168. The uricase variant polypeptide of any one of claims 162-164, wherein (i) the from 10 to 15 amino acid substitutions are from 14 to 15 amino acid substitutions.

169. The uricase variant polypeptide of any one of claims 162-164, wherein (i) the from 10 to 15 amino acid substitutions are 15 amino acid substitutions.

170. The uricase variant polypeptide of any one of claims 162-169, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, Q26T, L72T, F75L, G77E, S80D, T83E, one of V86K andV86T, one of S95G and S95E, K97D, one of I100E, I100N and I100S, one of Y105G, Y105A, Y105W and Y105S, V106M, one of N117G and N117E, V119H, R192T, G196D and R197D.

171. The uricase variant polypeptide of any one of claims 162-169, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, one of Q26T and Q26E, L72T, one of F75L and F75T, G77E, S80D, T83E, one of V86K, V86T and V86H, S95G, K97D, one of I100E, I100N and I100S, one of R101E and R101Q, one of Y105G, Y105A, Y105W and Y105S, one of V106M and V106G, F114L, N117G, one of V119E, V119R and V119N, K120S, E138R, G196D and R197D.

172. The uricase variant polypeptide of any one of claims 162-169, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, Q26T, L72T, F75L, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100N and I100S, one of Y105G, Y105A, Y105W and Y105S, V106M, N117G, G196D and R197D.

173. The uricase variant polypeptide of any one of claims 162-169, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : Q26T, L72T, F75L, T83E, one of V86KAty. Docket No.: INMD-220 / 01 WO PCT Application and V86T, K97D, one of I100E and I100N, one of Y105G and Y105A, V106M, N117G, G196D and R197D.

174. The uricase variant polypeptide of any one of claims 162-169, wherein (ii) the from one to 12 amino acid substitutions comprise from one to 11 amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: Q26T, F75L, T83E, one of V86K and V86T, K97D, I100E, Y105G, V106M, N117G, G196D and R197D.

175. The uricase variant polypeptide of any one of claims 162-169, wherein (ii) the from one to 12 amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : one of Q26T and Q26E, one of F75L and F75T, T83E, one of V86K, V86T and V86H, K97D, I100E, one of R101E and R101Q, one of Y105G, Y105W and Y105S, one of V106M and V106G, F114L, N117G, one of V119E, VI 19R and VI 19N, K120S, E138R, G196D and R197D.

176. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 2 to 12 amino acid substitutions.

177. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 3 to 12 amino acid substitutions.

178. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 4 to 12 amino acid substitutions.

179. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 5 to 12 amino acid substitutions.

180. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 6 to 12 amino acid substitutions.

181. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 7 to 12 amino acid substitutions.

182. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 8 to 12 amino acid substitutions.

183. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 9 to 12 amino acid substitutions.Aty. Docket No.: INMD-220 / 01 WO PCT Application184. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 1 to 11 amino acid substitutions.

185. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 2 to 11 amino acid substitutions.

186. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 3 to 11 amino acid substitutions.

187. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 4 to 11 amino acid substitutions.

188. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 5 to 11 amino acid substitutions.

189. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 6 to 11 amino acid substitutions.

190. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 1 to 10 amino acid substitutions.

191. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 2 to 10 amino acid substitutions.

192. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 3 to 10 amino acid substitutions.

193. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 4 to 10 amino acid substitutions.

194. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 5 to 10 amino acid substitutions.

195. The uricase variant polypeptide of any one of claims 162-175, wherein (ii) the from one to 12 amino acid substitutions comprise from 6 to 10 amino acid substitutions.

196. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

197. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationQ26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, F114L, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

198. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117G, V119E, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

199. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117G, V119N, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

200. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

201. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

202. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

203. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, R101Q, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

204. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, Y105W, V106G, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WOPCT Application205. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

206. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, A102V, V106M, K116M, N117G, V119R, K120S, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

207. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, R101E, A102V, Y105G, V106M, K116M, N117G, Y127H, E138R, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

208. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

209. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86H, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

210. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

211. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

212. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationQ26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95E, K97D, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

213. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

214. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26E, Y31H, L52T, N70H, F75T, K76S, T83E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

215. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

216. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, A102V, V106M, K116M, N117G, Y127H, K155G, F172W, G196D, R209W, S228G, I257K, L279T and N281Y.

217. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

218. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

219. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationQ26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

220. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

221. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

222. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

223. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

224. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

225. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100S, A102V, Y105S,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationV106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

226. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

227. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

228. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

229. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

230. The uricase variant polypeptide of claim 162 or 163, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

231. The uricase variant polypeptide of claim 162 or 164, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WO PCT Application232. The uricase variant polypeptide of claim 162 or 164, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

233. A uricase variant polypeptide, comprising (i) from 12 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO:1: V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, one of I257K and I257R, L279T and N281Y, and (ii) from one to nine amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105W, Y105S, Y105G and Y105A, one of N117G and N117E, VI 19H, R192T and R197D, wherein the uricase variant polypeptide 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.

234. The uricase variant polypeptide of claim 233, comprising (i) from 12 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257K, L279T and N281Y.

235. The uricase variant polypeptide of claim 233, comprising (i) from 12 to 18 amino acid substitutions selected from amino acid substitutions corresponding to the following amino substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, A102V, V106M, K116M, Y127H, K155G, F172W, R209W, S228G, I257R, L279T and N281Y.

236. The uricase variant polypeptide of any one of claims 233-235, wherein (i) the from 12 to 18 amino acid substitutions are from 13 to 18 amino acid substitutions.

237. The uricase variant polypeptide of any one of claims 233-235, wherein (i) the from 12 to 18 amino acid substitutions are from 14 to 18 amino acid substitutions.

238. The uricase variant polypeptide of any one of claims 233-235, wherein (i) the from 12 to 18 amino acid substitutions are from 15 to 18 amino acid substitutions.

239. The uricase variant polypeptide of any one of claims 233-235, wherein (i) the from 12 to 18 amino acid substitutions are from 16 to 18 amino acid substitutions.Aty. Docket No.: INMD-220 / 01 WO PCT Application240. The uricase variant polypeptide of any one of claims 233-235, wherein (i) the from 12 to 18 amino acid substitutions are from 17 to 18 amino acid substitutions.

241. The uricase variant polypeptide of any one of claims 233-235, wherein (i) the from 12 to 18 amino acid substitutions are 18 amino acid substitutions.

242. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105S, Y105G and Y105A, N117G, R192T and R197D.

243. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E and I100N, one of Y105S, Y105G and Y105A, N117G, R192T and R197D.

244. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, L72T, G77E, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E and I100N, one of Y105S, Y105G and Y105A, N117G, and R197D.

245. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E and I100N, one of Y105S, Y105G and Y105A, N117G, and R197D.

246. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, S80D, T83E, one of V86K and V86T, S95G, K97D, one of I100E and I100N, one of Y105G and Y105A, N117G, and R197D.

247. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding toAty. Docket No.: INMD-220 / 01 WO PCT Application the following amino acid substitutions of SEQ ID NO: 1 : L72T, S80D, T83E, one of V86K and V86T, K97D, one of I100E and I100N, one of Y105G and Y105A, N117G, and R197D.

248. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, one of N117G and N117E, and R197D.

249. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : T83E, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

250. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, one of N117G and N117E, and R197D.

251. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : S80D, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

252. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, one of N117G and N117E, and R197D.

253. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, T83E, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

254. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding toAtty. Docket No.: INMD-220 / 01 WOPCT Application the following amino acid substitutions of SEQ ID NO: 1 : H24R, G77E, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, one of N117G and N117E, and R197D.

255. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : H24R, G77E, T83E, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

256. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, T83E, one of V86K and V86T, S95G, K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, N117G, and R197D.

257. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, T83E, V86K, S95G, K97D, I100E, Y105G, N117G, and R197D.

258. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : L72T, T83E, V86T, S95G, K97D, I100E, Y105G, N117G, and R197D.

259. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions comprise from one to seven amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, one of V86K and V86T, one of S95G and K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, R192T, and R197D.

260. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions comprise from one to eight amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: S80D, T83E, one of V86K and V86T, one of S95G and K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, R192T, and R197D.Atty. Docket No.: INMD-220 / 01 WO PCT Application261. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions comprise from one to seven amino acid substitutions selected from amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO:1: L72T, one of V86K and V86T, one of S95G and K97D, one of I100E, I100S and I100N, one of Y105G, Y105A, Y105W and Y105S, R192T, and R197D.

262. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 9 amino acid substitutions.

263. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 9 amino acid substitutions.

264. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 4 to 9 amino acid substitutions.

265. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 5 to 9 amino acid substitutions.

266. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 6 to 9 amino acid substitutions.

267. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 7 to 9 amino acid substitutions.

268. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 6 to 8 amino acid substitutions.

269. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 8 amino acid substitutions.

270. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 7 amino acid substitutions.

271. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 6 amino acid substitutions.

272. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 1 to 5 amino acid substitutions.

273. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 2 to 9 amino acid substitutions.Atty. Docket No.: INMD-220 / 01 WOPCT Application274. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 3 to 8 amino acid substitutions.

275. The uricase variant polypeptide of any one of claims 233-261, wherein (ii) the from one to nine amino acid substitutions comprise from 4 to 7 amino acid substitutions.

276. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are 5, 6, 7, 8, or 9 amino acid substitutions, comprising amino acid substitutions corresponding to one of the groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table d.

277. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are 6, 7, 8, or 9 amino acid substitutions, comprising amino acid substitutions corresponding to one of the groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table C2.

278. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are 7, 8, or 9 amino acid substitutions, comprising amino acid substitutions corresponding to one of the groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table DI.

279. The uricase variant polypeptide of any one of claims 233-241, wherein (ii) the from one to nine amino acid substitutions are 8 or 9 amino acid substitutions, comprising amino acid substitutions corresponding to one of the groups of amino acid substitutions of SEQ ID NO: 1 set forth in Table D2.

280. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

281. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.Atty. Docket No.: INMD-220 / 01 WO PCT Application282. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

283. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

284. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R192T, R209W, S228G, I257K, L279T and N281Y.

285. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86T, S95G, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

286. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

287. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

288. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L,Atty. Docket No.: INMD-220 / 01 WOPCT ApplicationQ26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, I100S, A102V, Y105S, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

289. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

290. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, L72T, F75L, K76S, T83E, V86K, K97D, A102V, V106M, K116M, N117E, V119H, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

291. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

292. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, I100N, A102V, Y105A, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

293. The uricase variant polypeptide of claim 233 or 234, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, S80D, V86K, K97D, A102V, Y105W, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257K, L279T and N281Y.

294. The uricase variant polypeptide of claim 233 or 235, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, Q26T, Y31H, L52T, N70H, F75L, K76S, T83E, V86K, K97D, I100E, A102V, Y105G,Aty. Docket No.: INMD-220 / 01 WO PCT ApplicationV106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

295. The uricase variant polypeptide of claim 233 or 235, comprising amino acid substitutions corresponding to the following amino acid substitutions of SEQ ID NO: 1 : V22L, H24R, Q26T, Y31H, L52T, N70H, F75L, K76S, G77E, T83E, V86K, K97D, I100E, A102V, Y105G, V106M, K116M, N117G, Y127H, K155G, F172W, R197D, R209W, S228G, I257R, L279T and N281Y.

296. The uricase variant polypeptide of any one of claims 1-295, wherein the uricase variant polypeptide is a variant of a counterpart uricase protein, 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.

297. The uricase variant polypeptide of claim 296, wherein the counterpart uricase protein is a mammalian uricase protein.

298. The uricase variant polypeptide of claim 297, wherein the mammalian uricase protein is a dog uricase protein, a porcine uricase protein, a bovine uricase protein, an ovine uricase protein, or a baboon uricase protein.

299. The uricase variant polypeptide of claim 296 or 297, wherein the counterpart uricase protein is a chimeric uricase protein comprising two or more mammalian uricase domains.

300. The uricase variant polypeptide of claim 299, wherein the chimeric uricase protein is a chimeric pig-baboon uricase protein.

301. The uricase variant polypeptide of claim 300, wherein the chimeric pig-baboon uricase protein comprises the amino acid sequence of SEQ ID NO: 1.

302. The uricase variant polypeptide of claim 300, wherein the chimeric pig-baboon uricase protein comprises an amino acid sequence at least about 70% identical to SEQ ID NO: 1.

303. The uricase variant polypeptide of claim 300, wherein the chimeric pig-baboon uricase protein comprises an amino acid sequence at least about 80% identical to SEQ ID NO: 1.

304. The uricase variant polypeptide of claim 300, wherein the chimeric pig-baboon uricase protein comprises an amino acid sequence at least about 90% identical to SEQ ID NO: 1.Aty. Docket No.: INMD-220 / 01 WO PCT Application305. The uricase variant polypeptide of claim 300, wherein the chimeric pig-baboon uricase protein comprises an amino acid sequence that is from about 90% to about 100% identical to SEQ ID NO: 1.

306. The uricase variant polypeptide of claim 300, wherein the chimeric pig-baboon uricase protein comprises the amino acid sequence of SEQ ID NO:4.

307. The uricase variant polypeptide of claim 300, wherein the chimeric pig-baboon uricase protein comprises the amino acid sequence of SEQ ID NO:5.

308. The uricase variant polypeptide of any one of claims 1-307, wherein the counterpart uricase protein comprises a C-terminus truncation.

309. The uricase variant polypeptide of claim 308, wherein the C-terminus truncation comprises 4-13 amino acids.

310. The uricase variant polypeptide of any one of claims 1-309, wherein the counterpart uricase protein comprises an N-terminus truncation.

311. The uricase variant polypeptide of claim 310, wherein the N-terminus truncation comprises 4-13 amino acids.

312. The uricase variant polypeptide of any one of claims 1-311, comprising an amino acid sequence that is at least about 85% identical to SEQ ID NO: 1.

313. The uricase variant polypeptide of any one of claims 1-311, comprising an amino acid sequence that is at least about 90% identical to SEQ ID NO: 1.

314. The uricase variant polypeptide of any one of claimsl-311, comprising an amino acid sequence that is at least about 95% identical to SEQ ID NO: 1.

315. The uricase variant polypeptide of any one of claims 1-314, wherein the uricase variant polypeptide comprises an N-terminal methionine residue.

316. The uricase variant polypeptide of any one of claims 1-314, wherein the uricase variant polypeptide does not have an N-terminal methionine residue.

317. The uricase variant polypeptide of any one of claims 1-314, wherein the uricase variant polypeptide comprises a post-translational modification.

318. The uricase variant polypeptide of claim 317, wherein the post-translational modification is removal of an N-terminal methionine residue of the uricase variant polypeptide.Atty. Docket No.: INMD-220 / 01 WO PCT Application319. The uricase variant polypeptide of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 163, 165, 166, 169, 171, 172, 177, 183, 189, 190, 191, 204, 205, and 217.

320. The uricase variant polypeptide of claim 67, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 356, 360, 363, 366, 369, 371, 372, 375, 389, 390, 392, 417, 419, 422, 424, 426, 433, 460, 462, 524, 525, 563, 565, and 1408.

321. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:417.

322. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:419.

323. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:422.

324. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:424.

325. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:426.

326. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:433.

327. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:460.

328. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:462.

329. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:524.

330. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO: 525.

331. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:563.

332. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO:565.Aty. Docket No.: INMD-220 / 01 WO PCT Application333. The uricase variant polypeptide of claim 67, comprising the amino acid sequence of SEQ ID NO: 1408.

334. The uricase variant polypeptide of claim 162, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 583, 588, 591, 592, 597, 599, 601, 605, 606, 624, 625, 626, 630, 632, 633, 634, 635, 637, 647, 652, 656, 922, 924, 928, 931, 939, 940, 946, 949, 958, 961, 964, 979, 982, 988, 1409, and 1410.

335. The uricase variant polypeptide of claim 233, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 922, 924, 928, 931, 939, 940, 946, 949, 958, 961, 964, 979, 982, 988, 1409, and 1410.

336. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO:922.

337. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO: 928.

338. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO:931.

339. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO:939.

340. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO:940.

341. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO:949.

342. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO:958.

343. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO:961.

344. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO: 1409.

345. The uricase variant polypeptide of claim 233, comprising the amino acid sequence of SEQ ID NO: 1410.Atty. Docket No.: INMD-220 / 01 WO PCT Application346. A uricase variant polypeptide comprising an amino acid sequence selected from SEQ ID NOs:45-90.

347. A uricase variant polypeptide comprising an amino acid sequence selected from SEQ ID NOs:91-135.

348. A uricase variant polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 136-268.

349. A uricase variant polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 269-581 and 1408.

350. A uricase variant polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 582-1009, 1409, and 1410.

351. The uricase variant polypeptide of any one of claims 319-350, further comprising an N-terminal methionine residue.

352. The uricase variant polypeptide of any one of claims 1-351, wherein the uricase variant polypeptide is non-PEGylated.

353. The uricase variant polypeptide of any one of claims 1-351, wherein the uricase variant polypeptide is PEGylated.

354. The uricase variant polypeptide of any one of claims 1-353, wherein the uricase variant polypeptide is isolated.

355. The uricase variant polypeptide of any one of claims 1-354, further comprising a purification tag at the C-terminus.

356. The uricase variant polypeptide of any one of claims 1-355, further comprising a purification tag at the N-terminus.

357. The uricase variant polypeptide of claim 355 or 356, wherein the purification tag is a polyhistidine tag.

358. The uricase variant polypeptide of any one of claims 1-357, wherein the uricase variant polypeptide is a monomer.

359. The uricase variant polypeptide of any one of claims 1-357, wherein the uricase variant polypeptide is a homodimer.Aty. Docket No.: INMD-220 / 01 WO PCT Application360. The uricase variant polypeptide of any one of claims 1-357, wherein the uricase variant polypeptide is a homotetramer.

361. A uricase monomer comprising a uricase variant polypeptide of any one of claims 1- 357.

362. A uricase dimer comprising a first uricase monomer and a second uricase monomer, wherein the first uricase monomer is a uricase variant polypeptide of any one of claims 1-357.

363. The uricase dimer of claim 362, wherein the second uricase monomer is a uricase variant polypeptide of any one of claims 1-357.

364. The uricase dimer of claim 363, wherein the uricase dimer is a homodimer.

365. The uricase dimer of claim 363, wherein the uricase dimer is a heterodimer.

366. A uricase tetramer comprising a first, second, third and fourth uricase monomer, wherein the first uricase monomer is a uricase variant polypeptide of any one of claims 1-357.

367. The uricase tetramer of claim 366, wherein the second uricase monomer is a uricase variant polypeptide of any one of claims 1-357.

368. The uricase tetramer of claim 367, wherein the third uricase monomer is a uricase variant polypeptide of any one of claims 1-357.

369. The uricase tetramer of claim 368, wherein the fourth uricase monomer is a uricase variant polypeptide of any one of claims 1-357.

370. The uricase tetramer of any one of claims 366-369, wherein the uricase tetramer is a homotetramer.

371. The uricase tetramer of any one of claims 366-369, wherein the uricase tetramer is a heterotetramer.

372. A uricase conjugate comprising a first domain and a second domain, wherein the first domain comprises the uricase variant polypeptide of any one of claims 1-360, and the second domain comprises a first random coil polypeptide domain comprising at least about 50 amino acids.

373. The uricase conjugate of claim 372, wherein the uricase conjugate is a fusion protein of the first domain and the second domain.Aty. Docket No.: INMD-220 / 01 WO PCT Application374. The uricase conjugate of claim 373, wherein the first domain is C-terminal to the second domain.

375. The uricase conjugate of claim 373, wherein the first domain is N-terminal to the second domain.

376. The uricase conjugate of any one of claims 373-375, wherein an amino acid linker is present between the first domain and the second domain.

377. The uricase conjugate of claim 376, wherein the amino acid linker is from about two amino acids to about 5 amino acids long.

378. The uricase conjugate of claim 377, wherein the amino acid linker is two amino acids long.

379. The uricase conjugate of claim 378, wherein the amino acid linker is Gly-Ser.

380. The uricase conjugate of any one of claims 373-379, further comprising a third domain, wherein the third domain comprises a second random coil polypeptide domain comprising at least about 50 amino acids.

381. The uricase conjugate of claim 380, wherein the first domain is N-terminal to the second domain and C-terminal to the third domain.

382. The uricase conjugate of claim 381, wherein an amino acid linker is present between the first domain and the second domain.

383. The uricase conjugate of claim 381 or 382, wherein an amino acid linker is present between the first domain and the third domain.

384. The uricase conjugate of claim 382 or 383, wherein the amino acid linker is from about two amino acids to about 5 amino acids long.

385. The uricase conjugate of claim 384, wherein the amino acid linker is two amino acids long.

386. The uricase conjugate of claim 385, wherein the amino acid linker is Gly-Ser.

387. The uricase conjugate of any one of claims 372-386, wherein the second domain comprises an extended recombinant (XTEN) polypeptide.

388. The uricase conjugate of any one of claims 380-387, wherein the third domain comprises an extended recombinant (XTEN) polypeptide.Aty. Docket No.: INMD-220 / 01 WO PCT Application389. The uricase conjugate of claim 387 or 388, wherein the XTEN polypeptide comprises the amino acid sequence of SEQ ID NO: 12.

390. The uricase conjugate of claim 387 or 388, wherein the XTEN polypeptide comprises the amino acid sequence of SEQ ID NO:42.

391. The uricase conjugate of claim 387 or 388, wherein the XTEN polypeptide comprises the amino acid sequence of SEQ ID NO:43.

392. The uricase conjugate of claim 387 or 388, wherein the XTEN polypeptide comprises the amino acid sequence of SEQ ID NO:44.

393. The uricase conjugate of any one of claims 372-386, wherein the second domain comprises a Pro- Ala (PA) polypeptide.

394. The uricase conjugate of any one of claims 380-386 and 393, wherein the third domain comprises a Pro- Ala (PA) polypeptide.

395. The uricase conjugate of claim 393 or 394, wherein the PA polypeptide comprises the amino acid sequence of SEQ ID NO:25.

396. The uricase conjugate of any one of claims 372-386, wherein the second domain comprises a Pro-Ala-Thr (PAT) polypeptide.

397. The uricase conjugate of any one of claims 380-386 and 396, wherein the third domain comprises a Pro-Ala-Thr (PAT) polypeptide.

398. The uricase conjugate of claim 396 or 397, wherein the PAT polypeptide comprises the amino acid sequence of SEQ ID NO:27.

399. The uricase conjugate of claim 398, wherein the amino acid sequence of SEQ ID NO: 27 is encoded by the nucleotide sequence of SEQ ID NO:28.

400. The uricase conjugate of claim 396 or 397, wherein the PAT polypeptide comprises the amino acid sequence of SEQ ID NO:29.

401. The uricase conjugate of claim 400, wherein the amino acid sequence of SEQ ID NO: 29 is encoded by the nucleotide sequence of SEQ ID NO:30.

402. The uricase conjugate of claim 396 or 397, wherein the PAT polypeptide comprises the amino acid sequence of SEQ ID NO: 31.Aty. Docket No.: INMD-220 / 01 WO PCT Application403. The uricase conjugate of claim 402, wherein the amino acid sequence of SEQ ID NO: 31 is encoded by the nucleotide sequence of SEQ ID NO:32.

404. The uricase conjugate of any one of claims 372-386, wherein the second domain comprises a Pro- Ala-Ser (PAS) polypeptide.

405. The uricase conjugate of any one of claims 380-386 and 404, wherein the third domain comprises a Pro- Ala- Ser (PAS) polypeptide.

406. The uricase conjugate of claim 404 or 405, wherein the PAS polypeptide comprises an amino acid sequence of 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.

407. The uricase conjugate of claim 406, wherein the PAS polypeptide comprises the amino acid sequence of SEQ ID NO: 13.

408. The uricase conjugate of claim 407, wherein the amino acid sequence of SEQ ID NO: 13 is encoded by a nucleotide sequence selected from SEQ ID NOs: 1415-1445.

409. The uricase conjugate of claim 407, wherein the amino acid sequence of SEQ ID NO: 13 is encoded by a nucleotide sequence selected from SEQ ID NOs: 1426-1445.

410. The uricase conjugate of any one of claims 372-409, wherein the first random coil polypeptide domain comprises from about 60 amino acids to about 800 amino acids.

411. The uricase conjugate of any one of claims 380-410, wherein the second random coil polypeptide domain comprises from about 60 amino acids to about 800 amino acids.

412. The uricase conjugate of any one of claims 372-409, wherein the first random coil polypeptide domain comprises from about 60 amino acids to about 700 amino acids.

413. The uricase conjugate of any one of claims 380-412, wherein the second random coil polypeptide domain comprises from about 60 amino acids to about 700 amino acids.

414. The uricase conjugate of any one of claims 372-409, wherein the first random coil polypeptide domain comprises from about 60 amino acids to about 600 amino acids.

415. The uricase conjugate of any one of claims 380-414, wherein the second random coil polypeptide domain comprises from about 60 amino acids to about 600 amino acids.

416. The uricase conjugate of any one of claims 372-409, wherein the first random coil polypeptide domain comprises from about 60 amino acids to about 500 amino acids.Aty. Docket No.: INMD-220 / 01 WO PCT Application417. The uricase conjugate of any one of claims 380-416, wherein the second random coil polypeptide domain comprises from about 60 amino acids to about 500 amino acids.

418. The uricase conjugate of any one of claims 372-409, wherein the first random coil polypeptide domain comprises from about 60 amino acids to about 400 amino acids.

419. The uricase conjugate of any one of claims 380-418, wherein the second random coil polypeptide domain comprises from about 60 amino acids to about 400 amino acids.

420. The uricase conjugate of any one of claims 372-409, wherein the first random coil polypeptide domain comprises from about 60 amino acids to about 300 amino acids.

421. The uricase conjugate of any one of claims 380-420, wherein the second random coil polypeptide domain comprises from about 60 amino acids to about 300 amino acids.

422. The uricase conjugate of any one of claims 373-421, wherein the first domain does not include an N-terminal methionine residue.

423. The uricase conjugate of any one of claims 373-422, wherein the first random coil polypeptide domain does not include an N-terminal methionine residue.

424. The uricase conjugate of any one of claims 380-423, wherein the second random coil polypeptide domain does not include an N-terminal methionine residue.

425. The uricase conjugate of any one of claims 373-424, further comprising a purification tag at the C-terminus.

426. The uricase conjugate of any one of claims 373-425, further comprising a purification tag at the N-terminus.

427. The uricase conjugate of claim 425 or 426, wherein the purification tag is a polyhistidine tag.

428. The uricase conjugate of claim 380, 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: 1192-1263.

429. The uricase conjugate of claim 380, 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: 1264-1335.Aty. Docket No.: INMD-220 / 01 WO PCT Application430. The uricase conjugate of claim 380, 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: 1336-1407.

431. The uricase conjugate of any one of claims 372-430, wherein the uricase conjugate is a monomer.

432. The uricase conjugate of any one of claims 372-431, wherein the uricase conjugate is present within a homodimer.

433. The uricase conjugate of any one of claims 372-431, wherein the uricase conjugate is present within a homotetramer.

434. The uricase conjugate of any one of claims 372-430, wherein the uricase conjugate is a homotetramer.

435. The uricase conjugate of any one of claims 372-430, wherein the uricase conjugate is a homodimer.

436. The uricase conjugate of any one of claims 372-435, wherein the uricase conjugate is non-PEGylated.

437. The uricase conjugate of any one of claims 372-435, wherein the uricase conjugate is PEGylated.

438. An isolated nucleic acid which encodes the uricase variant polypeptide of any one of claims 1-360, or the uricase conjugate of any one of claims 372-437.

439. A nucleic acid vector comprising the nucleic acid of claim 438.

440. A host cell comprising the nucleic acid vector of claim 439.

441. A pharmaceutical composition comprising the uricase variant polypeptide of any one of claims 1-360, the uricase tetramer of any one of claims 366-371, or the uricase conjugate of any one of claims 372-437, and a pharmaceutically acceptable carrier.

442. 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 441.

443. The method of claim 442, wherein the uric acid levels are reduced in the plasma of the subject.Aty. Docket No.: INMD-220 / 01 WO PCT Application444. The method of claim 442 or 443, wherein the subject is a gout patient.

445. The method of claim 444, wherein the subject is a refractory gout patient.

446. The method of any one of claims 442-445, wherein the subject has been diagnosed with tumor lysis syndrome.

447. 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 441.

448. The method of claim 447, wherein the gout is refractory gout.

449. 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 441.

450. The method of any one of claims 442-449, wherein the subject is a human patient.

451. The method of claim 450, wherein the human patient is an adult human patient.

452. The method of any one of claims 442-451, wherein the pharmaceutical composition is administered parenterally.

453. The method of claim 452, wherein the pharmaceutical composition is administered intravenously.

454. The method of claim 452, wherein the pharmaceutical composition is administered subcutaneously.

455. A method of recombinantly producing the uricase variant polypeptide of any one of claims 1-360, comprising:(i) culturing a host cell comprising a nucleic acid vector comprising a nucleic acid sequence encoding the uricase variant polypeptide of any one of claims 1-360, 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 polypeptide and recombinant production of the uricase variant polypeptide by the host cell; and(ii) isolating the recombinantly produced uricase variant polypeptide.

456. A method of recombinantly producing the uricase conjugate of any one of claims 372- 437, 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 372-437, wherein the nucleic acidAtty. Docket No.: INMD-220 / 01 WO PCT Application 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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