Peg-urate oxidase conjugates and use thereof

IS 2 245 114 T3

DESCRIPTION

PEG-urate oxidase conjugates and their use.

Field of the invention

The present invention deals with the chemical modification of proteins to prolong their life in circulation and reduce their immunogenicity. More specifically, the invention addresses the conjugation of polyethylene glycol or polyoxyethylene with urate oxidases, which considerably eliminates the immunogenicity of urate oxidase without compromising its uricolytic activity.

Background of the invention

Statements in this section do not constitute an admission of prior technology, but rather reflect the inventors' subjective comments and interpretations of the state of the technology at the time this invention was made. Such interpretations may reflect the inventors' personal ideas, and therefore not known, which are not themselves part of the prior art.

Urate oxidases (uricases; EC 1.7.3.3) are enzymes that catalyze the oxidation of uric acid to a more soluble product, allantaine, a metabolite that is easier to excrete. Humans do not produce enzymatically active uricases as a result of various mutations in the gene for uricase acquired during the evolution of higher primates. Wu, X, et al., (1992) JMol Evol 34: 78-84. Consequently, in susceptible individuals, excessive levels of uric acid in the blood (hyperuricemia) and in the urine (hyperuricosuria) can lead to painful arthritis (gout), disfiguring urate deposits (tophi), and kidney failure. In some affected individuals, available medications such as allopurinol (an inhibitor of uric acid synthesis) cause treatment-limiting side effects or do not relieve symptoms satisfactorily. Hande, KR, et al., (1984) Am JMed 76: 47-56; Fam, AG, (1990) Bailliere's Clin Rheumatol 4: 177-192. Uricase injections can decrease hyperuricemia and hyperuricosuria, at least temporarily. However, since uricase is a foreign protein in humans, even the first injection of unaltered Aspergillus flavus protein has led to anaphylactic reactions in different percentages of patients under treatment (Pui, CH, et al., ( 1997) Leukemia 11: 1813-1816) and immune responses restrict its usefulness for chronic or intermittent treatments. Donadío, D, et al., (1981) Nouv Presse Méd 10: 711-712; Leaustic, M, et al., (1983) Rev Rhum Mal Osteoartic 50: 553554.

For several decades it has been recognized that the results of available treatments for hyperuricemia are less than optimal. Kissel, P, et al., (1968) Nature 217: 72-74. In the same way, the possibility that certain groups of patients with severe gout could benefit from a treatment with injectable uricase in a safe and effective way has been recognized for years. Davis, FF, et al., (1978) in GB Broun, et al., (Eds.) Enzyme Engineering, Vol. 4 (pp. 169-173) New York, Plenum Press; Nishimura, H, et al., (1979) Enzyme 24: 261-264; Nishimura, H, et al., (1981) Enzyme 26: 49-53; Davis, S, et al., (1981) Lancet 2 (8241): 281-283; Abuchowski, A, et al., (1981) J Pharmacol Exp Ther 219: 352-354; Chen, RH-L, et al., (1981) Biochim Biophys Acta 660: 293-298; Chua, CC, et al., (1988) Ann Int Med 109: 114-117; Greenberg, ML, et al., (1989) Anal Biochem 176: 290-293. Uricases derived from animal organs are practically insoluble in solvents that are compatible with safe administration by injection. US Patent 3,616,231. Certain uricases derived from plants or microorganisms are more soluble in medically acceptable solvents. However, the injection of microbial enzymes rapidly induces immune responses that can lead to life-threatening allergic reactions or inactivation or accelerated clearance of uricase from the circulation. Donadio, et al., (1981); Leaustic, et al., (1983). Enzymes based on deduced amino acid sequences of uricases from mammals, including pig and baboon, or from insects, such as Drosophila melanogaster or Drosophila pseudoobscura (Wallrath, LL, et al., (1990) Mol Cell Biol 10: 5114-5127 ), have not been advisable candidates for clinical use, due to immunogenicity problems and insolubility at physiological pH.

Covalent alterations of proteins with polyethylene glycol or polyoxyethylene (both known as PEG) have been used to increase the half-life of proteins and reduce immunogenicity. US Patents 4,179,337, 4,766,106 and 4,847,325; Saifer, MGP, et al., (1994) Adv Exp Med Biol 366: 377-387. The combination of high molecular weight PEG to produce conjugates with life cycles of prolonged circulation or reduced immunogenicity - while maintaining functional activity - was previously demonstrated for another enzyme, superoxide dismutase (Somack, R, et al., (1991) Free Rad Res Commun 12-13: 553-562; US Patents 5,283,317 and 5,468,478) and for other types of proteins, for example cytokines (Saifer, MGP, et al., (1997) Polym Preprints 38: 576577; Sherman, MR, et al., (1997) in JM Harris, et al., (Eds.), Poly (ethylene glycol) Chemistry and Biological Applications. ACS Symposium Series 680 (pp. 155-169) Washington, DC: American Chemical Society). Also, conjugates of uricase with polymers other than PEG have been described. US Patent 4,460,683.

In virtually all recorded attempts to PEGylate uricase (ie, covalently combine PEG with uricase), PEG was predominantly added to amino groups, including amino-terminal residues and available lysine residues. In the most commonly used uricases, the total number of lysines in each of the four identical subunits is between 25 (Aspergillus flavus (US Patent 5,382,518)) and 29 (pig (Wu, X, et al., (1989) Proc Natl Acad Sci USA 86: 9412-9416)). Some lysines are not available for PEGylation in

ES 2 245 114 T3 native conformation of the enzyme. The most common method of reducing uricase immunogenicity has been to combine large amounts of low molecular weight PEG chains. This has resulted in a large decrease in the enzymatic activity of the resulting conjugates.

Previous researchers used injected uricase to catalyze the conversion of uric acid to allantaine in vivo. See Pui, et al., (1997). This is the basis for the use in France and Italy of uricase derived from the fungus Aspergillus flavus (Uricozyme®) to prevent or provisionally correct hyperuricemia associated with cytotoxic therapy for hematologic malignancies and to temporarily reduce severe hyperuricemia in patients with gout. Potaux, L, et al., (1975) Nouv Presse Méd 4: 1109-1112; Legoux, R, et al., (1992) J Biol Chem 267: 85658570; US Patents 5,382,518 and 5,541,098. Due to its short circulation life, Uricozyme® requires daily injections and, furthermore, it is not very advisable for long-term therapies due to its immunogenicity.

A single intravenous injection of a Candida utilis-derived uricase preparation combined with 5 kDa PEG reduced serum urate to undetectable levels in five human subjects whose mean pre-injection serum urate concentration was 6.2 mg / dL, which is within the normal range (Davis, et al., (1981). Subjects were given an additional injection four weeks later, but their responses were not recorded. Using a relatively insensitive gel diffusion assay, no antibody to uricase was detected after the second (last) injection. This reference did not provide results on chronic or subchronic treatments in humans or animals used for experimental purposes.

A uricase preparation derived from Arthrobacter protoformiae combined with 5 kDa PEG was used to temporarily control hyperuricemia in a single lymphoma patient whose serum urate concentration before injection was 15 mg / dL, Chua, et al. , (1988). Due to the critical condition of the patient and the short duration of treatment (four injections in a 14-day period), it was not possible to assess the long-term efficacy or safety of the conjugate.

In this application, the term "immunogenicity" is understood as the induction of an immune response by injecting a preparation of PEG-modified or unmodified uricase (the antigen), while "antigenic" is understood as the reaction of an antigen with existing antibodies. Collectively, antigenic and immunogenicity are understood as "immunoreactivity". In previous investigations of PEG-uricase, immunoreactivity was evaluated by different methods, including: 1) the in vitro reaction of PEG-uricase with preformed antibodies; 2) measurement of induced antibody synthesis; and 3) accelerated clearance rates after repeated injections.

It is known that previous attempts were made to eliminate the immunogenicity of uricases derived from different sources by combining various amounts of PEG chains through various linkages, which had limited success. The first to discover PEG-uricases were Davis and Inada together with their collaborators. Davis, et al., (1978); US Patent 4,179,337; Nishimura, et al., (1979); Japanese patents 55-99189 and 62-55079. The conjugate described in the '337 patent was synthesized by reacting uricase of unknown origin with a 2,000-fold molar surplus of 750 Da of PEG, indicating that a large number of polymer molecules was similar to that added to each subunit of uricasa. The '337 patent addresses the combination of PEG or polypropylene glycol with molecular weights of 500 to 20,000 Da, preferably between 500 and 5,000 Da, to provide non-immunogenic, water-soluble, active conjugates of various polypeptide hormones and enzymes including oxidoreductases, of of which uricasa is one of three examples. In addition, the '337 patent highlights the combination of 10 to 100 polymer chains per enzyme molecule and the retention of at least 40% enzyme activity. Test results regarding the combination of PEG with the available amino groups of uricase, residual specific uricolytic activity or immunoreactivity of the conjugate were not reported.

The summary of the data derived from the 13 mentions related to uricase PEGylation is found in Table 1. Likewise, some of these results are presented graphically in Figures 1A-2B. Seven of these publications describe significant reductions in uricolytic activity measured in vitro generated by the combination of large amounts of PEG chains with the Candida utilis-derived uricase. The combination of large amounts of 5 kDa PEG chains with uricase from porcine liver gives similar results, as described in the publication by Chen and in the presentation at a symposium of the same group. Chen, et al., (1981); Davis, et al., (1978).

Among the studies summarized in Table 1, it was documented that immunoreactivity was reduced in seven of them, while it was eliminated in five of them thanks to PEGylation. In three of the last five studies, the elimination of immunoreactivity was associated with a marked decrease in uricolytic activity, to almost 15, 28 or 45% of its initial activity. Nishimura, et al., (1979) (15% activity); Chen, et al., (1981) (28% activity); Nishimura, et al., (1981) (45% activity). In the fourth report, it was documented that PEG was combined with 61% of the available lysine residues, however the residual specific activity Abuchowski, et al., (1981) was not expressed. However, a team of researchers consisting of two of the same scientists using the same methods documented that this over-combining left a residual activity of only 23-28%. Chen, et al., (1981). The 1981 publications by Abuchowski et al. and Chen et al. indicate that to significantly reduce uricase immunogenicity, PEG must be combined with approximately 60% of the available lysine residues (Table 1). In the first publication reporting elimination of immunoreactivity, excess PEG pooling, residual uricolytic activity, or the nature of the PEG-protein binding was not revealed. Veronese, FM, et

ES 2 245 114 T3 al., (1997) in JM Harris, et al., (Eds.), Poly (ethylene glycol) Chemistry and Biological Applications. ACS Symposium Series 680 (pp. 182-192) Washington, DC: American Chemical Society.

Conjugation of PEG with a small fraction of lysine residues in uricase reduced but did not eliminate its immunoreactivity in the case of experimental animals. Tsuji, J, et al., (1985) Int JImmunopharmacol 7: 725730 (28-45% of coupled amino groups); Yasuda, Y, et al., (1990) Chem Pharm Bull 38: 2053-2056 (38% of coupled amino groups). The residual urolytic activities of the corresponding adducts range from <33% (Tsuji, et al.) To 60% (Yasuda, et al.) Of their initial values. Tsuji, et al. they synthesized the PEG-uricase conjugates with 7.5 kDa and 10 kDa PEG, in addition to 5 kDa PEG. All the resulting conjugates were somehow immunogenic and antigenic, although they showed markedly reduced enzyme activities (Table 1; Figures 1A-1B).

A PEGylated preparation of uricase derived from Candida utilis that was safely administered twice to five humans was documented to retain only 11% of its initial activity. Davis, et al. (1981). Several years later, Arthrobacter protoformiae-derived PEG-modified uricase was administered four times to a patient with advanced lymphoma and severe hyperuricemia. Chua, et al., (1988). While the residual activity of this enzyme preparation was not measured, Chua, et al., Demonstrated the absence of anti-uricase antibodies in the patient's serum 26 days after the first injection of PEG-uricase, using the enzyme-linked immunosorbent assay (ELISA, enzyme-linked immunosorbent assay).

As summarized in Table 1, previous studies of PEGylated uricase show that catalytic activity is significantly reduced by combining a sufficient number of PEG chains to significantly reduce their immunoreactivity. Furthermore, most of the earlier PEG-uricase preparations were synthesized using cyanide chloride activated PEG, a triazine derivative (2,4,6-trichloro-1,3,5-triazine) that introduced new antigenic determinants, as well as inducing antibodies in rabbits. Tsuji, et al., (1985).

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IS 2 245 114 T3

TABLE 1

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IS 2 245 114 T3

TABLE 1 (continued)

Characteristics of PEG-uricases derived from previous studies.

<td>Uricasa fountain</td><td>Docking link</td><td>PEG molecular weight (kDa)</td><td>Percentage of Power Plants with PEG added</td><td>Activity uricolytic residual (%)</td><td>Antigenic or immunogenic Comments</td><td>Reference</td>

<td>Candida you use</td><td>Triazine according to Chen, R HL, et al., 1981</td><td> 5</td><td> 35 70</td><td>Undisclosed</td><td>PEG reduced immunogenicity in rabbits.</td><td>Savoca, KV, et al., (1984) Int Arch Allergy Appl Immunol 75: 58-61</td>

<td>Candida you use</td><td>Triazine</td><td> 5</td><td>Not revealed</td><td>Undisclosed</td><td>PEG-uricase was administered orally to chicken in liposomes (once)</td><td>Nishida, Y, et al., (1984) J Pharm Pharmacol 36: 354-355</td>

<td>Arthrobact er protoformi ae</td><td>Undisclosed</td><td> 5</td><td>Not revealed</td><td>Undisclosed</td><td>ELISA did not detect any antibodies 26 days after the first of four PEG-uricase injections.</td><td>Chua, CC, et al., 1988</td>

<td>Candida you use</td><td>Triazine peg₂</td><td>2x5</td><td> 10 12</td><td> 90 89</td><td>Undisclosed Undisclosed</td><td>Yasuda, Y, et al., 1990</td>

<td></td><td></td><td></td><td> 15</td><td> 80</td><td>Undisclosed</td><td></td>

<td></td><td></td><td></td><td> 21</td><td> 70</td><td>Undisclosed</td><td></td>

<td></td><td></td><td></td><td> 38</td><td> 60</td><td>The antigenic test with rabbit serum was reduced by 75%.</td><td></td>

<td>Candida you use</td><td>Triazine peg₂</td><td>2x5</td><td> 22</td><td> 68</td><td>A single injection. PEG increased the half-life of 1 hour ca. to 8 hours ca. in mice. PEG blocked elimination by liver, spleen, and kidney (study duration 24 h.)</td><td>Fujita, T, et al., 1991</td>

<td>Not revealed</td><td>PEG peg₂Link not reported</td><td>Not revealed</td><td>Not revealed It was reported to be the same as for PEG</td><td>Undisclosed Undisclosed</td><td>Immunogenicity was reduced in mice a 98% (PEG) or 100% (PEG₂)</td><td>Veronese, FM, et al., 1997</td>

Japanese patent 3-148298 to A Sano, et al., Describes modified proteins, including uricase, derivatized PEG having a molecular weight of 1-12 kDa that shows reduced antigenicity and "better and better" action as well as methods for making said derivatized peptides. However, there is no information on chain entourage, enzyme assays, biological tests, or the meaning of "better and longer." Patents

Japanese ES 2 245 114 T3 55-99189 and 62-55079, both from Y Inada, include conjugates of uricase prepared with PEG-triazine or bis-PEG-triazine (denoted as PEG₂ in table 1), respectively (Nishimura, et al., (1979 and 1981). In the first type of conjugate, the molecular weights of the PEGs are 2 kDa and 5 kDa, while in the second type of conjugate, only 5 kDa was used Nishimura et al. (1979) reported 15% recovery of uricolytic activity after modification of 43% of available lysines with 5 kDa linear PEGs, while Nishimura, et al., (1981) reported recovery of 31% or 45% of uricolytic activity after modification of 46% or 36% of lysines, respectively, with PEG2.

Summary of the invention

Previous studies show that when a significant reduction in uricase immunogenicity or antigenicity is achieved by PEGylation, it is invariably associated with a substantial loss of uricolytic activity. The safety, convenience and cost-effectiveness of biopharmaceuticals are adversely affected by reductions in their potencies and the resulting need to increase the administered dose. Therefore, there is a need for safe and effective alternative means of reducing elevated uric acid levels in body fluids, including blood and urine. The present invention features an essentially non-immunogenic PEG-uricase that retains all or nearly all of the unmodified enzyme activity.

One embodiment of the present invention is a urate oxidase (uricase) conjugate that retains at least about 75% of the uricolytic activity of unconjugated uricase and exhibits substantially reduced immunogenicity. This contribution includes a purified uricase in which each subunit can be covalently combined with an average of 2 to 10 PEG chains, which can be linear or branched, in which each PEG molecule can have a molecular weight of between 5 and 100 kDa. kDa. The uricase of this aspect of the invention can be recombinant. Whether or not it is recombinant, uricase can be of mammalian origin. In one aspect of this contribution, the uricase may be porcine, bovine, or ovine liver uricase. In another aspect of this contribution, uricase can be chimeric. Chimeric uricase may contain portions of porcine liver or baboon liver uricase. For example, the chimeric uricase may be pig-baboon chimeric uricase (PBC uricase) or porcine uricase containing the R291k and T301S mutations (PKS uricase) (see sequences in Figure 6 and the results of the Physiological and immunological studies of Figures 7-12). Alternatively, the uricase can be spleen and liver uricase in which tyrosine 97 is replaced by histidine, whereby the specific activity of uricase can be increased by at least about 60%. The uricase of the invention, whatever its origin, may also have a form that is truncated, either at the amino terminus, at the carboxyl terminus or at both termini. Also, uricase can be fungal or microbial uricase. In one aspect of this contribution, the fungal or microbial uricase can be a naturally occurring or recombinant form of uricase derived from Aspergillus flavus, Arthrobacter globiformis, or Candida utilis. Alternatively, the uricase may be an invertebrate uricase, such as a naturally occurring or recombinant form of uricase derived from Drosophila melanogaster or Drosophila pseudoobscura. Likewise, it is possible that this uricase of the invention is a plant uricase, for example a naturally occurring or recombinant form of uricase derived from the nodule of the soybean root (Glycine max). PEG can have an average molecular weight of between 5 kDa and 100 kDa; preferably, the PEG may have an average molecular weight of between 10 kDa and 60 kDa; Ideally, the PEG may have an average molecular weight of between 20 kDa and 40 kDa, for example 30 kDa. The average amount of the PEG covalently combined chains can be from 2 to 10 chains per uricase subunit; preferably, the average amount of the PEG covalently combined chains can be 3 to 8 chains per uricase subunit; ideally, the average amount of PEG covalently combined chains can be 4 to 6 chains per uricase subunit. In one aspect of this contribution, the uricase can be tetrameric. PEG chains can be covalently attached to uricase via urethane (carbamate) linkages, secondary amine linkages, and / or amide linkages. When uricase is a recombinant form of any of the uricases mentioned herein, the recombinant form may have substantially the sequence of the naturally occurring form.

Another contribution of the present invention is a pharmaceutical composition that serves to reduce uric acid levels in body fluids, which contain any of the PEG-uricase conjugates already described and a pharmaceutically acceptable carrier. The composition can be stabilized by lyophilization and dissolved immediately after reconstitution to offer advisable solutions for parenteral administration.

Also, the present invention features the use of PEG-urate oxidase to reduce uric acid levels in body fluids and tissues of a mammal. This use encompasses the administration to a mammal of an amount of PEG-uricase that effectively reduces uric acid. PEG-uricase can be a purified two or more subunit uricase in which each subunit can be covalently linked with an average of 2 to 10 straight or branched PEG chains, in which each PEG molecule can have an average molecular weight of between about 5 kDa and 100 kDa, in a pharmaceutically acceptable carrier. The mammal can be a human. The administration phase can be, for example, an intravenous, intradermal, subcutaneous, intramuscular or intraperitoneal injection; or an aerosol presentation for inhalation. Elevated levels of uric acid can be found in blood, urine, or other body fluids and tissues, as well as being related to gout, tophi, kidney failure, organ transplantation, or any other malignancy.

Other contributions of the present invention are a method for separating a tetrameric form of uricase derived from a solution containing multiple forms of uricase and the product of said method. First, the solution may contain tetrameric uricase and uricase aggregates. The method may include the following phases: applying the

ES 2 245 114 T3 solution to at least one separation column at a pH between almost 9 and 10.5, for example 10.2; recovering the eluate fractions and identifying those that may contain separated tetrameric uricase, wherein the fractions are basically free of uricase aggregates; and combining the fractions of the separated tetrameric uricase. The separation column can be based on ion exchange, size exclusion, or any other valid separation criteria. Similarly, the method may encompass an analysis of the fractions to determine the presence of tetrameric uricase or the absence of uricase aggregates. For example, such analysis may include high performance liquid chromatography (HPLC), other chromatography methods, light scattering, centrifugation, and / or electrophoresis. In one aspect of this addition, the purified tetrameric uricase may contain less than 10% uricase aggregates.

Brief description of the drawings

Figure 1A shows the retention of activity by PEGylated uricase derived from Candida utilis as a function of the number of PEG chains coupled per subunit.

Figure 1B shows the retention of activity by PEGylated uricase derived from Candida utilis as a function of the total mass of PEG coupled per subunit.

Figure 2A shows the retention of activity by PEGylated uricase derived from porcine liver as a function of the number of PEG chains coupled per subunit.

Figure 2B shows the retention of activity by PEGylated uricase derived from porcine liver as a function of the total mass of PEG coupled per subunit.

Figure 3A shows the retention of activity by pig-baboon chimeric PEGylated uricase (PBC: pig-baboon chimeric) as a function of the number of PEG chains coupled per subunit.

Figure 3B shows the retention of activity by PEGylated uricase PBC as a function of the total mass of PEG coupled per subunit.

Figure 4A shows the retention of activity by PEGylated uricase derived from Aspergillus flavus as a function of the number of PEG chains coupled per subunit.

Figure 4B shows the retention of activity by PEGylated uricase derived from Aspergillus flavus as a function of the total mass of PEG coupled per subunit.

Figure 5A shows the retention of activity by recombinant PEGylated uricase derived from the soybean root nodule as a function of the number of PEG chains coupled per subunit.

Figure 5B shows the retention of activity by recombinant PEGylated uricase derived from the soybean root nodule as a function of the total mass of PEG coupled per subunit.

Figure 6 shows the dedicated amino acid sequences of pig-baboon chimeric uricase (PBC uricase), PBC uricase that is truncated at the amino and carboxyl terminals (PBC-NT-CT), and porcine uricase containing the R291K and T301S mutations. (PKS uricase), compared to porcine and baboon sequences.

Figure 7 shows the uricase activity in mouse serum 24 hours after every four or five intraperitoneal injections of modified uricase-PEG PBC, relative to the value at 24 hours after the first injection.

Figure 8 shows the inverse relationship between the activity of PEG PBC-modified injected uricase in the serum of a mouse lacking uricase and the uric acid concentration in serum and urine.

Figure 9 shows a lower severity of a concentrating defect in urine in uricase-deficient (uox - / -) mice that were treated with modified uricase-PEG PBC.

Figure 10 shows the lesser severity of nephrogenic diabetes insipidus in uricase-deficient (uox - / -) mice that were treated with modified uricase-PEG PBC.

Figure 11 shows the lesser severity of uric acid-induced nephropathy, as observed through the magnetic resonance microscope, in uricase-deficient (uox - / -) mice that were treated with modified uricase-PEG PBC.

Figure 12 shows the accelerated clearance derived from circulation of BALB / c mice of injected PBC uricase octamer, compared to tetramer, when both are coupled with 5-6 10 kDa PEG chains per subunit.

IS 2 245 114 T3

Detailed description of preferred contributions

The present invention provides better conjugates of water soluble polymers, preferably polyethylene glycols or polyoxyethylenes, with uricases. Also, the invention offers improved conjugate pharmaceutical compositions. These conjugates are primarily non-immunogenic and retain at least 75%, preferably 85%, and ideally 95% or more of the uricolytic activity of the unmodified enzyme. Suggested uricases for conjugation with water-soluble polymers include naturally occurring urate oxidases isolated from bacteria, fungi, and plant or animal tissues, both vertebrates and invertebrates, as well as recombinant forms of uricase, including mutated, hybrid and / or variants. or truncated enzymatically active. Among the water-soluble polymers advisable for use in the present invention are the linear and branched polyethylene glycols or polyoxyethylenes, better known as PEG. Examples of branched PEGs are those subject to US Patent 5,643,575. A preferred example of linear PEGs is monomethoxyPEG, of general structure CH₃O- (CH₂CH₂OR)_nH, where n ranges from approximately 100 to 2,300.

A preferred uricase of mammalian origin is PBC recombinant uricase, composed of portions of pig liver and baboon liver sequences, Wu, et al., (1989), were the first to determine these. An example of such a chimeric uricase contains the first 225 amino acids derived from the porcine uricase sequence (SEQ ID NO: 1) and the last 79 amino acids derived from the baboon uricase sequence (SEQ ID NO: 2) (pig uricase- baboon or uricase PBC; see figure 6). Another example of this is that a uricase contains residues 7-225 of the porcine sequence (SEQ ID NO. 1) and residues 226-301 of the baboon sequence (SEQ ID NO. 2); which is equivalent to PBC uricase which is truncated at the amino and carboxyl terminals (PBC-NT-CT; see Figure 6). Another example of this is that chimeric uricase contains the first 288 amino acids derived from the porcine sequence (SEQ ID NO. 1) and the last 16 amino acids derived from the baboon sequence (SEQ ID NO.2). Because the latter sequence differs from the porcine sequence in only two positions, with a lysine (K) instead of arginine at residue 291 and a serine (S) instead of threonine at residue 301, this is referred to. mutation such as a porcine uricase-KS or PKS. Each of the uricases PKS, PBC, and PBC-NT-CT have one more lysine residue and thus one more potential PEGylation site than the porcine or baboon sequence.

The cDNAs for various mammalian uricases, including PBC uricase, PKS uricase, and recombinant baboon-like uricase were subcloned, and optimal conditions for expression in E. coli were determined using standard methods. See Erlich, HA, (Ed.) (1989) PCR Technology. Principles and Applications for DNA Amplification. New York: Stockton Press; Sambrook, J, et al., (1989) Molecular Cloning. A Laboratory Manual, Second Edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Recombinant uricases were extracted and purified, and their stability and activity were evaluated using a modification of standard assays. See Fridovich, I, (1965) JBiol Chem 240: 2491-2494; Nishimura, et al., (1979), and Example 1.

In one embodiment of the invention, uricase can be conjugated through a biologically stable, non-toxic, covalent bond with a relatively small number of PEG chains. Such linkages can include urethane (carbamate) linkages, secondary amine linkages, and amide linkages. Shearwater Polymers, Huntsville, AL markets several desirable activated PEGs for such conjugation.

For example, urethane linkages to uricase can be formed by incubating uricase in the presence of PEG-derived succinimidyl carbonate (SC) or 4-nitrophenyl carbonate (NPC). SC-PEG can be synthesized using the procedure described in US Patent 5,612,460, incorporated herein by reference. NPC-PEG can be synthesized by reacting PEG with 4-nitrophenyl chloroformate according to the methods described in Veronese, FM, et al., (1985) Appl Biochem Biotechnol 11: 141-152, and in US Patent 5,286,637, incorporated herein. present for reference. The methods described in the '637 patent are adapted to higher molecular weight PEGs by adjusting the concentrations of the reactants to maintain similar stoichiometry. An alternative method of synthesis of NPC-PEG is described in Büttner, W, et al., East German Patent Specification DD 279 486 A1.

Amide linkages to uricase can be obtained using an N-hydroxysuccinimide ester of a carboxylic acid derivative of PG (Shearwater Polymers). Secondary amine bonds can be formed using 2,2,2-trifluoroethanesulfonyl PEG (PEG tresyl; Shearwater Polymers) or by reductive alkylation using PEG aldehyde (Shearwater Polymers) and sodium cyanoborohydride.

In conjugates containing PEG with molecular weights between 5 kDa and 30 kDa, the maximum number of PEG chains that were combined per subunit, while retaining at least 75% of the uricolytic activity of the unmodified enzyme, has a range from an average of 2 chains for soybean uricase to more than 10 chains for PBC uricase (See test conditions in Example 1 and results in Figures 1A-5B). The last excess PEGylation corresponds to about one third of the total amino groups. In one embodiment of the invention, the average number of combined PEG chains per uricase subunit is between 2 and 10. In a preferred embodiment, the average number of combined PEG chains per uricase subunit is between 3 and 8. In a more preferred contribution, the average amount of PEG covalently coupled chains per uricase subunit is between 4 and 6. In another contribution, the molecular weight of PEG used for the combination reaction is between 5 kDa and 100 kDa , preferably between 10 kDa and 60 kDa, and ideally between 20 kDa and 40 kDa, for example 30 kDa.

IS 2 245 114 T3

There are several factors that can affect the choice of the optimal molecular weight and the number of PEG chains for combination with a certain form of uricase. In general, the reduction or elimination of immunogenicity without suffering a substantial loss of uricolytic activity may require the combination of relatively more lower molecular weight PEG chains, compared to the relatively few higher molecular weight PEG chains. For example, either 6 20 kDa PEG chains per subunit or 4 30 kDa PEG chains per subunit can give optimal results. Also, each different form of uricase can give different optimal results in relation to the size and number of chains. See Figures 1A-5B.

PEG conjugation converts all soluble and stable uricases into buffers for physiological pH, without the incorporation of an analogous substrate or inhibitor, as in the case of 8-azaxanthine which is used as a stabilizer in fungal uricase (Uricozyme^®) sold by Sanofi Winthrop in France and Italy. Two different PBC uricase conjugates, one with approximately 6 10 kDa PEG chains per subunit and the other with approximately 2 19 kDa PEG chains per subunit, retain significant activity after incubation in mouse serum for a period of time. greater than one month at 37 ° C. Furthermore, several of the conjugates of this invention recorded a circulating half-life, in the case of mice, greater than two days, compared to the half-lives of 8 or 24 hours reported for the PEG-modified uricases of mammals and microbes Chen, et al., (1981); Fuertges, F, et al., (1990) J Contr Release 11: 139-148; Fujita, T, et al., (1991) J Pharmacobiodyn 14: 623-629. The longer half-lives of injected protein drugs make them more cost-effective and can improve patient acceptance. Prolonged half-life is also an indication of products that are better tolerated by the body.

When PBC uricase PEG conjugates of the purified tetrameric form of the enzyme (four subunits of 35 kDa) were prepared, they demonstrated profoundly reduced immunogenicity in mice (Figure 7), compared to the moderate immunogenicity of PEG conjugates in forms larger amounts of the enzyme (eg, 35 kDa subunit octamers; see Figure 12) and very high immunogenicity in the unmodified enzyme. Mice deficient in uricase repeatedly injected with PEG-uricase of the present invention did not show hyperuricemia for more than two months and the structure and function of their kidneys were protected against damage caused by uric acid (Figures 8-11 ).

Injections of fully active conjugates of uricase PBC with 10 kDa PEG (Figures 3A-3B) significantly reduced hyperuricemia in homozygous, uricase-deficient mice (Figure 8). Urine uric acid levels were also significantly reduced in all uricase-deficient mice treated with modified uricase-PEG PBC. The uricase-deficient mice received a series of injections with a PEG-uricase preparation similar to those used to obtain the data in Figure 8. This treatment reduced the severity of a concentrating defect in the urine, as evidenced by measurements of urine osmolality under normal conditions and after a 12-hour period of water deprivation (Figure 9) and by their consumption of water and urine output, compared to corresponding measurements in genetically the same untreated mice. Similarly, it was shown that ten weeks of treatment, starting within the first ten days of life, in homozygous knockout mice lacking uricase (oux - / -) with a PEG-uricase of this invention decreased the severity of the disorder induced by the urate of the renal architecture, as seen by a magnetic resonance microscope (Figure 11). For more information on microscope methods, see Hedlund, LW, et al., (1991) FundAppl Toxicol 16: 787-797; Johnson, GA, et al., (1992) in JC Gore, (Ed.), Reviews of Magnetic Resonance in Medicine, Vol. 4 (pp. 187-219) New York: Pergamon Press.

Purified preparations of naturally occurring and recombinant uricases usually contain a mixture of enzyme aggregates, in addition to being in the tetrameric form (140 kDa). The percentage of each uricase preparation that is in the tetrameric form generally ranges from about 20% to 90%. Despite evidence that non-PEGylated aggregates of other proteins are highly immunogenic (see, for example, Moore, WV, et al., (1980) J Clin Endocrinol Metab 51: 691-697), earlier studies of PEG-uricase do not describe any efforts to limit the content of the aggregates, suggesting that the potential immunogenicity of the PEG-modified aggregates was not considered. Based on the observations of the inventors of the present invention, it appears that such aggregates were present in the enzyme preparations used for the previous synthesis of PEGuricase. Their presence can make the task of preparing non-immunogenic conjugates more difficult. Likewise, it appears that the large losses of uricolytic activity observed in previous efforts for PEGylated uricase were related to a large number of low molecular weight PEG chains that were combined. On the other hand, the uricase and PEGylation purification methods described herein allow the covalent incorporation of almost 10 PEG chains per subunit while retaining more than 75% of the uricolytic activity, at least for certain uricases, for example, pig-baboon chimeric uricase and enzyme derived from A. flavus (see Figures 3A and 4A)

In another preferred embodiment, substantially all aggregates of the tetrameric form of the enzyme can be removed by ion exchange or size exclusion chromatography at a pH between 9 and 10.5, preferably 10.2, prior to conjugation with PEG. of the resulting uricase preparation, substantially tetrameric. The molecular weight of uricase in each fraction derived from the preparation column can be monitored by any size analysis technique, including, for example, HPLC, conventional size exclusion chromatography, centrifugation, light scattering, capillary or capillary electrophoresis. gel in a non-denaturing buffer. In the case of tetrameric uricase isolated by size exclusion chromatography, fractions containing only the 140 kDa form of the enzyme can be pooled and used in conjugation with PEG. If

ES 2 245 114 T3 of tetrameric uricase isolated by ion exchange chromatography, the ion exchange column fractions can be analyzed for size to determine which fractions contain significant amounts of the tetrameric form with no detectable aggregates. Of the uricase thus assembled, at least 90% may be in the tetrameric form; undesirable aggregates may thus represent approximately 10%, 5%, 2% or less of the total uricase isolated.

The results presented here indicate that forms, even when extensively PEGylated, of PBC uricase larger than the tetramer are highly immunogenic in mice (Figure 12). Furthermore, in mice that received an injection of PEG conjugates of uricase aggregates, uricolytic activity in subsequent injections of PEGylated tetramers or PEGylated aggregates was rapidly cleared from the circulation. In contrast, conjugates prepared and derived from uricase containing less than 5% aggregates could be administered multiple times without accelerating their clearance rates (Figure 7) and without detectable antibody formation, as measured by a sensitive immunoassay of coupled enzymes. Furthermore, the use of highly purified tetrameric uricase distinguishes the improved conjugates of the present invention from the previously described PEG-uricase preparations. In contrast, the presence of a significant proportion (eg> 10%) of aggregates in the uricase preparations used by some other investigators may have led him to combine large amounts of PEG chains in order to suppress immunogenicity. Consequently, the enzymatic activity of the resulting conjugates was markedly reduced. In other contributions, the present invention expressly contemplates PEGylated uricase in a non-tetrameric form, as uricase dimers, while preparations of said conjugated uricase retain at least about 75% of their uricolytic activity and are essentially non-immunogenic.

In another contribution of the present invention, it is supplied with a mutated baboon liver uricase of unexpectedly increased potency, relative to that of the unmodified enzyme. This primary improved uricase was prepared using conventional recombinant DNA techniques. In particular, the substitution of an amino acid residue (tyrosine for histidine at position 97) in baboon uricase was not expected to result in a substantial increase in specific enzyme activity. When expressed in E. coli, this mutated protein was found to have at least 60% more specific activity than the recombinant baboon enzyme from which it was derived.

In another contribution, the specific activity was increased and / or the solubility of the non-PEGylated enzyme was improved when expressing truncated variants of chimeric porcine or pig-baboon uricases of which at least the first six amino acids of the amino terminals and / or at least the last three amino acids at the carboxyl terminal were removed from the expressed proteins (see Figure 6). Recombinant uricases with carboxyl-terminal truncation were able to improve solubility prior to PEGylation due to removal of the peroxisomal target sequence. See Miura, S, et al., (1994) Eur JBiochem 223: 141-146.

The PEG-uricase conjugates of the present invention are useful for reducing uric acid levels in body fluids and tissues of mammals, preferably in humans, and therefore can be used in the treatment of elevated uric acid levels. related to conditions including gout, tophi, kidney failure, organ transplantation, and malignancies. PEG-uricase conjugates can be injected into a mammal with elevated uric acid levels intravenously, subcutaneously, intradermally, intramuscularly or intraperitoneally among others. Alternatively, they can be converted to an aerosol and inhaled. See Patton, JS, (1996) Adv Drug Delivery Rev 19: 3-36 and US Patent 5,458,135. The effective dose of PEG-uricase of the present invention will depend on the level of uric acid, as well as the size of the individual. In one embodiment of this aspect of the invention, the PEG-uricase is administered in a pharmaceutically acceptable carrier or diluent in an amount ranging from about 10 pg to 1 g. In a preferred addition, the amount administered is between 100 pg and 500 mg. It is even more preferable that the administered amount of uricase is between 1 mg and 100 mg, for example 5 mg, 20 mg or 50 mg. The masses determined for the quantities of the doses with respect to the contributions refer to the quantity of the protein contained in the conjugate.

Pharmaceutical formulas containing PEG-uricase can be prepared using conventional techniques, for example, as described in Gennaro, AR (Ed.) (1990) Remington's Pharmaceutical Sciences, 18th Edition, Easton, PA: Mack Publishing Co. Among the recommended excipients For the preparation of injectable solutions, there are: phosphate buffered saline, Ringer's lactated solution, water, polyols and glycerol. Pharmaceutical compositions for parenteral injection contain aqueous or non-aqueous sterile pharmaceutically acceptable liquids, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just before use. These formulations may contain additional components, such as preservatives, solubilizers, wetting agents, emulsifiers, buffers, antioxidants, and diluents.

Also, PEG-uricase can be supplied as controlled release compositions for implantation in an individual in order to constantly control elevated uric acid levels in body fluids. For example, polylactic acid, polyglycolic acid, regenerated collagen, poly-L-lysine, sodium alginate, gellan gum, chitosan, agarose, multilamellar liposomes, and many other conventional depot formulations comprise bioerodible or biodegradable materials that can be formulated with biologically compositions. active. These materials, whether implanted or injected, progressively break down and release the active material into the surrounding tissue. For example, a method for encapsulating PEG-uricase comprises the method described in US Patent 5,653,974. The use of bioerodible, biodegradable or other depot formulations are expressly contemplated in the present invention. Also within the scope of the present invention is the use

ES 2 245 114 T3 of infusion pumps and matrix systems of traps for the release of PEG-uricase. PEG-uricase can also be contained in micelles or liposomes. The technology for encapsulating liposomes is well known in the field. See, for example, Lasic, D, et al., (Eds.) (1995) Stealth Liposomes. Boca Raton, FL: CRC Press.

The PEG-uricase pharmaceutical compositions of the invention will reduce the need for hemodialysis in patients at high risk of urate-induced renal failure, for example, organ transplant recipients (Venkataseshan, VS, et al., (1990) Nephron 56: 317-321) and patients with certain malignancies. In patients with large accumulations of crystalline urate (tophi), such pharmaceutical compositions will improve quality of life more rapidly than currently available treatments.

The following examples show the various aspects that have already been mentioned. These examples describe PEG-uricases prepared by combining activated (i.e., electrophilic) PEG derivatives of various sizes and compositions with naturally occurring porcine, fungal or bacterial uricases or with recombinant soybean, porcine or pig chimeric uricase- baboon. Results for activity, solubility, stability, pharmacokinetics, pharmacodynamics, and immunological studies are included. The data in Figures 8-11 provide evidence for the ability of the PEG-PBC modified uricase of this invention to correct hyperuricemia and hyperuricosuria, as well as to preserve renal structure and function in an animal model exhibiting hyperuricemia and hyperuricosuria which cause serious kidney damage (Wu, X, et al., (1994) Proc Natl Acad Sci USA 91: 742-746). These examples offer guidance to those of skill in the art regarding the production of non-immunogenic uricase conjugates that retain at least nearly 75% of the uricolytic activity of the unmodified enzyme.

Example 1

Purification of the tetrameric form of uricase

The tetrameric form of uricase (molecular weight ca. 140 kDa) was purified from a porcine liver uricase solution by preparative size exclusion or ion exchange chromatography, followed by analytical size exclusion chromatography. Porcine liver uricase was obtained from Sigma-Aldrich, St. Louis, MO, catalog # U2350 or U3377; or Boehringer Mannheim, Indianapolis, IN.

The preparative and analytical size exclusion chromatographies were performed with a pH of 10-10.5, preferably 10.2, in a 10 mM sodium carbonate buffer containing 0.1 M NaCl, on Superdex 200 columns that were previously calibrated. with proteins of known molecular weight. Superdex was obtained from Amersham Pharmacia, Piscataway, NJ. Any buffer that has the ability to maintain the desired pH and is compatible with the chemistry to be used for PEG coupling can be used. Such buffers are well known in the art. The ultraviolet absorbance of the eluate derived from the preparation column was monitored at 280 nm, while the uricase eluate portions corresponding to the molecular weight of the desired tetrameric form, but free of high molecular weight species, were collected for use. in non-immunogenic PEG-uricase as described in Example 2. Optionally, tetrameric forms of uricase can be isolated using another size exclusion medium such as Superose 12 (Amersham Pharmacia) or any other medium that is compatible with slightly alkaline solutions and has a suitable size fractionation range. Said means are obtained without problems, they are also well known in the field.

Ion exchange chromatography was carried out with a pH of 10-10.5, preferably 10.2, on Mono Q columns (Amersham Pharmacia, Piscataway, NJ) which was equilibrated with 0, sodium carbonate buffer. 1 M. Any buffer that is compatible with PEG coupling chemistry and has the ability to maintain the desired pH, at the ion concentration low enough to allow uricase uptake to the column, can be used. Such buffers are well known in the art. The ultraviolet absorbance of the eluate was monitored at 280 nm during the elution of uricase derived from the ion exchange resin by increasing the ion concentration of the applied buffer solution, for example by a linear gradient from 0 to 0.5 M NaCl. in the sodium carbonate buffer. Size exclusion HPLC was then used to identify fractions of the eluate containing the desired tetrameric form of uricase, without the detectable aggregates, for the synthesis of substantially non-immunogenic PEG-uricase. Optionally, the tetrameric form of uricase can be isolated using another ion exchange medium, such as Q-Sepharose (Amersham Pharmacia) or any other medium that is compatible with slightly alkaline solutions. Said means are obtained without problems, they are also well known in the field.

Uricase activity was tested using a modification of the standard methods. See, for example, Fridovich (1965); Nishimura, et al., (1979). Uric acid solutions were prepared daily in 50 mM sodium borate buffer, pH 9.2, to give final assay concentrations of 6-10 pM. The uricase preparations were diluted in this burate buffer containing bovine serum albumin (Sigma-Aldrich, St. Louis, MO, catalog # A-7030), so that the final albumin concentration in the assay was 0.1 mgr / mL. After mixing several dilutions of the enzyme with the substrate in a microtiter plate for a microplate reader, the rate of uric acid removal at 25 ° C was monitored at 292 nm every four seconds for 3 minutes. Of the samples in which between 10% and 40% of the substrate was consumed in a three minute period, at least 20 data points were used to calculate the maximum rate of reduction in absorption per minute. An international unit (IU) of uricase activity was defined as the amount of enzyme that consumes one micromole of uric acid per minute; specific activities are expressed as protein IU / mg. Some of the

Data regarding the relative uricase activities of Figures 1A-5B were obtained using 100 pM uric acid in the assay. Other results in relation to the uric acid velocity of 100 pM (Vi₀₀) were calculated from the values ​​of the Michaelis constant (K_M) and maximum speed (V_max) for the respective enzyme preparations, using the formula:

^V100 = 100 x V_max/ (KM + 100)

Where KM is expressed in micromole Example 2

PEG coupling for porcine tetrameric uricase

For a solution of tetrameric uricase in 0.1M sodium carbonate buffer, pH 10.2, 10-200 moles of an activated derivative of monomethoxyPEG, for example 4-nitrophenyl carbonate (NPC-PEG), of various sizes (5 kDa to 30 kDa) were added for each mole of uricase subunit (molecular weight 35 kDa). These and other recommended activated PEGs are available from Shearwater Polymers. Instructions for coupling these PEGs to proteins are included in the Shearwater Polymer catalog which can be found on their website, www.swpolymers.com, and in JM Harris, et al., (Eds.) (1997) Poly ( ethylene glycol) Chemistry and Biological Applications. ACS Symposium Series 680, Washington, DC: American Chemical Society. The coupling reaction was allowed to proceed at 0-8 ° C until the PEG coupling did not change significantly with time. Then the unreacted PEG was removed from the reaction product by chromatography and / or ultrafiltration.

The number of PEG chains coupled per uricase subunit was determined thanks to an adaptation of the methods described Kunitani, M, et al., (1991) J Chromatogr 588: 125-137; Saifer, et al., (1997) and Sherman, et al., (1997). Briefly, aliquots of PEGylation reaction mixtures or fractions derived from ion exchange or size exclusion preparation columns were characterized by size exclusion analysis HPLC on a TSK 5,000 PW column._XL at room temperature in 10 mM sodium carbonate buffer, pH 10.2, containing 0.1 M NaCl. The HPLC column was obtained from TosoHaas (Montgomeryville, PA). Proteins and PEG were monitored by ultraviolet absorbance and refractive index detectors. The protein volume of the conjugates was calculated from the ultraviolet absorbance compared to that of the appropriate standard of unmodified uricase. The amount of PEG in the conjugate was calculated from the area of ​​the refractive index peak point, corrected for the protein contribution to the refractive index, relative to the area of ​​the refractive index peak point of the appropriate PEG standard. .

Figure 2A shows the retention of activity by PEGylated porcine liver uricase as a function of the number of PEG chains coupled per subunit. Our data (a, □) are compared with Chen, et al., (1981). The data point within a large circle denotes a conjugate that Chen, et al., (1981) described as non-immunoreactive. As shown in Figure 2A, conjugates of porcine tetrameric uricase with up to 6 PEG chains of 30 kDa per subunit or up to 7 PEG chains of 5 kDa per subunit retained at least 75% of the enzyme activity. not modified. The apparent increase in specific activity with increasing numbers of 5 kDa or 30 kDa PEG chains (up to almost 4 chains per subunit) may reflect the relative insolubility or instability of the unmodified enzyme compared to the conjugates. As shown in Figure 2B, conjugates of porcine uricase with an average of more than 3 PEG 30 kDa chains per subunit contain a greater mass of PEG than that which Chen, et al., (1981) considered sufficient to rule out immunoreactivity.

Example 3

Properties of PEG conjugates of PBC-tetrameric recombinant uricase

Recombinant pig-baboon chimeric uricase (PBC) cDNA was subcloned into the expression vector pET3d (Novagen, Madison, WI) and the resulting plasmid construct was transformed and expressed in a copy of Escherichia coli BL21 (DE3) pLysS (Novagen ). These procedures were carried out using methods well known in the field of molecular biology. See Erlich (1989); Sambrook, et al., (1989); Ausubel, F, et al., (Eds.), (1997) Short Protocols in Molecular Biology. New York: John Wiley &amp; Sons.

Figure 6 shows the deduced amino acid sequence of PBC uricase (amino acids 1-225 of SEQ ID NO: 1) and amino acids 226-304 of SEQ ID NO: 2), compared to porcine sequences (SEQ ID NO: 1) and baboon (SEQ ID NO: 2). Residues in the sequence that differ from those in the porcine sequence are highlighted in bold. Wu, et al., (1989) determined the porcine and baboon sequences for the first time, something that has been confirmed by the authors of the present invention. SEQ ID NO. 1 is identical to GenBank Accession # p16164, except for the absence of the initial methionyl residue in the sequence. SEQ ID NO. 2 is identical to GenBank Accession # p25689, except for the absence of the initial methionyl residue and a change from threonine to histidine at residue 153 in the GenBank sequence (residue 154 of Figure 6).

IS 2 245 114 T3

The tetrameric form of uricase PBC was separated and coupled to PEG of different molecular weights as indicated in Examples 1 and 2. Conjugates prepared with 5 kDa, 10 kDa or 30 kDa PEG contained up to 10 PEG chains per subunit. Preparations with PEG of at least 10 kDa retained more than 95% of the initial specific activity of recombinant uricase (Figures 3A-3B).

The following properties of a tetrameric PBC uricase conjugate with approximately 6 PEG chains of 10 kDa per subunit were indicated in the indicated figures: the lack of immunogenicity (Figure 7) and the efficacy in uricase-deficient mice to 1) correct hyperuricemia and hyperuricosuria (Figures 8); 2) reduce the severity of a concentrating defect in urine (Figure 9); and 3) decrease the severity of nephrogenic diabetes insipidus. In addition, this PEG-uricase reduces the severity of kidney damage caused by uric acid, as observed by the magnetic resonance microscope (Figure 11).

Figure 7 shows the PBC uricase activity in mouse serum 24 hours after each of the four or five intraperitoneal injections of PEG-uricase relative to the value 24 hours after the first injection. PEG conjugates were made from three different PBC uricase preparations using two different techniques for PEG activation. A preparation (·) was tested in mice lacking uricase (uox - / -); the other two (Δ,) were tested in normal bAlb / C mice. The most immunoreactive preparation (Δ) was made from purified PBC uricase containing an unknown quantity of uricase aggregates coupled to an average of 7 PEG chains of 5 kDa per subunit, using the succinimidyl carbonate derivative of PEG ( SC-PEG). Zalipsky, US Patent 5,612,460, which is incorporated herein by reference. The moderately immunoreactive preparation () was prepared by coupling a PBC uricase preparation containing 11% aggregates to an average of 2 PEG chains of 19 kDa per subunit, using a 4-nitrophenyl carbonate derivative of PEG (NPC -PEG). Sherman, et al., (1997). The least immunoreactive conjugate (·) was prepared by coupling an average of 6 NPC-PEG chains of 10 kDa per subunit to a PBC uricase preparation containing &lt;5% uricase aggregates.

Figure 8 shows the inverse relationship between uric acid concentrations in serum and urine, as well as the activity of PEG-uricase injected into the serum of a mouse lacking uricase (uox - / -). Injections at time zero and after 72 hours contained 0.43 IU of PBC uricase conjugated to an average of 6 chains of 10 kDa PEG per enzyme subunit.

Figure 9 shows that treatment of uricase-deficient mice with modified uricase-PEG PBC decreased the severity of a concentrating defect in urine. The mean and standard deviation of the data relative to urine osmolality are shown in two mice that had one copy of the normal murine uricase gene (uox +/-), six homozygous uricase-deficient untreated mice (uox - / -) and six homozygous mice lacking uricase that received 10 injections between the third and 72nd day of life with 95 or 190 mlU of PEG-uricase. Mice in each of the genetic groups received water ad libitum (solid bars) or were deprived of water for a period of 12 hours (warped bars) prior to collection of their urine.

Figure 10 shows that treatment of uricase-deficient mice with PEG-PBC-modified uricase reduced the severity of nephrogenic diabetes insipidus, which is characterized by unusually high water intake and unusually high urine output. The genetic background of the mice and the treatment protocol were the same as in Figure 9. Average and standard deviation of daily water consumption (solid bars) and urine output (warped bars) are shown for three groups of six mice.

Figure 11 shows that treatment of uricase-deficient mice with modified uricase-PEG PBC decreased the severity of uric acid-induced nephropathy, as seen through the magnetic resonance microscope. The genetic background of the three groups of mice and the treatment protocol were the same as those used in Figures 9 and 10. The magnetic resonance microscope study was conducted at the Center for in vivo Microscopy, Duke University Medical Center, Durham, North Carolina.

In addition to the results summarized in Figures 8-11, it was shown that uric acid levels in the urine of all uricase-deficient mice decreased dramatically after treatment with modified uricase PEG PBC. Finally, Figure 12 shows that, unlike the PEG-modified tetrameric form of uricase PBC, the octameric form (molecular weight = 280 kDa), even if it is extensively PEGylated, is immunogenic in mice. This property is reflected in the accelerated clearance of the PEG-modified octamer over a period of 5 days after a single intraperitoneal injection. The same mice received another injection with the same dose of the same PEG-uricase preparations on days 8 and 15. Twenty four hours after the second and third injections, uricolytic activity was not detectable in the sera of mice injected with the PEGylated octamer, but it was easier to detect in the sera of the mice injected with the PEGylated tetramer. These findings, in combination with the accelerated removal of the PEGylated octamer that was observed after the first injection (Figure 12), support the utility of removing all major uricase forms from the tetramer prior to PEGylation of the enzyme.

IS 2 245 114 T3

Example 4

PEG conjugation of uricase derived from Candida utilis

Candida utilis derived uricase was obtained from Sigma-Aldrich (St. Louis, MO; catalog # U1878) or Worthington Biochemical Corporation (Freehold, NJ; catalog # URYW). Proceeding as described in examples 1 and 2, the tetrameric form was isolated and the PEG conjugates were synthesized with 5 kDa, 10 kDa or 30 kDa PEG (Figures 1A-1B). Figure 1A shows the retention of activity due to PEGylated uricase derived from Candida utilis as a function of the number of PEG chains coupled per subunit. Our data (a, ·, □) were compared with those of Nishimura, et al., (1979); Nishimura, et al., (1981); Chen, et al., (1981); Davis, et al., (1981); Tsuji, et al., (1985); Yasuda, et al., (1990) and Fujita, et al., (1991). The data illustrated by the large circles denote conjugates that Nishimura, et al., (1979 or 1981) reported as non-antigenic or that Chen, et al., (1981) reported as non-immunoreactive.

Figure 1B shows the retention of activity by PEGylated uricase derived from Candida utilis as a function of the total mass of PEG coupled per subunit. The data of the present inventors (a, ·, □) are compared with those that appear in the reports of Figure 1A. The data illustrated by the large circles have the same meaning as in Figure 1A.

As shown in Figure 1A and 1B, conjugates with an average of up to 6 PEG chains of 5 kDa or 30 kDa, or 9 PEG chains of 10 kDa per subunit retained at least 75% of the enzyme activity. not modified. The apparent increase in specific activity when an increasing number of 30 kDa PEG chains are incorporated (up to 5 or 6 chains per subunit) may reflect the relative insolubility or instability of the unmodified enzyme compared to the conjugates.

Example 5

PEG conjugation of uricase derived from Aspergillus flavus

Aspergillus flavus derived uricase was obtained from Sanofi Winthrop (Gentilly Cédex, France). Proceeding as described in Example 2, conjugates with PEGs of various molecular weights were synthesized (Figures 4A-4B). Conjugates made by coupling enzyme derived from A. flavus with an average of up to 12 5 kDa PEG chains or up to 7 30 kDa PEG chains per subunit retained at least 75% of the initial fungal uricase specific activity.

Example 6

PEG conjugation of soy derived uricase

Recombinant uricase derived from the soybean root nodule (also known as nodulin 35) was prepared and purified according to the method of Kahn and Tipton (Kahn, K, et al., (1997) Biochemistry 36: 4731-4738) and Dr. Tipton (University of Missouri, Columbia, MO) supplied it. Proceeding as described in Examples 1 and 2, the tetrameric form was isolated and the conjugates were prepared with PEG of various molecular weights (Figures 5A-5B). Unlike Candida utilis-derived uricase (Figure 1A), porcine uricase (Figure 2A), pig-baboon chimeric uricase (Figure 3A), and Aspergillus flavus-derived uricase (Figure 4A), the soybean enzyme tolerated coupling of only about 2 PEG chains of 5 kDa or 30 kDa per subunit with retention of at least 75% of initial uricolytic activity.

Example 7

PEG conjugation of uricase derived from Anthrobacter globiformis

Anthrobacter globiformis-derived uricase was obtained from Sigma-Aldrich (catalog # U7128). See Japanese patent 9-154581. Proceeding as described in Examples 1 and 2, the tetrameric form was isolated and conjugates with 5 kDa and 30 kDa PEG were prepared. While conjugates with an average of more than 3 5 kDa PEG chains per subunit retained less than 60% of the initial specific activity, conjugates with an average of approximately 2 30 kDa PEG chains per subunit retained at least 85% of the initial specific activity.

Example 8

PEG conjugation of porcine uricases and PBC truncated at the amino terminus

Porcine uricases and recombinant PBCs derived from which the first six amino acids were removed at the amino terminus were expressed in E. Coli and purified therein by means of standard techniques, as described in Example 3. Proceeding as described in Examples 1 and 2, PEG conjugates of amino truncated uricases are synthesized to produce substantially non-immunogenic conjugates that retain at least 75% of the initial specific activity.

IS 2 245 114 T3

Example 9

PEG conjugation of porcine uricases and PBC truncated at the carboxyl terminus or at the amino and carboxyl termini

Porcine uricases and recombinant PBC derived from which the last three amino acids were removed at the carboxyl terminal were expressed in E. coli and purified therein by means of standard techniques, as described in Example 3. Removal of the carboxyl terminal can increase the solubility of unmodified enzymes by removing the signal from the peroxisomal target. See Miura, et al., (1994). Proceeding as described in Examples 1 and 2, PEG conjugates of carboxyl truncated uricases are synthesized to produce substantially non-immunogenic conjugates that retain at least 75% of the initial specific activity. Figure 6 depicts the recombinant PBC uricase sequence truncated by six residues at the amino terminus and three residues at the carboxyl terminus (PBC-NT-CT). This uricase was expressed, purified, and PEGylated as described in Examples 1, 2, and 3 to produce substantially non-immunogenic conjugates that retain at least 75% of the initial specific activity.

Example 10

PEG conjugation of porcine uricase mutants containing increased number of PEG binding sites

Recombinant porcine uricases were prepared according to Example 3, in which the potential number of PEG binding sites was increased by replacing one or more arginine residues with lysine. See Hershfield, MS, et al., (1991) Proc Natl Acad Sci USA 88: 7185-7189. The amino acid sequence of an example of a mutant (uricase PKS) is shown in Figure 6, in which arginine at residue 291 is replaced by lysine and threonine at residue 301 by serine. Proceeding as described in Examples 1 and 2, PEG is conjugated to this uricase to produce substantially non-immunogenic conjugates that retain at least 75% of the initial recombinant uricase specific activity.

Example 11

PEG conjugation of a recombinant baboon uricase mutant

Using standard molecular biology methods, as in Example 3, recombinant baboon uricase is constructed having an amino acid substitution (histidine for tyrosine) at position 97 (see baboon sequence in Figure 6). Proceeding as described in Examples 1 and 2, PEG conjugates of tetrameric form of the recombinant baboon uricase mutant are synthesized to produce conjugates of substantially reduced immunogenicity that retain at least 75% of the initial specific activity of recombinant uricase.

Example 12

Immunogenicity of PEG conjugates derived from Candida utilis, Aspergillus flavus and Arthrobacter globiformis

The uricases of Candida utilis, Aspergillus flavus and Arthrobacter globiformis were obtained as described in Examples 4, 5 and 7, respectively. Proceeding as described in Examples 1 and 2, the PEG conjugates are synthesized with PEGs of 5 kDa, 10 kDa, 20 kDa or 30 kDa. The immunogenicity of these conjugates is significantly reduced or eliminated.