Untitled record

The present invention relates to genetically modified proteins having uricolytic activity. More particularly, the present invention relates to proteins consisting of truncated urate oxidases and processes for their preparation.

The terms "urate oxidase" and "uricase" are used interchangeably with each other. Urate oxidases (uricases; EC 1.7.3.3) are enzymes that catalyze the oxidation of uric acid to a more soluble product, allantoin. Allantoin is a purine metabolism product that is easier to select. No enzymatically active uricase is produced in humans, which is the result of numerous mutations in the uricase gene during the evolution of higher primates. Wu, X., et al., J. Mol. Evol. 34: 78-84 (1992), which is incorporated herein by reference in its entirety. Consequently, in susceptible individuals uric acid in excess blood! concentration (hyperuricemia) can lead to painful arthritis (gout), malformation of urate deposits (tophus) and kidney failure. In some affected individuals, the available active substances - such as allopurinol (an inhibitor of uric acid synthesis) results in treatment-limiting, confounding effects or does not adequately alleviate these conditions (Hande, KR, et al., 1984, Am. J. Med. 7: 47-56; Wood, AG Bailliere's Clin. Rheumatol. 4, 177-192 (1990), which is 18662 KB

<img file="HU0700730A2_D0001.tif" />

- 2 ▼ must be considered in their entirety as part of the teaching].

Uricase injections can reduce, at least temporarily, hyperuricemia and hyperuricosuria. Because uricase is a foreign protein in the human body, in a few percent of treated patients, unmodified protein from Aspergillus flavus induced anaphylactic reactions even after the first injection [Pu, CH. et al., Leukemia 11: 1813-1816 (1997), which is incorporated herein by reference in its entirety; and the immune responses elicited by it limit the applicability of chronic or intermittent treatment [Donadio, D., et al., Nouv. Presse Med 10: 711-712 (1981); Leaustic, M., et al., Rev. Rhum. Today. Osteoartic. 50, 553-554 (1983), which are incorporated herein by reference in their entirety.

The sub-optimal performance of available treatments for hyperuricemia has been recognized decades ago [cf. Kissel, P. et al., Natúré 217, 72-74 (1968), which is to be considered in its entirety as part of the teachings]. Similarly, it has been recognized many years ago that certain groups of patients with severe gout may benefit from a safe and effective form of injectable uricase. Davis, FF et al., In: Enzyme Engineering, 4. Vol. 169-173. Broun, GB et al., New York, Plenum Press, 1978; Nishimura, H., et al., Enzyme 24: 261-264 (1979);

Nishimura, H., et al., Enzyme 26, 49-53 (1981); Davis, S..

Et al., Chain 2 (8241), 281-283 (1981); Abuchowski, A., et al., J. Pharmacol. Exp. Ther. 219: 352-354 (1981); Chen, RH-L. et al., Biochim. Biophys. Acta 660: 293-298 (1981); Chua, CC, et al., Ann. Int. Med. 109: 114-117 (1988); Greenberg, ML, et al., Anal. Biochem. 176, 290293 (1989)], all of which are to be considered as part of the teachings. Animal uricases are almost insoluble in solvents compatible with safe injection. U.S. Patent No. 3,616,231, which is hereby incorporated by reference in its entirety. Certain uricases from plants or microorganisms exhibit higher solubility in pharmaceutically acceptable solvents, but injection of microbial enzymes can rapidly induce immune responses that may cause life-threatening allergic reactions or induce uricase inactivation and / or circulatory clearance. (Donadio et al., 1981); Leaustic et al. (1983)]. Of mammals (e.g. pigs or baboons) or insects (e.g. Enzymes based on the deduced amino acid sequence of uricases from Drosophila melanogaster or Drosophila pseudoobscura (Wallrath, LL et al., Mol. Cell. Biol. 10: 5114-5127 (1990)], due to immunogenicity problems and their insolubility at physiological pH, have not proved to be suitable candidates for clinical use.

Previously, researchers have investigated uric acid into allantoin in vivo

<img file="HU0700730A2_D0002.tif" />

▼ was injected with uricase [cf. Púi et al. (1997)]. This is the basis for the use of Aspergillus flavus fungal uricase (Uricozyme®) in France and Italy to prevent or temporarily correct hyperuricemia associated with the cytotoxic therapy of haematological malignancies and to temporarily reduce severe hyperuricemia in gout patients. Potaux, L., et al., Nouv. Presse Med. 4, 1109 (1975); Legoux, R., et al., J. Bioi. Chem. 267: 8565-8570 (1992); and U.S. Patent Nos. 5,382,518 and 5,541,098, each of which is hereby incorporated by reference in its entirety. Because Uricozyme® is short circulating in the circulation, it requires daily injection and, due to its immunogenicity, is not suitable for long-term therapy.

Certain uricases may be advantageously used in the preparation of conjugates with polyethylene glycol or polyethylene oxide (both hereinafter referred to as "PEGs") to produce pharmaceutically effective uricase forms with increased half-life and reduced immunogenicity. U.S. Patent Nos. 4,179,337; 4,776,106; 4,847,325; and 6,576,235; and U.S. Patent Application Publication No. US2003 / 0082786A1, which are incorporated herein by reference in their entirety. Conjugates of uricase with polymers other than PEG have also been disclosed (see U.S. Pat. No. 4,460,683).

- 5 patent descriptions, which are to be considered in their entirety as part of the teaching).

In almost all published attempts to PEGylate uricase (i.e., covalently linking PEG to uricase), PEG is predominantly linked to amino groups (including the N-terminal amino acid and accessible lysines). In the commonly used uricases, the total amount of lysine in each of the four identical subunits is 25 (in Aspergillus flavus; U.S. Patent No. 5,382,518, which is hereby incorporated by reference in its entirety) and 29 [porcine; ld. Wu, X., et al., Proc. Natl. Acad. Sci. USA 86, 9412-9416 (1989), which is incorporated herein by reference in its entirety. In the native conformation of the enzyme, some lysines are not available for PEGylation. The most common way to reduce the immunogenicity of uricase is by coupling a large number of low molecular weight PEG strands to uricase, which in each case resulted in a significant reduction in the enzymatic activity of the conjugates formed.

Single intravenous injection of a 5 kD PEG-linked formulation of Candida utilis uricase in five subjects (pre-injection serum urate concentration of 6.2 mg / dL, which was within normal range) reduced serum urate levels to undetectable levels [Davis et al. (1981)]. Patients were given another vaccine four weeks later, but no reactions were reported. The second (and

After the last injection, no antibodies against uricase were detected by a relatively insensitive gel diffusion assay. The results of chronic or subchronic treatment of humans or experimental animals are not disclosed in this reference.

Uricase from Arthrobacter protoformiae is 5 kD

Its PEG-linked formulation was used to temporarily control hyperuricemia of a lymphoma patient (pre-injection serum urate concentration 15 mg / dL) [Chua et al. (1988)]. The critical condition of the patient and the short duration of treatment (four injections over 14 days) did not allow an evaluation of the sustained efficacy and safety of the conjugate.

Modification of each uricase subunit with 2-10 strands of high molecular weight PEG (&amp; gt; 5 kD - 120 kD) afforded improved immunological recognition (Saifer et al., U.S. Patent No. 6,576,235); Adv. Exp Med Bioi. 366: 377-387 (1994), each of which is to be considered as part of the teachings. This solution resulted in retention of more than 75% (after PEGylation) of the uricase of various species (after PEGylation); improved uricase circulation time; and allowed repeated injection of the enzyme without inducing antibodies in mice or rabbits.

Hershfield and Kelly (WO 00/08196; U.S. Pat. No. 60/095489).

- 7 Dalmatian Announcements, which are intended to be incorporated herein in their entirety, have developed methods for the production of recombinant uricase proteins from mammalian species with an optimal number of PEGylation sites. PCR techniques have been used to increase the number of lysines available at selected sites in the enzyme, and the enzyme has been designed to reduce, after proper PEGylation, the recognition by the immune system while substantially retaining its uricolytic activity. Some of the uricase proteins they generate have been truncated at the C-terminus and / or N-terminus. The authors have provided no guidance on other specific genetically induced modifications of the protein.

As used herein, "immunogenicity" refers to the induction of an immune response by injection of a PEG-modified or unmodified uricase (as an antigen), while "antigenicity refers to the reaction of an antigen with pre-existing antibodies. Antigenicity and immunogenicity are collectively referred to as "immunoreactivity. In previous experiments on the study of PEG-uricase, immunoreactivity was evaluated by a variety of methods, such as: 1) reacting PEG-uricase with pre-generated antibodies in vitro; 2) determining induced antibody synthesis; and 3) accelerated clearance after repeated injections.

Previous attempts to reduce the immunogenicity of uricases from various sources by coupling different numbers of PEG strands through different linker components have been unsuccessful. PEGuricases were first discovered by FF Davis, Y. Inada, and co-workers [cf. Davis et al. (1978); United States Patent 4,179,337; Nishimura et al. (1979); Japanese Patent Laid-Open No. 55-99189 62-55079; which sources are to be considered in their entirety as part of the teaching]. The conjugate disclosed in U.S. Patent No. 4,179,337 was synthesized by reacting a 2,000-fold molecular weight of a uricase of unspecified origin and a 750 D molecular weight PEG, suggesting that each uricase moiety was likely to have a large number of polymer molecules attached. U.S. Patent 4,179,337 discloses active, water-soluble and non-immunogenic conjugates of various polypeptide hormones and enzymes, including oxidoreductases, three of which are uricase, three to about 20,000 D (preferably about 500- 5,000 D) PEG or poly (propylene glycol) coupling has been discovered. In addition, said US patent emphasizes the coupling of 10-100 polymer fibers per enzyme molecule and retaining at least 40% of the enzymatic activity. Test results on the extent of PEG binding to the accessible amino groups of uricase, the residual specific uricolytic activity and the immunoreactivity of the conjugate have not been reported.

··· ··· * .

• · « · · ·

Previous publications have reported that the coupling of different numbers of PEG filaments to uricase from Candida utilis resulted in a significant reduction in in vitro uricolytic activity. Linking a large number of 5 kD PEGs to uricase from porcine liver

<td colspan="4">gave similar results [cf. Chen's publication and</td><td rowspan="2">same al.</td>

<td>on this</td><td colspan="2">a group symposium report;</td><td>Chen and</td>

<td> (1981</td><td>); Davis et al. (1978)].</td><td></td><td></td><td></td>

<td colspan="2">Seven previous investigations</td><td>the</td><td>uricase</td><td>immune-</td>

of PEGylation, while five additional studies reported a complete loss of immunoreactivity. In three of these five studies, the loss of immune reactivity was associated with a significant reduction (to 15%, 28% and 45% of the original activity) of uricolytic activity [Nishimura et al., 1979 (15% activity). ); Chen et al., 1981 (28% activity); Nishimura et al., 1981 (45% activity). In the fourth study, PEG was linked to 61% of the available lysine, but no residual specific activity was found [Abuchowski et al. (1981)]. However, a research team (two of the same scientists using the same procedures) reported elsewhere that this level of coupling results in a residual activity of only 23% to 28% [Chen et al. (1981)]. Abuchowski et al., And Chen et al. The 1981 publication shows that, in order to significantly reduce the immunogenicity of uricase, the available lysines • ··· • · «« « 60% need to be switched to PEG. In Fifth Publication (which reported the elimination of uricase immunoreactivity), the degree of PEG-coupling, residual uricolytic activity, and nature of PEGprotein binding were not described [Veronese, FM et al. (1997) in: JM Harris et al. (ed.) ·: “Poly (ethylene glycol) Chemistry and Biological Applications, ÁCS Symposium Series 680 (182-192), Washington, DC: American Chemical Society, which is to be considered in full part of the teaching].

Although PEG was coupled to a minor proportion of uricase leases, the conjugate produced in experimental animals, although decreased, did not completely disappear (Tsuji, J. et al., Int. J. Immunopharmacol 7: 725-730 (1985)). in its scope it shall be considered as part of the teaching; PEGylation of 28-45% of amino groups]; Yasuda et al., Chem. Pharm. Bull. 38, 2053-2056 (1990), which is to be read in its entirety as part of the teaching; PEGylation of 38% of amino groups]. The residual uricolytic activity of the corresponding adducts ranged from less than 33% of their initial activity (Tsuji et al.) To 60% (Yasuda et al.). Tsuji et al. In addition to 5 kD PEG, PEG uricase conjugates were synthesized using 7.5 Kd and 10 kD PEG. Each of the conjugates generated showed some degree of immunogenicity and antigenicity, but had a significantly reduced enzymatic activity.

Davis et al. (1981) reported that the PEGylated preparation of uricase from Candida utilis11 (which was safely administered twice in five humans) retained only 11% of the initial activity. Years later, PEGylated uricase from Arthrobacter protoformiae was administered to a single patient with advanced lymphoma and severe hyperuricemia four times [Chua et al. (1988)]. Although the residual activity of the latter enzyme preparation was not measured, Chua et al. the patient's serum was detected 26 days after the first injection of PEG-uricase using an enzyme-linked immunosorbent assay (ELISA), and the absence of antibodies against uricase was detected.

Previous studies with PEGylated uricase showed that catalytic activity was significantly reduced by coupling sufficient PEG filaments with enzymes to significantly reduce immunoreactivity. In addition, most prior formulations of PEG-uricase have been synthesized using cyanuric chloride-activated PEG [cyanuric chloride is a triazine derivative (2,4,6-trichloro-1,3,5-triazine) that has been shown in rabbits to be a novel antigen. -determinates delivery and induces antibody production; ld. Tsuji et al. (1985)].

Japanese Patent 3-148298 (Sano et al., Which is incorporated herein by reference in its entirety) discloses modified proteins, including uricase, which have been derivatized with 1-12 kD PEG and which have reduced antigenicity and " improved long-term effects; further explored this ♦ ·· ·

- Methods of making 12 derivatized peptides. However, the number of PEGs used for derivatization, the enzyme assays and biological assays used, and the meaning of "improved sustained expression" were not disclosed. Japanese Patent Nos. 55-99189 and 62-55079 (both written by Y. Inada), which are incorporated herein by reference in their entirety, include PEG-triazine and bis-PEG-triazine (abbreviated as PEG).₂), uricase conjugates have been disclosed [cf. Nishimura et al., 1979 and 1981]. In the first type of conjugate, PEGs had molecular weights of 2 kD and 5 kD, whereas in the second, only 5 kD of PEG was used. Nishimura et al. (1979) described a 15% retention of uricolytic activity after modification of the available lysines with 5 kD linear PEG, whereas 46% or 36% of lysines had PEG₂after modification of the uricolytic activity was reported in 31% and 45%, respectively. Nishimura et al. (1981)].

Previously studied uricase proteins are natural or recombinant proteins, but studies using SDS-PAGE analysis or western blot showed the presence of unexpected low molecular weight peptides that appeared to be degradation products and increased with time. The present invention relates to mutant recombinant uricase proteins which have truncated sequence and enhanced structural stability.

The present invention provides novel recombinant uricase proteins. In one embodiment, you will find * ··· ».

- The proteins of the 13 copies have a truncated sequence and contain mutagenized amino acids as compared to naturally occurring uricase proteins. In preferred embodiments of the invention, mutations occur at or around amino acids 7, 46, 291, and 301. Conservative mutations in any part of the peptide are also within the scope of the invention.

The present invention provides a mutant recombinant uricase which is truncated by 1-20 amino acids while retaining the uricolytic activity of the naturally occurring uricase. Truncations occur at or near the terminus of the sequences, and the protein contains the terminal amino acids. These mutations and truncations may enhance the stability of the protein carrying such mutations.

In another embodiment of the invention, there is provided a means for metabolizing uric acid comprising a novel recombinant uricase protein having uricolytic activity. As used herein, uricolytic activity refers to the enzymatic conversion of uric acid to allantoin.

The present invention further provides a host cell capable of producing a uricase truncated with 1 to 20 amino acids, which contains mutagenized amino acids and retains its uricolytic activity.

In one embodiment, isolated truncated mammalian uricase is disclosed which - at the N-terminus, the C-terminus, or both - is ca. 1-13 amino acids are truncated ».» «,

<img file="HU0700730A2_D0003.tif" />

14 amino acid sequences of mammalian uricase, ca. and at position 46 it contains an amino acid substitution. In preferred embodiments of the invention, the N-terminal amino acid of the uricase comprises alanine, glycine, proline, or threonine. We also uncover a uricase in which ca. In position 46 it is replaced by threonine or alanine. In one embodiment, the uricase comprises the amino acid sequence of SEQ ID NO: 8. In another embodiment, the uricase is conjugated to a polymer to form, for example, a polyethylene glycoluricase conjugate. In preferred embodiments of the invention, the polyethylene glycol uricase conjugates contain from 2 to 12 (preferably 3-10) polyethylene glycol molecules on each uricase subunit. In preferred embodiments, each polyethylene glycol molecule in the polyethylene glycol-uricase conjugate has a solubility of about 10%. Between 1 kD and 100 kD; approx. Between 1 kD and 50 kD; approx. 5 kD to 20 kD; or approx. It has a molecular weight of 10 kD. Further disclosed are pharmaceutical compositions containing a uricase of the invention, including a polyethylene glycol uricase conjugate. In one embodiment, the pharmaceutical composition is suitable for repeated administration.

There is further disclosed a method of reducing uric acid levels in a biological fluid of a patient in need thereof comprising administering to the patient a pharmaceutical composition comprising the uricase of the invention. In one preferred embodiment, the blood uric acid is lowered.

In one embodiment of the invention, the uricase comprises a peptide comprising the sequence from position 44 to position 56 (SEQ ID NO: 14) of the porcine KS-AN uricase.

In one embodiment, the uricase protein comprises an N-terminal methionine. In a preferred embodiment, the uricase comprises the amino acid sequence set forth in SEQ ID NO: 7.

In addition, isolated nucleic acids comprising a nucleic acid sequence encoding a uricase of the present invention (e.g., uricases comprising the amino acid sequence set forth in SEQ ID NOs: 7, 8, 12, or 13) are disclosed. In one embodiment, the isolated nucleic acid is operably linked to a heterologous promoter (e.g., osmB promoter). Vectors containing nucleic acids encoding the uricases of the invention and host cells comprising such vectors are further disclosed. In one embodiment of the invention, the nucleic acid comprises SEQ ID NO: 7. In addition, methods of producing uricase are disclosed, comprising culturing a host cell of the invention under conditions that facilitate the production of uricase by the host cell and isolating the uricase produced.

The following is a brief description of the figures.

Figure 1 shows the structure of plasmid pOUR-P-AN-ks-1. The numbers beside the restriction sites represent the nucleotide positions relative to the Haell site (position 1). Rest16 restriction sites lost during cloning are indicated in parentheses.

Figure 2 shows the DNA sequence of porcine KS-AN uricase (Figure 9).

SEQ ID NO: 7) and its deduced amino acid sequence (SEQ ID NO: 7). Amino acid numbering is given relative to the complete sequence of porcine uricase. Following the initial methionine, the aspartic acid at position 7 of the porcine yeast sequence is replaced by threonine. The restriction sites used to perform the various steps of subcloning are shown in the figure. The 3 'untranslated sequence is in lowercase. The translational stop codon is marked with an asterisk.

Figure 3 shows the relative alignment of the deduced amino acid sequences of different recombinant uricase sequences (porcine (SEQ ID NO: 11), PBC-ANC (SEQ ID NO: 12) and Porcine KS-ΔΝ (SEQ ID NO: 7)). . Asterisks indicate positions where the porcine KSΔΝ amino acids differ from the published sequence of porcine uricase, while circles indicate positions where the porcine KSΔΝ amino acids differ from the PBC-ΔΝ sequence. The dashed lines indicate amino acid deletions.

Figure 4 shows porcine uricase and Figure 1-3. The results of the SDS-PAGE analysis of the highly purified uricase variants described in Examples 1 to 8 are shown. The date of production (month / year) and the number of bands corresponding to each sample are given below: Lane 1: Molecular Weight · · · · · · · · · · · · · · · · · · · · · · · · · · · · • · · ·····

- 17 - ........... * markers; Lane 2: porcine KS-ΔΝ (7/98); Lane 3: pig (9/98); Lane 4: porcine KS (6/99); Lane 5: porcine KS (6/99);

6th lane: porcine ΔΝ (6/99); Lane 7: porcine KS-ΔΝ (7/99); 8th

lane: porcine KS-ΔΝ (8/99). The weight of the molecular weight markers is shown on the Y-axis and the number of bands at the top of the figure.

Figure 5 depicts the pharmacokinetic profiles of PEGylated (9x10 kD) porcine KS-AN-uricase following intramuscular (IM), subcutaneous (SC) and intravenous (IV) injection in rats by monitoring enzymatic activity in blood samples. . Plasma samples collected at the indicated times were determined for uricase activity by a colorimetric assay. Activity values ​​(mAU = milli-absorbance unit) represent the rate of enzymatic reaction per 1 μΐ blood sample. The bioavailability of the injected uricase (amount of active compound circulating relative to the amount injected intravenously) was calculated from the area under the graph.

Figure 6 shows the pharmacokinetic profiles of PEGylated (9x10 kD) porcine KS-AN-uricase after intramuscular (IM), subcutaneous (SC) and intravenous (IV) injection in rabbits, as measured by monitoring enzymatic activity in blood samples. . Plasma samples collected at the indicated times were determined for uricase activity by a colorimetric assay. Activity values ​​(mAU = milli-absorbance unit) for the enzymatic reaction per 1 μί blood sample · · · · · · · · · · · · · · · · · · · · · · · &lt;·· * "" * · · · *

- They represent 18 geese. The bioavailability of the injected uricase (amount of active compound circulating relative to the amount injected intravenously) was calculated from the area under the graph.

Figure 7 depicts the pharmacokinetic profiles of PEGylated (9x10 kD) porcine KS-AN-uricase after intramuscular (IM), subcutaneous (SC) and intravenous (IV) injection in dogs, as measured by monitoring enzymatic activity in blood samples. . Plasma samples collected at the indicated times were determined for uricase activity by a colorimetric assay. Activity values ​​(mAU = milli-absorbance unit) represent the rate of enzymatic reaction per 1 μΐ blood sample. The bioavailability of the injected uricase (amount of active compound circulating relative to the amount injected intravenously) was calculated from the area under the graph.

Figure 8 depicts the pharmacokinetic profiles of PEGylated (9x10 kD) porcine KS-AN uricase after intramuscular (IM), subcutaneous (SC) and intravenous (IV) injection in pigs, which are monitored by monitoring enzymatic activity in blood samples. . Plasma samples collected at the indicated times were determined for uricase activity by a colorimetric assay. Activity values ​​(mAU = milli-absorbance unit) represent the rate of enzymatic reaction per 1 μΐ blood sample. The bioavailability of the injected uricase (relative to the amount of circulating active substance compared to the amount injected by the intravenous injection) is &lt;RTI ID = 12.1&gt; int19 &lt;/RTI&gt; · · · · · · · · · · · · · · · · · area of ​​the graph.

Previous studies have found that when a significant reduction in the immunogenicity and / or antigenicity of uricase was achieved by PEGylation, this resulted in a significant reduction in uricolytic activity in all cases. The safety, comfort, and cost-effectiveness of biological drugs are adversely affected by the need to reduce their efficacy and the resulting dose increase. As such, safe and effective alternatives to increased levels of uric acid in body fluids (including blood) are needed. The present invention provides a recombinant mutant uricase which is truncated at its N- or C-terminus (or both) by 1-20 amino acids and substantially retains the uricolytic activity of the naturally occurring uricase.

As used herein, the term "uricase, unless otherwise indicated, refers to each subunit as well as the tetramer.

As used herein, the term "mutagenized uricase" refers to uricase molecules in which some amino acids are replaced by other amino acids.

As used herein, the term "conservative mutation" refers to a mutation affecting one or more amino acids at or near a position that does not substantially modify the behavior of the protein. In a preferred embodiment of the invention, the at least one conservative one is a conservative one. a mutated uricase has the same uricase activity as a non-mutated uricase. In other embodiments, the activity of the uricase carrying at least one conservative mutation is substantially the same (within 5%, within 10%, or within 30%).

Conservative amino acid substitution is defined as a change in the amino acid composition based on the amino acid exchange of a peptide, polypeptide, or protein (or fragment thereof). In specific embodiments of the invention, uricase carries two, three, or four conservative substitutions. Substitution has generally similar properties (e.g. acidity, basicity, aromaticity, size, positive or negative charge, polarity, apolarity), so that such substitutions do not significantly affect the properties of the peptide, polypeptide or protein (e.g. charge, IEF, affinity, avidity, conformation, solubility) or activity. Conservative amino acid substitutions typically occur within the following amino acid residues:

glycine (G), alanine (A), valine (V), leucine (L) and isoleucine (I);

- aspartic acid (D) and glutamic acid (Ξ);

- alanine (A), serine (S) and threonine (T);

histidine (H), lysine (K) and arginine (R);

- asparagine (N) and glutamine (Q);

- phenylalanine (F), tyrosine (Y) and tryptophan (W).

<img file="HU0700730A2_D0004.tif" />

• · · ····· ·«·»··« · · ·

A protein bearing one or more conservative substitutions retains its structural stability and its ability to catalyze reaction, even if its DNA sequence is not identical to its original protein.

As used herein, the term "truncated uricase" refers to uricase molecules whose primary amino acid sequence has been truncated. Possible truncations may occur at or near the N and / or C terminals. As a result of specific truncations of this type, the amino acid residues (naturally occurring at the N- and / or C-terminus) of the naturally occurring protein are present in the truncated protein. N-terminal truncations are shown in Figures 1, 2, 3, 4, 5 or 6. position. Preferably, the N-terminal truncations begin at position 2, resulting in the retention of the N-terminal methionine. This can be removed by post-translational modification of methionine. In preferred embodiments of the invention, the N-terminal methionine is removed after the uricase is produced. In one preferred embodiment, the methionine is removed by endogenous bacterial aminopeptidase.

One or more amino acid sequences were deleted from the truncated uricase relative to the entire sequence. A protein containing a truncated uricase may include any amino acid sequence in addition to the truncated uricase sequence, but may not be a protein that comprises a uricase sequence containing an additional wild-type amino acid sequence. In other words: a protein containing truncated uricase »· ♦ * *» · in which the truncation is as described in Figure 6. does not contain an amino acid at position 6 directly from the truncated uricase at the N-terminus.

Unless otherwise indicated (with specific reference to another sequence or SEQ ID NO:), the numbering of the amino acids in the uricases of the invention is given relative to the position of the amino acids in the porcine uricase sequence. The amino acid sequence of the porcine uricase and the numbering of the amino acids comprising this sequence are shown in Figure 3. As used herein, the term "from position X to position Y" used for amino acids or nucleic acids refers to a continuous sequence that starts at position X and ends at position Y and includes amino acids or nucleic acids at position X or Y, respectively.

Uricase genes and proteins have been identified in several mammalian species (such as pigs, baboons, rats, rabbits, mice and rhesus monkeys). The sequence of the various uricase proteins is given by reference to publicly accessible database registry numbers as follows: gi | 50403728 | sp | P25689; gi1205136341dbjIBAB91555.1; giI176610 | gbIAAA35395.1; giI20513654 | BOM | BAB91557.1; gi | 47523606 | ref | NP_999435.1; gi166785091ref | NP_033500.1; gi | 57 463 | emb | CAA31490.1;

gi1201273951ref | NP_446220.1;

giI137107 | sp | P11645;

· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·–––––– · · To · To ; gi | 2 07 619 | gb | AAA42318.1;

gi | 26340770 | BOM | BAC34047.1; and gi | 57459 | emb | CAA30378.1. Each of these sequences, as well as their annotations in the databases, accessible through the National Center for Biotechnology Information (NCBI), should be considered in their entirety as part of the teaching.

In one embodiment, the uricase is truncated at the N-terminus by 4-13 amino acids.

In another embodiment, the uricase is truncated at the C-terminus by 4-13 amino acids. In another embodiment, the uricase is truncated at 4 to 13 amino acids at both the C and the N-terminus.

In one embodiment, the uricase is truncated at the N-terminus by 6 amino acids. In another embodiment, the uricase is truncated at the C-terminus by 6 amino acids. In another embodiment, the uricase is truncated by 6 amino acids at both the C and the N-terminus.

In a preferred embodiment, the uricase protein of the invention comprises the amino acid sequence from position 13 to position 292 of the porcine uricase amino acid sequence (SEQ ID NO: 11). In a preferred embodiment, the uricase protein comprises an amino acid sequence from position 8 to position 287 of the PBC-ANC amino acid sequence (SEQ ID NO: 12). In another preferred embodiment, the uricase protein comprises an amino acid sequence from position 8 to position 287 of the porcine KS-ΔΝ amino acid sequence (SEQ ID NO: 7).

In another embodiment of the invention, the uricase protein comprises an amino acid sequence from position 44 to position 56 of the porcine KS-ΔΝ amino acid sequence (SEQ ID NO: 14). This region of uricase is homologous to sequences within the 'tunneling fold' ('T-fold') domain of the uricase and contains a mutation at position 46 (relative to the native porcine uricase sequence). Surprisingly, this mutation does not significantly affect the uricase activity of the protein.

According to one embodiment of the invention,

Amino acids at positions 46, 291, and 301 have been mutagenized. In preferred embodiments of the invention, amino acids 7, 46, 291 and 301 themselves have been mutagenized.

In preferred embodiments of the invention, the protein is encoded by a nucleic acid encoding an N-terminal methionine. Preferably, the N-terminal methionine is followed by a codon that permits the removal of this N-terminal methionine by bacterial methionine aminopeptidase (MAP) (Ben-Bassat and Bauer, Naturre 326, 315 (1987), which is incorporated herein by reference in its entirety). . The amino acids that allow the most complete removal of the N-terminal methionine are: alanine, glycine, proline, serine and threonine.

In one embodiment of the invention, the amino acids at or around position 7 and / or 46 are replaced by threonine. The enzymatic activity of the truncated uricases produced by these mutations is, surprisingly, similar to that of the non-truncated enzyme. In another embodiment of the invention, the amino acid mutations include substitutions at positions 7, 46, 291, and 301, respectively, of threonine, threonine, lysine, or serine.

Truncated mammalian uricases of the invention

They may also contain methionine as the N-terminal amino acid; in this case, an amino acid is present at the last position which allows the removal of the N-terminal methionine by bacterial methionine aminopeptidase (MAP). The amino acids that allow the most complete removal of the N-terminal methionine are:

alanine, glycine, proline, serine and threonine. In a preferred embodiment of the invention, the uricase comprises two N-terminal amino acids, one of which is methionine, followed by alanine, glycine, proline, serine or threonine.

In another embodiment of the invention, the substituted amino acids are replaced by threonine.

In one embodiment, the uricase is a mammalian uricase.

In another embodiment of the invention, the mammalian uricase comprises a sequence of porcine, bovine, ovine or baboon liver uricase.

• · · ·

In another embodiment, the uricase is a chimeric uricase formed from two or more mammalian uricases.

In another embodiment of the invention, the mammalian uricases may be porcine, bovine, ovine or baboon liver uricases.

In another embodiment of the invention, uricase comprises SEQ ID NO: 8.

In another embodiment of the invention, the uricase comprises SEQ ID NO: 13.

The present invention provides nucleic acids encoding uricase comprising SEQ ID NO: 10.

In one embodiment, the uricase comprises a fungal or microbial uricase.

In a further embodiment of the invention, the fungal or microbial uricase is Aspergillus flavus, Arthrobacter globiformis or Candida utilis uricase.

In another embodiment of the invention, the uricase comprises uricase from an invertebrate animal.

In another embodiment of the invention, the invertebrate uricase is Drosophila melanogaster or Drosophila pseudoobscura uricase.

In another embodiment of the invention, the uricase comprises uricase of plant origin.

In a further embodiment of the invention, the uricase of plant origin is uricase derived from Glycine max root tubers.

The present invention also provides a nucleic acid sequence encoding uricase.

We further disclose a vector comprising a nucleic acid sequence encoding uricase.

In a preferred embodiment, the uricase is isolated. In another preferred embodiment, the uricase is purified. In another preferred embodiment, the uricase is isolated and purified.

The present invention also provides a host cell comprising a vector.

In addition, a method for producing a nucleic acid sequence is disclosed, which process comprises modifying a nucleic acid sequence containing a non-truncated uricase by PCR (polymerase chain reaction) technique. It is known to those skilled in the art that preparation of a desired nucleic acid sequence by polymerase chain reaction can be accomplished using synthetic primer oligonucleotides that are complementary to the target DNA regions (one on each strand). The primer oligonucleotides are added to the target DNA (which need not be pure) in the presence of excess deoxynucleotides and Taq polymerase (a thermostable DNA polymerase). During PCR, the target DNA is repeatedly denatured (at about 90 ° C) in a series of temperature cycles (typically 30 cycles), hybridized to the primer oligonucleotides (usually at 50-60 ° C), and the "primer strand" longer than the primers. • »»

28 ppm (72 ° C). Because the "daughter strands themselves serve as templates in subsequent cycles, DNA fragments complementing both primers are amplified exponentially (and not linearly).

The present invention provides a method of producing a mutant recombinant uricase comprising transfecting a host cell with a vector to express the host cell uricase, isolating the mutant recombinant uricase from the host cell, isolating the purified mutant recombinant uricase (e.g., mutant recombinant uricase. For example, uricase can be prepared using the methods disclosed in WO 00/08196 (which is incorporated herein by reference in its entirety).

It can be isolated and purified by methods well known to those skilled in the art of uricase. The expressed polypeptides of the invention are generally isolated in substantially pure form. The polypeptides are preferably isolated in a purity of at least 80% w / w, more preferably at least 95% w / w, most preferably at least 99% w / w. Purification is generally carried out, for example, by standard ammonium sulfate fractionation, SDS-PAGE electrophoresis, and affinity chromatography. Preferably, uricase utilizes a cationic surfactant such as cetylpyridinium chloride (CPC) in U.S. Patent Application 60/670520, issued April 11, 2005, to Purification Of Proteins With Cationic.

- 29 Surfactant; attorney registration number: 103864.146644; which source is to be considered as part of the teaching) is isolated.

In a preferred embodiment of the invention, the host cell is subjected to treatment to induce the production of mutant recombinant uricase. It is known to those skilled in the art that transfection of cells with a vector can usually be accomplished using calcium precipitated DNA, although other methods (e.g. electroporation) may be employed.

In one embodiment of the invention, the vector is under the control of an osmotic pressure sensitive promoter. The promoter is a DNA region for which RNA polymerase has been used

- before initiating transcription of DNA into RNA

- binds. The osmotic pressure-sensitive promoter initiates transcription as a result of increased cellular osmotic pressure sensed by the cell.

In one embodiment, the promoter is a modified osmB promoter.

In preferred embodiments of the invention, the uricase of the invention is a polymer conjugated uricase.

In another embodiment, the invention provides a pharmaceutical composition comprising uricase. In one embodiment, the composition is a uricase solution. In a preferred embodiment, the solution is sterile and suitable for injection. In one embodiment, the composition of the invention comprises uricase dissolved in phosphate buffered saline. Another embodiment wi ·· »* ··

- 30 rins are packaged in a vial, which is optionally closed with a plunger stopper. In preferred embodiments of the invention, the composition comprises the uricase solution in a concentration of 2 to 16 mg uricase / ml solution, 4 to 12 mg uricase / ml solution or 6 to 10 mg uricase / ml solution. In one preferred embodiment, the composition comprises uricase at a concentration of 8 mg / ml. Preferably, the weight of the uricase is measured relative to the weight of the protein.

Effective dosage regimens for the compositions of the invention may be determined by one of ordinary skill in the art. Indicators suitable for evaluating the effectiveness of a given scheme are well known to those skilled in the art. Examples of such indicators include normalizing plasma uric acid (PUA) levels and reducing or maintaining PUA to 6.8 mg / dL or less. In one preferred embodiment, the level of PUA of the patient to be treated with the composition of the invention is 6 mg / ml or less for at least 70%, at least 80%, or at least 90% of the total duration of treatment. For example, for a treatment period of 24 weeks, the patient's PUA level is 6 mg / dL or less in at least 80% of the 24-week treatment period, i.e. at least 134.4 days (= 24 weeks x 7 days / week x 0.8). exceeding 6 mg / dl.

In certain embodiments of the invention, a solution of 0.5 to 24 mg of uricase is administered once every 2 to 4 weeks. By any of the methods well known to those skilled in the art of uricase administration (e.g., intravenous, intramuscular, or subcutaneous) **^Α“3 · · ·” 1 &lt;· · · · · · · · 1 ”. For intravenous administration, 0.5 to 12 mg uricase is preferably administered, whereas for subcutaneous administration, 4 to 24 mg uricase is preferred. In a preferred embodiment of the invention, uricase is administered by intravenous infusion for 30-240 minutes. In one embodiment, 8 mg of uricase is administered once every two weeks. In certain embodiments of the invention, the infusion is administered using 100-500 mL saline. In one preferred embodiment, a solution of 8 mg of uricase is administered once every 2 or 4 weeks for 120 minutes and is preferably dissolved in 250 ml of saline for infusion. In certain embodiments of the invention, the uricase is administered over a period of 3 months, β months, 8 months, or 12 months. In other embodiments, the treatment period is 12 weeks, 24 weeks, 36 weeks or 48 weeks. In a preferred embodiment, the treatment is administered over a long period of time, e.g. for two years or longer (up to the end of the patient's life). In addition, multiple treatment periods may be applied, which may be replaced by periods without treatment; for example, after six months of treatment, three months of treatment break, then again of six months of treatment, etc.

In certain embodiments of the invention, anti-inflammatory compounds may be used prophylactically to avoid or reduce infusion reactions resulting from uricase administration. In one embodiment, at least one corticosteroid, at least one antihistamine, and at least one NSAID or at least one corticosteroid, at least one anti-histamine, and at least one NSAID or * · * · «· I are combinations of these. Preferred corticosteroids for this purpose include betamethasone, budesonide, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone and triamcinolone. Preferred NSAIDs include ibuprofen, indomethacin, naproxen, aspirin, acetomiphen, celecoxib and valdecoxib. Examples of preferred antihistamines are: azatadine, brompheniramine, cetirizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexchlorpheniramine, dicmenhydrinate, diphenhydramine, doxylamine, fexofenadine, hydroxyzin, loratadine.

In one embodiment, the antihistamine is fexofenadine, the NSAID is acetaminophen, and the corticosteroid is hydrocortisone and / or prednisone. Prior to administration of the uricase solution for infusion, a combination of all three of these compounds is preferably administered (not necessarily simultaneously). In a preferred embodiment of the invention, the NSAID and the antihistamine are administered orally 1-4 hours prior to the uricase infusion. The applicable dose of fexofenadine is ca. 30-180 mg, ca. 40-150 mg, ca. 50-120 mg, ca. 60-90 mg, ca. 60 mg, preferably 60 mg. A suitable dose of acetaminophen is ca. 500-1500 mg, ca. 700-1200mg, approx. 800-1100 mg, ca. 1000 mg, preferably 1000 mg. A suitable dose of hydrocortisone is about. 100-500 mg, ca. 150-300 mg, ca. 200 mg, preferably 200 mg. In one embodiment, the antihistamine is not diphenhydamine. In another embodiment, the NSAID is non-acetaminophen. In one preferred embodiment, 50 mg of fexofenadine is administered orally the night before the uricase infusion, followed by 60 mg of fexofenadine and 1000 mg of acetaminophen orally the following morning, followed by 200 mg of hydrocortisone just prior to the infusion of the uricase solution. In one embodiment, prednisone is administered on the day before the uricase administration, preferably in the evening. A suitable dose of prednisone is 5 to 50 mg, preferably 20 mg. In some embodiments, prophylactic treatments to prevent or reduce infusion reactions are performed on patients in treatment (or prior to treatment) with uricase (including both PEGylated uricase and non-PEGylated uricase). In other embodiments of the invention, these prophylactic treatments are administered to patients in treatment (or prior to treatment) with therapeutic peptides other than uricase (which may be PEGylated or non-PEGylated).

In one embodiment, the pharmaceutical composition comprises a polymer conjugated modified uricase that retains its uricolytic activity. In one preferred embodiment, the polymer-uricase conjugates are prepared as disclosed in WO 01/59078 and U.S. Patent Application Serial No. 09/501730, which are incorporated herein by reference in their entirety.

In another embodiment of the invention, the &amp; quot; 4&gt; «*« ·.

<img file="HU0700730A2_D0005.tif" />

the polymer used for conjugation is selected from the group consisting of polyethylene glycol, dextran, polypropylene glycol, hydroxypropylmethylcellulose, carboxymethylcellulose, polyvinylpyrrolidone and polyvinyl alcohol.

In another embodiment of the invention, the composition comprises from 2 to 12, preferably from 3 to 10, polymer molecules for each uricase subunit.

In another embodiment, each polymer molecule has a molecular weight of about. 1 kD to approx. Up to 100 kD.

In another embodiment of the present invention, each polymer molecule has a molecular weight of about. 1 kD approx. Up to 50 kD. In a preferred embodiment of the invention, each polymer molecule has a molecular weight of about. 5-20 kD, approx. 8-15 kD, approx. 10-12 kD, preferably approx. 10 kD. In another preferred embodiment, each polymer molecule has a molecular weight of about. 5 kD to approx. Up to 20 kD. In a particularly preferred embodiment of the invention, each polymer molecule has a molecular weight of 10 kD. A mixture of molecules of different molecular weights! are also applicable. In one embodiment, the composition is suitable for repeated administration.

In another embodiment of the invention, the polymer conjugate of uricase comprises a urethane bond, a secondary amine bond, or an amide bond.

The present invention further provides a cell capable of producing a uricase comprising the amino acid sequence of a recombinant uricase having a uricase having 1-20 amino acids.

THE"" .

»·« Has truncated, contains mutagenized amino acids and has uricolytic activity.

We also disclose a means for uric acid metabolism using uricase.

In addition, the use of a uricase formulation to reduce uric acid levels in a biological fluid is disclosed.

In one embodiment of the invention, the uricase composition is used to lower blood uric acid levels.

In addition, novel nucleic acid molecules encoding uricase polypeptides are disclosed. The manipulations leading to their creation are well known to those skilled in the art. For example, uricase nucleic acid sequences can be modified by any of several methods known in the art [Maniatis, T.: "Molecular Cloning," Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1990). The sequence may be cleaved at appropriate sites using restriction endonuclease (s) and, if desired, further enzymatically modified, isolated and ligated in vitro. When creating a gene encoding uricase, care must be taken to keep the modified gene in its proper translation reading phase (without interruption by the translation stop codon). In addition, the nucleic acid sequence encoding uricase may be mutagenized in vitro or in vivo, for example, to generate and / or digest translation, initiation and / or termination sequences, to generate variants of the coding regions and / or to create new restriction endonuclease recognition sites. or further in vitro modification. Any of the mutagenesis techniques known in the art can be used, including, but not limited to, in vitro site-specific mutagenesis [Hutchinson, C., et al., J. Bioi. Chem. 253, 6551 (1978)], the use of TAB® linkers (Pharmacia), etc.

The nucleotide sequence encoding the uricase protein may be inserted into a suitable expression vector, i.e., a vector containing the elements necessary for transcription and translation of the inserted protein coding sequence. A variety of host-vector systems can be used to express the protein coding sequence. Examples include, but are not limited to: mammalian cell systems infected with a virus (e.g., vaccinia, adenovirus, etc.); virus (e.g. baculovirus) infected insect cell systems; microorganisms containing yeast vector, e.g. yeast; or bacteria transformed with bacteriophage DNA, plasmid DNA or cosmid DNA. The expression elements of these vectors may vary in their strength and specificity. Depending on the host-vector system used, any of a number of transcriptional and translational elements suitable for this purpose may be used.

Any of the known methods for inserting DNA fragments into a vector may be used to generate expression vectors comprising the appropriate transcriptional / translational regulatory signals and the coding sequence of the protein. Such methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination techniques (genetic recombination). The expression of the nucleic acid sequence encoding the uricase protein may be regulated by another nucleic acid sequence, whereby the uricase protein is produced in the host transformed with the recombinant DNA molecule. For example, uricase expression may be regulated by any promoter / enhancer sequence known in the art. Promoter to regulate uricase expression includes, among others, the early promoter region of SV40 (Bernoist and Chambon, Natur. 290, 304-310 (1981)), the 3 'long long repetitive sequence of Rous sarcoma virus [Yamamoto et al., Cell. 22, 787797 (1980)], the thymidine kinase promoter of the herpesvirus (Wagner et al., Proc. Natl. Acad. Sci. USA 78, 44-1445 (1981)], regulatory sequences of the metallothionine gene (Brinster et al., Natur. 296, 39-42 (1982)); prokaryotic expression vectors, e.g. the β-lactamase promoter [Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-373 (1978)], the tac promoter [DeBOer et al., Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)] and the osmB promoter. In preferred embodiments of the invention, the nucleic acid comprises a nucleic acid sequence encoding uricase operably linked to a heterologous promoter.

Following the preparation and isolation of a recombinant DNA molecule containing a coding nucleic acid, a number of well known techniques can be used to amplify it. Once the appropriate host system and growth conditions have been established, the recombinant expression vectors can be amplified and replicated.

- 38 - ..... * '' can be produced in large quantities. As mentioned above, suitable expression vectors include, but are not limited to, the following vectors and derivatives thereof: viruses that infect humans or animals, such as viruses. vaccinia virus and adenovirus; insect viruses, e.g. baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda); and plasmid and cosmid vectors.

In addition, a host cell strain can be chosen in which expression of the inserted sequences is modified or the gene product is modified and matured to a desired degree. The expression regulated by certain promoters can be enhanced in the presence of certain inducers, so that the expression of the genetically engineered uricase protein can be regulated. In addition, translational and post-translational maturation and modification of proteins (e.g. glycosylation, cleavage) occurs through specific and specific mechanisms in different host cells. Appropriate cell lines or host cell systems may be selected to provide the desired modifications and maturation of the expressed foreign protein. Different vector / host cell expression systems may promote maturation reactions (e.g., proteolytic cleavage) to varying degrees.

In preferred embodiments of the invention, expression of uricase in E. coli is preferably accomplished using vectors containing the osmB promoter.

• · • · · · · * · • · · · · • ··· ··· · · · · · • · · «····

- 39 - ...........

First example

Construction of a gene and expression plasmid capable of expressing urease

Recombinant porcine uricase (urate oxidase), porcine KS-AN (an N-terminally truncated porcine uricase protein substituted with amino acids 291 and 301 in lysine and serine) in strain W310 F-E. coli K-12 We expressed. A series of plasmids were generated which resulted in pOUR-P-AN-ks1, which was able to regulate the efficient expression of uricase after transformation of E. coli cells. Isolation and subcloning of urease oDNA from porcine and baboon liver

Uricase cDNAs were prepared from porcine and baboon liver by isolation and subclonation with the appropriate RNA. Porous cellular RNA was extracted from porcine and baboon livers. Erlich, HA: PCR Technology; Principles and

Application Fora DNA Amplification (1988); Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989); Ausubel, FM et al., Current protocols in molecular biology (1998)] followed by reverse transcription using the First-Strand cDNA Synthesis kit (Pharmacia Biotech). PCR amplification was performed using Taq DNA polymerase (Gibco BRL, Life Technologies).

The synthetic primer oligonucleotides used for PCR amplification of porcine and baboon urate oxidase (uricase) are shown in Table 1.

• · · · · · • · · · · • ·· · · · ···« · • · · ····· ···«·*· · ·· ·

- 40 Table 1

Primer primer oligonucleotides used for PCR amplification of uricase cDNA

O x ü. Shed. antisense 5 'gcgctctagaagcttccatggTCACAGCCTTGAAGTCAGC 3' (SEQ ID NO: 2) 5 ^ "gcgcgaattccATGGCTCATTACCGTAATGACTACA 3 '(SEQ ID NO: 1)

Baboon (D3H) liver uricase:

sense 5 'gcgcgaattccATGGCCCACTACCATAACAACTAT 3' (SEQ ID NO: 3) antisense 5 'gcgcccatggtctagaTCACAGTCTTGAAGACAACTTCCT 3' (SEQ ID NO: 4)

The restriction enzyme recognition site sequences (inserted at the end of the primers (indicated in lowercase in Table 1)) were sense EcoRI and Ncol (porcine and baboon) and antisense Ncol, Hindin and Xbal (porcine) and Xbal and Ncol (porcine). The third GAC codon (aspartic acid) in the baboon sense primer (aspartic acid) was replaced by the CAC codon (histidine) located at this position of the pseudogenic coding sequence for human urate oxidase. The recombinant baboon uricase construct generated using these primer oligonucleotides was called D3H baboon uricase.

The porcine uricase PCR product was digested with EcoRI and HindIII and cloned into the pUC18 vector to generate plasmid pUC18. The D3H baboon uricase PCR product was directly cloned into the pCR ™ II vector using TA Cloning ™ (Invitrogen, Carlsbad, CA) to give plasmid pCR ™ IID3H baboon uricase.

• · ···· ·· ·» · • · · · · · · • ··· ··· ···· · • · · ····· ·«··«· « ·« ·

The ligated cDNAs were used to transform E. coli XL1-Blue strain (Stratagene, La Jolla, CA). Plasmid DNA containing the cloned uricase cDNA was prepared and clones containing the published uricase DNA coding sequences other than the D3H substitution in baboon uricase (see Table 1) were selected and isolated. In the selected pCR ™ II-D3H baboon uricase clone, the pCR ™ II sequences were located immediately adjacent to the uricase stop codon resulting from the deletion of the PCR-inserted sequences. Consequently, the Xba I and Nco I restriction sites from the 3 'untranslated region have been removed, allowing for targeted cloning using the Nco I site at the 5' end of the PCR product and the Bam HI site from the pCR ™ II vector.

Subcloning of uricase cDNA into pET expression vectors

Subcloning of baboon uricase

The D3H baboon cDNA containing the full-length uricase coding sequence was introduced into the pET-3d expression vector (Novagen, Madison, WI). Plasmid pCR ™ II-D3H babanuricase was digested with NcoI and BamHI endonucleases and the 960 bp fragment was isolated. The pET-3d expression plasmid was also digested with NcoI and BamHI and a 4600 bp fragment was isolated. The two fragments were ligated with the plasmid pET-3d-D3H baboon.

Subcloning of porcine baboon chimeric uricase

To provide increased expression, stability and activity of the recombinant gene, porcine baboon chimeric (PBC) uricase was generated. To generate PBC, the 4936 bp Ncol / Apalf fragment of the pET-3d-D3H baboon clone was isolated and the isolated fragment was ligated with the 624 bp Ncol / Apalf fragment isolated from pUC18 swine uricase plasmid to give pET-3d-PBC. The PBC uricase cDNA is porcine uricase 1-225. codons, and baboon uricase, linked in the same reading frame as before, 226-304. consists of its codons.

Subcloning of porcine KS-uricase

Swine KS-uricase was created to add a lysine which could serve as an additional PEGylation site. "KS stands for Substitution of arginine for lysine (R291K) at position 291 for porcine uricase. In addition, threonine at position 301 was replaced with serine (T301S). To generate the swine KS-uricase plasmid, the 4696 bp NcoI / NdeI fragment of the pET-3d-D3H baboon plasmid was isolated and ligated to the 864 bp NcoI / NdeI fragment isolated from the pUC18 porcine uricase, whereby the pET-3dser Plasmid KS was obtained. The porcine KSuricase sequence thus generated is the porcine uricase 1-288. codons and baboon uricase, linked in the same reading phase, to 289-304. consists of its codons.

Subcloning of the urease sequence under the control of the osmB promoter

The uricase gene was subcloned into an expression vector containing the osmB promoter, following the procedure disclosed in U.S. Patent No. 5,795,776 (which is incorporated herein by reference in its entirety). to be considered as part of the teaching). This vector allows for the induction of protein expression by high osmotic pressure or culture aging. The pMFOA-18 expression plasmid contains an osmB promoter, a ribosome binding site sequence (rbs) and a transcription termination sequence (space), and provides ampicillin resistance (AmpR) and expresses recombinant human acetylcholine esterase (AChE).

Subcloning of D3H babanuricase

Plasmid pMF0A-18 was digested with NcoI and BamHI endonucleases and the larger fragment was isolated. The pET-3d-D3H baboon construct was also digested with NcoI and BamHI endonucleases, and the 960 kb fragment (which contained the D3H baboon uricase gene) was isolated. The two fragments were ligated together to give plasmid pMFOU18.

The plasmid pMFXT133 contains an osmB promoter, a ribosome binding site sequence (E. coli deo operon), a transcription termination sequence (E. coli TrypA), a recombinant factor Xa inhibitor polypeptide (FXaI), and tetracycline resistance (T). To replace the antibiotic resistance genes, the baboon uricase gene was inserted into this plasmid. Plasmid pMFOU18 was digested with NcoI, filled up, then digested with Xhol and a 1030 bp fragment was isolated. Plasmid pMFXT133 was digested with NdeI endonuclease, and the following was used:

44, then digested with XhoI endonuclease and isolated the larger fragment. The baboon uricase expression vector pURBA was ligated to each of the two isolated fragments.

Subcloning of porcine baboon chimeric uricase

Plasmid pURBA16 was digested with the ApaI and AlwNI endonucleases and a 2320 bp fragment was isolated. Plasmid pMFXT133 was digested with NdeI endonuclease, filled up, then digested with AlwNI endonuclease and a 620 bp fragment was isolated. The pET-3d-PBC construct was digested with Xbalendonuclease, filled up, then digested with Apalendonuclease, and the 710 kb fragment was isolated. These three isolated fragments were ligated to each other by the plasmid pUR-PB, which expresses PBC-uricase under the control of the osmB promoter and rbs and T7-rbs derived from the pET-3d vector.

T7-rbs was further cleaved. Plasmid pURPB was digested with NcoI endonuclease, filled in, then digested with AlwNI endonuclease, and the 3000 kb fragment was isolated. Plasmid pMFXT133 was digested with NdeI endonuclease, filled up, then digested with AlwNI endonuclease and a 620 bp fragment was isolated which was ligated with the former fragment to the plasmid pDUR-PB expressing the PBCter by osmB.

Construction of plasmid pOUR-PB-ANC

Several changes that are significant in the stability of the recombinant enzyme have been introduced into the uricase cDNA.

- resulted in a 45 degree increase. Plasmid pOUR-PBC-ANC was constructed in which both the N-terminal six amino acid maturation peptide and the C-terminal tripeptide (which function as in vivo peroxisomal delivery signals) were removed. This was accomplished by PCR amplification using the PBC sequence in pDUR-PB and the specific primer oligonucleotides shown in Table 2.

Second spreadsheet

Primer oligonucleotides used for PCR amplification of PBC-ANC uricase

PBC uricase ANC:

sense

5 'gcgcatATGACTTACAAAAAGAATGATGAGGTAGAG 3' (SEQ ID NO: 5)

antisense

5'ccgtctagaTTAAGACAACTTCCTCTTGACTGTACCAGTAATTTTTCCGTATGG3 '(SEQ ID NO: 6)

In Table 2, restriction enzyme recognition sites inserted at the end of the primer oligonucleotides are shown in bold and non-coding regions are shown in lower case. You are Ndel, you are Xbal antisense. The antisense primer was also used to eliminate an internal Ndel recognition site by inserting a point mutation (underlined) (which does not affect the amino acid sequence), which facilitated subcloning using Ndel endonuclease.

Created by PCR amplification of pDUR-PB plasmid * ·· * · a · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

900 The bp fragment was cleaved with NdeI and XbaI endonucleases and isolated. The resulting fragment was inserted into a "deo expression plasmid (pDBAST-RAT-N) containing deo-P1P2 promoter and rbs from E. coli and constitutively expresses the human recombinant insulin precursor. The plasmid was digested with Nde I and Xba I endonuclease, the 4035 kb fragment was isolated and ligated to the PBC-uricase PCR product. The pDÜR-PB-ANC construct thus created was E. coli K-12S &lt;J&gt; 733 (F-cytR strA), which expressed high levels of active truncated uricase.

The doubly truncated PBC-ANC sequence was also expressed under the control of the osmB promoter. Plasmid pDURPB-ANC was digested with AlwNI and NdeI endonuclease and a 3459 bp fragment was isolated. Plasmid pMFXT133 described above was also digested with NdeI and AlwNI and a 660 bp fragment was isolated. The two fragments were ligated to each other by the plasmid pOUR-PB-ANC, E. coli K-12 strain W3110F and resulted in high expression of active truncated uricase.

Construction of the uricase expression plasmid pOUR-P-AN-ks-1

This plasmid was designed to improve the activity and stability of the recombinant enzyme. Porcine KSΔΝ uricase was truncated only at its N-terminus (ΔΝ, where six amino acid N-terminal maturation peptides were removed) and carries mutations S46T, R291K and T301S. In Figure 46.

position threonine, which is the result of PCR-47 amplification and conservative mutation during cloning. At position 291 arginine is replaced by lysine and at position 301 serine is substituted for threonine (both derived from the baboon uricase sequence). As mentioned above, the R291K and T301S substitutions are collectively designated as "KS." The additional lysine provides another potential PEGylation site.

To generate plasmid pOUR-β-AN-ks-1 (Figure 1), plasmid pOUR-PB-ANC was digested with the ApaI and XbaI endonucleases and a fragment of 3873 bp was isolated. Plasmid pET-3dPKS (construction see Figure 4) was digested with ApaI and SpeI endonucleases and a 270 bp fragment was isolated. The SpeI cleavage left a 5'CTAG overhang which could be efficiently ligated to the Xba I cleaved DNA fragments. The two fragments were ligated together to give plasmid pOUR-P-AN-ks-1. After ligation, the Spel and Xbal recognition sites were lost (their positions are shown in brackets in Figure 1). The pOUR-Δ-ΔΝks-1 construct was introduced into E. coli strain K-12 W3110F '(prototroph, ATCC 27325). The resulting porcine KS-AN uricase, which was expressed under the control of the osmB promoter, resulted in a high level of recombinant enzyme with high activity and stability.

Figure 1 shows the structure of plasmid pOUR-β-AN-ks-1. The numbers next to the restriction sites represent the nucleotide positions relative to the Haell site (position 1). Restriction sites lost during cloning are indicated in parentheses. The Pig-KS • · • · · · · · · · · · · · · · ·

- The plasmid pOUR-P-AN-ks-1 encoding 48 Δ ur-uricase has a length of 4143 base pairs (bp) and consists of the following elements:

First A 113 bp DNA fragment extending from nucleotide 1 to the NdeI recognition site (position 113), including the osmB promoter and the ribosome binding site (rbs).

Second 932 bp DNA fragment extending from the NdeI recognition site (position 113) to the SpeI / XbaI site (position 1045), including the 900 bp coding region of the porcine KS-ΔΝ (N-terminally truncated, 291 and 301) a nucleic acid sequence encoding a porcine uricase protein substituted at lysine and serine) and a 32 bp flanking sequence derived from pCR ™ II (TA cloning site 5 'to the Spel / Xbal site).

Third 25 bp Multiple Cloning Site Sequence (MCS) stretching from the Spel / XbaI site (position 1045) to the HindIII recognition site (position 1070).

4th A 40 bp synthetic oligonucleotide containing the TrpA transcription termination sequence (space) extending from position 1070 (HindIII recognition site) to position 1110 (AatlI recognition site).

5th A 1519 bp DNA fragment stretching from the AatlI recognition site (position 1110) to the MscI / Scal site (position 2629) of the pBR322 plasmid, including the tetracycline resistance gene (TetR).

6th 1514 bp DNA fragment from the Scal site (position 2629) of the plasmid pBR322 to the Haell site (position 4143).

* *

- 49 positions), including the DNA replication origin.

Figure 2 shows the DNA sequence and the deduced amino acid sequence of Swine-KS-AN-uricase. Amino acid numbering is given relative to the complete sequence of porcine uricase. Following the initial methionine, threonine was inserted in place of aspartic acid in the porcine uricase sequence. This threonine allows the removal of methionine by bacterial aminopeptidases. The gap in the amino acid sequence indicates the deleted N-terminal maturation peptide. The restriction sites (ApaI, NdeI, BamHI, EcoRI and SpeI) used to perform the various steps of subcloning are shown in the figure. The 3 'untranslated sequence (indicated in lower case) is derived from the pCR ™ II sequence. The translational stop codon is marked with an asterisk.

Figure 3 shows the relative alignment of the amino acid sequences of different recombinant uricase sequences. The first line shows the sequence of the porcine uricase, which contains the entire amino acid sequence. The second row shows the sequence of the double-truncated porcine chimeric uricase (PBC-ANC), while the third row shows the porcine-KS-AN-uricase sequence which is truncated at its N-terminus only and carries mutations S46T and R291K and T301S. (the latter two representing the baboon origin of the C-terminus of the uricase coding sequence). Asterisks indicate positions where the porcine KS-ΔΝ amino acids differ from the sera · · · · · · · · · · · · · · »« ·

- from the published sequence of 50 bovine uricases, while the circles indicate positions where the porcine KS-ΔΝ amino acids differ from the PBC-ΔΝ (porcine baboon chimeric) sequence. The dashed lines indicate amino acid deletions.

Native baboon, porcine and rabbit uricase (carrying the Y97H mutation) and porcine / baboon chimeric (PBC) cDNA were prepared for cloning into E. coli. Clones expressing high levels of uricase variants were generated that were selected to contain all W3110 F "E. coli and expression is regulated by the osmB promoter. Plasmid DNAs were sequenced

It was verified by DNA sequencing and restriction enzyme analysis and the cells were cultured.

Cross-ligation between PBCANC and porcine KS was performed to generate truncated uricases (porcine-ΔΝ and porcine-KS-ΔΝ) following cleavage with Apai and Xbal and Apai + Spel restriction endonucleases. It will be appreciated that these truncated mutants may retain their activity, since the six N-terminal amino acids, the so-called. Neither the maturation peptide (1-2) nor the C-terminal tripeptide, the peroxisomal targeting signal (3-5), have a function that would significantly affect enzymatic activity, and it is likely that these sequences may be immunogenic. Clones which expressed very high levels of uricase variants were selected.

<img file="HU0700730A2_D0006.tif" />

. example

Transformation of an expression plasmid into a bacterial host cell

The plasmid pOUR-P-AN-ks-1 expression plasmid was introduced into E. coli strain K-12 W3110 F. Bacterial cells were grown in Luria medium (LB) to mid-log stage for preparation for transformation, harvested by centrifugation, washed with cold water and ca.

It was suspended in 10% aqueous glycerol at a cell density of 3x10 10 cells / ml. The cells were stored in aliquots at -70 ° C. Plasmid DNA was precipitated in ethanol and dissolved in water.

The bacterial cells and the plasmid DNA were mixed and transformed using a Gene Pulser Type II (BIORAD) high-voltage electroporation procedure (Trevors et al., "Electrotransformation of Bacteria by Plasmid DNA, in Ed. DC Chang, BM Chassy, ​​JA Saunders and AE Sowers), pp. 265-290. pp. Academic Press

Inc., San Diego (1992); Hanahan et al., Meth. Enzymol. 204: 63-113 (1991)]. Transformed cells in SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO4₄20 mM glucose), incubated at 37 ° C for one hour, selected for tetracycline resistance, and a high expression clone was selected.

Third example

Production of recombinant uricase

Bacteria transformed as described above ···

- Cultured in 52 glucose medium (pH 7.2 ± 0.2) at approximately 37 ° C. In the final 5-6 hours of culture, KCl was added to the medium at a final concentration of 0.3 M and culturing was continued to accumulate uricase.

In bacterial cells, the recombinant uricase accumulates in the form of an insoluble precipitate similar to the inclusion bodies. The cell suspension was washed by centrifugation and resuspended in 50 mM Tris buffer (pH 8.0) containing 10 mM EDTA, and the final volume of the suspension was approx. We added it 40 times.

The bacterial cells were digested under high pressure using lysozyme and the inclusion bodies containing the recombinant uricase were isolated by centrifugation. The lysosomal treatment (2000-3000 units / ml) was carried out at pH 8.0, 7 ± 3 ° C for 16-20 hours with stirring. The pellet was washed with water and stored at -20 ° C until use.

The enriched inclusion bodies were resuspended in 50 mM NaHCCh buffer (pH 10.3 ± 0.1) for further processing. The resulting suspension was incubated overnight at room temperature to solubilize the uricase in the inclusion bodies and clarified by centrifugation.

The uricase was subjected to further purification by several chromatographic steps. The first chromatographic step was performed on a QSepharose FF column. The loaded column was washed with 150 mM sodium chloride bicarbonate buffer and the uricase eluted with 250 mM sodium chloride bicarbonate buffer. Thereafter, the uricase »« * »· * ·« · · ··· · ···

Xanthine-agarose resin (Sigma) was used to remove minor contamination in 4 «··« formulations. The eluate from the Q-Sepharose FF column was diluted with 50 mM glycine buffer, pH 10.3 + 0.1 to a protein concentration of approximately 0.25 mg / ml and loaded onto the column. The column was washed with bicarbonate buffer (pH 10.3 + 0.1) containing 100 mM NaCl and the uricase was eluted with the same buffer used with the washing but supplemented with 60 μΜ xanthine. At this stage, the uricase was subjected to repeated purification on a Q-Sepharose column to remove aggregated forms.

The purity of each of the uricase formulations was found to be greater than 95% as determined by size selection chromatography. Aggregate forms in each formulation were detected using a Superdex 200 column in a ratio of less than 0.5%.

Table 3 summarizes the purification steps of porcine KSAN in inclusion bodies from 25 L fermentation broth.

Third spreadsheet

Purification of porcine KSAN uricase

<td>Cleaning step</td><td>Protein (Mg)</td><td>Activity (E)</td><td>Specific activity (E / mg)</td>

<td>Unblocking</td><td> 12 748</td><td> 47 226</td><td> 3,7</td>

<td>Clear solution</td><td> 11 045</td><td> 44 858</td><td> 4,1</td>

<td>Q-Sepharose I - main file</td><td> 7 590</td><td> 32 316</td><td> 4,3</td>

<td>Xanthine agarose is the main stock</td><td> 4 860</td><td> 26 361</td><td> 5,4</td>

<td>Q-Sepahrose II - main stock</td><td> 4 438</td><td> 22 982</td><td> 5,2</td>

<td>30 kD UF retentate</td><td> 4 262</td><td> 27 556</td><td> 6, 5</td>

4th example

Properties of recombinant uricases

5DS PAGE

SDS-PAGE analysis of the highly purified uricase variants (see Figure 4) resulted in a fairly representative pattern. Samples were stored in carbonate buffer (pH 10.3) at 4 ° C for several months. For the full-length variants (porcine uricase, porcine KS and PBC), two major degradation products (about 20 kD and about 15 kD) were accumulated. This observation suggests that at least one notch cleaves the uricase subunit molecule. In the case of clones truncated at the N-terminus, a different degradation pattern was found, as with rabbit uricase, but to a lesser extent. The N-terminal of the rabbit uricase resembles the truncated clone. The uricase fragments formed during purification and storage are N-terminal

4 «·« • »· * * ·« «I

The sequences of ♦♦ · · »· ** 4 4 * •« · * · »« ♦ * - »· Ο» * · «· were determined.

Peptidszekvenálás

N-terminal sequencing of uricase preparations was performed using the Edman degradation procedure for ten cycles. Recombinant porcine uricase (full-length clone) showed higher levels of degradation fragments than porcine KS-ΔΝ. The derived sites of the cleavages resulting in degradation fragments were as follows:

1) Main position at position 168 with the sequence:

--QSG ^ FEGFI2) Secondary position at position 142 having the sequence:

The above sequences of IRNGPPVIA do not indicate any known proteolytic cleavage. However, the cleavage may be the result of protolysis or a chemical reaction. Surprisingly, N-terminal truncated uricases are more stable than non-N-terminal truncated uricases. The stability of PBC-ANC is similar to that of other ΔΝ molecules, but is lower compared to non-N-terminally truncated PBC. Efficiency

Uricase activity was measured by UV. The enzymatic reaction rate was determined by the decrease in absorbance at 292 nm resulting from the oxidation of uric acid to allantoin. An activity unit is defined as the amount of uricase required to oxidize one pmole of uric acid per minute at 25 ° C under defined conditions. The efficacy of uricase is expressed as activity units / mg protein (E / mg).

extinction coefficient of mM uric acid at 292 nm 12.2 mM '¹cm '¹. Accordingly, 1 pmol of uric acid / ml of the reaction mixture had an oxidation of 12.2 mA₂92 resulted in a decrease in absorbance. The change in absorbance over time (AA₂₉₂/ min) was derived from the linear portion of the curve.

Protein concentration was modified by the Bradford method [Macart and Gerbaut, Clin. Chim. Acta 122: 93-101 (1982)]. The specific activity (potency) of uricase was calculated by dividing the activity (E / ml) by the protein concentration (mg / ml). The enzymatic activity results for the various uricases are summarized in Table 4. The results of the commercially available formulations are shown in the table as reference values. The results show that their enzymatic activity is not significantly affected by truncation of the uricase proteins.

- 57 ··* ·»· ·*&lt;

4th spreadsheet

Summary of kinetic parameters of recombinant and native uricases

<td>JVA. t u</td><td>- .. - · x oj_ j- ac concentration (Mg / ml) ⁽¹⁾</td><td>úpccxxxhud activity (U / mg) ^{(2) 3 4}</td><td>-P7P -, -, PdÍL (μΜ urinary- acid)</td><td>^{r &lt;}kat (1 minute)</td>

<td colspan="5">recombinant</td>

<td>Pig</td><td> 0,49</td><td> 7,41</td><td> 4,39</td><td> 905</td>

<td>Pork ΔΝ</td><td> 0, 54</td><td> 7,68</td><td> 4,04</td><td> 822</td>

<td>Pig-KS</td><td> 0,33</td><td> 7,16</td><td> 5,27</td><td> 1085</td>

<td>Pig-KS-ΔΝ</td><td> 1,14</td><td> 6, 20</td><td> 3, 98</td><td> 972</td>

<td>PBC</td><td> 0,76</td><td> 3,86</td><td> 4,87</td><td> 662</td>

<td>PBC ANC</td><td> 0,55</td><td> 3,85</td><td> 4,3</td><td> 580</td>

<td>Rabbit</td><td> 0,44</td><td> 3,07</td><td> 4,14</td><td> 522</td>

<td colspan="5">native</td>

<td>Pig (Sigma)</td><td> 2,70</td><td> 3,26^Uí</td><td> 5,85</td><td> 901</td>

<td>A. flavus (Merck)</td><td> 1, 95</td><td> 0, 97^(Jl</td><td> 23,54</td><td> 671</td>

⁽¹⁾ Protein concentration was determined by absorbance at 278 nm using an extinction coefficient of 11.3 for 10 mg / ml uricase solution (Mahler, 1963).

⁽²⁾ 1 unit of uricase activity is defined as the amount of enzyme which oxidizes 1 pmole of uric acid to allantoin per minute at 25 ° C.

⁽³⁾ Specific activity values ​​were derived from Lineweaver-Burk graphs (at substrate concentrations of 60 μΜ).

⁽⁴⁾ The reaction mixtures consisted of various combinations of the following stock solutions:

100 mM sodium borate buffer (pH 9.2);

300 50 mM sodium borate buffer (pH 9.2) containing μΜ uric acid;

50 mM sodium borate buffer (pH 9.2) containing mg / ml BSA.

^&lt;5&gt; AK_kat to calculate the value, the Vmax (calculated from the corresponding Lineweaver-Burk diagram) is divided by the concentration of uricase in the reaction mixture (expressed in molar equivalents based on the tetramer molecular weight of the uricases).

5th example

Conjugation of urease to m-PEG (PEGylation)

The conjugation of porcine KS-AN uricase was performed using m-PEG-NPC (monomethoxy poly (ethylene glycol) nitrophenyl carbonate). The conditions leading to the presence of 2-12 strands of 5, 10 or 20 kD PEG per uricase subunit were created and the m-PEG-NPC was gradually added to the protein solution. After the addition of PEG, the uricase / m-PEG-NPC reaction mixture was incubated at 2-8 ° C for 16-18 hours, until the conjugation of the maximum amount of unbound m-PEG strand to uricase did not occur.

The number of PEG fibers per PEG uricase monomer was determined by Superose 6 size exclusion chromatography (SEC) using PEG and uricase standards. The number of bound PEG strands per subunit was calculated using the following equation:

PEG fiber / = 3.42 x PEG (μg) subunit in injected sample protein (μρ)

Concentration of PEG and protein units in the PEG-uricase sample was determined by size exclusion chromatography (SEC), sequential ultraviolet (UV) and refractive index (R1) detectors [Kunitani et al. (1991)]. Three calibration curves were prepared: one protein curve (absorbance at 220 nm); another protein curve (measured by refractive index); and a PEG curve (measured by refractive index). PEG uricase samples were then analyzed using the same system. The concentrations of PEG and protein relative to the calibration curves were calculated using the UV and RI peak area values ​​obtained for the experimental samples. The index of 3.42 is the ratio of the molecular weight of the uricase monomer (34.192 D) to the molecular weight of the 10 kDa PEG.

PEG bound to uricase increased the solubility of uricase in physiological pH solutions. Table 5 shows the variability between different batches of PEGylated porcine KS-AN uricase. In general, there is an inverse relationship between the number of PEG fibers bound to uricase and the remaining specific activity (SA) of the enzyme.

• · « · ·

5th spreadsheet

Enzymatic activity of PEGylated porcine KS-AN-uricase conjugates

Γττ: ι --- ι --- ι -——— ·.-Ι-1

<td>iVVii J SA CJ GL C. item</td><td>i La molecule- crowd (KDa)</td><td>Γ fibers / urikáz- sub-unit</td><td>Ul ± jvcl L SA (U / mg)</td><td>Sri (as a percentage of control)</td>

<td>Pig-KS ΔΝ</td><td></td><td></td><td> 8,2</td><td> 100</td>

<td> 1-17 #</td><td> 5</td><td> 9,7</td><td> 5,8</td><td> 70,4</td>

<td>LP-17</td><td> 10</td><td> 2,3</td><td> 7,8</td><td> 94,6</td>

<td> 1-15 #</td><td> 10</td><td> 5,1</td><td> 6, 4</td><td> 77,9</td>

<td> 13 #</td><td> 10</td><td> 6, 4</td><td> 6, 3</td><td> 76, 9</td>

<td> 14 #</td><td> 10</td><td> 6, 5</td><td> 6, 4</td><td> 77,5</td>

<td> 5-15 #</td><td> 10</td><td>00 co</td><td> 5,4</td><td> 65, 3</td>

<td> 5-17 #</td><td> 10</td><td> 11,3</td><td> 4,5</td><td> 55,3</td>

<td> 4-17 #</td><td> 10</td><td> 11,8</td><td> 4,4</td><td> 53, 9</td>

<td> 1-18 #</td><td> 20</td><td> 11,5</td><td> 4,5</td><td> 54,4</td>

6th example

PEGylation of urease with 1000 D and 100 000 D PEG

Porcine KS-AN uricase was conjugated as described in Example 5 using 1000 D and 100,000 D m-PEG-NPC. The conditions resulting in the presence of 2-12 strands of PEG per Uricase subunit were used. After the addition of PEG, the uricase / m-PEG-NPC reaction mixture was incubated at 2-8 ° C for 16-18 hours, until the conjugation of the maximum amount of unbound m-PEG fiber to uricase did not occur.

PEG fibers per PEG uricase monomer! number was determined as described above.

Uricase-bound PEG increased uricase physiology • · ················································ · ·· «· · ·· ·

- solubility in solutions of pH 61i.

7th example

Pharmacokinetics of PEG-conjugated porcine KS-AN uricase

Biological experiments were performed to determine the optimal size and extent of PEGylation to achieve therapeutic benefit.

In rat pharmacokinetic studies, 0.4 mg (2 units) / kg body weight of unchanged uricase was injected intravenously in the circulation on days 1 and 8 of the experiment. It showed a half-life of 10 minutes. However, clearance rate studies in rats with the 2-11 x 10 kD PEG / porcine SKΔΝ-uricase conjugate following nine weekly injections showed that clearance was not correlated with the number of PEG filaments (in this order) and remained relatively constant throughout the study (see Figure 2)

6th table; half-life: approx. 30 hours). Differences from week to week remained within the experimental margin of error. Similar results were obtained after nine injections of uricase conjugates with 10x5kD and 10x20 kD PEG filaments. The results indicate that irrespective of the degree of PEGylation of uricase (in this order of magnitude), similar biological effects were observed in the rat model.

·· ···· «· · · · • ·· · ·· ·· • ··· · · · · · 4 · · ··«·»«· * * »*’**»’

6th spreadsheet

Half-life of PEGylated porcine KS-AN uricase formulations in rats il.Ca (number of PEG filaments per uricase subunit)

<td></td><td>5kDPEG</td><td colspan="5">10d PEG</td><td>20kD PEG</td>

<td>Seven</td><td>lOx</td><td>2x</td><td>5x</td><td>7x</td><td>9x</td><td>IIx</td><td>lOx</td>

<td> 1.</td><td> 25, 7</td><td> 29, 4</td><td> 37,7</td><td> 37, 6</td><td> 36, 9</td><td> 31,4</td><td> 21,6</td>

<td></td><td> ± 1,7</td><td> ± 3,4</td><td> ± 3,1</td><td> ± 3,9</td><td> ± 4,3</td><td> ± 4,3</td><td> ± 1,5</td>

<td></td><td> (5)</td><td> (5)</td><td> (5)</td><td> (5)</td><td> (5)</td><td> (5)</td><td> (5)</td>

<td> 2.</td><td></td><td></td><td></td><td> 26, 7 ± 3,0 (5)</td><td> 28,4 ± 1,6 (5)</td><td></td><td></td>

<td> 3.</td><td> 27,5</td><td> 29,0</td><td> 29, 9</td><td> 32,7</td><td> 26, 3</td><td> 11,8</td><td> 14,5</td>

<td></td><td> ± 3,8</td><td> ± 2,6</td><td> +</td><td> ± 11,1</td><td> ± 4,7</td><td> + 3,3</td><td> ± 2,7</td>

<td></td><td> (5)</td><td> (5)</td><td> 11,7 (5)</td><td> (5)</td><td> (5)</td><td> (5)</td><td> (5)</td>

<td> 4 .</td><td></td><td></td><td> 27,1 ± 5,3 (5)</td><td> 18,4 ± 2,2 (4)</td><td> 19, 7 ± 5,6 (4)</td><td></td><td></td>

<td> 5.</td><td> 28,6</td><td> 22,5</td><td> 34,3</td><td> 37,3</td><td> 30,4</td><td> 30,5</td><td> 19, 3</td>

<td></td><td> + 1,7</td><td> ± 2,7</td><td> ± 3,9</td><td> ± 3,0</td><td> ± 3,6</td><td> + 1,3</td><td> ± 2,5</td>

<td></td><td> (5)</td><td> (5)</td><td> (4)</td><td> (5)</td><td> (5)</td><td> (5)</td><td> (5)</td>

<td> 6.</td><td></td><td></td><td> 35,4 ± 3,1 (14)</td><td> 27,1 ± 3,6 (13)</td><td> 30, 7 ±2,9 (13)</td><td></td><td></td>

<td> 7.</td><td> 16, 5 ±4,9 (5)</td><td> 32,5 ± 4,3 (5)</td><td></td><td></td><td></td><td> 16, 12 ± 2,7 (5)</td><td> 25, 8 ± 2,5 (5)</td>

<td> 8.</td><td></td><td> -</td><td> -</td><td> -</td><td> -</td><td> -</td><td></td>

<td> 9.</td><td> 36, 8</td><td> 28,7</td><td> 34,0</td><td> 24,2</td><td> 31, 0</td><td> 29, 3</td><td> 26,7</td>

<td></td><td> ± 4,0</td><td> ± 2,7</td><td> ± 2,4</td><td> ± 3,4</td><td> ± 2,6</td><td> + 1,4</td><td> ± 0,5</td>

<td></td><td> (15)</td><td> (15)</td><td> (13)</td><td> (13)</td><td> (13)</td><td> (15)</td><td> (15)</td>

In Table 6, the results are hourly ± standard deviation values.

- We entered 63 as sulfur. The numbers in parentheses represent the number of animals tested.

Rats were injected intravenously weekly with 0.4 mg / kg PEGylated porcine KS-AN uricases as indicated in the table. Initially, each group consisted of 15 rats, each of which was sub-divided into subgroups of five animals. During the experiment several rats died due to anesthesia. Half-lives were determined by measuring uricase activity (colorimetric assay) in plasma samples taken 5 minutes, and 6, 24 and 48 hours after injection.

Table 5 shows the batch number of PEGylated uricases used in the assay.

The results of bioavailability studies with 6x5 kD PEG / porcine-KS-AN uricase conjugate in rabbits indicate that after first injection, the circulatory half-life was 98.2 ± 1.8 hours (iv) and the bioavailability was intramuscular (im ) and 71% and 52% after subcutaneous (sc) injection, respectively. However, the second im and sc all injectable anti-uricase antibody titers were detected after injection, and clearance was accelerated after further injections. The same conjugates were injected into rats to give a half-life of 26 ± 1.6 hours (iv), while the bioavailability after im and sc injection was 33% and 22%, respectively.

The results of experiments with 9x10 kD PEG / porcine-KS-AN-uricase conjugate in rats indicate that the circulating half-life after the first (iv) injection was 42.4 hours and the bioavailability after im and sc injection was 28.9%, and 14.5% (see Figure 5 and Table 7). Following the fourth injection, the circulatory half-life was 32.1 ± 2.4 hours, while the bioavailability after IM and SC injection was 26.1% and 14.9%, respectively.

Based on results of similar pharmacokinetic studies with 9x10 kD PEG / porcine KS-AN uricase conjugate in rabbits, no acceleration of clearance was observed following injection of this conjugate (four injections every two weeks). The circulating half-life in these animals was 88.5 hours after the first injection (iv), and the bioavailability after im and sc injection was 98.3% and 84.4% (see Figure 6 and Table 7). ). After the fourth injection, the circulatory half-life was 141.1 ± 15.4 hours and the bioavailability was 85% (im) and 83% (sc), respectively.

Similar bioavailability studies with 9x10 kD PEG / porcine KS-AN-uricase conjugate were performed in beagle dogs (two males and two females per group). The circulating half-life after the first iv injection was 70 ± 11 hours, whereas the bioavailability after im and sc injection was 69.5% and 50.4% (see Fig. 7).

and Table 7).

9x10 kD PEG / porcine-KS-AN-uricase conjugate was also tested in pigs. Three animals per group were used for iv, sc and im injections. Following the first iv injection, a circulating half-life of 178 ± 24 hours was recorded, while the bioavailability after im and sc injections was 71.6% and 76.8%, respectively (see Figure 8 and Table 7).

7th spreadsheet

Results of pharmacokinetic studies with 9x10 kD PEG / porcine KS-AN uricase conjugate

<td rowspan="2">injections number</td><td>Half-life (Hours)</td><td colspan="2">bioavailability</td>

<td>arc</td><td>im</td><td>sc</td>

<td>rats</td><td></td><td></td><td></td>

<td> 1</td><td> 42,4 ± 4,3</td><td> 28,9%</td><td> 14,5%</td>

<td> 2</td><td> 24,1 + 5,0</td><td> 28,9%</td><td> 14,5%</td>

<td> 4</td><td> 32,1 ± 2,4</td><td> 26, 1%</td><td> 14,9%</td>

<td>rabbits</td><td></td><td></td><td></td>

<td> 1</td><td> 88,5 ± 8,9</td><td> 98,3%</td><td> 84,4%</td>

<td> 2</td><td> 45,7 ± 40,6</td><td> 100%</td><td> 100%</td>

<td> 4</td><td> 141,1 ± 15,4</td><td> 85%</td><td> 83%</td>

<td>Dogs</td><td></td><td></td><td></td>

<td> 1</td><td> 70,0 ± 11,7</td><td> 69, 5%</td><td> 50, 4%</td>

<td>pigs</td><td></td><td></td><td></td>

<td> 1</td><td> 178 ± 24</td><td> 71, 6%</td><td> 76, 8%</td>

The 9x10 kD PEG / porcine-KS-AN-uricase conjugate by Bolton &amp; Hunter ¹²⁵After iodination with I-isotope, absorption, distribution, metabolism and excretion assays (ADME) were performed. The radiolabeled conjugate was injected into seven groups of rats (four rats per group, two males, two females). The distribution of radioactivity was examined one hour after injection and every 24 hours for seven days. We killed all the groups,

<img file="HU0700730A2_D0007.tif" />

The organs of the animals were excised and analyzed. The seventh group was housed in a metabolic cage from which urine and faeces were collected. The distribution of the substance in the body of the animals was evaluated based on the radioactivity in each organ and the number of counts (kidney, liver, lung and spleen) available for precipitation with TCA (bound protein normalized to organ size). None of the excised organs exhibited more specific radioactivity than the others, indicating that there was no significant accumulation, for example in the liver or kidney. By day 7, 70% of the radioactivity was selected.

8th example

Results of clinical trials

To determine the urate response, pharmacokinetics and safety profile of PEG-uricase (Puricase®, Savient Pharmaceuticals) in patients with hyperuricaemia and severe gout, who are unresponsive to or unsuitable for conventional therapy, we have randomized, open-label, multi-center, parallel groups. The median duration of the disease was 14 years and 70% of the subjects included one or more tophus.

In the study, 41 patients (mean age: 58.1 years) were randomized to receive intravenous PEG-uricase conjugate for 12 weeks in one of the following four dosage regimens: 4 mg (7 patients) every two weeks; 8 mg every 8 weeks (8 patients); every 4 weeks 8 mg (13 · ·

- 67 patients); 12 mg (13 patients) every four weeks. Plasma uricase activity and urate levels were measured at regular intervals. From the analysis of uricase activity and urate level, pharmacokinetic parameters, mean plasma urate concentration and time to blood plasma urate concentration up to 6 mg / dl were calculated.

Patients treated with PEG-uricase conjugate 8 mg every two weeks showed the greatest decrease in plasma urate level (PUA); PUA levels were below 6 mg / dl in 92% of the treatment duration (pre-treatment plasma urate level was 9.1 mg / dL, compared to 1.4 mg / dl mean plasma levels over a 12-week treatment period).

In other groups treated with PEG-uricase conjugate, a significant and sustained decrease in plasma urate levels was also observed: 86% of the treatment duration had a PUA level below 6 mg / dL (pre-treatment PUA level 9%). 1 mg / dl, as opposed to an average PUA level of 2.6 mg / dl over a 12-week treatment period); in the 12 mg conjugate-treated group, 84% of the duration of treatment had PUA levels below 6 mg / dL (here the pre-treatment PUA level was 8.5 mg / dL, as opposed to the 12-week treatment duration

2.6 mg / dL average PUA level); and 73% of the duration of treatment had a PUA level below 6 mg / dL (pre-treatment PUA 7.6 mg / dL versus 12 weeks of treatment) in 73% of patients treated with conjugate every 4 weeks. , With an average PUA level of 2 mg / dl).

In the first 24 hours after administration of the PEG-uricase conjugate, the maximum percentage reduction from baseline in plasma urea was 72% (p = 0.0002) in the 4 mg / 2 week regimen; 94% (p &lt;0.0001) in the 8 mg / 2 week group; 87% (p &lt;0.0001) in the 8 mg / 4 week treatment group; and 93% (p &lt;0.0001) in the 12 mg / 4 week treatment group.

During the 12-week treatment period, the percentage reduction from baseline in plasma ururate levels in the 4 mg / 2 week regimen was 38% (p = 0.0002); 86% (p &lt;0.0001) in the 8 mg / 2 week group; 58% (p = 0.0003) in the 8 mg / 4 week treatment group; and 67% (p &lt;0.0001) in the 12 mg / 4 week treatment group.

Surprisingly, some of the patients treated with PEG-uricase conjugate experienced infusion-related events, i.e., an infusion reaction. These reactions occurred in 14% of all infusions.

All references cited in the description of the invention are to be understood in their entirety as part of the teaching.

As will be apparent to those skilled in the art, many modifications and variations can be made to the practice of the present invention without departing from the spirit of the invention. The specific embodiments we have disclosed are merely exemplary; the invention is clearly defined by the appended claims.

• « 9 » · · ·

To translate the free text snippets of the sequence list:

For SEQ ID NO: 1:

&lt;223&gt; Porcine liver uricase (senses)

For SEQ ID NO: 2:

&lt;223&gt; Porcine liver uricase (antisense)

For SEQ ID NO: 3:

&lt;223&gt; Baboon (D3H) liver uricase (sense)

For SEQ ID NO: 4:

&lt;223&gt; Baboon (D3H) liver uricase (antisense)

For SEQ ID NO: 5:

&lt;223&gt; PBC-DeltaNC Uricase (Sense)

For SEQ ID NO: 6:

&lt;223&gt; PBC-DeltaNC Uricase (antisense)

For SEQ ID NO: 7:

&lt;223&gt; Pig-KS-DeltaN

For SEQ ID NO: 8:

&lt;223&gt; Swine-KS-DeltaN (without Met)

For SEQ ID NO: 9:

&lt;223&gt; Pig-KS-DeltaNA

For SEQ ID NO: 10:

&lt;223&gt; Pig-KS-DeltaN (no beginner ATG)

For SEQ ID NO: 12:

&lt;223&gt; PBC-DeltaN

For SEQ ID NO: 13:

&lt;223&gt; PBC-DeltaN (Beginner Without Met)

For SEQ ID NO: 14:

&lt;223&gt; PBC-DeltaN (fragment 44-56 of PBC-DeltaNC)