Untitled record

Urate oxidase version and application

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, excessive blood uric acid levels (hyperuricemia) can lead to painful arthritis (gout), malformed urate deposits (tophus), and renal 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: 6, 47-56; Wood, AG Bailliere's Clin. Rheumatol. _4, 177-192 (1990), source 18661 KB «« ·

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much to their full extent is to be regarded 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 it causes 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 present invention relates to mutant recombinant uricase proteins having a truncated amino acid sequence and increased structural stability.

The present invention provides novel recombinant uricase proteins. The proteins of the invention have truncated amino acid sequences and contain amino acids mutagenized compared to naturally occurring uricase proteins.

In one embodiment, the invention provides a novel mutant recombinant uricase comprising the amino acid sequence set forth in SEQ ID NO: 8. Another embodiment of the present invention is SE 3 f

mutant recombinant uricase which

13th contains the amino acid sequence shown as SEQ ID NO.

We also disclose a means for metabolizing uric acid, which comprises a novel recombinant uricase protein having uricolytic activity. As used herein, uricolytic activity refers to the enzymatic conversion of uric acid to allantoin.

In another embodiment, the uricase of the invention is amino acid sequence SEQ ID NO: 8-287 of SEQ ID NO: 7 or 12. contains the amino acid sequence. In another embodiment, the invention provides uricases comprising the amino acid sequence set forth in SEQ ID NO: 8 or 13. In one embodiment, the uricase comprises an N-terminal amino acid which may be alanine, glycine, proline, serine or threonine. In a preferred embodiment of the invention, uricase is used

It contains methionine as the N-terminal amino acid.

In addition, isolated nucleic acids comprising the nucleic acid sequence encoding the uricase of the invention are disclosed. In one embodiment, the nucleic acid encoding uricase is operably linked to a heterologous promoter (e.g., the osmB promoter). Vectors containing nucleic acids encoding the uricases of the invention and host cells comprising such vectors are further disclosed. In addition, methods for producing uricase are disclosed

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culturing the 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). Restriction sites lost during cloning are indicated in parentheses.

Figure 2 shows the DNA sequence (SEQ ID NO: 9) and the deduced amino acid sequence (SEQ ID NO: 7) of Swine KS-AN uricase. Amino acid numbering is given relative to the complete sequence of porcine uricase. Following the initial methionine, aspartic acid at position 7 of the porcine uricase 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 the circles represent the positions • · ·

<img file="HU0700729A2_D0001.tif" />

- 5 Τ in which the porcine-KS-ΔΝ 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 SDS-PAGE analysis of the highly purified uricase variants described in Examples 1 to 8 are shown. The date of manufacture (month / year) and the number of bands corresponding to each sample are given below: Lane 1: molecular weight 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); Lane 6: porcine ΔΝ (6/99); Lane 7: porcine KS-ΔΝ (7/99); Lane 8: 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 depicts the pharmacokinetic profiles of PEGylated (9x10 kD) porcine KS-AN uricase after 6 intramuscular (IM), subcutaneous (SC) and intravenous (IV) injections in rabbits, which are monitored by monitoring enzymatic activity in blood samples. a. 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 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: Pharmacokinetics of PEGylated (9x10 kD) porcine KS-AN uricase following intramuscular (IM), subcutaneous (SC) and intravenous (IV) injection in pigs.

<img file="HU0700729A2_D0002.tif" />

Figure 7 shows profiles of 7 tics as determined 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.

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 consequent need to increase the dose. 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.

In one preferred embodiment, the uricase comprises N-temrinal 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. In such a case, an amino acid is present at the last-to-last position that 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.

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 generating a nucleic acid sequence is disclosed which is a non-truncated uricase-containing nucleic acid sequence PCR (polymerase 9

<img file="HU0700729A2_D0003.tif" />

chain reaction). 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 the PCR, the target DNA is repeatedly denatured (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" is stretched from the primers. 72 ° C). Because the filaments 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 techniques disclosed in WO 00/08196 and U.S. Patent Application Publication No. 60 / 095,489, which are incorporated herein by reference in their entirety.

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.

The uricase can be isolated and purified by any of the methods well known to those skilled in the art. 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). United States Patent Application 60/670520, entitled Purification Of Proteins With Cationic Surfactant;

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 the RNA polymerase has a r-ar ·

- 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.

The uricase of the invention may also be a polymer conjugated uricase; for example, polyethylene glycol conjugated uricase (PEGylated 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. In another embodiment, the composition is packaged in a vial, optionally closed with a puncture-resistant rubber 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 normalization of plasma uric acid (PUA) levels and PUA

<img file="HU0700729A2_D0004.tif" />

Reduction or maintenance 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. The uricase may be administered by any of the methods well known to those skilled in the art (for example, intravenously, intramuscularly or subcutaneously). 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, 6 months, 8 months or 12 months. Other Embodiments Wed ·· »

- 13 rins for a treatment period of 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 combinations thereof are administered. 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, dimenhydrinate, diphenhydramine, doxylamine, fexofenadine, hydroxyzin, loratadine.

In one embodiment of the invention, the antihistamine is fexofenadine, the NSAID is α-acetaminophen, and the corticosteroid is hydrocortisone and / or prednisone.

<img file="HU0700729A2_D0005.tif" />

- 14 employed. 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, 60 mg of fexofenadine is administered orally the night before the uricase infusion, and 60 mg of fexofenadine and 1000 mg of acetaminophen are administered 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 certain embodiments, prophylactic treatments with uricase (including PEGylated uricase and non-

PEGylated uricase is administered to patients in (or prior to) treatment. In other embodiments of the invention, these prophylactic k

<img file="HU0700729A2_D0006.tif" />

Treatments with therapeutic peptides other than uricase (which may be PEGylated or non-PEGylated) are administered to patients in treatment (or prior to treatment).

In one embodiment, the pharmaceutical composition comprises a polymer-conjugated uricase which retains its uricolytic activity. In one preferred embodiment, the uricase is

PEGylated uricase.

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 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.

• «»· «

• · · ··«· ·»*

- 16 •·« ···

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.

In one embodiment of the invention, the composition is suitable for repeated administration.

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 the uricase of the invention are disclosed. The manipulations leading to their creation are well known to those skilled in the art. For example, uricase nucleic acid sequences may 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 can be cleaved at appropriate sites using restriction endonuclease (s), and then desired. · ·· '* ···

<img file="HU0700729A2_D0007.tif" />

case, further enzymatic modification, isolation and ligation 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).

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, synthesis techniques, and in vivo recombinant 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 produced in large quantities. As mentioned above, • · ♦

-included expression vectors include, but are not limited to, the following vectors and derivatives thereof: human or animal infectious viruses 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.

First Example 1: Construction of a gene and expression plasmid capable of expressing yyrase

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 uricase cDNA 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.

<img file="HU0700729A2_D0008.tif" />

*·· ·-.

First spreadsheet

Primer oligonucleotides used for PCR amplification of uricase cDNA, _z'- -. .......... - - Vol

Seitesmaj uricase:

sense 5 'gcgcgaattccATGGCTCATTACCGTAATGACTACA 3' (SEQ ID NO: 1) antisense 5 'gcgctctagaagcttccatggTCACAGCCTTGAAGTCAGC 3' (SEQ ID NO: 2)

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 (Baboon) The third GAC codon (aspartic acid) in the baboon sense primer was replaced by the CAC codon (histidine) located at this position in the human urate oxidase pseudogenic coding sequence. 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-babanuricase PCR product was directly cloned into the pCR ™ II vector using TA Cloning ™ (Invitrogen, Carlsbad, CA), whereby the pCR ™ II1 '·· * ·· ~&gt;

» » ··**

Plasmid D3H-babanuricase was obtained.

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 using 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 porcine 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-3ds 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 yyrase 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). This vector allows for the induction of protein expression by high osmotic pressure or culture aging. The pMFOA-18 expression plasmid contains the osmB promoter, ribosome binding site sequence (rbs) and 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 deoperoperon), a transcription termination sequence (F. 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, with:

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

Subcloning of porcine baboon chimeric uricase

Plasmid pURBA A1 was digested with 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 in, 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. · ·························• · Increased growth. 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 is shown by the PBC sequence in the plasmid pDUR-PB as well as in Figure 2. was performed by PCR amplification using the specific primer oligonucleotides shown in Table II.

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 plasmid pDUR-PB ·· ···· ·· · ·

Λ · · · · · ·. · 9 · ··· · · · · · «······ · ··

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 expressing 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 resulting pDUR-PB-ANC construct was constructed by E. coli K-12S? 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 endonucleases 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 together to give plasmid pOUR-PB-ANC, E. coli K-12 W3110F 'and resulted in high expression of the 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. At position 46, serine is replaced by threonine, which is PCR28

<img file="HU0700729A2_D0009.tif" />

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, expressed under the control of the osmB promoter, resulted in a high amount of recombinant enzyme with excellent activity and stability.

Figure 1 shows the structure of plasmid pOUR-P-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. Pig-KS • · · · · · · · · · · · · · · ·

Plasmid pOUR-P-AN-ks-1 encoding ΔΝ-uricase is 4143 base pairs (bp) in length 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).

V • · ···· ·· · · «· · · · · ·. ··· · «· · · · · · · · · · · · · · · · · ·

- 30 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 only N-terminally and carries mutations S46T, 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 in the serV • · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·_v aaa ·· aaaaa • · · · P · «« · »'» · ·· · *

- from the published sequence of 31 porcine 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.

To generate truncated uricases (porcine-ΔΝ and porcine-KS-ΔΝ), cross-ligation between PBCANC and porcine-KS was performed after 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.

·· · · «· · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · plan List How are you? example

Transformation of an expression plasmid into a bacterial host cell

The expression plasmid pOUR-P-AN-ks-1 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. 3xlO¹⁰ cells / ml at a cell density of 10% aqueous glycerol. 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 high-voltage electroporation procedure using a Gene Pulser Type II (BIORAD) (Trevors et al., "Electrotransformation of Bacteria by Plasmid DNA, 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 MgCl₂10 mM MgSCG, 20 mM glucose), incubated at 37 ° C for one hour, selected for tetracycline resistance, and selected a high expression clone.

• ·

- 33 • ··· • · *

Third example

Production of recombinant uricase

Bacteria transformed as described above were grown in glucose-containing 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. Subsequently, Xanthine-agarose resin (Sigma) was used to remove a small amount of impurity in the uricase preparation. 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

SDS-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 N-terminal sequences of the uricase fragments formed during purification and storage 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:

--irn4gppviThe above sequences 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 t »í • X * · ♦ * · * ·

The potency of 37 uricases 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 truncation of uricase proteins does not significantly affect their enzymatic activity.

<img file="HU0700729A2_D0010.tif" />

* ··. spreadsheet

Summary of kinetic parameters of recombinant and native uricases

<td>-77 - Z-1 u ixKdZO K</td><td>_. . -&gt; · ι ΟΓ ZOOxddZ '' concentration (Mg / ml) ⁽¹⁾</td><td>Specific activity (U / mg) ⁽²⁾</td><td>--- Γ7Π-1 ml (μΜ urinary- acid)</td><td>- / L. \ .........-₃Nkat '' (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 ΔΝΟ</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^U)</td><td> 5,85</td><td> 901</td>

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

^(1&gt; 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);

«·. 4 4 * «*« * «« »» · · • · «« • t) ♦ * · • · · «· &amp;

·«· «♦»

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.

ⁱ⁵⁾ 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-NAN-uricase was performed using m-PEG-NPC (monomethoxy-poly (ethylene glycol) -ni.-trophenyl 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 bound PEG fibers per subunit! number was calculated using the following equation:

PEG fiber / = 3.42 x PEG (gg) subunit in injected sample protein (pg) • · ···; * ». · »· &lt;*« ·· «« * t ·· * · * «·» · ·· &lt;* f 4 * · «•« ti · ♦ · · 9 &lt;* «

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. To calculate concentrations of PEG and protein relative to calibration curves, the UV and

Area values ​​below the RI peak were used. 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.

- 41 • · 4 · - * »·« · * · • «*» &lt;· », ϊ.

5th spreadsheet

Enzymatic activity of PEGylated porcine KS-AN-uricase conjugates

<td>Koiij barking lot</td><td>PEG molecule- crowd (KDa)</td><td>PEG fibers / urikáz- sub-unit</td><td>Unkaz SA (U / mg)</td><td>SA (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> 7 7,9</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> 8,8</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 urease 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 .- &lt;*&gt; ♦ * »*

«« »&lt;· * · · · Ν« ♦ · *&gt; «4,.» ·. '

- solubility in 42 pH solutions.

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.

f * »·· * w» · '' í

6th spreadsheet

Half-life of PEGylated porcine KS-AN-uricase formulations in rats

Rate of Change (number of PEG strands 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 shown as hour ± standard deviation.

<img file="HU0700729A2_D0011.tif" />

given 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 after injection, significant rabbit anti-uricase antibody titer was detected in all rabbits 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 * 9 · ·*· · the

The circulatory half-life after the first 45 (iv) injections was 42.4 hours and the bioavailability after im and sc injections 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). In these animals, the circulatory half-life was 88.5 hours after the first injection (iv), and the bioavailability after im and sc injections was 98.3% and 84.4% (see Figure 6 and Fig. 7). table). 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 recorded after the first iv injection was 70 ± 11 hours, while the bioavailability after im and sc injection was 69.5% and 50.4%, respectively (see Figure 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">Number of injections</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 groups, · ·

47 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.

During the study, 41 patients (mean age: 58.1 years) were randomized to receive intravenous doses of PEGuricase conjugate for 12 weeks in one of four dosage regimens: 4 mg (7 patients) every two weeks; 8 mg every 8 weeks (8 patients); 8 mg every four weeks (13 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 treatment duration had a PUA level of less than 6 mg / dL (pre-treatment PUA 7.6 mg / dL versus the 12-week treatment period) in 73% of patients treated with conjugate every 4 weeks. · · · · · · · · · · · · · · ·_ete calculated with an average PUA level of 4.2 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 mg / 4 week schedule; 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.

• · ·

- 50 Translation of the free-text sections of the Sequence Listing:

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)