SOLUBLE HYALURONIDASE GLYCOPROTEIN (sHASEGP), PROCESS FOR PREPARING THE SAME, USES AND PHARMACEUTICAL COMPOSITIONS COMPRISING THEREOF

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DESCRIPTION

Soluble hyaluronidase glycoprotein (sHASEGP), process to prepare it, uses and pharmaceutical compositions that comprise it.

Cross reference with related requests

This application claims priority in accordance with 35 USC § 119 (e) over US Serial Number 60 / 452,360, filed March 5, 2003.

Background of the invention

Field of the invention

The present invention relates generally to soluble neutral pH active hyaluronidase glycoproteins (sHASEGP), portions thereof, particularly hyaluronidase domains. More specifically, the invention relates to chemical modifications, pharmaceutical compositions, expression plasmids, methods for the manufacture and therapeutic methods using the hyaluronidase glycoproteins and domains thereof and the encoding nucleic acid molecules for the therapeutic modification of glycosaminoglycans in the treatment of a disease and for the use to increase the diffusion of other molecules injected of less 200 nanometers in diameter in an animal.

Background information

Glycosaminoglycans (GAGs) are complex linear polysaccharides of the extracellular matrix (ECM). GAGs are characterized by repeating disaccharide structures of an N-substituted hexosamine and uronic acid, [hyaluronan (HA), chondroitin sulfate (CS), chondroitin (C), dermatan sulfate (DS), heparan sulfate (HS) , heparin (H)], or a galactose, [keratan sulfate (KS)]. Except for HA, all are covalently bound to core proteins. GAGs with their core proteins are structurally called proteoglycans (PGs).

Hyaluronan (HA) is found in mammals predominantly in connective tissues, skin, cartilage, and in synovial fluid. Hyaluronan is also the main constituent of the vitreous humor of the eye. In connective tissue, the hydration water associated with hyaluronan generates spaces between tissues, thus generating an environment that leads to cell movement and proliferation. Hyaluronan plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, embryonic development, wound healing, angiogenesis and oncogenesis (Toole 1991 Cell Bioll Extracell. Matrix, Hay (ed.), Plenum Press, New York, 1384-1386; Bertrand et al. 1992 Int. J. Cancer 52: 1-6; Knudson et al, 1993 FASEB J. 7: 1233-1241). Furthermore, hyaluronan levels correlate with tumor aggressiveness (Ozello et al. 1960 Cancer Res. 20: 600-604; Takeuchi et al. 1976, Cancer Res. 36: 2133-2139; Kimata et al. 1983 Cancer Res. 43: 13471354).

HA is found in the extracellular matrix of many cells, especially in soft connective tissues. HA has been assigned various physiological functions, such as in water and plasma protein homeostasis (Laurent TC et al (1992) FASEB J 6: 2397-2404). HA production is increased in proliferating cells and may play a role in mitosis. It has also been implicated in locomotion and cell migration. HA appears to play important roles in cell regulation, development, and differentiation (Laurent et al, above).

HA has been used in clinical medicine. Its rheological and tissue protective properties have proven useful in ophthalmic surgery to protect the corneal endothelium during cataract surgery. Serum HA is diagnostic of liver disease and various inflammatory conditions, such as rhe6umatoid arthritis. Interstitial edema caused by accumulation of HA can cause dysfunction in various organs (Laurent et al, previously).

The protein interactions of hyaluronan are also involved in the structure of the extracellular matrix or "ground substance".

Hyaluronidases are a group of enzymes active at neutral pH and at acidic pH that are found throughout the animal kingdom. Hyaluronidases vary with respect to substrate specificity and mechanism of action.

There are three general classes of hyaluronidases:

1. Mammalian-type hyaluronidases, (EC 3.2.1.35) which are endo-beta-N-acetylhexosaminidases with tetrasaccharides and hexasaccharides as the main end products. They have both hydrolytic and transglycosidase activities and can degrade hyaluronan and chondroitin sulfates (CS), specifically C4-S and C6-S.

2. Bacterial hyaluronidases (EC 4.2.99.1) degrade hyaluronan and to varying degrees CS and DS. They are endobeta-N-acetylhexosaminidases that work through a beta-elimination reaction that mainly produces disaccharide end products.

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3. Hyaluronidases (EC 3.2.1.36) from leeches, other parasites, and crustaceans are endo-beta-glucuronidases that generate tetrasaccharide and hexasaccharide end products through hydrolysis of the beta 1-3 bond.

Mammalian hyaluronidases can be further divided into two groups: enzymes active at neutral pH and active at acidic pH. There are six hyaluronidase-like genes in the human genome, HYAL1, HYAL2, HYAL3, HYAL4, HYALP1, and PH20 / SPAM1. HYALP1 is a pseudogene and HYAL3 has not been shown to possess enzymatic activity towards any known substrates. HYAL4 is a chondroitinase and lacks activity towards hyaluronan. HYAL1 is the prototypical acidic pH active enzyme and PH20 is the prototypical neutral pH active enzyme. Hyaluronidases active at acidic pH, such as HYAL1 and HYAL2, lack catalytic activity at neutral pH. For example, HYAL1 has no in vitro catalytic activity above pH 4.5 (Frost et al Anal Biochemistry, 1997). HYAL2 is an acidic pH active enzyme with very low specific activity in vitro.

Hyaluronidase-like enzymes can also be characterized by those that are immobilized on the plasma membrane through a glycosylphosphatidylinositol anchor such as human HYAL2 and human PH20 (Danilkovitch-Miagkova, et al. Proc Natl Acad Sci USA. April 15, 2003 ; 100 (8): 4580-5, Phelps et al., Science 1988) and those that are soluble, such as human HYAL1 (Frost et al, Biochem Biophys Res Commun. 1997 Jul 9; 236 (1): 10 -5). However, there are variations between species: for example, bovine PH20 binds very weakly to the plasma membrane and is not anchored by a phospholipase-sensitive anchor (Lalancette et al, Biol Reprod., 2001 Aug; 65 (2): 628 -36.). This unique characteristic of bovine hyaluronidase has allowed the use of soluble bovine testes hyaluronidase enzyme as an extract for clinical use (Wydase®, Hyalase®). Other PH20 species are lipid-anchored enzymes that are not insoluble without the use of detergents or lipases. For example, human PH20 is anchored to the plasma membrane through a GPI anchor. Attempts to prepare human PH20 DNA constructs that would not introduce a lipid anchor into the polypeptide resulted in a catalytically inactive enzyme or an insoluble enzyme (Arming et al Eur J Biochem. 1997 Aug 1; 247 (3): 810 -4). The naturally occurring macaque sperm hyaluronidase is found in both soluble and membrane-bound forms. While the 64 kDa membrane-bound form possesses enzymatic activity at pH 7.0, the 54 kDa form is only active at pH 4.0 (Cherr et al, Dev Biol. 1996 Apr 10; 175 (1) : 142-53). Therefore, soluble forms of PH20 often lack enzymatic activity under neutral conditions.

Chondroitinases are enzymes found throughout the animal kingdom. These enzymes break down glycosaminoglycans through an endoglycosidase reaction. Specific examples of known chondroitinases include chondroitinase ABC (obtained from Proteus vulgaris; Japanese Patent Application Laid-Open No. 6153947, T. Yamagata, H. Saito, O. Habuchi and S. Suzuki, J. Biol. Chem., 243, 1523 (1968), S. Suzuki, H. Saito, T. Yamagata, K. Anno, N. Seno, Y. Kawai, and T. Furuhashi, J. Biol. Chem., 243, 1543 (1968)), chondroitinase AC (obtained from Flavobacterium heparinum; T. Yamagata, H. Saito, O. Habuchi and S. Suzuki, J. Biol. Chem., 243, 1523 (1968)), chondroitinase AC II (obtained from Arthrobacter aurescens; K. Hiyama and S. Okada, J. Biol. Chem., 250, 1824 (1975), K. Hiyama and S. Okada, J. Biochem. ( Tokyo), 80, 1201 (1976)), hyaluronidase ACIII (obtained from Flavobacterium sp. Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, Keiichi Yoshida, Kiyoshi Morikawa and Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1989)), chondroitinase B (obtained from Flavobacterium eparinum; YM Michelacci and CP Dietrich, Biochem. Biophys. Res. Commun., 56 Res. Commun., Res. Commun. 973 (1974), YM Michelacci and CP Dietrich, Biochem. J., 151, 121 (1975), Kenichi Maeyama, AkiraTawada, Akiko Ueno and Keiichi Yoshida, Seikagaku, 57, 1189 (1985)), chondroitinase C (obtained from Flavobacterium sp. Hp102; Hirofumi Miyazono, Hiroshi Kikuchi, Keiichi Yoshida, Kiyoshi Morikawa and Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1939)) and the like.

Glycoproteins are composed of a polypeptide chain covalently linked to one or more carbohydrate moieties. There are two broad categories of glycoproteins that possess carbohydrates coupled through their N-glycosidic or O-glycosidic bonds to their constituent protein N- and O-linked glycans bind to polypeptides through asparagine-N-acetyl-D-glucosamine bonds and serine (threonine) -N-acetyl-D-galactosamine, respectively. Complex N-linked oligosaccharides do not contain terminal mannose residues. They contain only terminal N-acetylglucosamine, galactose and / or sialic acid residues. Hybrid oligosaccharides contain terminal mannose residues, as well as terminal N-acetylglucosamine, galactose and / or sialic acid residues.

With N-linked glycoproteins, an oligosaccharide precursor binds to the amino group of asparagine during peptide synthesis in the endoplasmic reticulum. The oligosaccharide residue is then processed sequentially by a series of specific enzymes that delete and add sugar residues. Processing occurs in the endoplasmic reticulum and continues through the cis-, medial-, and trans-Golgi apparatus.

Summary of the invention

Provided herein are members of the family of soluble hyaluronidase glycoproteins active at neutral pH, particularly the soluble human PH-20 hyaluronidase proteins (also referred to herein as sHASEGP). The sHASEGP provided herein is a member of the sHASEGP family, referred to herein as sHASEGP. The soluble hyaluronidase domain and uses thereof are also provided.

The invention is based on the discovery that a neutral pH active soluble hyaluronidase activity can be produced in high yield in a mammalian expression system by introduction of nucleic acids lacking amino acids encoding a narrow region at the carboxy terminal end of the PH20 cDNA

ES 2 335 005 T3 human. Additional modifications of sHASEGP are also provided to increase secretion through the use of non-native leader peptides. In addition, methods are provided for modifying sHASEGP to extend its half-life by masking the protein with polyethylene glycol and post-translational modifications to native glycosylation. Previous attempts to generate a secreted human sHASEGP active at neutral pH were unsuccessful. It was concluded that truncations of the human sHASEGP polypeptide resulted in both a loss of enzymatic activity at neutral pH and an inability of cells to secrete the recombinant protein in mammalian expression systems (Arming, et al Eur J Biochem, August 1, 1997; 247 (3): 810-4). Generating a secreted sHASEGP that acts at neutral pH is critical for commercial production and therapeutic utility as hyaluronidase. The invention, described in this document, overcomes these challenges.

In a first aspect, the present invention provides a substantially purified glycoprotein comprising a neutral pH active soluble hyaluronidase polypeptide containing at least one N-linked sugar residue, the N-linked sugar residue being covalently linked to an asparagine residue of the polypeptide; the substantially purified glycoprotein comprises an amino acid sequence included in SEQ ID NO: 1 or a sequence having an amino acid sequence identity of at least about 91% with an amino acid sequence included in SEQ ID NO: 1 ; and the substantially purified glycoprotein is soluble. The studies shown in this document demonstrate that human PH20 requires N-linked glycans for catalytic activity, while bovine and bee venom hyaluronidases remain active without such N-linked glycans. A human hyaluronidase domain devoid of linked residues a N is catalytically inactive. Therefore, classical recombinant DNA technology does not allow the production of a catalytically active human sHASEGP, unlike bee venom HASEGP, which can be produced in E. coli.

The invention includes methods and cells for the generation of an N-linked sHASEGP glycoprotein polypeptide by using a cell capable of introducing said N-linked sugar moieties or by introducing said N-linked moieties into a sHASEGP polypeptide. Methods for properly identifying glycosylated sHASEGP are further described.

Modifications of sHASEGP are provided to further extend the half-life. Chemical modifications of a sHASEGP are provided with polymers such as polyethylene glycol and dextran. These modifications protect sHASEGP from elimination from the circulation and the immune system, as well as glycosylation receptors for mannose and asialoglycoprotein. Also provided are methods for attaching to specific functional groups, such as glycosylation sites, positively charged amino acids, and cysteines.

Also provided herein are assays to identify effectors, such as compounds, including small molecules, and conditions, such as pH, temperature, and ionic strength, that modulate sHASEGP activation, expression, or activity. In exemplary assays, the effects of test compounds on the ability of a hyaluronidase domain of sHASEGP to cleave a known substrate, typically a glycosaminoglycan or proteoglycan, are evaluated. Agents, generally compounds, particularly small molecules, that modulate the activity of the hyaluronidase domain are candidate compounds for modulating the activity of sHASEGP. Hyaluronidase domains can also be used to produce hyaluronidase-specific antibodies with function-altering activity. The hyaluronidase domains provided herein include, but are not limited to, the N-terminal glycosyl hydrolase domain with truncated C-terminal portions thereof exhibiting in vitro catalytic activity.

Also provided are nucleic acid molecules that encode the hyaluronidase proteins and domains. Nucleic acid molecules are provided that encode a soluble hyaluronidase domain or catalytically active portions thereof and also those that encode the full-length sHASEGP. The nucleic acid encoding the hyaluronidase domain and the downstream nucleic acid are set forth in SEQ ID NO: 6; and the hyaluronidase domain of sHASEGP is set forth in SEQ ID NO: 1 (amino acids 35-464). The protein sequence and the nucleic acid sequence encoding the full-length sHASEGP are set forth in SEQ ID NOS: 1 and 6.

Also provided are nucleic acid molecules that hybridize to said sHASEGP-encoding nucleic acid along its full length or along at least about 70%, 80%, or 90% of the full length and encode the hyaluronidase domain or a portion of it. Hybridization is generally carried out under conditions of at least low stringency, generally at least moderate and with high frequency.

The isolated nucleic acid fragment is DNA, including genomic or cDNA, or it is RNA or it can include other components, such as peptide nucleic acid or other nucleotide analogs. The isolated nucleic acid can include additional components, such as heterologous or native promoters and other transcriptional and translational regulatory sequences, these genes can be linked to other genes, such as reporter genes or other reporter genes or genes that encode reporters.

Also provided is an isolated nucleic acid molecule that includes the sequence of molecules that is complementary to the nucleotide sequence encoding sHASEGP or the portion thereof.

Also provided are fragments thereof or oligonucleotides that can be used as probes or primers and that contain at least about 10, 14, 16 nucleotides, generally less than 1000 or less than or equal to 100, set forth in SEQ ID NO: 6 (or complementary to it); or contain at least approximate

ES 2 335 005 T3 30 nucleotides (or the complementary thereof) or contain oligonucleotides that hybridize along their entire length (or at least approximately 70, 80 or 90% of it) with any of said fragments or oligonucleotides. The length of the fragments is a function of the purpose for which they are used and / or the complexity of the genome of interest. Probes and primers generally contain less than about 50, 150, or 500 nucleotides.

Plasmids containing any of the nucleic acid molecules provided herein are also provided. Cells containing the plasmids are also provided. Such cells include, but are not limited to, bacterial cells, yeast cells, fungal cells, plant cells, insect cells, and animal cells.

Enhanced mammalian expression systems using signal leaders capable of efficient sHASEGP secretion are also provided. An example of such an efficient secretory leader peptide amino acid sequence and fusion protein with sHASEGP is found in SEQ ID NOs: 43 and 46.

Also provided is a method of producing a substantially purified glycoprotein comprising introducing a nucleic acid encoding a polypeptide of the invention operably linked to a suitable promoter into a cell capable of incorporating N-linked sugar moieties into the polypeptide; culturing the cell under conditions whereby an encoded polypeptide is expressed by the cell; and recovering the expressed polypeptide or polypeptides.

Also provided are cells, generally eukaryotic cells, such as mammalian cells and yeast cells, in which the sHASEGP polypeptide is expressed on the surface of the cells. Such cells are used in drug screening assays to identify compounds that modulate the activity of the sHASEGP polypeptide. These assays, including in vitro binding assays, and transcription-based assays in which signal transduction mediated directly or indirectly, such as by activation of progrowth factors, by sHASEGP is evaluated.

Also provided are peptides encoded by such nucleic acid molecules. Those polypeptides include the hyaluronidase domain of sHASEGP or a polypeptide with amino acid changes such that the hyaluronidase specificity and / or activity remains substantially unchanged. In particular, a substantially purified mammalian sHASEGP glycoprotein is provided that includes a secreted form catalytically active at neutral pH.

The invention also includes a hyaluronidase catalytic domain and may further include other domains. SHASEGP can form homodimers and can also form heterodimers with some other protein, such as a membrane-bound protein. Also provided is a substantially purified glycoprotein that includes an amino acid sequence that has an identity of at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the sHASEGP. , the percent identity being determined using conventional gap penalties and algorithms that maximize percent identity.

Splicing variants of sHASEGP are contemplated herein, particularly those with a catalytically active hyaluronidase domain.

In other embodiments, substantially purified polypeptides are provided that include a hyaluronidase domain of a sHASEGP polypeptide or a catalytically active portion thereof, but do not include the complete amino acid sequence set forth in SEQ ID NO: 1. Among these are polypeptides that include an amino acid sequence that has at least 95% or 100% sequence identity to SEQ ID NO: 1 or 3.

In a specific embodiment, a nucleic acid is provided that encodes a eukaryotic hyaluronidase glycoprotein designated sHASEGP. In particular, the nucleic acid includes the nucleotide sequence set forth in SEQ ID NO: 6, particularly set forth as nucleotides 106-1446 of SEQ ID NO: 6 or a portion thereof that encodes a catalytically active polypeptide.

Also provided are nucleic acid molecules that hybridize under conditions of at least low stringency, generally moderate stringency, more typically high stringency to SEQ ID NO: 6 or degenerate sequences thereof.

In one embodiment, the isolated nucleic acid fragment hybridizes with a nucleic acid molecule containing the nucleotide sequence set forth in SEQ ID NO: 6 (or degenerate sequences thereof) under high stringency conditions. A full-length sHASEGP is set forth in SEQ ID NO: 1 and is encoded by SEQ ID NO: 6 or degenerate sequences thereof.

Muteins of the hyaluronidase domain of sHASEGP are also provided, particularly muteins in which the Cys residue in the hyaluronidase domain that is free, that is, does not form disulfide bonds with any other Cys residues in the hyaluronidase domain, is replaced with another substitution of amino acid, typically, but not necessarily, with a conservative amino acid substitution or a substitution that does not eliminate activity, and muteins in which a specific glycosylation site (s) is removed.

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Provided herein are sHASEGP polypeptides, including but not limited to splice variants thereof and nucleic acids encoding sHASEGP and domains, derivatives, and analogs thereof. Also provided are single chain secreted hyaluronidase glycoproteins having an N-terminal end functionally equivalent to that generated by activating a signal peptidase to form sHASEGP. There are seven potential N-linked glycosylation sites at N82, N166, n235, N254, N368, N393, N490 of sHASEGP as exemplified in SEQ ID NO: 1. Disulfide bonds are formed between Cys residues C60-C351 and Cys residues C224 to C238 to form the core hyaluronidase domain. However, additional cysteines are required at the carboxy terminus for enzymatic catalytic activity at neutral pH, so that the sHASEGP from amino acids 36 to Cys 464 in SEQ ID NO: 1 comprises the minimally active human sHASEGP hyaluronidase domain. . Therefore, the N-490 N-linked glycosylation site is not necessary for proper sHASEGP activity.

The N-linked glycosylation of sHASEGP is critical for its catalytic activity and stability. While alteration of the type of glycan that a glycoprotein modifies can have drastic effects on the antigenicity, structural folding, solubility, and stability of a protein, it is believed that most enzymes do not require glycosylation for optimal enzyme activity. Therefore, sHASEGPs are unique in this regard, so that removal of N-linked glycosylation can result in almost complete inactivation of hyaluronidase activity. The presence of N-linked glycans is critical for generating an active sHASEGP. Suitable protein expression systems are included for the introduction of critical N-linked glycosylation residues into sHASEGP. In addition, the introduction of deglycosylated sHASEGP polypeptide in the presence of extracts capable of introducing N-linked glycans is included. In one aspect of the invention, a complex glycosylation terminally protected with sialation is described, although others terminally protected with free mannose residues are also contemplated. . Preferably, sialic acid residues are found at the terminal N-linked glycosylation residues in sHASEGP.

N-linked oligosaccharides are included in several main types (oligomannose, complex, hybrid, sulfated), all having 3-GlcNAc-GlcNAc (Man) nuclei linked via the amide nitrogen of Asn residues that are included in -Asn- sequences. Xaa-Thr / Ser- (where Xaa is not Pro). Glycosylation at an -AsnXaa-Cys- site for coagulation protein C. N-linked sites are often assigned indirectly by the appearance of a "blank" cycle during sequencing. Positive identification can be made after release of the oligosaccharide by PNGase F, which converts glycosylated Asn to Asp. After release by PNGase F, the N-linked oligosaccharides can be purified using Bio-Gel P-6 chromatography, the combination of oligosaccharides being subjected to preparative high pH anion exchange chromatography (HPAEC) (Townsend et al., (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide isomers can be resolved using HPAEC. Fucose residues will shift elution positions earlier in the HPAEC chromatogram, while additional sialic acid residues will increase retention time. Simultaneous treatment of glycoproteins whose oligosaccharide structures are known (eg, bovine fetuin, α-1 acid glycoprotein, ovalbumin, RNase B, transferrin) can facilitate the assignment of oligosaccharide peaks. The collected oligosaccharides can be characterized by a combination of compositional and methylation bond analysis (Waeghe et al; (1983), Carbohydr Res. 123, 281-304), with the anomeric configurations assigned by NMR spectroscopy (Van Halbeek (1993) in Methods Enzymol 230).

Formulations of sHASEGP are also provided. SHASEGP can be formulated in lyophilized forms and stabilized solutions. Formulations containing specific metal ions, such as calcium, magnesium, or sodium are useful for optimal activity at neutral pH. In addition to stabilized solution formulations, slow release formulations for prolonged removal of glycosaminoglycans are contemplated herein. Also provided herein are kits that provide prepackaged syringes of sHASEGP for the administration of small volumes of sHASEGP for intraocular surgical procedures and other small volume procedures. Balanced salt formulations are also provided for ex vivo use in artificial reproductive technology procedures.

The use of sHASEGP in the removal of glycosaminoglycans is also provided. SHASEGPs open channels in the interstitial space through the degradation of glycosaminoglycans that allow the diffusion of molecules smaller than 500 nm in size. These channels remain for a period of 24-48 hours depending on the dose and the formulation. Such channels can be used to facilitate the diffusion of exogenously added molecules such as fluids, small molecules, proteins, nucleic acids and gene therapy vectors and other molecules smaller than 500 nm in size.

SHASEGPS can also be used to remove excess glycosaminoglycans such as those that appear after ischemia-reperfusion, inflammation, arteriosclerosis, edema, cancer, spinal cord injury, and other forms of scarring. In some cases, sHASEGPs can be delivered systemically by intravenous infusion. This can be useful when local access is not readily available, such as the heart or brain, or in the case of a disseminated neoplasm, where the disease is throughout the body. Supersialated sHASEGPs are preferable to increase serum half-life and distribution over native hyaluronidase enzymes lacking terminal sialic acids.

In some cases, such as spinal cord injury, glaucoma, and cosmetic treatments, a sustained supply is preferred.

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In other indications, a single short-acting dose is preferable. The temporary elimination of glycosaminoglycans can be used to increase the delivery of solutions and drugs in interstitial spaces. This can be useful for the diffusion of anesthesia and for the administration of therapeutic fluids, molecules and proteins. Subcutaneous and intramuscular administration of molecules in the presence of sHASEGP also facilitates their systemic distribution more rapidly. Such methods are very useful when intravenous access is not available or when a more rapid systemic delivery of molecules is necessary. The delivery of other large molecules, such as Factor VIII, which are poorly bioavailable after subcutaneous administration, can be injected with sHASEGP to increase their availability.

Also provided are uses of sHASEGP for enzymatic removal of the cumulus matrix surrounding the oocytes. Elimination of the cumulus matrix using a purified sHASEGP without the toxic contaminants of hyaluronidase obtained from extracts allows a smoother recovery of the oocyte with higher viabilities. Furthermore, sHASEGP can be prepared without the use of extracts from cattle or other organisms that carry viruses and other pathogens such as transmissible spongiform encephalopathies.

Small volume injections of sHASEGP can also be used for intraocular use for small spaces. SHASEGP can be injected into the anterior chamber of the eye to remove excess viscoelastic substrates that are delivered during surgery. Intraocular injection of sHASEGP may also be used to reduce infraocular pressure in glaucoma, to dissolve vitreous aggregates, or "detachments," to clear a vitreous hemorrhage, to treat macular degeneration, to promote vitreoretinal detachment in diabetic retinopathy and mixed with other enzymes to promote corneal reshaping along with corrective lenses. It will be recognized that in some cases, the use of a long lasting sHASEGP such as a pegylated sHASEGP will be desirable.

Co-formulations of sHASEGP with other substances can be imagined for injectable pens for small volumes or rapid subcutaneous administration. Examples such as Epipen can be formulated^®, insulin and other fluids. Methods of the invention include administration of the sHASEGP polypeptide or pharmaceutical compositions containing sHASEGP prior to, concurrently with or after the administration of other therapeutic molecules. The sHASEGP can be administered at a site different from the therapeutic molecule's administration site or the sHASEGP can be administered at the same site as the therapeutic molecule's administration site.

Therefore, provided herein is a family of eukaryotic neutral pH active secreted hyaluronidase glycoproteins called sHASEGP and functional domains, especially hyaluronidase (or catalytic) domains thereof, muteins and other derivatives and analogs thereof. Also provided herein are nucleic acids encoding sHASEGPs. Further provided are formulations and uses of such sHASEGPs for treating disease and for use as tissue modifying enzymes.

Brief description of the drawings

Figure 1 is a vector map of the sHASEGP vector HZ24.

Detailed description of the invention

A. Definitions

Unless otherwise defined, all technical and scientific terms used in this document have the same meaning as is commonly understood by one of ordinary skill in the art to which the invention or inventions pertain. All patents, patent applications, published applications and publications, Genbank sequences, web pages and other published materials referenced throughout the description of this document, unless otherwise indicated, are incorporated by reference in its entirety. In the event that there is a plurality of definitions for the terms in this document, those in this section prevail.

When referring to a URL or other identifier or address of this type, it is understood that said identifiers may change and that particular information on the internet may come and go, but equivalent information can be found by searching the internet. Reference to them proves the availability and public dissemination of such information.

As used herein, abbreviations for any protecting group, amino acid, and other compounds are, unless otherwise indicated, in accordance with commonly used, recognized abbreviations, or the TUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11: 942-944).

As used herein, eukaryotic hyaluronidase refers to a diverse family of glycosaminoglycan endoglucosaminidases in which a glutamate residue in the hyaluronidase hydrolyzes the beta 1,4 bonds of hyaluronan and chondroitin sulfates through an acid-catalytic mechanism. base.

Of particular interest are sHASEGPs of mammalian origin, including humans. Those skilled in the art recognize that, in general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al., (1987) Molecular Biology of the Gene, 4^to Edition, The Benjamin / Cummings Pub. Co., P. 224).

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As used herein, a "membrane-anchored sHASEGP" refers to a family of membrane-anchored hyaluronidases that share common structural features as described herein.

As used herein, a soluble hyaluronidase refers to a polypeptide characterized by its solubility under physiological conditions. Soluble HASEGP can be differentiated for example by its partition in the aqueous phase of a Triton X-114 solution heated to 37 ° C (Bordier et al J Biol Chem., February 25, 1981; 256 (4): 16047). On the other hand, the lipid-anchored HASEGP will partition into the detergent-rich phase, but will partition into the aqueous or poor-detergent phase after treatment with phospholipase C.

Therefore, reference, for example, to "sHASEGP" includes all glycoproteins encoded by the sHASEGP family of genes including, but not limited to: human sHASEGP, mouse sHASEGP, or an equivalent molecule obtained from or from any other source. has prepared synthetically or that presents the same activity. The coding nucleic acid molecule sequences and the encoded amino acid sequences of exemplary sHASEGP and / or domains thereof are set forth, for example, in SEQ ID NO: 4. The term also includes sHASeGp with amino acid substitutions that do not they substantially alter the activity of each limb and also include splice variants thereof. Suitable substitutions are known to those skilled in the art, including, but not necessarily, conservative amino acid substitutions, and can be made without eliminating biological activity, such as catalytic activity, of the resulting molecule.

As used herein, a sHASEGP, each time it is referred to herein, includes a polypeptide that includes the amino acid sequence included in SEQ ID NO: 1; or an amino acid sequence that has a sequence identity of at least about 91% to the amino acid sequence set forth in SEQ ID NO: 1.

In particular, the sHASEGP polypeptide with the hyaluronidase domains indicated in SEQ ID NO: 4 is described. The polypeptide is a one or two chain polypeptide. Smaller portions thereof are also provided which retain hyaluronidase activity. The hyaluronidase domains of sHASEGPs vary in size and constitution, including insertions and deletions in surface loops. Therefore, for the purposes of this document, the catalytic domain is a portion of a sHASEGP, as defined herein, and is homologous to a domain of other hyaluronidase-like sequences, such as HYAL1, HYAL2, HYAL3, which have been previously identified; however, it was not recognized that an isolated single chain form of the human hyaluronidase domain could function in in vitro assays. The aspartate and glutamate residues necessary for activity are present in conserved motifs.

As used herein, a "neutral hyaluronidase domain of a soluble sHASEGP" refers to a beta-1,4-endoglucosaminidase domain of a sHASEGP that exhibits hyaluronidase activity at neutral pH, is soluble under conditions as described, and shares homology. and structural features with the hyaluronidase domains of the glycosyl hydrolase family but contains additional sequences at the carboxy terminus that are necessary for activity at neutral pH. Thus, it is at least the minimal portion of the domain that exhibits hyaluronidase activity as assessed by standard in vitro assays and remains soluble. Such hyaluronidase domains and catalytically active portions thereof are contemplated herein. Also provided are truncated forms of the hyaluronidase domain that include the smallest fragment thereof that acts catalytically as a single chain form.

A hyaluronidase domain of a sHASEGP, whenever referred to herein, includes at least one or all of or any combination of or a catalytically active portion of: an N-linked glycoprotein polypeptide that includes the exposed amino acid sequence in SEQ ID NO: 1; a polypeptide encoded by a nucleotide sequence that hybridizes under conditions of low, moderate or high stringency to the nucleotide sequence set forth in SEQ ID NO: 6; a polypeptide that includes the amino acid sequence set forth in SEQ ID N: 1; a polypeptide that includes an amino acid sequence that has a sequence identity of at least about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the amino acid sequence set forth in SEQ ID NO: 1; and / or a hyaluronidase domain of a polypeptide encoded by a splice variant of sHASEGP.

Therefore, for the purposes of this document, the hyaluronidase domain is a portion of a sHASEGP, as defined herein, and is homologous to a domain of other sHASEGPs. As with the larger class of enzymes in the hyaluronidase family, the catalytic domains of sHASEGP share a high degree of amino acid sequence identity. The Asp and Glu residues required for activity are present in conserved motifs.

By active form is meant an active form in vivo and / or in vitro. As described herein, the hyaluronidase domain can also exist as a soluble secreted glycoprotein. It is shown herein that, at least in vitro, the single chain forms of the sHASEGPs and the catalytic domains or enzymatically active portions thereof (typically C-terminal truncations) exhibit hyaluronidase activity. Therefore, isolated forms of the hyaluronidase domains of sHASEGP and their use in in vitro drug screening assays for the identification of agents that modulate the activity thereof are provided herein.

As used herein, the catalytically active domain of a sHASEGP refers to the neutral pH active endoglucosaminidase domain as defined by in vitro activity towards a glycosaminoglycan substrate.

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The sHASEGPs of interest include those that are active against chondroitin sulfates and chondroitin sulfate proteoglycans (CSPGs) in vivo and in vitro; and those that are active against hyaluronan. As used herein, a human sHASEGP is one encoded by a nucleic acid, such as DNA, present in the genome of a human, including all allelic variants and conservative variations provided they are not variants found in other mammals. .

As used herein, a nucleic acid encoding a hyaluronidase domain or active catalytic portion of a "sHASEGP" is to be construed as referring to a nucleic acid encoding only the detailed single-chain hyaluronidase domain or an active portion thereof, and not the other contiguous portions of the sHASEGP as a continuous sequence.

As used herein, the terms "disease" or "disorder" refer to a pathological condition in an organism that is the result of, for example, an infection or genetic defect, and that is characterized by identifiable symptoms.

As used herein, a "splice variant" refers to a variant produced by differential processing of a primary transcript of genomic nucleic acid, such as DNA, that results in more than one type of mRNA. Splicing variants of sHASEGP are provided herein.

As used herein, the hyaluronidase domain of a sHASEGP protein refers to the hyaluronidase domain of a sHASEGP that exhibits endoglucosaminidase activity at neutral pH. Therefore, it is at least the minimal portion of the protein that exhibits endoglucosaminidase activity as assessed by standard in vitro assays. Exemplary human hyaluronidase domains include at least a sufficient portion of the amino acid sequences set forth in SEQ ID NO: 4 that exhibit endoglucosaminidase activity.

Also contemplated are nucleic acid molecules that encode a polypeptide that has endoglucosaminidase activity in an in vitro hyaluronidase assay and that have a sequence identity of at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with the length completeness of a hyaluronidase domain of a sHASEGP polypeptide, or that hybridizes along its entire length or along at least about 70%, 80% or 90% of the full length with a nucleic acid encoding a hyaluronidase domain, particularly under conditions of moderate, generally high stringency.

For hyaluronidase domains, residues in the N-terminal region can be critical but not sufficient for their activity. The hyaluronidase domain of sHASEGP is shown herein to be catalytically active. Therefore, the hyaluronidase domain generally requires the N-terminal amino acids thereof for its activity; the C-terminal portion can be truncated to the last cysteine ​​residue although it requires additional amino acids to be optimally active. The amount that can be removed can be determined empirically by testing the polypeptide for hyaluronidase activity in an in vitro assay evaluating catalytic cleavage.

Therefore, smaller portions of the hyaluronidase domains are contemplated, particularly the single chain domains thereof that retain hyaluronidase activity. Said smaller versions are generally C-terminal truncated versions of the hyaluronidase domains. Hyaluronidase domains vary in size and constitution, including insertions and deletions in surface loops. Said domains have a conserved structure, including at least one structural feature, such as the proton donor and / or other features of the hyaluronidase domains of endoglucosaminidases. Therefore, for the purposes of this document, the hyaluronidase domain is a single-chain portion of a sHASEGP, as defined herein, but is homologous in its structural characteristics and in the retention of a sequence similarity or homology with the hyaluronidase domain of other hyaluronidase-like sequences. The glycoprotein exhibits hyaluronidase activity as a single chain.

As used herein, by "homologue" is meant a nucleic acid sequence identity greater than 25%, such as 25%, 40%, 60%, 70%, 80%, 90%, or 95%. If necessary, the percent homology will be specified. The terms "homology" and "identity" are often used interchangeably. In general, the sequences are aligned so that the highest order of matches is obtained (see, for example, Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part /, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; Carillo et al. (1988) et al. (1988) Slam J Applied Math 48]: 1073).

Using sequence identity, the number of conserved amino acids is determined by conventional alignment algorithm programs and used with the default gap penalties set by each vendor. Substantially homologous nucleic acid molecules would typically hybridize to moderate stringency or high stringency along the entire length of the nucleic acid or along at least about 70%, 80%, or 90% of the nucleic acid molecule of full length of interest. Also contemplated are nucleic acid molecules that contain degenerate codons in place of codons in the nucleic acid molecule that hybridizes.

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It can be determined whether any two nucleic acid molecules have nucleotide sequences that have an "identity" of at least, for example, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. using known computer algorithms such as the "FASTA" program, using for example the default parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444 (other programs include the GCG program package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), BLASTP, BLAStN, FASTA (Atschul, [S] [F.], [ et al, J MOLEC BIOL 215]: 403 (1990), Guide to Huge Computers, Martin J. Bishop, [ED.,] Academic Press, San Diego, 1994, and [CARRILLO ETA /.] (1988) SIAM J Applied Math 48: 1073) For example, the BLAST function from the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include the DNASTAR “MEGALIGN” PROGRAM (Madison, WI) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison, WI). Percent homology or identity of proteins and / or nucleic acid molecules can be determined, eg, by comparison of sequence information using a GAP computer program, eg, Needleman et al. (1970), J Mol Biol. 48: 443, as reviewed by Smith and Waterman Adv. Appl. Mathematics (1981) 2: 482). In summary, the GAP program defines similarity as the number of aligned symbols (ie, nucleotides or amino acids) that are similar, divided by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program may include: (1) a unitary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al (1986) Nucl. Acids Res. 14: 6745, as described by Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in each gap; and (3) no penalty for terminal gaps. Therefore, as used herein, the term "identity" represents a comparison between a test polypeptide or polynucleotide and a reference one.

As used herein, the term "at least 90% identity with" refers to percentages of identity from 90 to 99.99 to reference polypeptides. An identity at a level of 90% or more is indicative of the fact that, assuming for exemplification purposes, a test and reference polynucleotide length of 100 amino acids is compared. No more than 10% (ie, 10 out of 100) of the amino acids in the test polypeptide differ from those in the reference polypeptide. Similar comparisons can be made between test and reference polynucleotides. Such differences can be represented as point mutations randomly distributed along the full length of an amino acid sequence or they can be grouped at one or more locations of variable length up to the maximum allowable, for example, a difference of 10/100 amino acids (an identity of about 90%). Differences are defined as nucleic acid or amino acid substitutions or deletions. At the level of homologies or identities above approximately 85-90%, the result should be independent of the program and the adjustment of parameters by gaps; Such high levels of identity can be easily assessed, often without relying on a computer program.

As used herein, a primer refers to an oligonucleotide containing two or more deoxyribonucleotides or ribonucleotides, typically more than three, from which the synthesis of a primer extension product can be initiated. Experimental conditions leading to synthesis include the presence of nucleoside triphosphate and an agent for polymerization and extension, such as a DNA polymerase, and a suitable buffer, temperature, and pH.

As used herein, animals include any animal, such as, but not limited to, goats, cows, deer, sheep, rodents, pigs, and humans. Non-human animals exclude human beings as the contemplated animal. The sHASEGPs provided in this document are of any origin, animal, plant, prokaryotic and fungal. Most sHASEGPs are of animal origin, including mammalian origin.

As used herein, gene therapy involves the transfer of a heterologous nucleic acid, such as DNA, into certain cells, target cells of a mammal, particularly a human, with a disorder or conditions for which such therapy is sought. . Nucleic acid, such as DNA, is introduced into selected target cells such that heterologous nucleic acid, such as DNA, is expressed and a therapeutic product encoded by it is produced.

Alternatively, the heterologous nucleic acid, such as DNA, may in some way mediate the expression of DNA encoding the therapeutic product, or it may encode a product, such as a peptide or RNA that in some way directly or indirectly mediates the expression of a therapeutic product. Gene therapy can also be used to deliver a nucleic acid that encodes a gene product that replaces a defective gene or complements a gene product produced by the mammal or cell into which it is introduced. The introduced nucleic acid may encode a therapeutic compound, such as a growth factor inhibitor thereof, or a tumor necrosis factor or inhibitor thereof, such as a receptor therefore, not normally produced in the mammalian host or not produced in therapeutically effective amounts or at a therapeutically useful time. The heterologous nucleic acid, such as DNA, encoding the therapeutic product can be modified prior to introduction into the cells of the affected host to increase or otherwise alter the product or the expression thereof. Gene therapy may also involve the delivery of an inhibitor or repressor or other modulator of gene expression.

As used herein, a heterologous nucleic acid is a nucleic acid that (if DNA encodes RNA) and proteins that are not normally produced in vivo by the cell in which it is expressed or that mediates or encodes mediators that alter expression. of an endogenous nucleic acid, such as DNA, affecting transcription, translation or other

ES 2 335 005 T3 adjustable biochemical processes. A heterologous nucleic acid, such as DNA, can also be referred to as a foreign nucleic acid, such as DNA. Any nucleic acid, such as DNA that one skilled in the art would recognize or consider heterologous or foreign to the cell in which it is expressed is included herein by the term heterologous nucleic acid; heterologous nucleic acid includes an exogenously added nucleic acid that is also expressed endogenously. Examples of heterologous nucleic acids include, but are not limited to, a nucleic acid that encodes traceable marker proteins, such as a protein that confers drug resistance, a nucleic acid that encodes therapeutically effective substances, such as anticancer agents, enzymes, and hormones, and a nucleic acid such as DNA, which encodes other types of proteins, such as antibodies. Antibodies that are encoded by the heterologous nucleic acid can be secreted or expressed on the surface of the cell into which the heterologous nucleic acid has been introduced.

Heterologous nucleic acid is generally not endogenous to the cell into which it is introduced, but has been obtained from another cell or has been prepared synthetically.

Generally, but not necessarily, such nucleic acid encodes RNA and proteins that are not normally produced by the cell in which it is expressed.

As used herein, a therapeutically effective product is a product that is encoded by a heterologous nucleic acid, typically DNA, that upon introduction of the nucleic acid into a host, a product is expressed that ameliorates or eliminates the symptoms, manifestations of an inherited or acquired disease, or that cures the disease.

As used herein, reporting that a glycoprotein consists essentially of the hyaluronidase domain means that the only sHASEGP portion of the polypeptide is a hyaluronidase domain or an active catalytic portion thereof. The polypeptide can optionally and generally will include additional amino acid sequences not derived from sHASEGP.

As used herein, a domain refers to a portion of a molecule, eg, glycoproteins or the encoding nucleic acids, that is structurally and / or functionally different from other portions of the molecule.

As used herein, hyaluronidase refers to an enzyme that catalyzes the hydrolysis of glycosaminoglycans.

For clarity, reference to hyaluronidase refers to all forms, and particular forms will be specifically designated. For the purposes of this document, the hyaluronidase domain includes the membrane-bound and soluble forms of a sHASEGP protein.

As used herein, nucleic acids include DNA, RNA, and analogs thereof, including peptide nucleic acids (PNAs) and a mixture thereof. Nucleic acids can be single or double stranded. When referring to probes or primers, optionally labeled with a detectable label such as a fluorescent label or radiolabel, single stranded molecules are contemplated. Such molecules are typically of a length such that their target is statistically unique or low copy number (typically less than 5, generally less than 3) to probe or prime a library. Generally a probe or primer contains at least 14,16 or 30 contiguous positions of sequence complementarity with or identity with a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids in length.

As used herein, a nucleic acid encoding a fragment or portion of a sHASEGP refers to a nucleic acid encoding only the fragment or reported portion of sHASEGP and not the other contiguous portions of the sHASEGP.

As used herein, an operative linkage of a heterologous nucleic acid with nucleotide regulatory and effector sequences, such as promoters, enhancers, transcriptional and translational termination sites, and other signal sequences refers to the relationship between said nucleic acid, such as DNA, and such nucleotide sequences. For example, the operative binding of heterologous DNA to a promoter refers to the physical relationship between the DNA and the promoter such that the transcription of said DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds, and transcribes. DNA in reading phase. Therefore, the terms "operably linked" or "functionally associated" refer to the functional relationship of a nucleic acid, such as DNA, with regulatory and effector nucleotide sequences such as promoters, enhancers, transcriptional and translational termination sites, and other signal sequences. For example, the operative binding of DNA to a promoter refers to the physical and functional relationship between the DNA and the promoter such that the transcription of said DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to and transcribes DNA. To optimize expression and / or transcription in vitro it may be necessary to remove, add or alter 5 'untranslated portions of the clones to remove potentially inappropriate alternative extra translation initiation (i.e. start) codons or other sequences that may interfere with or reduce expression, at the level of transcription or translation. Alternatively, consensus ribosome binding sites can be inserted (see, eg, Kozak J. Biol. Chem. 266: 19867-19870 (1991)) immediately 5 'of the start codon and can increase expression. How desirable (or necessary) such a modification is can be empirically determined.

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As used herein, a sequence complementary to at least a portion of an RNA, relative to antisense oligonucleotides, refers to a sequence that has sufficient complementarity to be capable of hybridizing to the RNA, generally under moderate stringency conditions. u high, forming a stable duplex; In the case of double-stranded sHASEGP antisense nucleic acids, therefore a single strand of the DNA duplex (or dsRNA) can be tested or the formation of a triplex can be tested. The ability to hybridize depends on the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the nucleic acid that hybridizes, the more base mismatches with an RNA encoding sHASEGP it can contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can determine a tolerable degree of mismatch by using standard procedures to determine the melting point of the hybridized complex.

For the purposes of this document, amino acid substitutions can be made in any of the sHASEGPs and hyaluronidase domains thereof as long as the resulting protein exhibits hyaluronidase activity. Contemplated amino acid substitutions include conservative substitutions, such as those set forth in Table 1, that do not eliminate proteolytic activity. As described herein, substitutions that alter properties of proteins are also contemplated, such as removal of cleavage sites and other such sites; such substitutions are generally not conservative but can easily be made by those skilled in the art.

Suitable conservative amino acid substitutions are known to those of skill in the art and can generally be made without altering the biological activity, eg, the enzymatic activity, of the resulting molecule. Those skilled in the art recognize that, in general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. Biology of the Gene, 4^to Edition, 1987, The Benjamin / Cummings Pub. Co., P. 224). Also included in the definition is the catalytically active fragment of a sHASEGP, particularly, a single chain hyaluronidase portion. Conservative amino acid substitutions are made, for example, in accordance with those set forth in Table 1 as follows:

Table 1 Original remainder Conservative substitution Ala (A) Gly; Ser, Abu Arg (R), Lys, orn Asn (N) Gln; His Cys (C) Ser Gin (Q) Asn Glu (E) ASP Gly (G) Ala; Pro His (H) Asn; Gin He (I), Leu, Val, Met; Nle; Nva Leu (L), Val, Met; Nle; Nv Lys (K) Arg; Gin; Glu Met (M) Leu, Tyr; Ile; NLe Val Ornithine Lys, Arg Phe (F) Met; Leu, Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) ILE; Leu; Met; Nle; Nv. Other substitutions are also allowed and can be determined empirically or in accordance with known conservative substitutions.

As used herein, Abu is 2-aminobutyric acid; Orn is ornithine. As used herein, amino acids that appear in the various amino acid sequences that appear herein are identified according to their well-known three-letter or one-letter abbreviations. The nucleotides that appear in the various DNA fragments are designated by the standard single letter designations routinely used in the art.

As used herein, a nucleotide sequence-based probe or primer described herein includes at least 10, 14, typically at least 16 contiguous positions of the nucleotide sequence of SEQ ID NO: 6, and probes of at least 30, 50 or 100 contiguous positions of the nucleotide sequence of SEQ ID NO: 6. The length of the probe or primer for single hybridization is a function of the complexity of the genome of interest.

As used herein, amelioration of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any reduction, whether of permanent or temporary or transitory duration, that can be attributed to or associated with the administration of the composition. .

As used herein, antisense polynucleotides refer to synthetic sequences of nucleotide bases complementary to mRNA or to the sense strand of double-stranded DNA. The mixture of sense and antisense polynucleotides under appropriate conditions leads to the union of the two molecules or hybridization. When these polynucleotides bind to (hybridize with) mRNA, inhibition of protein synthesis (translation) occurs. When these polynucleotides bind to double-stranded DNA, inhibition of RNA synthesis (transcription) occurs.

The resulting inhibition of translation and / or transcription leads to an inhibition of the synthesis of the protein encoded by the sense strand. The antisense nucleic acid molecule typically contains a sufficient number of nucleotides to specifically bind to a target nucleic acid, generally at least 5 contiguous nucleotides, often at least 14 or 16 or 30 contiguous nucleotides or modified nucleotides complementary to the coding portion of a nucleic acid molecule that encodes a gene of interest, eg, a nucleic acid that encodes a single-chain hyaluronidase domain of a sHASEGP.

As used herein, a matrix refers to a group of elements, such as antibodies, that contains three or more members. An addressable matrix is ​​one in which the members of the matrix can be identified, typically, by their position on a solid phase support. Therefore, in general the members of the matrix are immobilized at separate identifiable loci on the surface of a solid phase.

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As used herein, an "antibody" refers to an immunoglobulin, either naturally occurring or produced partially or fully synthetically, that includes any derivative thereof that retains the specific binding ability of the antibody. Thus, an antibody includes any protein that has a binding domain that is homologous or substantially homologous to an immunoglobulin-binding domain. Antibodies include members of any immunoglobulin claim, including IgG, IgM, IgA, IgD, and IgE.

As used herein, an "antibody fragment" refers to any derivative of an antibody that is less than full length, which retains at least a portion of the specific binding ability of the full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab ', F (AB) 2, single chain Fv (scFV), FV, dsFV diabodies, and Fd fragments. The fragment can include multiple chains linked together, such as by disulfide bridges. An antibody fragment generally contains at least about 50 amino acids and typically at least 200 amino acids.

As used herein, an Fv antibody fragment is composed of a variable heavy domain (VH) and a variable light domain linked by non-covalent interactions.

As used herein, a "dsFV" refers to an Fv with an intermolecular disulfide bond generated by genetic engineering.

As used herein, an F (AB) 2 fragment is an antibody fragment that is the result of digestion of an immunoglobulin with pepsin at pH 4.0-4.5; it can be expressed recombinantly to produce the equivalent fragment.

As used herein, Fab fragments are antibody fragments that are the result of digestion of an immunoglobulin with papain; they can be expressed recombinantly to produce the equivalent fragment.

As used herein, scFVs refer to antibody fragments containing a V variable light chain and a variable heavy chain (VH) covalently connected by a polypeptide linker in any order. The linker is of such length that the two variable domains are linked without substantial interference. Linkers included are (Gly-Ser) n residues with some Glu or Lys residues dispersed throughout to increase solubility.

As used herein, humanized antibodies refer to antibodies that are modified to include human amino acid sequences so that administration to a human does not elicit an immune response. Methods for the preparation of such antibodies are known. For example, to produce such antibodies, the hybridoma or another prokaryotic or eukaryotic cell such as an E. coli or a CHO cell, which expresses that monoclonal antibody is altered by recombinant DNA techniques to express an antibody in which the amino acid composition of the non-variable region is based on human antibodies. Computer programs have been designed to identify these regions.

As used herein, the diabodies are dimeric scFVs; diabodies typically have shorter peptide linkers than scFVs and generally dimerize.

As used herein, production by recombinant means using recombinant DNA methods refers to the use of well known molecular biology methods to express proteins encoded by cloned DNA.

As used herein, the term "evaluate" is intended to include quantitative and qualitative determination in the sense of obtaining an absolute value for the activity of a sHASEGP, or a domain thereof, present in the sample, and also of obtaining an index, proportion, percentage, visual or other value indicative of the level of activity. The evaluation can be direct or indirect and of course it is not necessary that the chemical species detected are actually the proteolysis product itself, but rather they can be for example a derivative thereof or some additional substance.

As used herein, "biological activity" refers to the in vivo activities of a compound or physiological responses that are the result of in vivo administration of a compound, composition, or other mixture. Biological activity, therefore, includes the therapeutic effects and pharmaceutical activity of such compounds, compositions, and mixtures. Biological activities can be observed in in vitro systems designed to test or use such activities. Therefore, for the purposes of this document, the biological activity of a luciferase is its oxygenase activity, whereby, upon oxidation of a substrate, light is produced.

As used herein, "functional activity" refers to a polypeptide or portion thereof that exhibits one or more activities associated with a full-length protein.

Functional activities include, but are not limited to, biological activity, enzymatic or catalytic activity, antigenicity (ability to bind or compete with a polypeptide for binding to an anti-polypeptide antibody), immunogenicity, ability to form multimers, ability to specifically bind to a receptor or ligand for the polypeptide.

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As used herein, a "conjugate" refers to the compounds provided herein that include one or more sHASEGPs, including a sHASEGP, particularly single chain hyaluronidase domains thereof, and one or more targeting agents. These conjugates include those produced by recombinant means such as fusion proteins, those produced by chemical means, such as by chemical coupling through, for example, coupling to sulfhydryl groups, and those produced by any other method by which at least one sHASEGP, or a domain thereof, is linked directly or indirectly via a linker or linkers to a targeting agent.

As used herein, a targeting agent is any moiety, such as a protein or an effective portion thereof, that provides specific binding of the conjugate to a cell surface receptor that can internalize the conjugate or the sHASEGP portion of the same. A targeting agent can also be one that promotes or facilitates, for example, isolation or affinity purification of the conjugate; the binding of the conjugate to a surface; or the detection of the conjugate or complexes containing the conjugate.

As used herein, an "antibody conjugate" refers to a conjugate in which the targeting agent is an antibody.

As used herein, a derivative or analog of a molecule refers to a portion derived from or a modified version of the molecule.

As used herein, an effective amount of a compound to treat a particular disease is an amount that is sufficient to ameliorate or in some way reduce the symptoms associated with the disease. Said amount can be administered as a single dosage or it can be administered according to a regimen, whereby it is effective. The amount can cure the disease but is typically administered to improve the symptoms of the disease. Repeated administration may be necessary to achieve the desired improvement in symptoms.

As used herein the term equivalent, when referring to two nucleic acid sequences means that the two sequences in question encode the same amino acid sequence or equivalent proteins. When the term equivalent is used in relation to two proteins or peptides, it means that the two proteins or peptides have substantially the same amino acid sequence with only amino acid substitutions (such as, but not limited to, conservative changes such as those set forth in Table 1 above) that do not substantially alter the activity or function of the protein or peptide. When the term "equivalent" refers to a property, the property need not be present to the same degree (eg, two peptides may exhibit different rates of the same type of enzyme activity), but the activities are usually substantially the same. The term "complementary", when referring to two nucleotide sequences, means that the two nucleotide sequences are capable of hybridizing, typically with less than 25%, 15%, 5%, or 0% mismatch between opposing nucleotides. If necessary, the percentage of complementarity will be specified. Typically the two molecules are selected so that they will hybridize under conditions of high stringency.

As used herein, an agent that modulates the activity of a protein or the expression of a gene or nucleic acid decreases or increases or otherwise alters the activity of the protein or, in some way, positively or negatively regulates or alters in some way. otherwise the expression of nucleic acid in a cell.

As used herein, an inhibitor of the activity of a sHASEGP includes any substance that prevents or decreases the production, modification or post-translational modifications, maturation or membrane localization of the sHASEGP or any substance that interferes with or decreases the proteolytic efficacy of the same, particularly in a single stranded form in an in vitro screening assay.

As used herein, a method of treating or preventing a neoplastic disease refers to any of the symptoms, such as tumor, metastasis thereof, vascularization of tumors, or other parameters by which the disease is characterized, it is reduced, improved, prevented, placed in a state of remission or maintained in a state of remission. It also means that the hallmarks of neoplastic disease and metastasis can be removed, reduced, or prevented by treatment. Non-limiting examples of characteristic hallmarks include uncontrolled degradation of the basement membrane and proximal extracellular matrix, migration, division, and organization of endothelial cells into new functional capillaries, and the persistence of such functional capillaries.

As used herein, the pharmaceutically acceptable salts, esters, or other derivatives of the conjugates include any salt, ester, or derivative that can be readily prepared by those skilled in this art using known methods for such derivatization and that produce compounds that can be administered to animals or humans without substantial toxic effects and which are active pharmaceutical ingredients or prodrugs.

As used herein, a prodrug is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically, or therapeutically active form of the compound. To produce a prodrug, the pharmaceutical active compound is modified so that the active compound is regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or transport characteristics of a drug, to mask side effects or toxicity, to improve taste

ES 2 335 005 T3 of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and of drug metabolism in vivo, those skilled in the art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach , Oxford University Press, New York, pages 388-392).

As used herein, a drug identified by the screening methods provided herein refers to any compound that is a candidate for use as a therapeutic compound or a candidate for the design of a therapeutic compound. Such compounds can be small molecules, including small organic molecules, peptides, peptidomimetics, antisense or dsRNA molecules, such as RNAi, antibodies, antibody fragments, recombinant antibodies, and other such compounds that can serve as drug candidates or candidate compounds.

As used herein, a peptidomimetic is a compound that mimics the conformation and certain stereochemical characteristics of the biologically active form of a particular peptide. In general, peptidomimetics are designed to mimic certain desirable properties of a compound, but not the undesirable properties, such as flexibility, that lead to a loss of a biologically active conformation and bond breaking. Peptidomimetics can be prepared from biologically active compounds by substituting certain groups or bonds that contribute to undesirable properties with bioisoesters. Bioisoesters are known to those of skill in the art. For example, the methylene bioisoter CH2S has been used as an amide substitution in enkephalin analogs (see, for example, Spatola (1983) pp. 267-357 in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, Weistein, Ed. volume 7, Marcel Dekker, New York). Morphine, which can be administered orally, is a compound that is a peptidomimetic of the endorphin peptide. For the purposes of this document, cyclic peptides are included among the peptidomimetics.

As used herein, a promoter region or promoter element refers to a segment of DNA or RNA that controls the transcription of the DNA or RNA to which it is operably linked. The promoter region includes specific sequences that are sufficient for the recognition of an RNA polymerase, its binding, and the initiation of transcription.

This portion of the promoter region is called the promoter. In addition, the promoter region includes sequences that modulate this RNA polymerase recognition, binding, and transcription initiation activity. These sequences can be cis-acting or can be sensitive to trans-acting factors. Promoters, depending on the nature of regulation, can be constitutive or regulated. Exemplary promoters contemplated for use in prokaryotes include the bacteriophage T7 and T3 promoters.

As used herein, a "receptor" refers to a molecule that has an affinity for a given ligand. Receptors can be naturally-occurring or synthetic molecules. Receptors may also be referred to in the art as antiligands. As used herein, the terms receptor and antiligand are used interchangeably. Receptors can be used in their unaltered state or as aggregates with other species. Receptors can be attached, covalently or non-covalently, or in physical contact with, to a binding member, directly or indirectly through a specific binding substance or linker. Examples of receptors include, but are not limited to: antibodies, cell membrane receptors, surface receptors and internalizing receptors, monoclonal antibodies, and antisera reactive with specific antigenic determinants such as viruses, cells or other materials, drugs, polynucleotides, nucleic acids, peptides, factors, lectins, sugars, polysaccharides, cells, cell membranes, and organelles.

Examples of receptors and applications using such receptors include, but are not limited to: a) enzymes: specific transport proteins or enzymes essential for the survival of microorganisms that could serve as targets for selection of antibiotics [ligand]; b) antibodies: the identification of a ligand binding site on an antibody molecule that combines with the epitope of an antigen of interest can be investigated; determining a sequence that mimics an antigenic epitope can lead to the development of vaccines in which the immunogen is based on one or more of said sequences or lead to the development of related diagnostic agents or compounds useful in therapeutic treatments such as autoimmune diseases; c) nucleic acids: identification of ligands, such as proteins or RNA, binding sites; d) catalytic polypeptides: polymers, including polypeptides that are capable of promoting a chemical reaction that involves the conversion of one or more reagents with one or more products; Such polypeptides generally include a specific binding site for at least one reactant or reaction intermediate and an active functionality close to the binding site, wherein the functionality is capable of chemically modifying the bound reagent (see, for example, US Pat. United States No. 5,215,899); e) hormone receptors: the determination of the ligands that bind with high affinity to a receptor is useful in the development of hormone replacement therapies, for example, the identification of ligands that bind to said receptors can lead to the development of drugs to control blood pressure; and f) opiate receptors: the determination of ligands that bind to opiate receptors in the brain is useful in the development of less addictive substitutes for morphine and related drugs.

As used herein, a sample refers to anything that may contain an analyte for which an analyte assay is desired. The sample can be a biological sample, such as a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, sperm, amniotic fluid, or the like. Biological tissues are

ES 2 335 005 T3 cell aggregates, usually of a particular class together with their intercellular substance that forms one of the structural materials of a human, animal, plant, bacterial, fungal or viral structure including connective, epithelial, muscular and nervous tissues. Examples of biological tissues also include organs, tumors, lymph nodes, arteries, and individual cells.

As used in this document: Hybridization stringency in determining percent mismatch is as follows: 1) high stringency: 0.1 x SSPE, 0.1% SDS, 65 ° C 2) medium stringency : 0.2x SSPE, 0.1% SDS, 50 ° C 3) Low stringency: 1.0x SSPE, 0.1% SDS, 50 ° C. Those skilled in this art know that the washing step selects stable hybrids and they also know the ingredients of SSPE (see, for example, Sambrook, E, F, Fritsch, T, Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold spring Harbor Laboratory Press 1989 Vol 3, p. B. 13, see also numerous catalogs describing commonly used laboratory solutions). The SSPE is 0.18 NaCl phosphate buffered at pH 7.4. Furthermore, those skilled in the art recognize that the stability of hybrids is determined by the Tm, which is a function of the sodium ion concentration and the temperature (Tm = 81.5 ° C - 16.6 + 0.41 (% G + C) - 600 / L)), so that the only parameters in the critical washing conditions for the stability of the hybrid are the concentration of sodium ion in the SSPE (or SSC) and the temperature.

It is understood that equivalent stringency can be achieved using alternative buffers, salts and temperatures. By way of example and not limitation, procedures using low stringency conditions are as follows (see also Shilo and Weinberg, Proc. Natl. Acad Sci USA 78: 6789-6792 (1981)): Filters containing DNA are pretreated for 6 hours at 40 ° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5 ), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 jug / ml denatured salmon sperm DNA. The SSC (10x) is 1.5 M sodium chloride and 0.15 M sodium citrate adjusted to pH 7.

Hybridizations are performed in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 VG / M sperm DNA, 10% dextran sulfate (p / v) and a 5-20 x 10 32P labeled probe is used⁶ cpm. Filters were incubated in hybridization mix for 18-20 hours at 40 ° C and then washed for 1.5 hours at 55 ° C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA and 0.1% SDS. The wash solution is replaced with fresh solution and incubated for an additional 1.5 hours at 60 ° C. Filters were blotted dry and exposed to autoradiography. If necessary, the filters are washed a third time at 65-68 ° C and re-exposed to film. Other low stringency conditions that can be used are well known in the art, eg, as used for cross hybridizations between species).

By way of example, and not limitation, procedures using moderate stringency conditions include, for example, but not limited to, procedures using such mild stringency conditions as follows: Filters containing DNA are pretreated for 6 hours at 55 ° C in a solution containing 6X SSC, 5X Denhart's solution, 0.5% SDS and 100 jug / ml denatured salmon sperm DNA. Hybridizations are performed in the same solution and probe labeled with 32P 5-20 X 10 is used.⁶. Filters are incubated in hybridization mix for 18-20 hours at 55 ° C and then washed twice for 30 minutes at 60 ° C in a solution containing 1X SSC and 0.1% SDS. Filters are blotted dry and exposed to autoradiography. Other conditions of moderate stringency that can be used are well known in the art. The filters are washed at 37 ° C for 1 hour in a solution containing 2X SSC, 0.1% SDS.

By way of example and not limitation, the procedures using high stringency conditions are as follows: Pre-hybridization of DNA-containing filters is performed for 8 hours to overnight at 65 ° C in 6X SSC compound buffer , 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA and DNA from denatured salmon sperm 500 jug / ml. Filters hybridize for 48 hours at 65 ° C in prehybridization mix containing 100 jug / ml denatured salmon sperm DNA and 5-20 X 10 32P-labeled probe.⁶ CPM. Filters are washed at 37 ° C for 1 hour in a solution containing 2X SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA. This is followed by washing in 0.1X SSC at 50 ° C for 45 minutes prior to autoradiography. Other high stringency conditions that can be used are well known in the art.

The term "substantially identical or substantially homologous or similar" varies with context as understood by those skilled in the relevant art, and generally refers to an identity of at least 60% or 70%, preferably it refers to at least 80%, 85% or more preferably at least 90% and more preferably at least 95%.

As used herein, "substantially identical to a product" means "substantially similar" so that the property of interest remains sufficiently unchanged that the substantially identical product can be used in place of the product.

As used herein, "substantially pure" means sufficiently homogeneous to appear free of readily detectable impurities as determined by conventional analytical methods, such as thin layer chromatography (TLC), gel electrophoresis, and high performance liquid chromatography (HPLC). , used by those skilled in the art to evaluate such purity, or sufficiently pure so that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purification of compounds are known to those skilled in the art to produce

ES 2 335 005 T3 cir substantially chemically pure compounds. A substantially chemically pure compound can however be a mixture of stereoisomers or isomers. In such cases, further purification can increase the specific activity of the compound.

As used herein, a "target cell" refers to a cell that expresses a sHASEGP in vivo.

As used herein, a test substance (or test compound) refers to a chemically defined compound (e.g., organic molecules, inorganic molecules, organic / inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides or hybrids between these molecules such as glycoproteins, etc.) or mixtures of compounds (for example, a library of test compounds, natural extracts or culture supernatants, etc.) whose effect on a sHASEGP, particularly a single chain form that includes the hyaluronidase domain or a sufficient portion thereof for activity, as determined by an in vitro method, such as the assays provided herein.

As used herein, the terms "therapeutic agent, therapeutic regimen, radioprotective, or chemotherapeutic" refer to conventional drugs and pharmacological therapies, including vaccines, that are known to those of skill in the art. Radiation therapy agents are well known in the art.

As used herein, "treatment" refers to any way in which the symptoms of a condition, disorder, or disease are improved or otherwise beneficially altered.

Treatment also includes any pharmaceutical use of the compositions herein.

As used herein, a vector (or plasmid) refers to discrete elements that are used to introduce a heterologous nucleic acid into cells for expression or replication thereof. Vectors typically remain episomal, but can be designed to achieve integration of a gene or portion thereof into a chromosome of the genome. Vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes, are also contemplated. The selection and use of such vehicles is well known to those of skill in the art. An expression vector includes vectors capable of expressing DNA that is operably linked with regulatory sequences, such as promoter regions, that are capable of achieving expression of said DNA fragments. Thus, an "expression vector" refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that, upon introduction into an appropriate host cell, results in expression of the DNA. cloned. Appropriate expression vectors are well known to those of skill in the art and include those that can replicate in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those that integrate into the genome of host cells.

As used herein, a protein binding sequence refers to a protein or peptide sequence that is capable of specific binding to another protein or peptide sequences, generally to a set of proteins or peptide sequences or to a protein or sequence. particular peptide.

As used herein, an epitopic marker refers to a short stretch of amino acid residues that corresponds to an epitope to facilitate subsequent biochemical and immunological analysis of the epitope-tagged protein or peptide. Epitope tagging is accomplished by including the sequence of the epitope marker in the coding sequence for a protein in an appropriate expression vector. Epitope-tagged proteins can be affinity purified using highly specific antibodies raised against the tags.

As used herein, a metal binding sequence refers to a protein or peptide sequence that is capable of specific binding to metal ions, generally to a set of metal ions or to a particular metal ion.

As used herein, a combination refers to any association between two or more items.

As used herein, a composition refers to any mixture. It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous, or any combination thereof.

As used herein, a fluid refers to any composition that can flow. Fluids therefore include compositions that are in the form of semisolids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

As used herein, a "cell extract" refers to a preparation or fraction that is prepared from a lysed or broken cell.

As used herein, an agent is said to be randomly selected when the agent is randomly selected without regard to the specific sequences involved in the association of a protein alone or with its associated substrates, binding partners, etc. An example of randomly selected agents is the use of a chemical library, a peptide combinatorial library, or a culture broth of a conditioned organism or medium.

ES 2 335 005 T3

As used herein, an agent is said to be rationally selected or designed when the agent is selected on a non-random basis that takes into account the sequence of the target site and / or its conformation in relation to the agent of action. . As described in the Examples, there are proposed binding sites for hyaluronidase and (catalytic) sites on glycoprotein having SEQ ID NO: 1 or SEQ ID NO: 4. Agents can be rationally selected or rationally designed using the peptide sequences that make up these sites. For example, a rationally selected peptide agent can be a peptide whose amino acid sequence is identical to ATP or to calmodulin binding sites or domains.

Oligosaccharides are considered to have a reducing end and a non-reducing end, regardless of whether the saccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are represented herein with the non-reducing end on the left and the reducing end on the right. All oligosaccharides described in this document are described by the name or abbreviation for the non-reducing saccharide (e.g. Gal) followed by the glycosidic bond configuration (alpha or beta), the ring bond, the reducing saccharide ring position involved in the linkage and then the name or abbreviation of the reducing saccharide (eg, GlcNAc). The bond between two sugars can be expressed, for example, as 2,3, 2 ^ 3 or (2,3). Each saccharide is a pyranose.

As used herein, an "N-linked sugar residue" refers to an oligosaccharide linked to a sHASEGP through the amide nitrogen of Asn residues. N-linked oligosaccharides are included in several main types (oligomannose, complex, hybrid, sulfated), all having 3-GlcNAc-GlcNAc (Man) nuclei linked through the amide nitrogen of Asn residues that are included in -Asn sequences. -Xaa-Thr / Ser- (where Xaa is not Pro). N-linked sites are often assigned indirectly by the appearance of a "blank" cycle during sequencing. Positive identification can be made after release of the oligosaccharide by PNGase F, which converts glycosylated Asn to Asp. After release by PNGase F, the N-linked oligosaccharides can be purified using Bio-Gel P-6 chromatography, the combination of oligosaccharides being subjected to preparative high pH anion exchange chromatography (HPAEC) (Townsend et al., (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide isomers can be resolved using HPAEC. Fucose residues will shift elution positions earlier in the HPAEC chromatogram, while additional sialic acid residues will increase retention time. Simultaneous treatment of glycoproteins whose oligosaccharide structures are known (eg, bovine fetuin, α-1 acid glycoprotein, ovalbumin, RNase B, transferrin) can facilitate the assignment of oligosaccharide peaks. The collected oligosaccharides can be characterized by a combination of compositional and methylation bond analysis (Waeghe et. Al; (1983), Carbohydr Res. 123, 281-304), with the anomeric configurations assigned by NMR spectroscopy (Van Halbeek (1993 ) in Methods Enzymol 230).

Alternatively, oligosaccharides can be identified by fluorescence-assisted carbohydrate electrophoresis (FACE) Callewaert et al. (2001) Glycobiology 11, 275-281.

As used herein, the term "sialic acid" refers to any member of a family of nine carbon carboxylated sugars. The most common member of the sialic acid family is N-acetylneuraminic acid (2-keto-5-acetamido-3,5-dideoxy-D-glycero-D-galactononulopyrans-1-ionic acid (often abbreviated as Neu5Ac, NeuAc, or NANA). A second member of the family is N-glycolyl-neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl group of NeuAc is hydroxylated. A third member of the sialic acid family is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et al. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et al. (1990 ) J. Biol. Chem. 265: 21811-21819 Also includes 9-substituted sialic acids such as 9-O-Ci-C₆ acyl-Neu5Ac such as 9-O-lactyl-Neu5Ac or 9-O-acetyl-Neu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido9-deoxy-Neu5Ac. For a review of the sialic acid family see, for example, Varki (1992) Glycobiology 2: 25-40; Sialic Acids: Chemistry, Metabolism and Function, R. Schauer, Ed. (Springer-Verlag, NY (1992)). The synthesis and use of sialic acid compounds in a sialation process is described in international application WO 92/16640, published October 1, 1992.

As used herein, PNGase refers to an Asparagine Peptide-specific N-glycosidase F, such as Flavobacterium maningoseptum peptide-N-glycosidase F. PNGase enzymes are characterized by their specificity towards N-linked rather than O-linked oligosaccharides. Characterization of PNGase efficacy can be defined by either SDS PAGE electrophoresis or fluorescence-assisted carbohydrate electrophoresis.

As used herein, a "substantially terminated sialation" refers to N-linked oligosaccharides that end with a sialic acid residue as the terminal sugar. Terminal sialic acids can be identified by FACE analysis of carbohydrates released after neuraminidase treatment.

The circulatory life of glycoproteins in blood is highly dependent on the composition and structure of their N-linked carbohydrate groups. This fact is of direct importance for therapeutic glycoproteins that are intended to be administered parenterally. In general, the maximum circulatory half-life of a glycoprotein requires that its N-linked carbohydrate groups end in the NeuAc-Gal-GlcNAc sequence. Without terminal sialic acid (NeuAc) the glycoprotein is rapidly cleared from the blood by a mechanism involving recognition of the underlying N-acetylgalactosamine (GalNAc) or galactose (Gal) residues (Goochee et al. (1991) Biol / Technology 9 : 1347-1355). For this reason, ensuring the presence of terminal sialic acid in N-linked carbohydrate groups of therapeutic glycoproteins is an important consideration for their commercial development.

ES 2 335 005 T3

Circulating glycoproteins are exposed to sialidases (or neuraminidase) that can remove terminal sialic acid residues. Typically, removal of sialic acid exposes galactose residues and these residues are recognized and bound by specific galactose receptors on hepatocytes (reviewed in Ashwell and Harford (1982) Ann. Rev. Biochem. 51: 531). The liver also contains other specific sugar receptors that mediate the removal of glycoproteins from the circulation. The specificities of these receptors also include N-acetylglucosamine, mannose, fucose, and phosphomannose. Glycoproteins cleared by galactose receptors on hepatocytes undergo substantial degradation and then enter the bile; glycoproteins cleared by the Kupffer cell mannose receptor enter the reticuloendothelial system (reviewed in Ashwell and Harford (1982) Ann. Rev. Biochem. 51:53).

As used herein, the term Active at Neutral pH refers to a sHASEGP glycoprotein with catalytic activity towards a glycosaminoglycan substrate in vitro at a pH of between 5 and 8 under salt conditions less than 150 mM and buffering potency less than 50 mM.

As used herein, a stabilized solution refers to a sHASEGP that retains more than 60% of its initial activity after storage at room temperature for 30 days.

As used herein, unless otherwise specified, a unit is expressed in turbidity reducing units (TRU). A TRU is defined as the amount of hyauloronidase activity necessary to reduce the turbidity of an acidified hyaluronic acid solution and is equivalent to USP / National Formulary Units (NF XIII) (NFU). The ELISA-type enzyme assay described in this document can be related to the TRU, NFU and USP units through a standard curve of a hyaluronidase sample (for example, USP or WHO standard) standardized through the USP. , the enzymatic activities determined by the ELISA-type enzyme assay are actually relative TRUs, since the enzyme activity is not actually measured using the turbidimetric assay (Dorfman et al., 1948, J. Biol. Chem. 172: 367).

As used herein, potency is defined by the amount of sHASEGP protein necessary to degrade a substrate in vitro based on a Turbidity Reducing Unit or Relative Turbidity Reducing Unit.

As used herein, specific activity refers to units of activity per mg of protein. The amount of sHASEGP protein is obtained by absorbing a sHASEGP solution at 280 nm assuming a molar extinction coefficient of approximately 1.7, in units of M^-1 cm^-1.

Polyethylene glycol (PEG) has been widely used in biomaterials, in biotechnology and medicine mainly because PEG is a biocompatible, non-toxic, non-immunogenic and water soluble polymer (Zhao and Harris, ACS Symposium Series 680: 458-72, 1997). In the area of ​​drug delivery, PEG derivatives have been widely used in covalent binding (ie, "PEGylation") to proteins to reduce immunogenicity, proteolysis, and renal clearance and to increase solubility (Zalipsky, Adv. Drug Del . Rev. 16: 157-82, 1995). Similarly, PEG has been linked to relatively hydrophobic low molecular weight drugs to increase solubility, reduce toxicity, and alter biodistribution. Typically, PEGylated drugs are injected as solutions.

A closely related application is the synthesis of degradable cross-linked PEG networks or formulations for use in drug delivery since much of the same chemistry used in the design of degradable soluble drug carriers can also be used in the design of degradable gels ( Sawhney et al., Macromolecules 26: 581-87, 1993). It is also known that intermolecular complexes can be formed by mixtures of solutions of two complementary polymers. Said complexes are generally stabilized by electrostatic interactions (polyanion-polycation) and / or hydrogen bonds (polyacid-polybase) between the polymers involved and / or by hydrophobic interactions between the polymers in an aqueous environment (Krupers et al., Eur. Polym J. 32: 785-790, 1996). For example, mixing solutions of polyacrylic acid (PAAc) and polyethylene oxide (PEO) under the appropriate conditions results in the formation of complexes based largely on the formation of hydrogen bonds. Dissociation of these complexes under physiological conditions has been used for the delivery of free (ie, non-PEGylated) drugs. In addition, complementary polymer complexes have been formed from both homopolymers and copolymers.

In one aspect, polyethylene glycol has a molecular weight ranging from about 3 kD to about 50 kD and preferably from about 5 kD to about 30 kD. Covalent attachment of the PEG to the drug (known as "PEGylation") can be achieved by known chemical synthesis techniques. For example, in one aspect of the present invention PEGylation of a protein can be achieved by reacting NHS-activated PEG with the protein under suitable reaction conditions.

Although numerous reactions have been described for PEGylation, those that are most generally applicable confer addressability, use mild reaction conditions, and do not require extensive downstream processing to remove toxic catalysts or by-products. For example, monomethoxyPEG (mPEG) has only one reactive terminal hydroxyl and therefore its use somewhat limits the heterogeneity of the resulting PEGprotein product mixture. Activation of the hydroxyl group at the end of the polymer opposite the terminal methoxy group is generally necessary to achieve efficient protein PEGylation, the goal being to make the derivatized PEG more susceptible to nucleophilic attack. The attack nucleophile is usually the epsilon amino group of a lysyl residue, but other amines may also react (for example, alpha N-terminyl amine or ring amines

ES 2 335 005 T3 of histidine) if local conditions are favorable. More targeted binding is possible in proteins that contain a single lysine or cysteine. The latter residue can be targeted by a PEG-maleimide for specific thiol modification. Alternatively, a hydrazide PEG can react with periodate oxidized sHASEGP and reduce in the presence of NaCNBH₃. More specifically, PEGylated CMP sugars can be reacted with sHASEGP in the presence of appropriate glycosyltransferases. One technique is the "PEGylation" technique in which various polymeric molecules are coupled to the polypeptide in question. When using this technique the immune system has difficulty recognizing the epitopes on the surface of the polypeptide responsible for the formation of antibodies, thereby reducing the immune response. For polypeptides introduced directly into the circulatory system of the human body to give a particular physiological effect (i.e. pharmaceutical compounds) the typical potential immune response is an IgG and / or IgM response, whereas polypeptides that are inhaled through the system respiratory (ie, industrial polypeptide) can potentially cause an IgE response (ie, an allergic response). One of the theories that explain the reduced immune response is that the polymeric molecule or molecules protect epitopes on the surface of the polypeptide responsible for the immune response that drives the formation of antibodies. Another theory or at least a partial factor is that the heavier the conjugate, the less immune response is obtained.

Polymeric molecules coupled to the polypeptide can be any suitable polymeric molecule with a molecular weight as defined in accordance with the invention, including natural and synthetic homopolymers, such as polyols (i.e., poly-OH), polyamines (i.e., poly-OH). NH₂) and polycarboxylic acids (i.e. poly-COOH) and additional heteropolymers, i.e. polymers comprising one or more different coupling groups, for example a hydroxyl group and amine groups.

Examples of suitable polymeric molecules include polymeric molecules selected from the group comprising polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polypropylene glycols (PEG), methoxypolyethylene glycols (mPEG) and polypropylene glycols, PEG-glycidyl ethers (Epox-PEG ), PEG-oxycarbonylimidizadol (CDI-PEG) branched polyethylene glycols (PEG), polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, poly-D, L-amino acids, Anhydrous polyethylene-co-maleic acid, anhydrous polystyrene-co-malic acid, dextrans including carboxymethyldextrans, heparin, homologous albumin, cellulose, including methylcellulose, carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxyethylcellulose carboxymethylcellulose such as hydrocellulose hydroxypropyls, starchrolyses such as hydrocellulose hydroxypropyl and hydroxypropyl starches, glycogen, agarose and derivatives thereof, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates and biopolymers.

Preferred polymeric molecules are non-toxic polymeric molecules such as (m) polyethylene glycol (mPEG), which further requires relatively simple chemistry for their covalent coupling to binding groups on the surface of the enzyme.

Generally observed polyalkylene oxides (PAO), such as polyethylene oxides, such as PEG and especially mPEG are the preferred polymeric molecules since these polymeric molecules, compared to polysaccharides such as dextran, pullulan and the like, have few groups reagents capable of cross-linking, being undesirable.

B. sHASEGP tissue expression profiles

Although previously thought to be testis specific, human sHASEGP is expressed in multiple tissues in humans when more sensitive techniques such as RT-PCR are used. The sHASEGP transcript is found in marrow (brain), microvascular endothelium, prostate, breast, retina, combined human melanocytes, fetal heart, and pregnant uterus. SHASEGP is also expressed in germ cell tumors. RT-PCR-based detection of sHASEGP transcripts is generally necessary to detect levels in tissues other than testis.

ES 2 335 005 T3

C. Assays for enzymatic activity of sHASEGP

Turbidometric microtiter assay to determine hyaluronidase activity

Hyaluronidase activity can be detected by means of a modified turbidimetric assay in acidified serum solution. The required reagents are as follows:

<td>2X UV sterilized deionized water or sterile water for irrigation</td><td>Braun</td><td>R5000-01</td>

<td>Hylumed Medical - Sodium Hyaluronate, High Molecular Weight HA</td><td>Genzyme Advanced Biomaterials</td><td> 4876</td>

<td>Hyaluronidase Reference Standard</td><td>USP</td><td> 31200</td>

<td>Potassium Acetate, Granular, USP, ACS</td><td>JTBaker</td><td> 2914-01</td>

<td>Acetic Acid, Glacial, 99 +%</td><td>Sigma</td><td>A-6283</td>

<td>Monobasic Sodium Phosphate Monohydrate, USP Granular</td><td>Mallinkrodt</td><td> 7774</td>

<td>Anhydrous Dibasic Sodium Phosphate, USP</td><td>Mallinkrodt</td><td> 7771</td>

<td>Sodium Chloride, Crystals, GR, ACS</td><td>EMScience</td><td>SX0420-5</td>

<td>Enzymatic Gelatin Hydrolyzate</td><td>Sigma</td><td>G-0262</td>

<td>Horse serum, donor herd, cell culture assay, hybridoma culture assayed, United States origin</td><td>Sigma</td><td>H-1270</td>

<td>20% Human Serum Albumin</td><td>Griffols</td><td></td>

<td>Hydrochloric Acid, ACS Reagent</td><td>Sigma</td><td>H-7020</td>

<td>Calcium Chloride, Dihydrate, Granular USP, -FCC</td><td>JTBaker</td><td> 1336-01</td>

The following reagents are prepared: Acetate Buffer Solution - 14.0 g of potassium acetate and 25.0 ml of glacial acetic acid in water to make 1000 ml. Phosphate Buffer Solution - 2.5 g of monobasic sodium phosphate, 1.0 g of anhydrous dibasic sodium phosphate and 8.2 g of sodium chloride in water to make 1000 ml. Enzyme Diluent Stock Solution- 500 ml of Phosphate Buffer Solution with 500 ml of water. Enzyme Diluent Working Solution - 33 mg of hydrolyzed gelatin in 50 ml of enzyme diluent stock solution prepared 2 hours before use. Sample Stabilization Buffer Solution ("SSB" Solution) - 125 μl of a 20% Human Serum Albumin Solution and 50 μl of a 1 M Calcium Chloride solution in 50 ml of Enzyme Diluent Working Solution and mix thoroughly. Serum Stock Solution - Dilute 1 volume of Horse Serum with 9 volumes of Acetate Buffer Solution. Adjust with 4 N hydrochloric acid to a pH of 3.1 and allow the solution to stand at room temperature for 18 to 24 h. Store the solution at 4 ° C and use within 30 days. Serum Working Solution - 10 ml of Serum Stock Solution in 30 ml of Acetate Buffer Solution adjusted to room temperature. Hyaluronic Acid Stock Solution - Sodium Hyaluronic Acid at a concentration of 5.0 mg / ml in water. Hyaluronic Acid Working Solution - 0.75 ml of Hyaluronic Acid Stock Solution in 4.25 ml of Phosphate Buffer Solution. Conventional Stock Solution - One container of USP Reference Standard Hyaluronidase at a concentration of 1000 Units / ml in water, aliquoted into 50 µl portions and stored at -20 ° C. Conventional Working Solution - 40 µl of Conventional Stock Solution in 960 µl of cold Enzyme Diluent Working Solution to obtain a solution having a known concentration of 40 Units / ml, prepared immediately before use in the assay.

ES 2 335 005 T3

All enzyme samples are diluted in a 96-well "Low Protein Binding" plate according to the following guidelines:

a) The maximum sensitivity range of this assay is between 10-30 Units / ml. To minimize the number of times an assay must be repeated to obtain in-range results, first determine the approximate number of total units / ml for the sample and then select a dilution (absolute number) so that the final concentration is approximately 20 Units / ml.

b) The Minimum Sample volumes required to perform an assay are as follows: FPLC Fractions = 50 µl, Tissue Culture Supernatants = 1 ml, Purified Material / Concentrate / Final Stage = 10 µl.

c) For samples with serial dilutions, 1:10 dilutions are made in the 96-well "Low Protein Binding" plate in triplicate by pipetting 360 µl of the “SSB” solution and 40 µl of the sample in each well.

For the preparation of the USP Standard, the USP Standard Curve is prepared in the "Low Protein Binding" 96-well plate as follows:

USP Pattern Curve

<td>Wells:</td><td>Pattern:</td><td>Sol. Of Enzyme Diluent (in μΙ):</td><td>Sol. Of Conventional Work (in μΙ):</td><td>Final Conc. (In Units / ml):</td>

<td>A1-A3</td><td>StO1</td><td> 0</td><td> 100</td><td> 40</td>

<td>B1-B3</td><td>St02</td><td> 20</td><td> 80</td><td> 32</td>

<td>C1-C3</td><td>St03</td><td> 40</td><td> 60</td><td> 24</td>

<td>D1-D3</td><td>St04</td><td> 60</td><td> 40</td><td> 16</td>

<td>E1-E3</td><td>St05</td><td> 80</td><td> 20</td><td> 8</td>

<td>F1-F3</td><td>St06</td><td> 90</td><td> 10</td><td> 4</td>

<td>G1-G3</td><td>St07</td><td> 100</td><td> 0</td><td> 0</td>

For the preparation of the Hyaluronic Acid control in columns 1-3, prepare the HA Control in the 96-well "Flat Bottom" plate as follows:

_____________________ HA Controls: _______________ Acid Working Sol. Diluent Working Sol. Wells: Control: Hyaluronic (in μΙ): _______ Enzymatic (in μΙ): ____________

H1-H3 Co01 0 60

The Reaction Plate: pipet 30 µl per well of Hyaluronic Acid Working Solution using a 50 µl 8-channel transfer pipet into a 96-well "Flat Bottom" microtiter plate, leaving wells H1-H3 empty. . 60 µl / well of Enzyme Diluent Working Solution is pipetted into wells H1-H3 of the same plate as the HA control.

Serum Working Solution: 40 ml of Serum Working Solution are dispensed into a transfer tray and close to the thermoblock.

Prewarming stage: Once both plates have been prepared, the Low Protein Binding 96-well plate containing the diluted samples, standards, controls, and the flat-bottom 96-well plate containing the Hyaluronic Acid Working Solution They are placed in a thermoblock and allowed to heat for 5 min at 37 ° C.

The Reaction is initiated by adding Enzyme to Substrate: 30 µl of the enzyme plate in all wells of column # 1 of the 96-well flat-bottom plate (containing the substrate) using an 8-channel pipet. of 5-50 pl. The Enzyme / Substrate reaction mix is ​​aspirated 5 times (drawing the solution up and down with the blot for the first 15 seconds to ensure a complete sample mix.

ES 2 335 005 T3 after mixing the enzyme and substrate, the tips are ejected and a new set of tips is loaded into the transfer pipette for the next column. A timer is restarted and at time (t) = 0:30, this process is repeated for column 2. In the next 30 second interval (t) = 1:00, this process is repeated for column 3. This process is repeated by scrolling from left to right across the plate, every 30 seconds until all wells contain both enzyme and substrate.

Stopping the reaction: When the timer reaches 6 minutes (t) = 6:00, 240 µl of the Serum Working Solution is pipetted into each well, using a 50-300 µl 8-channel transfer pipet on the column. 1 of the 96-well flat bottom plate from the adjacent 50 ml Reagent Pool. The mixture is aspirated 3 times (drawing the solution up and down with the transfer pipet) during the first 10 seconds to ensure thorough mixing. The process is repeated every 30 seconds, advancing from column 1 to 12.

After completion of the last column (column 12), the reaction plate is removed from the thermoblock and the plate is placed on the plate reader reading tray at 640 nM. A linear curve fit is generated from the standard curve that allows extrapolation of the test samples.

Alternative Assays for Hiauloronidase

Biotinylated Hyaluronan Microtiter Assay

The free carboxyl groups or gluoronic acid residues of Hyaluronan are biotinylated in a one-step reaction using biotin-hydrazide (Pierce), Sulfo NHS (Pierce) and 1-Ethyldimethylaminopropyl-carbodiimide (Sigma). This biotinylated HA substrate is covalently coupled to a 96-well microtiter plate in a second reaction. At the completion of the enzymatic reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read on a standard ELISA plate reader. Since the substrate is covalently bound to the microtiter plate, artifacts such as pH-dependent shift of the biotinylated substrate do not appear. The sensitivity allows rapid measurement of hyaluronidase activity from cultured cells and biological samples with an inter-assay variation of less than 10%.

The specific activity of hyaluronidase is expressed in turbidity reducing units (TRU). A TRU is defined as the amount of hyaluronidase activity necessary to reduce the turbidity of an acidified hyaluronic acid solution and is equivalent to USP / National Formulary Units (NF XIII) (NFU). The ELISA-type enzyme assay used for purification is related to the TRU, NFU and USP units. through a standard curve of a hyaluronidase sample (eg USP) standardized through USP. Therefore, the enzymatic activities determined by the ELISA-type enzyme assay are actually relative TRU, since the enzyme activity is not actually measured using the turbidimetric assay (Dorfman et al., 1948, J. Biol. Chem. 172: 367 ).

Many hyaluronidase tests have been based on the measurement of the generation of new reducing N-acetylamino groups (Bonner and Cantey, Clin. Chim. Acta 13: 746-752, 1966), or loss of viscosity (De Salegui et al., Arch. Biochem. Biophys. 121: 548-554,1967) or turbidity (Dorfinan and Ott, J. Biol. Chem. 172: 367,1948). With purified substrates, all these methods are sufficient for the determination of the presence or absence of endoglucosamide activity.

Substantially purified glycosaminoglycan substrates can also be used for a Gel Displacement Assay. Glycosaminoglycans are mixed with combining sHASEGP to test for endoglucosidase activity, which results in a change in substrate motility within the gel. Chondroitin sulfate 4 and 6, dermatan sulfate, heparan sulfate can be obtained from Sigma Chemical. Hyaluronan can be obtained from human umbilical cord in CNI. Each test substrate is diluted to 0.1 mg / ml in a buffer range of pH 3.5-7.5. 10 µl samples of purified sHASEGP or conditioned media from cells expressing sHASEGP are also mixed with 90 µl of assay substrate in the desired buffer and incubated for 3 hours at 37 ° C. After incubation the samples are neutralized with sample buffer (Tris EDTA pH 8.0, Bromophenol Blue and glycerol) followed by electrophoresis. Glycosaminoglycans are detected by staining the gels in 0.5% Alcian Blue in 3% Glacial Acetic Acid overnight, followed by fading in 7% Glacial Acetic Acid. Degradation is determined by comparison of substrate motility in the presence and absence of enzyme.

Hyaluronidase activity can also be detected by substrate gel zymography (Guentenhoner et al., 1992, Matrix 388-396). In this assay, a sample is applied to an SDS-PAGE gel containing hyaluronic acid and the proteins in the sample are separated by electrophoresis. The gel is then incubated in an enzyme assay buffer and subsequently stained for hyaulonic acid in the gel. Hyaluronidase activity is visualized as a cleared area on the substrate gel.

D. Identification and isolation of sHASEGP polypeptide genes

The sHASEGP polypeptide gene and / or domains thereof can be obtained by methods well known in the art for DNA isolation. Any method known to those of skill in the art for the identification of nucleic acids encoding desired genes can be used. Any method available in the art can be used

ES 2 335 005 T3 to obtain a full-length cDNA (ie, including the entire coding region) or genomic DNA clone that encodes a sHASEGP polypeptide. For example, polymerase chain reaction (PCR) can be used to amplify a sequence that is expressed in normal tissues, for example, nucleic acids encoding a sHASEGP polypeptide (SEQ ID NO: 1 and 2) in a library of genomic or cDNA. Oligonucleotide primers that hybridize to sequences at the 3 'and 5' termini of the sequences identified as primers can be used to PCR amplify sequences from a nucleic acid sample (RNA or DNA, generally a cDNA library) from an appropriate source (eg testis, prostate, breast).

PCR can be performed, for example, using a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). The DNA that is amplified can include mRNA, or cDNA, or genomic DNA from any eukaryotic species. One can choose to synthesize several different degenerate primers for use in PCR reactions.

It is also possible to vary the stringency of the hybridization conditions used in priming PCR reactions to amplify nucleic acid homologs (for example, to obtain sHASEGP polypeptide sequences from species other than humans or to obtain human sequences with homology to sHASEGP polypeptide), allowing greater or lesser degrees of nucleotide sequence similarity between the known nucleotide sequence and the nucleic acid homologue being isolated. For cross hybridization between species, conditions of low stringency to moderate stringency are used. For hybridization within the same species, moderately stringent to highly stringent conditions are used. Conditions can be determined empirically.

After successful amplification of nucleic acid containing all or a portion of the identified sHASEGP polypeptide sequence or a nucleic acid encoding all or a portion of a sHASEGP polypeptide homolog, that segment can be molecularly cloned and sequenced, and used as a probe to isolate a complete genomic or cDNA clone. This in turn allows the determination of the complete nucleotide sequence of the gene, the analysis of its expression and the production of its protein product for functional analysis. Once the nucleotide sequence is determined, an open reading frame encoding the protein product of the sHASEGP polypeptide gene can be determined by any method well known in the art for determining open reading frames, for example, using publicly available computer programs. for nucleotide sequence analysis. Once an open reading frame is defined, it is routine to determine the amino acid sequence of the protein encoded by the open reading frame. In this way, the nucleotide sequences of the entire sHASEGP polypeptide genes, as well as the amino acid sequences of sHASEGP polypeptide proteins and the like, can be identified.

Any eukaryotic cell can potentially serve as the nucleic acid source for molecular cloning of the sHASEGP polypeptide gene. Nucleic acids can be isolated from vertebrates, mammals, humans, pigs, bovines, felines, birds, equines, canines, as well as sources of additional primates, insects, plants, and other organisms. DNA can be obtained by standard procedures known in the art from cloned DNA (eg, a DNA "library"), by chemical synthesis, by cDNA cloning, or by cloning of genomic DNA or fragments thereof, purified to starting from the desired cell (see, for example, Sambrook et al. 1989, Molecular Cloning, A Laboratory Manual, 2^to Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, DM Ed., 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, UK Vol. 1,11. Clones derived from genomic DNA can contain intronic and regulatory DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. For any source, the gene is cloned into a suitable vector for its propagation.

In molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will code for the desired gene.

DNA can be cleaved at specific sites using various restriction enzymes.

Alternatively, DNase in the presence of manganese can be used to fragment the DNA or the DNA can be physically broken, for example, by sonication. The linear DNA fragments can then be separated according to size by conventional techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.

Once DNA fragments are generated, identification of the specific DNA fragment that contains the desired gene can be accomplished in a number of ways.

For example, a portion of the sHASEGP polypeptide gene (of any species) (eg, a PCR amplification product obtained as described above or an oligonucleotide having a sequence of a portion of the known nucleotide sequence) or its Specific RNA, or a fragment thereof, can be purified and labeled and the generated DNA fragments can be screened by nucleic acid hybridization with the labeled probe (Benton and Davis, Science 196: 180 (1977); Grunstein and Hogness, Proc. Natl. Acad. Sci. USA. 72: 3961 (1975)). DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion (s) and comparison of fragment sizes with those expected according to a known restriction map, if such a map is available, or by DNA sequence analysis and comparison. with the known nucleotide sequence of sHASEGP polypeptide. Additional selection can be made based on the properties of the gene. Alternatively,

ES 2 335 005 T3 the presence of the gene can be detected by assays based on the physical, chemical or immunological properties of its expressed product. For example, cDNA clones or DNA clones can be selected that select by hybridization the appropriate mRNA that produces a protein that, for example, has electrophoretic migration, isoelectric concentration behavior, proteolytic digestion maps, antigenic properties, hyaluronidase activity similar. or identical. If an anti-sHASEGP polypeptide antibody is available, the protein can be identified by binding of labeled antibody to clones putatively synthesizing the sHASEGP polypeptide in an ELISA-like procedure (enzyme-linked immunosorbent assay).

Alternatives for isolating genomic sHASEGP polypeptide DNA include, but are not limited to, chemically synthesizing the gene sequence from a known sequence or converting the cDNA to mRNA encoding the sHASEGP polypeptide.

For example, RNA for cloning sHASEGP polypeptide gene cDNA can be isolated from cells expressing the protein. The identified and isolated nucleic acids can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives or plasmids such as plasmid derivatives pBR322 or pUC or the Bluescript vector (Stratagene, La Jolla, CA). Insertion into a cloning vector can be achieved, for example, by ligation of the DNA fragment into a cloning vector having complementary cohesive terminal ends.

If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any desired site can be produced by ligation of nucleotide sequences (linkers) at the terminal ends of the DNA; These linked linkers can include specific chemically synthesized oligonucleotides that encode restriction endonuclease recognition sequences. In an alternative method, the excised vector and the sHASEGP polypeptide gene can be modified by homopolymeric tailing.

Recombinant molecules can be introduced into host cells by transformation, transfection, infection, electroporation, calcium precipitation, and other methods, so that many copies of the gene sequence are generated.

In specific embodiments, transformation of host cells with recombinant DNA molecules incorporating the isolated sHASEGP polypeptide gene, cDNA, or synthesized DNA sequences, allows the generation of multiple copies of the gene.

Therefore, the gene can be obtained in large quantities by culturing transformants, isolating the recombinant DNA molecules from the transformants, and, when necessary, recovering the inserted gene from the isolated recombinant DNA.

E. Vectors, plasmids, and cells containing nucleic acids encoding a sHASEGP polypeptide or hyaluronidase domain thereof and expression of sHASEGP polypeptide vectors and cells

For recombinant expression of one or more of the sHASEGP polypeptides, the nucleic acid containing all or a portion of the nucleotide sequence encoding the sHASEGP polypeptide can be inserted into an appropriate expression vector, i.e., a vector containing the elements required for transfection and translation of the inserted protein coding sequence. The necessary transcription and translation signals can also be supplied by the native promoter for sHASEGP genes and / or their flanking regions.

Also provided are vectors containing a nucleic acid encoding sHASEGPs that can be introduced into an expression system capable of producing a soluble, neutral pH active sHASEGP.

Cells containing the vectors are also provided. Cells include eukaryotic and prokaryotic cells and vectors suitable for use therein.

Eukaryotic cells, including dihydrofolate reductase (DG44) deficient Chinese Hamster Ovary Cells, are provided containing the vectors. Suitable cells include yeast cells, fungal cells, plant cells, insect cells, and animal cells. The cells are used to produce a sHASEGP polypeptide or hyaluronidase domain thereof by (a) culturing the cells described above under conditions whereby the encoded sHASEGP polypeptide or hyaluronidase domain of the sHASEGP polypeptide is expressed by the cell, and then (b) recovery of the expressed hyaluronidase domain protein. In exemplified embodiments, the hyaluronidase domain is secreted into the medium.

In one embodiment, vectors are provided that include a nucleotide sequence that encodes a polypeptide that has hyaluronidase activity and contains all or a portion of only the hyaluronidase domain, or multiple copies thereof, of a sHASEGP protein. Vectors are also provided that comprise a nucleotide sequence encoding the hyaluronidase domain and additional portions of a sHASEGP protein of up to, and in.

ES 2 335 005 T3 containing, a full-length sHASEGP protein, as well as multiple copies thereof. Vectors can be selected for expression of the sHASEGP protein or hyaluronidase domain thereof in the cell or such that the sHASEGP protein is expressed as a secreted protein. Alternatively, the vectors can include the signals necessary for the secretion of encoded proteins. When the hyaluronidase domain is expressed, the nucleic acid is linked to a nucleic acid that encodes a secretion signal, such as the Saccharomyces cerevisiae conjugation factor signal sequence or a portion thereof, or the native signal sequence.

To generate a soluble sHASEGP active at neutral pH, cells capable of introducing N-linked glycosylation are necessary. In the preferred embodiment, dihydrofolate reductase-deficient Chinese Hamster Ovary mammalian cells such as DG44 are electroporated with a plasmid encoding a strong mammalian promoter, such as CMV, a nucleic acid encoding a sHASEGP followed by a internal ribosome entry site, the mouse dihydrofolate reductase gene, and the SV40 polyadenylation sequence, as shown in SEQ ID NO: 51. Said cells are then cultured in a chemically defined medium in the absence of hypoxanthine and thymidine, followed by further gene amplification with increasing concentrations of methotrexate.

A variety of host-vector systems can be used to express the protein coding sequence. These include, but are not limited to, mammalian cell systems infected with viruses, eg, vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (eg, baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Vector expression elements vary in their potencies and specificities. Depending on the vector-host system used, any one of a number of suitable transcription and translation elements can be used. Note that bacterial expression of sHASEGP DNA will not by itself result in catalytically active sHASEGP, but when combined with the appropriate glycosylation machinery it can be artificially glycosylated as such.

Any method known to those of skill in the art for inserting nucleic acid fragments into a vector can be used to construct expression vectors that contain a chimeric gene that contains the appropriate transcriptional / translational control signals and protein coding sequences. These methods can include in vitro recombinant and synthetic DNA techniques and in vivo recombinant (genetic recombination). The expression of nucleic acid sequences encoding sHASEGP polypeptide or domains, derivatives, fragments or homologues thereof can be regulated by a second nucleic acid sequence such that the genes or fragments thereof are expressed in a host transformed with the molecule or recombinant DNA molecules. For example, the expression of the proteins can be controlled by any promoter / enhancer known in the art. In a specific embodiment, the promoter is not native to the genes for the sHASEGP polypeptide. Promoters that can be used include, but are not limited to, the SV40 early promoter (Bernoist and Chambon, Nature 290: 304-310 (1981), the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22: 787-797 (1980), the herpes thymidine kinase promoter (Wagner et al. Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39-42 (1982)); prokaryotic expression vectors such as the, β-Lactamase promoter (VillaKamaroff et al., Proc. Natl. Acad. Sci. USA 75: 3727-3731 1978)) or the TAC Promoter (Deboer et al, Proc. Natl. Acad. Sci. USA 80: 21-25 (1983)); see also "Useful Proteins from Recombinant Bacteria": in Scientific American 242: 79-94 (1980)); Plant expression vectors containing the opaline synthetase promoter (HerrarEstrella et al. Nature 303: 209-213 (1984)) or the cauliflower mosaic virus 35S RNA promoter (Garder et al. Nucleic Acids RES. 9: 2871 (1981)), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al., Nature 310: 115-120 (1984)); yeast and other fungal promoter elements such as the Gal4 promoter, alcohol dehydrogenase promoter, phosphoglycerol kinase promoter, alkaline phosphatase promoter, and the following tissue-specific animal transcription control regions that The following have been used in transgenic animals: elastase I gene control region, which is active in pancreatic atinar cells (Swift et al, Cell 38: 639-646 (1984); Omitz et al, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986); Macdonald, Hepatology 7: 425-515 (1987)); insulin gene control region, which is active in pancreatic beta cells (Hanahan et al, Nature 315: 115-122 (1985)), immunoglobulin gene control region, which is active in lymphoid cells (Grosschedl et al. al, Cell 38: 647-658 (1984); Adams et al. Nature 318: 533-538 (1985); Alexander et al, Mol. Cell Biol. 7: 1436-1444 (1987)), mouse mammary tumor virus control region, which is active in testicular, mammary, lymphoid and mast cell cells (Leder et al, Cell 45: 485-495 (1986)), region control region of the albumin gene, which is active in the liver (PINCKERT et al, Genes and Devel. 1: 268-276 (1987)), control region of the alpha-fetoprotein gene, which is active in liver (Krumlauf et al. al, Mol. Cell. Biol. 5: 1639-1648 (1985); Hammer et al, Science 235: 53-58 1987)), control region of the alpha-1 antitrypsin gene, which is active in the liver (Kelsey et al. Genes and Devel. 1: 161-171 (1987)), control region of the betaglobin gene, which is active in myeloid cells (Mogram et al. Nature 315: 338-340 (1985); Kollias et al, Cell 46: 89-94 (1986)), gene control region of myelin basic protein, which is active in oligodendrocytes in the brain (Readhead et al. Cell 48: 703-712 (1987)), myosin 2 light chain gene control region, which is active in skeletal muscle (Sani, Nature 314: 283-286 (1985)), and gene control region of gonadotropin-releasing hormone, which is active in gonadotrophic cells of the hypothalamus (Mason et al. Science 234: 1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoter operably linked to nucleic acids encoding a sHASEGP polypeptide or a domain, fragment, derivative or homolog thereof, one or more origins of replication and, optionally, one or more markers of selection (eg, an antibiotic resistance gene).

ES 2 335 005 T3

Specific start signals may also be necessary for efficient translation of a sHASEGP sequence. These signals include the ATG start codon and adjacent sequences. In cases where sHASEGP, its start codon and its upstream sequences are inserted into the appropriate expression vector, additional translational control signals may not be necessary. However, in cases where only the coding sequence or a portion thereof is inserted, exogenous transcriptional control signals must be provided including the ATG start codon. Furthermore, the start codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous start codons and transcriptional elements can be of various origins, both natural and synthetic. The efficiency of expression can be increased by including enhancers appropriate to the cell system used (Scharf D et al (1994) Results Probl Cell Differ 20: 125-62; Bittner et al (1987) Methods in Enzymol 153: 516- 544).

In addition, a host cell strain can be selected for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired manner. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that clears a "prepro" form of the protein may also be important for correct insertion, folding, and / or function. Different host cells such as CHO (DG44, DXB 11, CHO-K1), HeLa, MDCK, 293, WI83, etc. they have specific cellular machinery and characteristic mechanisms for said post-translational activities and can be selected to ensure correct modification and processing of the foreign protein introduced.

For long-term high-throughput production of recombinant proteins, stable expression is preferred. For example, cell lines stably expressing sHASEGP can be transformed using expression vectors containing viral origins of replication or endogenous expression elements and a selection marker gene. After vector introduction, cells can be allowed to grow for 1-2 days in enriched medium before they are switched to selective medium. The purpose of the selection marker is to confer resistance to selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequences. Resistant clusters of stably transformed cells can proliferate using tissue culture techniques appropriate for the cell type.

Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase genes (Wigler M et al (1977) Cell 11: 22332) and adenine phosphoribosyltransferase (Lowy I et al (1980) Cell 22: 817-23), which can be used in TK- or APRT- cells, respectively. Furthermore, resistance to antimetabolites, antibiotics or herbicides can be used as the basis for selection. For example, DHFR conferring resistance to methotrexate (Wigler M et al (1980) Proc Natl Acad Sci 77: 3567-70); the npt that confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin Fetal (1981) J Mol Biol 150: 1-14) and als or pat, which confer resistance to chlorsulfuron and phosphofinothricin acetyltransferase, respectively (Murry, previously) . Additional selection genes have been described, for example, trpB which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol instead of histidine (Hartman SC and RC Mulligan (1988) Proc Natl Acad Sci 85: 8047-51). Recently, the use of visible markers has gained popularity with markers such as anthocyanins, beta glucuronidase and its substrate, GUS and luciferase and its substrate, luciferin, being widely used not only to identify transformants, but also to quantify the amount of transient protein expression. or stable attributable to a specific vector system (Rhodes CA et al (1995) Methods Mol Biol 55: 121-131).

Identification of transformants containing the polynucleotide sequence

Although the presence / absence of marker gene expression suggests that the gene of interest is also present, the presence and expression of an active sHASEGP should be confirmed. For example, if sHASEGP is inserted into a marker gene sequence, recombinant cells containing sHASEGP can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sHASEGP sequence under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of sHASEGP in tandem as well. Detection of an appropriately glycosylated neutral pH active sHASEGP can be determined by testing conditioned media for sHASEGP enzymatic activity under appropriate conditions.

SHASEGP purification

Host cells transformed with a sHASEGP nucleotide sequence can be cultured under conditions suitable for expression and recovery of the purified protein from cell culture. The protein produced by a recombinant cell is preferably secreted, but may be contained intracellularly depending on the sequence and / or the vector used. As will be understood by those of skill in the art, expression vectors containing sHASEGP can be designed with signal sequences that facilitate direct secretion of sHASEGP through a prokaryotic or eukaryotic cell membrane. Other recombinant constructs can bind sHASEGP to a nucleotide sequence encoding a polypeptide domain that will facilitate the purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12: 441-53; see discussion of protein-containing vectors below. of fusion).

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SHASEGP can also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such purification facilitation domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain used in the FLAGS affinity purification / extension system (Immunex Corp, Seattle Wash). The inclusion of cleavable linker sequences such as Factor XA or enterokinase (Invitrogen, San Diego Calif.) Between the purification domain and the sHASEGP is useful to facilitate purification. Such an expression vector provides for the expression of a fusion protein comprising a sHASEGP and containing a nucleic acid encoding 6 histidine residues followed by thioredoxin and an enterokinase cleavage site. Histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography, as described in Porath et al (1992) Protein Expression and Purification 3: 263-281), while the enterokinase cleavage site provides a means to purify chemokine from the fusion protein.

In addition to recombinant production, sHASEGP fragments can be produced by direct peptide synthesis using solid phase techniques (see Stewart et al (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco; Merrifield J (1963) J Am Chem Soc 85: 2149-2154). In vitro protein synthesis can be performed using manual techniques or by automation. Automatic synthesis can be accomplished, for example, using an Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City Calif.) According to the instructions provided by the manufacturer. Various fragments of sHASEGP can be chemically synthesized separately and combined using chemical methods to produce the full-length molecule.

Expression vectors containing the coding sequences, or portions thereof, of a sHASEGP polypeptide are generated, for example, by subcloning the coding portions into the EcoR1 restriction site of each of the three PGEX vectors (glutathione S -transferase (Smith and Johnson, Gene 7: 3140 (1988)) This allows the expression of products in the correct reading frame. Exemplary vectors and systems for the expression of the hyaluronidase domains of sHASEGP polypeptides include the well-known Pichia vectors (available, for example, from Invitrogen, San Diego, CA), particularly those designed for secretion of the encoded proteins. The protein can also be expressed cytoplasmically, such as in inclusion bodies. An exemplary vector is described in the examples.

Plasmids for transformation of E. coli cells include, for example, pET expression vectors (see, US Patent 4,952,496; available from Novagen, Madison, WI; see also Novagen published literature that describes the system).

Such plasmids include pET 11a, which contains the T7 lac promoter, the T7 terminator, the lac operator of

Inducible E. coli and the lac repressor gene; pET 12A-C, containing the T7 promoter, T7 terminator, and the E. coli OMPT secretion signal; and pET 15B and PET19B (Novagen, Madison, Wi), which contain a His-tagged leader sequence for use in purification with a His column and a thrombin cleavage site that allows cleavage after purification on the column; the lac promoter region of T7 and the terminator of T7.

Vectors are introduced into host cells, such as Pichia cells and bacterial cells, such as E. coli, and proteins are expressed therein. Exemplary Pichia strains include, for example, GS115. Exemplary bacterial hosts contain chromosomal copies of DNA encoding T7 aRn polymerase operably linked to an inducible promoter, such as the LACUV promoter (see, US Patent No. 4,952,496). Such hosts include, but are not limited to, the lysogenic E. coli strain BL21 (DE3).

The sHASEGP domains, derivatives, and analogs can be produced by various methods known in the art. For example, once a recombinant cell is identified that expresses a sHASEGP polypeptide or a domain, fragment, or derivative thereof, the individual gene product can be isolated and analyzed. This is accomplished by assays based on the physical and / or functional properties of the protein, including, but not limited to, radioactive labeling of the product followed by analysis by gel electrophoresis, immunoassay, cross-linking with labeled product, and proteolytic activity assays.

SHASEGP polypeptides can be isolated and purified by standard methods known in the art (from natural sources or recombinant host cells expressing the complexes or proteins), including, but not limited to, column chromatography (eg, ion exchange, affinity, gel exclusion, high pressure reverse phase and fast liquid protein), differential centrifugation, differential solubility or by any other conventional technique used for protein purification.

In one embodiment, a sHASEGP can be purified to homogeneity from chemically defined conditioned media of DG44 cells transfected with HZ24 and amplified with methotrexate by 1) tangential flow diafiltration, 2) binding and elution from anion exchange chromatography. , 3) flow chromatography through phenylsepharose, 4) binding and elution from phenylboronate chromatography, and 4) binding and elution with hydroxyaptathia chromatography.

Functional properties can be evaluated using any suitable assay known in the art.

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Alternatively, once a sHASEGP polypeptide or its domain or derivative is identified, the amino acid sequence of the protein can be deduced from the nucleotide sequence of the gene that encodes it. As a result, the protein or its domain or derivative can be synthesized by conventional chemical methods known in the art (eg, see Hunkapiller et al, Nature 310: 105-111 (1984)), followed by in vitro glycosylation.

Manipulations of sHASEGP polypeptide sequences can be performed at the protein level. Also contemplated herein are sHASEGP polypeptide proteins, domains thereof, derivatives or analogs or fragments thereof, which are differentially modified during or after translation, eg, by glycosylation, acetylation, phosphorylation, amidation, pegylation. , derivatization by known protecting / blocking groups, proteolytic cleavage, binding to an antibody molecule or other cellular ligand.

Any of numerous chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8, NABH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin and other agents of this type.

In addition, domains, analogs, and derivatives of a sHASEGP polypeptide can be chemically synthesized. For example, a peptide that corresponds to a portion of a sHASEGP polypeptide that includes the desired domain or that mediates the desired activity in vitro can be synthesized using a peptide synthesizer.

In addition, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the sHASEGP polypeptide sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, E-ABU, e-Ahx, 6-aminohexanoic acid, Aib , 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, / -Lalanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, camethyl amino acids, na-methyl amino acids, and amino acid analogs in general. Also, the amino acid can be d (dextrorotatory) or ol (levorotatory).

In cases in which natural products are suspected to be mutants or are isolated from new species, the amino acid sequence of the sHASEGP polypeptide isolated from the natural source, as well as those expressed in vitro or from expression vectors synthesized in vivo or In vitro can be determined from DNA sequence analysis or, alternatively, by direct sequencing of the isolated protein. Such analysis can be performed by manual sequencing or by use of an automated amino acid sequencer.

Modifications - A variety of modifications of sHASEGP polypeptides and domains are contemplated herein. A nucleic acid molecule encoding sHASEGP can be modified by any of numerous strategies known in the art (Sambrook et al. (1990), Molecular Cloning, A Laboratory Manual, 2^toed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Sequences can be cleaved at appropriate sites with restriction endonucleases, followed by further enzymatic modification if desired, isolated and ligated in vitro. In producing the gene encoding a sHASEGP domain, derivative or analog, care must be taken to ensure that the modified gene preserves the original translational reading frame, not interrupted by translation termination signals, in the gene region in the the desired activity is encoded.

Furthermore, nucleic acid molecules encoding sHASEGP can be mutated in vitro or in vivo to generate and / or destroy translation, start and / or stop sequences or to generate variations in coding regions and / or form new restriction endonuclease sites. or destroy pre-existing ones, to facilitate further in vitro modification. Furthermore, as described herein, muteins with primary sequence alterations such as substitutions of Cys residues and removal or addition of glycosylation sites are contemplated; the sHASEGP of SEQ ID NO: 1 has seven potential glycosylation sites. Such mutations can be effected by any strategy for mutagenesis known in the art including, but not limited to, chemical mutagenesis and site-directed mutagenesis in vitro (Hutchinson et al., J. Biol. Chem. 253: 6551-6558 (1978)), use of TABE linkers (Pharmacia). In one embodiment, for example, a sHASEGP polypeptide or domain thereof is modified to include a fluorescent marker. In other specific embodiments, the sHASEGP polypeptide is modified to have a heterobifunctional reagent, and such heterobifunctional reagents can be used to cross-link the members of the complex.

In addition, domains, analogs, and derivatives of a sHASEGP can be chemically synthesized. For example, a peptide corresponding to a portion of a sHASEGP that includes the desired domain or that mediates the desired activity can be synthesized in vitro using a peptide synthesizer. In addition, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the sHASEGP sequence. Non-classical amino acids include, but are not limited to, the D-isomers of the common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, S-ABU, e-Ahx, 6-aminohexanoic acid, Aib , 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, - -alanine, fluoroamino acids, designer amino acids such as thi-methyl-amino acids, ca-methyl-amino acids, na-methyl-amino acids, and amino acid analogs in general. Also, the amino acid can be d (dextrorotatory) or ol (levorotatory).

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F. Generation of a functionally active glycosylated sHASEGP with N-linked sugar residues

Properly N-glycosylated human sHASEGP is required to generate a catalytically stable protein. N-linked glycosylation of sHASEGP can be accomplished by various techniques. Glycosylation of sHASEGP can be achieved by introducing nucleic acids encoding sHASEGP into cells of eukaryotic origin capable of appropriate N-linked glycosylation or, alternatively, by contacting the sHASEGP polypeptide with cell-free extracts or purified enzymes capable of introducing the linked sugar residues. to N desired.

Selecting an expression system

Eukaryotic cell expression systems vary in the degree and type of glycosylation they introduce into an ectopically expressed polypeptide. CHO cells are, for example, highly efficient for the introduction of N-linked glycosylation into an active sHASEGP polypeptide.

Additional eukaryotic expression systems that introduce N-linked glycosylation to generate a functional sHASEGP product by introducing a human sHASEGP expression plasmid into such cells and assaying for activity at neutral pH. Appropriate N-linked glycosylation can be determined by FACE analysis of oligosaccharides released by PNGASA. Catalytically active sHASEGP glycosylation profiles are further provided herein. Verification of glycosylation can also be performed by sHASEGP treatment of said cells with PNGASA-F or by culturing said cells in tunicamycin, followed by introduction of nucleic acids encoding sHASEGP.

N-glycosylation of sHASEGP polypeptide in vitro. The sHASEGP polypeptide can be N-glycosylated by contacting a sHASEGP polypeptide with cell-free extracts containing an activity capable of transferring N-linked sugars to sHASEGP polypeptide, such as canine microsomal membranes or through coupled transcription and translation, as available from the market (Promega Madison WI).

Oligosaccharides are considered to have a reducing end and a non-reducing end, regardless of whether the saccharide at the reducing end is in fact a reducing sugar. In accordance with accepted nomenclature, oligosaccharides are represented herein with the non-reducing end on the left and the reducing end on the right. All oligosaccharides described in this document are described by the name or abbreviation for the non-reducing saccharide (e.g. Gal), followed by the glycosidic bond configuration (alpha or beta), the ring bond, the saccharide ring position reducing agent involved in the binding, and then the name or abbreviation of the reducing saccharide (eg, GlcNAc). The bond between two sugars can be expressed, for example, as 2,3, 2 ^ 3 or (2,3). Each saccharide is a pyranose.

As used herein, the "N-linked sugar moiety" refers to an oligosaccharide attached to a sHASEGP via the amide nitrogen of Asn moieties. N-linked oligosaccharides are included in several main types (oligomannose, complex, hybrid, sulfated), all having 3-GlcNac-GlcNAc (Man) nuclei linked via the amide nitrogen of Asn residues that are included in -Asn sequences -Xaa-Thr / Ser- (where Xaa is not Pro). N-linked sites are often assigned indirectly by the appearance of a "blank" cycle during sequencing. Positive identification can be made after release of the oligosaccharide by PNGase F, which converts glycosylated Asn to Asp. After release by PNGase F, the N-linked oligosaccharides can be purified using Bio-Gel P-6 chromatography, the combination of oligosaccharides being subjected to preparative high pH anion exchange chromatography (HPAEC) (Townsend et al., (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide isomers can be resolved using HPAEC. Fucose residues will shift elution positions earlier in the HPAEC chromatogram, while additional sialic acid residues will increase retention time. Simultaneous treatment of glycoproteins whose oligosaccharide structures are known (eg, bovine fetuin, α-1 acid glycoprotein, ovalbumin, RNase B, transferrin) can facilitate the assignment of oligosaccharide peaks. The collected oligosaccharides can be characterized by a combination of compositional and methylation bond analysis (Waeghe et. Al; (1983), Carbohydr Res. 123, 281-304), with the anomeric configurations assigned by NMR spectroscopy (Van Halbeek (1993 ) in Methods Enzymol 230).

Alternatively, oligosaccharides can be identified by fluorescence-assisted carbohydrate electrophoresis (FACE) Callewaert et al. (2001) Glycobiology 11, 275-281.

G. Detection and characterization of N-linked sugar residues in sHASEGP

Determining whether a protein is in fact glycosylated is the initial stage in glycan analysis of glycoproteins. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has become the method of choice as the final step before protein sequencing. Glycosylated proteins frequently migrate as diffuse bands by SDS-PAGE. A marked decrease in bandwidth and a change in migration position after treatment with peptide-N4- (N-acetyl-D-glucosaminyl) asparagine amidase (PNGase F) is considered diagnostic of N-linked glycosylation. other types of glycosylation are predominant, other strategies must be used. Lectin transfer methods provide a strategy that is independent of glycosylation class (N versus O). Lectins, carbo-binding proteins

ES 2 335 005 T3 hydrates from various plant tissues, have both high affinity and narrow specificity for a wide range of defined sugar epitopes found on glycoprotein glycans (Cummings, RD (1994) Methods in Enzymol. 230, 66 -86). When conjugated to biotin or digoxigenin, they can be easily identified on membrane blots through a colorimetric reaction using avidin or alkaline phosphatase-conjugated anti-digoxigenin antibodies (Haselbeck, et al. (1993) Methods in Mol. Biol. 141, 161-173), analogous to secondary antibody-alkaline phosphatase reactions used in Western blots. Screening with a panel of lectins with well-defined specificity can provide considerable information about the carbohydrate complement of a glycoprotein. Importantly, the amplification of color development is high enough that 10-50 ng of a glycoprotein can be easily seen on a membrane blot of an SDS-PAGE. Although lectins have a very high affinity for their cognate ligands, some show significant avidity for structurally related epitopes. Therefore, it is important to carefully note the possibility of cross-reactivity when selecting a panel of lectins and to apply those that have the highest probability of individually differentiating complex, hybrid, and mannose-rich N-linked glycans from O-linked structures.

Monosaccharide analysis can also be used to determine whether sHASEGP is glycosylated and, as in the case of lectin analysis, provides additional information on structural characteristics. The quantitative analysis of the composition of monosaccharides i) identifies glycosylated proteins, ii) provides the molar ratio of individual sugars to protein, iii) suggests, in some cases, the presence of classes of oligosaccharides, iv) is the first stage in the design of a structural elucidation strategy and v) provides a measure of production consistency for recombinant glycoprotein therapeutic compounds. In recent years, high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) has been widely used to determine monosaccharide composition (Townsend, et al. (1995) in Carbohydrate Analysis: High-performance liquid chromatography and capillary electrophoresis (Z. E1 Rassi ed.) pp. 181-209). More recently, fluorophore-based labeling methods have been introduced and many are available in kit form. A different advantage of fluorescent methods is an increase in sensitivity (50 times). A potential disadvantage is that different monosaccharides can demonstrate different selectivity for the fluorophore during the coupling reaction, in the hydrolyzate, or in the external standard mixture. However, the increased sensitivity and the ability to identify which monosaccharides are present from a small portion of the total amount of available glycoprotein, as well as the potential for increased sensitivity using laser-induced fluorescence makes this strategy attractive. .

Analysis of the monosaccharide composition of small amounts of sHASEGP is best performed on PVDF (PSQ) membranes after electroblotting (Weitzhandler et al, (1993) J. Biol. Chem. 268, 51215130) or if aliquots are to be analyzed smaller on point transfers. PVDF is an ideal matrix for carbohydrate analysis since neither mono- nor oligosaccharides bind to the membrane, once released by acid or enzymatic hydrolysis.

FACE analysis is an effective means of detecting glycosylation profiles of sHASEGP. Profiling N-linked oligosaccharides FACE® (Prozyme) with 30% oligosaccharide gels is such a mechanism. Oligosaccharides cleaved from 100 pg glycoproteins by enzymatic digestion with N-Glycanase (also known as PNGase), labeled using the ANTS fluorophore and separated by electrophoresis can be used for the detection of glycosylation profiles of sHASEGP. The relative positions of the oligosaccharide bands are determined by processing the sample and dilutions of the sample alongside a standard oligosaccharide ladder that designated the migration distance in units of Degree of Polymerization (DP).

H. Screening methods to identify compounds that modulate sHASEGP activity

Various types of assays are exemplified and described herein. It is understood that hyaluronidase domains can be used in other assays. It is shown herein, however, that the hyaluronidase domains exhibit catalytic activity.

As such they are ideal for in vitro screening assays.

They can also be used in binding assays.

The full-length zymogens of sHASEGP, activated enzymes, and hyaluronidase domains are contemplated for use in any screening assay known to those of skill in the art, including those provided herein. Therefore, the following description, if it relates to hyaluronidase assays, is intended to apply to the use of a single chain hyaluronidase domain or a catalytically active portion thereof of any hyaluronidase, including a sHASEGP. Other assays such as binding assays are provided herein, particularly for use with a sHASEGP, including any variants, such as splice variants thereof.

1. Catalytic assays for identification of agents that modulate the hyaluronidase activity of a sHASEGP protein. Provided herein are methods for identifying a modulator of the catalytic activity of a sHASEGP, particularly a single chain hyaluronidase domain or catalytically active portion thereof.

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The methods can be practiced by: contacting the sHASEGP, a full-length zymogen or activated form and, particularly, a single chain domain thereof with a sHASEGP substrate in the presence of a test substance and detection of the substrate proteolysis, by which the activity of sHASEGP is evaluated, and comparison of the activity with a control. For example, a control can be the sHASEGP activity assessed by contacting a sHASEGP, including a full-length zymogen or activated form and, particularly, a single-chain domain thereof, particularly a single-chain domain thereof. , with a sHASEGP substrate and detection of substrate proteolysis, for which sHASEGP activity is evaluated. The results in the presence and absence of the test compounds will be compared. A difference in activity indicates that the test substance modulates the activity of sHASEGP. Activators of sHASEGP activation cleavage are also contemplated; these trials are discussed below.

In one embodiment, a plurality of test substances are simultaneously scanned in the above screening method. In another embodiment, sHASEGP is isolated from a target cell as a means of further identifying agents that are potentially specific for the target cell.

In another embodiment, a test substance is a therapeutic compound, and whereby a difference in sHASEGP activity measured in the presence and absence of the test substance indicates that the target cell responds to the therapeutic compound.

One method includes the steps of (a) contacting the sHASEGP polypeptide or hyaluronidase domain thereof with one or a plurality of test compounds under conditions conducive to interaction between the ligand and the compounds; and (b) identifying one or more compounds in the plurality that specifically bind to the ligand.

Another method provided herein includes the steps of a) contacting a sHASEGP polypeptide or hyaluronidase domain thereof with a sHASEGP polypeptide substrate and detecting substrate degradation, whereby the activity of the sHASEGP polypeptide is assessed; b) contacting the sHASEGP polypeptide with a sHASEGP polypeptide substrate in the presence of a test substance and detecting degradation of the substrate, whereby the activity of the sHASEGP polypeptide is evaluated; and c) comparing the activity of the sHASEGP polypeptide evaluated in steps a) and b), so that if the activity measured in step a) differs from the activity measured in step b) it indicates that the test substance modulates the activity of the polypeptide. sHASEGP.

In another embodiment, a plurality of the test substances are simultaneously scanned. When comparing the activity of a sHASEGP polypeptide in the presence and absence of a test substance to assess whether the test substance is a modulator of the sHASEGP polypeptide, it is not necessary to assess the activity in parallel, although such a parallel measurement is typical. It is possible to measure the activity of the sHASEGP polypeptide at a time point and compare the measured activity with a historical value of the activity of the sHASEGP polypeptide.

For example, the activity of the sHASEGP polypeptide in the presence of a test substance can be measured and compared to a historical value of the activity of the sHASEGP polypeptide previously measured in the absence of the test substance and vice versa. This can be accomplished, for example, by providing the activity of the sHASEGP polypeptide in a package insert or booklet provided with a kit for performing the assay.

Methods for selecting substrates for a particular sHASEGP are described in the Examples and particular hyaluronidase assays are exemplified.

Combinations and kits are provided containing the combinations optionally including instructions for performing the assays. The combinations include a sHASEGP polypeptide and a sHASEGP polypeptide substrate to be tested; and, optionally, reagents to detect proteolysis of the substrate. Substrates, which can be chromogenic or fluorogenic molecules including glycosaminoglycans subject to proteolysis by a particular sHASEGP polypeptide, can be empirically identified by testing the ability of the sHASEGP polypeptide to cleave the test substrate. The substrates that cleave most efficiently, ie, at the lowest concentrations and / or at the highest rate, or under desirable conditions, are identified.

A kit containing the combination described above is further provided herein. The kit optionally includes instructions for identifying a modulator of the activity of a sHASEGP polypeptide. Any sHASEGP polypeptide is targeted to identify modulators of its activity.

2. Bonding tests. Also provided herein are methods for the identification and isolation of agents, particularly compounds that bind to sHASEGP. The assays are designed to identify agents that bind to the isolated hyaluronidase domain (or a protein, other than a sHASEGP polypeptide, that contains the hyaluronidase domain of a sHASEGP polypeptide) and to the activated form, including the activated form derived from the full-length zymogen. or an extended hyaluronidase domain. The identified compounds are candidate compounds or candidate compounds for the identification of compounds for treatments of disorders and diseases involving aberrant hyaluronidase activity. The sHASEGP polypeptides used in the methods include any sHASEGP polypeptide as defined herein, including the single chain hyaluronidase domain of sHASEGP or a proteolytically active portion thereof.

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A variety of methods are provided herein. These methods can be performed in solution or solid phase reactions in which the sHASEGP polypeptide (s) or hyaluronidase domain (s) thereof are linked directly or indirectly via a linker to a solid support. Screening assays are described in the Examples and these assays have been used to identify candidate compounds.

For the purposes of this document, all of the binding assays described above for sHASEGP are provided.

Provided herein are methods of identifying an agent, such as a compound, that specifically binds to a single-chain hyaluronidase domain of sHASEGP, a full-length activated sHASEGP, or a double-chain hyaluronidase domain thereof. The method can be performed by (a) contacting the sHASEGP with one or a plurality of test agents under conditions that lead to binding between the sHASEGP and an agent; and (b) identifying one or more agents within the plurality that specifically bind to the sHASEGP.

For example, in performing such methods the sHASEGP polypeptide is mixed with a potential binding partner or an extract or fraction of a cell under conditions that allow the association of potential binding partners with the polypeptide. After mixing, the peptides, polypeptides, proteins, or other molecules that have been associated with a sHASEGP are separated from the mixture. The binding partner that binds to the sHASEGP can then be removed and further analyzed. To identify and isolate a binding partner, the entire protein can be used, eg, the described full-length protein of SEQ ID NO: 1. Alternatively, a fragment of the protein can be used.

A variety of methods can be used to obtain cell extracts or body fluids such as blood, serum, urine, sweat, synovial fluid, CSF, and other such fluids.

For example, cells can be disrupted using physical or chemical disruption methods. Examples of physical disruption methods include, but are not limited to, sonication and mechanical disruption. Examples of chemical lysis methods include, but are not limited to, detergent lysis and enzymatic lysis. One of ordinary skill can easily adapt methods for preparing cell extracts to obtain extracts for use in the present methods.

Once an extract of a cell is prepared, the extract is mixed with the sHASEGP under conditions in which association of the protein with the binding partner can occur. A variety of conditions can be used, including conditions that resemble conditions found in the cytoplasm of a human cell or in a body fluid, such as blood. Characteristics such as osmolarity, pH, temperature and the concentration of cell extract used can be varied to optimize the association of the protein with the binding partner. Similarly, methods are known for the isolation of molecules of interest from body fluids.

After mixing under appropriate conditions, the bound complex is separated from the mixture. A variety of techniques can be used to separate the mixture. For example, antibodies specific for a sHASEGP can be used to immunoprecipitate the binding partner complex. Alternatively, conventional chemical separation techniques such as chromatography and density / sedimentation centrifugation can be used.

After removing the unassociated cellular constituents in the extract, the binding partner can be dissociated from the complex using conventional methods. For example, dissociation can be achieved by altering the salt concentration or pH of the mixture.

To help separate the associated binding partner pairs from the mixed extract, the sHASEGPs can be immobilized on a solid support. For example, the protein can be bound to a matrix of nitrocellulose or acrylic beads. Binding of the protein or a fragment thereof to a solid support helps to separate peptide / binding partner pairs from other constituents found in the extract. The identified binding partners can be a single protein or a complex made up of two or more proteins.

Alternatively, the nucleic acid molecules encoding single-chain hyaluronidases can be used in a yeast two-hybrid system. The yeast two-hybrid system has been used to identify other protein-partner pairs and can be readily adapted to employ the nucleic acid molecules described herein.

Another in vitro binding assay, particularly for a sHASEGP, uses a mixture of a polypeptide containing at least the catalytic domain of one of these proteins and one or more candidate binding targets or substrates. After incubating the mixture under appropriate conditions, the ability of the sHASEGP or a polypeptide fragment thereof containing the catalytic domain to bind or interact with the candidate substrate is evaluated. For cell-free binding assays, one of the components includes or is coupled to a detectable marker. The marker can provide direct detection, such as radioactivity, luminescence, electron or optical density, etc. or an indirect detection such as an epitopic marker, an enzyme, etc. A variety of methods can be employed to detect the marker depending on the nature of the marker and other test components. For example, the label can be detected bound to the solid substrate, or a portion of the bound complex containing the label can be separated from the solid substrate and the label thereafter detected.

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3. Detection of signal transduction sHASEGP, which is a membrane-anchored protein, may be involved directly or indirectly in signal transduction directly as a cell surface receptor or indirectly by activating proteins, such as progrowth factors that can initiate transduction. of signals.

Furthermore, secreted sHASEGP, such as the soluble domain of sHASEGP that is described in SEQ ID NO: 4, may be involved in signal transduction directly by binding to or interacting with a cell surface receptor or indirectly by activation of proteins. , such as progrowth factors that can initiate signal transduction. Assays to assess signal transduction are well known to those of skill in the art and can be adapted for use with the sHASEGP polypeptide.

Assays are provided to identify agents that affect or alter directly or indirectly mediated signal transduction, such as by activation of a progrowth factor by a sHASEGP, particularly of full length or a portion sufficient to anchor to the extracellular domain or a functional portion thereof. of a sHASEGP on the surface of a cell. Such assays include, for example, transcription-based assays in which the modulation of a transduced signal is assessed by detecting an effect on the expression of a reporter gene (see, for example, US Patent No. 5,436,128 ).

Four. Methods to Identify Agents that Modulate the Expression of a Nucleic Acid Encoding a sHASEGP. Another embodiment provides methods for identifying agents that modulate the expression of a nucleic acid encoding a sHASEGP. Such assays use any available means of control for changes in the level of expression of nucleic acids encoding a sHASEGP.

Assay formats can be used to monitor the ability of the agent to modulate the expression of a nucleic acid encoding a sHASEGP. For example, mRNA expression can be controlled directly by hybridization to nucleic acids. In addition, enzymatic assays such as those described can be used to detect agents that modulate sHASEGP expression.

Cell lines are exposed to the agent to be tested under appropriate conditions and for an appropriate time and total RNA or mRNA is isolated by standard procedures (see, for example, Sambrook et al (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2^to Ed. Cold Spring Harbor Laboratory Press). Probes to detect differences in RNA expression levels between cells exposed to the agent and control cells can be prepared from the nucleic acids. It is typical, but not necessary, to design probes that hybridize only to target nucleic acids under conditions of high stringency. Highly complementary nucleic acid hybrids are only formed under high stringency conditions. Therefore, the stringency of the assay conditions determines the amount of complementarity that should exist between two nucleic acid strands to form a hybrid. The stringency should be selected to maximize the difference in stability between the probe: target hybrid and the potential probe: non-target hybrids.

For example, N- and C-terminal fragments of sHASEGP can be expressed in bacteria and used to search for proteins that bind to these fragments. Fusion proteins, such as His or GST tag fusion with the N- or C-terminal regions of the sHASEGP can be prepared for use as a substrate. These fusion proteins can be coupled to, for example, Glutathione-Sepharose beads and then probed with cell lysates or body fluids. Before lysis, cells or body fluids can be treated with a candidate agent that can modulate a sHASEGP or proteins that interact with domains thereon. Binding of lysate proteins to fusion proteins can be resolved by SDS-PAGE, isolated, and identified by protein sequencing or mass spectroscopy, as is known in the art.

Antibody probes are prepared by immunization of suitable mammalian hosts in appropriate immunization protocols, using the peptides, polypeptides, or proteins if they are of sufficient length (e.g., 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13,14,15,20,25, 30, 35,40 or more consecutive amino acids) of the sHASEGP polypeptide or if required to increase immunogenicity, conjugated with suitable vehicles. Methods for preparing immunogenic conjugates with carriers, such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or other carrier proteins are well known in the art. In some circumstances, direct conjugation may be effective using, for example, carbodiimide reagents; in other cases, binding reagents such as those supplied by Pierce Chemical Co., Rockford, IL may be desirable to provide accessibility to the hapten. Hapten peptides can be amino- or carboxy-terminal extended with a Cys residue or intercalated with cysteine ​​residues, for example, to facilitate binding to a carrier.

Administration of the immunogens is generally carried out by injection over a suitable period of time and with the use of suitable adjuvants, as is generally understood in the art. During the immunization program, antibody titers are taken to determine the ability to form antibodies.

Anti-peptide antibodies can be generated using synthetic peptides that correspond, for example, to the carboxy terminal amino acids of sHASEGP.

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Synthetic peptides can be as small as 1-3 amino acids in length, generally at least 4 or more amino acid residues in length. The peptides can be coupled to KLH using conventional methods and can be used to immunize animals, such as rabbits or ungulates. Polyclonal antibodies can then be purified, for example, using Actigel beads containing the covalently linked peptide.

Although the polyclonal antisera produced in this way may be satisfactory for some applications, the use of monoclonal preparations is generally used for pharmaceutical compositions. Immortalized cell lines that secrete the desired monoclonal antibodies can be prepared using the conventional method of Kohler et al., (Nature 256: 495-7 (1975)) or modifications that achieve immortalization of lymphocytes or splenocytes, as are generally known. Immortalized cell lines secreting the desired antibodies are screened by an immunoassay in which the antigen is the peptide, polypeptide, or protein hapten.

When the appropriate culture of immortalized cells secreting the desired antibody is identified, the cells can be cultured in vitro or by in vivo production by ascites fluid. Of particular interest are monoclonal antibodies that recognize the catalytic domain or activation cleavage site (region) of a sHASEGP.

Antibodies or fragments can also be produced. Regions that specifically bind to desired receptor regions can also be produced in the context of chimeras of multiple species origin.

The agents that are tested in the above method can be randomly selected or rationally selected or designed.

The agents can be, as examples, peptides, small molecules, and carbohydrates. A specialist can easily recognize that there are no limits when it comes to the structural nature of agents.

Peptide agents can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as known in the art. Furthermore, DNA encoding these peptides can be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. Production using solid phase peptide synthesis is necessary if non-gene encoded amino acids are to be included.

I. Treatment methods

SHASEGP identified by the methods herein are used to treat or prevent abnormal accumulations of sHASEGP substrates in an animal, particularly a mammal, including a human. In one embodiment, the method includes administering to a mammal an effective amount of a sHASEGP glycoprotein, whereby the disease or disorder is treated or prevented.

In another embodiment, a sHASEGP inhibitor can be used in treating an excessive amount of hyaluronidase activity at neutral pH. The treated mammal can be a human. The inhibitors provided herein are those identified by screening assays. In addition, antibodies and antisense nucleic acids or double stranded RNA (dsRNA), such as RNAi, are contemplated.

1. Antisense Treatment: In a specific embodiment, as described hereinbefore, sHASEGP polypeptide function is reduced or inhibited by sHASEGP polypeptide antisense nucleic acids to treat or prevent excessive chondroitinase activity. Therapeutic or prophylactic use of nucleic acids of at least six nucleotides, generally up to about 150 nucleotides, that are antisense to a gene or cDNA encoding a sHASEGP polypeptide or a portion thereof is provided. A sHASEGP polypeptide "antisense" nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a portion of a sHASEGP polypeptide RNA (generally mRNA) by virtue of some sequence complementarity and generally under conditions of high rigor. The antisense nucleic acid can be complementary to a coding and / or non-coding region of a sHASEGP polypeptide mRNA. Such antisense nucleic acids have utility as therapeutic agents that reduce or inhibit the function of the sHASEGP polypeptide and can be used in the treatment or prevention of disorders such as those described above.

SHASEGP polypeptide antisense nucleic acids are at least six nucleotides and are generally oligonucleotides (ranging from 6 to about 150 nucleotides, including 6 to 50 nucleotides). The antisense molecule can be complementary to all or a portion of the hyaluronidase domain. For example, the oligonucleotide is at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or at least 125 nucleotides. The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single or double stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone. The oligonucleotide can include other added groups such as peptides or agents that facilitate transport across the cell membrane (see, eg, Letsinger et al., Proc. Natl. Acad. Sci. USA 86: 6553-6556 (1989) ; Lemaitre et al., Proc. Natl. Acad. Sci. USA 84: 648-652 (1987); PCT Publication No. WO 88/09810, published December 15, 1988) or blood-brain barrier (see, for example, PCT Publication No. WO 89/10134, published April 25, 1988), triggered cleavage agents by hybridization (see, for example, Krol et al., BioTechniques 6: 958-976 (1988)) or intercalating agents (see, for example, Zon. Pharm. Res. 5: 539-549 (1988)).

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The sHASEGP polypeptide antisense nucleic acid is generally an oligonucleotide, typically single stranded DNA or RNA or an analog thereof or mixtures thereof. For example, the oligonucleotide includes a sequence antisense to a portion of a nucleic acid that encodes a human sHASEGP polypeptide. The oligonucleotide can be modified at any position in its structure with substituents generally known in the art.

The sHASEGP polypeptide antisense oligonucleotide may include at least one modified base moiety that is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine , 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosine, 5- apos-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, keosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, Uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-n-2-carboxypropyl) uracil, (ACP3) w and 2,6-diaminopurine.

In another embodiment, the oligonucleotide includes at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. The oligonucleotide can include at least one modified phosphate backbone selected from a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphorodiamidate, a methyl phosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

The oligonucleotide can be an α-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which the strands run parallel to each other (Gautier et al., Nucl. Acids Res. 15: 6625-6641 (1987)).

The oligonucleotide can be conjugated to another molecule, such as, but not limited to, a peptide; hybridization-triggered crosslinking agent, transport agent, or a hybridization-triggered cleavage agent. The oligonucleotides can be synthesized by conventional methods known in the art, for example, using an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al., Nucl. Acids Res. 16: 3209 (1988)), methylphosphonate oligonucleotides can be prepared using controlled pore size glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. USA 85: 74487451 (1988) ), etc. In a specific embodiment, the sHASEGP polypeptide antisense oligonucleotide includes catalytic RNA or a ribozyme (see, for example, International PCT Application WO 90/11364, published October 4, 1990; Saber et al., Science 247: 1222- 1225 (1990)). In another embodiment, the oligonucleotide is a 2'-O-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15: 6131-6148 (1987)) or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett 215: 327-330 (1987)).

Alternatively, the oligonucleotide can be double stranded RNA (dsRNA) such as RNAi.

In an alternative embodiment, the sHASEGP polypeptide antisense nucleic acid is produced intracellularly by transcription from an exogenous sequence.

For example, a vector can be introduced in vivo so that it is taken up by a cell, the vector or a portion thereof being transcribed within said cell, producing an antisense nucleic acid (RNA). Such a vector would contain a sequence encoding the sHASEGP polypeptide antisense nucleic acid. Such a vector can remain episomal or chromosomally integrated as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art used for replication and expression in mammalian cells. Expression of the sequence encoding the sHASEGP polypeptide antisense RNA can be any known promoter known in the art to act in mammalian cells, including humans. Said promoters can be inducible or constitutive. Such promoters include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, Nature 290: 304-310 (1981), the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al. , Ce // 22: 787-797 (1980), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39-42 (1982)), etc.

Antisense nucleic acids include sequence complementarity with at least a portion of an RNA transcript of a sHASEGP polypeptide gene, including a human sHASEGP polypeptide gene. Absolute complementarity is not necessary. The amount of sHASEGP polypeptide antisense nucleic acid that is effective in treating or preventing neoplastic disease depends on the nature of the disease and can be determined empirically by standard clinical techniques.

Where possible, it is desirable to determine antisense cytotoxicity in cells in vitro, and then in useful animal model systems prior to testing and use in humans.

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2. RNA interference. Interference RNA (RNAi) (see, for example, Chuang et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4985) can be used to inhibit the expression of a gene encoding a sHASEGP. Interfering RNA fragments (RNAi), particularly double-stranded RNAi (bc), can be used to generate loss of function of sHASEGP. Methods are known regarding the use of RNAi to silence genes in organisms including mammals, C. elegans, Drosophila, and plants and humans (see, for example, Fire et al. (1998) Nature 391: 806811; Fire (1999) Trends Genet. 15: 358-363; Sharp (2001) Genes Dev. 15: 485- 490; Hammond et al. (2001) Nature Rev, Genet. 2: 110-119; Tuschl (2001) Chem. Biochem. 2: 239-245; Hamilton et al. (1999) Science 286: 950-952; Hammond et al. (2000) Nature 404: 293-296; Zamore et al. (2000) Cell 101: 25-33; Bernstein et al. (2001) Nature 409: 363-366; Elbashir et al. (2001) Genes Dev. 15: 188-200; Elbashir et al. (2001) Nature 411: 494-498; PCT International Application No. WO 01/29058; PCT International Application No. WO 99/32619).

Constructs expressing double-stranded RNA (dsRNA) are introduced into a host, such as an animal or plant using a replicable vector that remains episomal or integrates into the genome. By selecting the appropriate sequences, dsRNA expression can interfere with the accumulation of endogenous mRNA encoding a sHASEGP. RNAi can also be used to inhibit expression in vitro.

The regions include at least about 21 (or 21) nucleotides that are selective (ie, unique) for sHASEGP that are used to prepare RNAi. Smaller fragments of about 21 nucleotides can be transformed directly (ie, in vitro or in vivo) into cells; Larger dsRNA RNAi molecules are generally introduced using vectors that encode them. The dsRNA molecules are at least 21 bp in length or longer, such as 50, 100, 150, 200 and longer. Methods, reagents, and protocols for introducing nucleic acid molecules into cells in vitro and in vivo are known to those of skill in the art.

3. Gene therapy. In an exemplary embodiment, nucleic acids that include a nucleotide sequence encoding a sHASEGP polypeptide or functional domains or derivatives thereof, are administered to promote sHASEGP polypeptide function through gene therapy. Gene therapy refers to a therapy performed by administering a nucleic acid to a subject. In this embodiment, the nucleic acid produces its encoded protein that mediates a therapeutic effect by promoting the function of the sHASEGP polypeptide. Any of the methods for gene therapy available in the art can be used (see, Goldspiel et al., Clinical Pharmacy 12: 488505 (1993); Wu and Wu, Biotherapy 3: 87-95 (1991); Tolstoshev, An. Rev. Pharmacol Toxicol 32: 573-596 (1993), Mulligan, Science 260: 926-932 (1993), and Morgan and Anderson, An. Rev. Biochem. 62: 191-217 (1993); TIBTECH 115: 155-215 (1993). For example, a therapeutic composition for gene therapy includes a nucleic acid encoding a sHASEGP polypeptide that is part of an expression vector that expresses a sHASEGP polypeptide or chimeric domain, fragment, or protein thereof in a suitable host. In particular, said nucleic acid has a promoter operably linked to the coding region of the sHASEGP polypeptide, the promoter being inducible or constitutive and, optionally, tissue specific. In another particular embodiment, a nucleic acid molecule is used in which the sHASEGP polypeptide coding sequences and any other desired sequence are flanked by regions that promote homologous recombination at a desired site in the genome, thereby providing intrachromosomal expression. of the nucleic acid of a sHASEGP protein (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al., Nature 342: 435-438 (1989)).

Delivery of the nucleic acid to a patient can be direct, in which case the patient is exposed directly to the nucleic acid or vector carrying the nucleic acid, or indirect, in which case the cells are first transformed with the nucleic acid in vitro, they are then transplanted into the patient. These two strategies are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid is administered directly in vivo, where it is expressed to produce the encoded product. This can be achieved by any of numerous methods known in the art, for example, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, for example by infection, using defective or attenuated retroviral vector or another viral vector (see, US Patent No. 4,980,286) or by direct injection of naked DNA or by use of microparticle bombardment (eg, gene gun; Biolística, DuPont) or coating with lipids or cell surface receptors or transfection agents, encapsulation in liposomes, microparticles or microcapsules or by administration thereof bound with a peptide known to be introduced into the nucleus, by administration thereof bound with a ligand undergoing receptor-mediated endocytosis (see, eg, Wu and Wu, J. Biol. Chem, 262: 4429-4432 (1987)) (which can be used to target cell types that specifically express the receptors), etc. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand is a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to prevent lysosomal degradation. In yet another embodiment, the nucleic acid can be targeted in vivo for cell-specific uptake and expression by targeting a specific receptor (see, for example, PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al.) ; WO 92/22635 dated December 23, 1992 (Wilson et al.); WO 92/20316 dated November 26, 1992 (Findeis et al.); WO 93/14188 dated July 22, 1993 (Clarke et al.), WO 93/20221 dated October 14, 1993 (Young)). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into host cell DNA for expression by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86: 8932-8935 (1989); Zijistra et al., Nature 342: 435-438 (1989)).

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In a specific embodiment, a viral vector containing the sHASEGP polypeptide nucleic acid is used. For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. The sHASEGP polypeptide nucleic acid to be used in gene therapy is cloned into the vector, which facilitates delivery of the gene to the patient. More details about retroviral vectors can be found in Boesen et al., Biotherapy 6: 291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells to make stem cells more resistant to chemotherapy.

Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest. 93: 644-651 (1994); Kiem et al., Blood 83: 1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4: 129141 (1993); and Grossman and Wilson, Curr. Opin. In Genetics AndDevel. 3: 110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to the respiratory epithelium. Adenoviruses naturally infect the respiratory epithelium, where they cause mild illness. Other targets for adenovirus-based delivery systems are liver, central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being able to infect non-dividing cells. Kozarsky and Wilson, Current Opinion in Genetics and Development 3: 499503 (1993) present a review of adenovirus-based gene therapy. Bout et al., Human Gene Therapy 5: 310 (1994) demonstrate the use of adenovirus vectors to transfer genes to the respiratory epithelium of rhesus monkeys. Other cases of the use of adenovirus in gene therapy can be found in Rosenfeld et al., Science 252: 431-434 (1991); Rosenfeld et al., Cell 68: 143-155 (1992); and Mastrangeli et al., J. Clin. Invest. 91: 225-234 (1993).

Adeno-associated viruses (AAVs) have also been proposed for use in gene therapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204: 289-300 (1993).

Another strategy for gene therapy involves transferring a gene to cells in tissue culture by methods such as electroporation, lipofection, calcium phosphate-mediated transfection, or viral infection. Typically, the method of transfer includes transferring a selection marker to cells. The cells are then placed on selection to isolate cells that have taken up and are expressing the transferred gene. These cells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior to in vivo administration of the resulting recombinant cell. Such introduction can be accomplished by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, gene transfer. mediated by microcells, spheroplast fusion, etc. Numerous procedures for introducing foreign genes into cells are known in the art (see, for example, Loeffler and Behr, Meth. Enzymol. 217: 599618 (1993); Cohen et al., Meth. Enzymol. 217: 618-644 (1993); Cline, Pharmac. Ther. 29: 69-92 (1985)) and can be used as long as the necessary developmental and physiological functions of the recipient cells are not disturbed. The technique should provide for stable transfer of the nucleic acid into the cell, so that the nucleic acid can be expressed by the cell and can generally be inherited and expressed by its cell line.

The resulting recombinant cells can be delivered to a patient by various methods known in the art. In one embodiment, epithelial cells are injected, for example, subcutaneously. In another embodiment, recombinant skin cells can be applied as a skin graft onto the patient. Recombinant blood cells (eg, stem cells or hematopoietic progenitors) can be administered intravenously. The number of cells intended for use depends on the desired effect, the condition of the patient, etc. and can be determined by one of ordinary skill in the art.

Cells into which a nucleic acid can be introduced for the purposes of gene therapy include any available desired cell type and include, but are not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem cells or progenitors, for example, such as stem cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, and other sources thereof.

For example, a cell used for gene therapy is autologous to the patient. In one embodiment where recombinant cells are used in gene therapy, a sHASEGP polypeptide nucleic acid is introduced into the cells so that it can be expressed by the cells or their progeny, and then the recombinant cells are administered in vivo for an effect. therapeutic. In a specific embodiment, stem or progenitor cells are used. Any stem and / or progenitor cell that can be isolated or maintained in vitro can potentially be used in accordance with this embodiment.

Such stem cells include, but are not limited to, hematopoietic stem cells (HSC), stem cells from epithelial tissues such as the skin and lining of the intestine, embryonic cardiac muscle cells, liver stem cells (PCT Publication WO 94/08598, dated April 28, 1994) and neural stem cells (Stemple and Anderson, Cell 71: 973-985 (1992)).

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Epithelial stem cells (ESCs) or keratinocytes can be obtained from tissues such as the skin and the lining of the intestine by known procedures (Rheinwald, Meth. Cell Bio. 21 A: 229 (1980)). In stratified epithelial tissue, such as skin, stem cell mitosis renewal occurs within the germ layer, the layer closest to the basal lamina. Stem cells within the lining of the intestine provide a rapid turnover rate for this tissue. ESCs or keratinocytes obtained from the skin or gut lining of a patient or donor can be cultured in tissue culture (Rheinwald, Meth. Cell Bio. 21 A: 229 (1980); Pittelkow and Scott, Cano. Clinic Proc. 61 : 771 (1986)). If ESCs are provided by a donor, a method of suppressing host reactivity to a graft may also be used (eg, irradiation, administration of drugs or antibodies to promote moderate immunosuppression).

With respect to hematopoietic stem cells (HSCs), any technique that provides for the in vitro isolation, propagation and maintenance of HSCs can be used in this embodiment. Techniques by which it can be achieved include (a) the isolation and establishment of HSC cultures from bone marrow cells isolated from the future host or donor or (b) the use of previously established long-term HSC cultures. , which can be allergenic or xenogeneic.

Non-autologous HSCs are generally used with a method of suppressing immune reactions against transplantation of the future host / patient. In a particular embodiment, human bone marrow cells can be obtained from the posterior iliac crest by needle aspiration (see, for example, Kodo et al., J. Clin. Invest. 73: 13771384 (1984)). For example, hSc can be prepared in highly enriched or substantially pure form. This enrichment can be achieved before, during or after long-term cultivation and can be accomplished by any method known in the art. Long-term cultures of bone marrow cells can be established and maintained using, for example, modified Dexter cell culture techniques (Dexter et al., J. Cell Physiol. 91: 335 (1977)) or Witlock culture techniques -Witte (Witlock and Witte, Proc. Natl. Acad. Sci. USA 79: 3608-3612 (1982)).

In a specific embodiment, the nucleic acid to be introduced for the purposes of gene therapy includes an inducible promoter operably linked to the coding region, such that expression of the nucleic acid can be controlled by controlling the presence or absence of the inducer of the appropriate transcription.

3. Prodrugs- A method of treating tumors is provided. The method is performed by administering a prodrug that is cleaved at a specific site by a HASEGP to release an active drug or a precursor that can be converted to an active drug in vivo. Upon contact with a cell that expresses sHASEGP activity, the prodrug becomes an active drug. The prodrug can be a conjugate containing an active agent, such as an antitumor drug, such as a cytotoxic agent or other therapeutic agent (TA), bound to a substrate for the target sHASEGP, such that the drug or agent is inactive or unable to enter a cell in the conjugate, but becomes activated upon cleavage. The prodrug, for example, may contain a chondroitin sulfate molecule, typically a relatively short one of less than about 20 disaccharide units, that is catalytically cleaved by the target sHASEGP. Cytotoxic agents include, but are not limited to, alkylating agents, antiproliferative agents, and tubulin binding agents. Others include vinca drugs, mitomycins, bleomycins, and taxanes.

Pharmaceutical compositions and modes of administration

1. Components of compositions

Provided herein are pharmaceutical compositions containing an active sHASEGP. Also provided are combinations of compounds that modulate the activity of a sHASEGP polypeptide and another treatment or compound for the treatment of a hyaluronidase disorder, such as an antibody compound.

The sHASEGP polypeptide and a second agent can be packaged as separate compositions for administration together or sequentially or intermittently. Alternatively they can be provided as a single composition for administration or as two compositions for administration as a single composition. The combinations can be packaged as kits.

2. Formulations and Route of Administration

The sHASEGP polypeptides and the soluble human hyaluronidase domain thereof provided herein can be formulated as pharmaceutical compositions, typically for single dosage administration. The concentrations of the polypeptides in the formulations are effective to deliver an amount, after administration, that is effective for the desired treatment. Typically, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of a sHASEGP polypeptide, soluble human hyaluronidase domains thereof, or a mixture thereof is dissolved, suspended, and expressed or otherwise mixed in a selected vehicle at an effective concentration so that the condition treated is relieved or improved.

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Suitable pharmaceutical carriers or excipients for the administration of the sHASEGP or soluble human hyaluronidase domains thereof provided herein include any such vehicle known to those skilled in the art to be suitable for the particular mode of administration.

In addition, the polypeptides can be formulated as the only pharmaceutically active ingredient in the composition or they can be combined with other active ingredients. Liposomal suspensions, including tissue targeted liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in US Patent No. 4,522,811.

The active sHASEGP or soluble human hyaluronidase domain thereof is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects in the treated patient. The therapeutically effective concentration can be determined empirically by testing the polypeptides in known in vitro and in vivo systems such as by use of the assays provided herein or see, for example, Taliani et al. (1996) Anal. Biochem. 240: 60-67, Filocamo et al. (1997) J. Virology 71: 1417-1427, Sudo et al. (1996) Antiviral Res. 32: 9-18, Buffard et al. (1995) Virology 209: 52-59, Bianchi et al. (1996) Anal. Biochem. 237: 239-244, Hamatake et al. (1996) Intervirology 39: 249-258, Steinkiihler et al. (1998) Biochem. 37: 8899-8905, D'Souza et al. (1995) J. Gen. Virol. 76: 1729-1736, Takeshita et al. (1997) Anal. Biochem. 247: 242-246; see also, for example Shimizu et al. (1994) J. Virol. 68: 8406-8408; Mizutani et al. (1996) J. Virol. 70: 7219-7223, Mizutani et al. (1996) Biochem. Biophys. Res. Commun. 227: 822826, Lu et al. (1996) Proc. Natl. Acad. Sci. (USA) 93: 1412-1417, Hahm et al. (1996) Virology 226: 318-326, Ito et al. (1996) J. Gen. Virol. 77 \ 1043-1054, Mizutani et al. (1995) Biochem. Biophys. Res. Commun. 212: 906-911, Cho et al. (1997) J. Viral. Meth. 65: 201-207, and then extrapolated from these for human dosages.

Typically, a therapeutically effective dosage is contemplated. The amounts administered can be on the order of 0.001 to 1 mg / ml, including about 0.005-0.05 mg / ml and about 0.01 mg / ml of blood volume. Dosage unit dosage forms are prepared to provide from about 1 mg to about 1000 mg, including from about 10 to about 500 mg, and including about 25-75 mg of the essential active ingredient or a combination of essential ingredients per unit dosage form. . The exact dosage can be determined empirically.

In some cases, a high unit dose of human sHASEGP is preferable. For example, with intravenous administration of sHASEGP sHASEGP concentrations of 500-100,000 units per ml are preferable. The lyophilized formulations of sHASEGP are also ideal for storage of large unit doses of sHASEGP. Lyophilized vials of 200,000 units of sHASEGP are contemplated for intravenous delivery.

High concentration doses are also contemplated for the delivery of small volumes of sHASEGP. Administration of 10-100 μl of 5000 units / ml sHASEGP is contemplated for injection into the anterior chamber to dissolve viscoelastic substances previously administered during cataract and phakic intraocular lens implantation surgeries. Small volume injections of doses of 50-200 U / ml are also contemplated for intravitreal procedures such as the treatment of vitreous hemorrhage or vitreoretinal detachment in diabetic retinopathy.

The active ingredient can be administered at one time or it can be divided into several smaller doses to be administered at intervals of time. It is understood that the exact dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known test protocols or by extrapolation of in vivo or in vitro test data. It should be noted that concentrations and dosage values ​​may also vary with the severity of the condition being relieved. It should further be understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual needs and professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth in This document is exemplary only and is not intended to limit the scope or use of the claimed compositions and combinations containing them.

Pharmaceutically acceptable derivatives include acids, salts, esters, hydrates, solvates, and prodrug forms. The derivative is typically selected so that its pharmacokinetic properties are superior to the corresponding neutral pH active sHASEGP or soluble human hyaluronidase domain thereof.

Therefore, effective concentrations or amounts of one or more of the polypeptides herein or pharmaceutically acceptable derivatives thereof are mixed with a suitable pharmaceutical carrier or excipient for systemic, topical, or local administration to form pharmaceutical compositions. The sHASEGP polypeptides or soluble human hyaluronidase domains thereof are included in an amount effective to ameliorate or treat the disorder for which treatment is contemplated. The concentration of active polypeptide in the composition depends on the rates of absorption, inactivation, excretion of the active polypeptide, the dosage schedule, the amount administered, the particular formulation, as well as other factors known to those of skill in the art.

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Therapeutic agents for use in the methods can be administered by any route known to those skilled in the art, such as, but not limited to, topical, intraarticular, intracisternal, intraocular, intraventricular, intrathecal, intravenous, intramuscular, intraperitoneal, intradermal. , intratracheal, as well as by any combination of any two or more of them. Dry powder lung formulations can also be envisaged.

The most suitable route of administration will vary depending on the intended use, such as, for example, use as a delivery agent to facilitate subcutaneous delivery of fluids, uses to reduce intraocular pressure in the eyes of glaucoma patients receiving viscoelastic compounds, or use as a "spreading agent" to increase chemotherapeutic activity and the site of interest, such as a particular internal organ, a tumor growth, an intraocular cavity and the epidermis. Modes of administration include, but are not limited to, topical, local, intraarticular, intracisternal, intraocular, intraventricular, intrathecal, intravenous, intramuscular, intratracheal, intraperitoneal, intradermal, and by a combination of any two or more thereof. For example, for the treatment of various cancers, such as squamous cell carcinoma, breast cancer, urinary bladder cancer, and gastrointestinal cancer, local administration, including administration at the site of tumor growth (e.g., intrathecally, intraventricular or intracisternal) provides the advantage that the therapeutic agent can be administered in a high concentration without risk of the complications that can accompany the systemic administration of a therapeutic agent.

Suitable pharmaceutical and cosmetic vehicles or excipients for the administration of sHASEGP polypeptides or a soluble human hyaluronidase domain thereof provided herein include any such vehicle known to those skilled in the art to be suitable for the particular mode of administration. Furthermore, the polypeptides can be formulated as the only pharmaceutically active ingredient in the composition or they can be combined with other active ingredients that do not alter the desired action, or with materials that complement the desired action known to those skilled in the art. For example, the sHASEGP polypeptides provided herein can be used as a delivery or "propagation" agent in combination with a second active compound, such as a therapeutically effective agent, including, but not limited to, a drug or a prodrug, to facilitate the supply or to increase the activity of the second active ingredient. In a particular embodiment, a sHASEGP polypeptide or a soluble human hyaluronidase domain thereof can be co-formulated with an anesthetic agent, such as Lignocaine, Bupivacaine or a mixture of the two and, optionally, a hormonal agent, such as epinephrine to decrease or interrupt the uptake of blood during ophthalmic surgery. A sHASEGP polypeptide or a soluble human hyaluronidase domain thereof can also be coformulated with various chemotherapeutic agents, such as a toxin or a tumor necrosis factor, to increase the activity of the chemotherapeutic and / or the accessibility of the target tumors to the chemotherapeutic. The active compound is included in the vehicle in an amount sufficient to exert a therapeutically useful effect in the absence of serious toxic effects in the treated individual. The effective concentration can be determined empirically by testing the compounds using in vitro and in vivo systems including the animal models described herein.

Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent, such as water for injection, saline, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, and other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for adjusting tonicity, including, but not limited to, sodium chloride, calcium chloride, magnesium chloride, dextrose, glycerol, or boric acid. Parenteral preparations can be included in ampoules, disposable syringes, or single or multi-dose vials made of glass, plastic, or other suitable material.

The sHASEGP polypeptides or soluble human hyaluronidase domains thereof may be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active or to produce a prodrug. The form of the resulting mixture depends on several factors, including the desired mode of administration and the solubility of the polypeptide in the selected excipient or vehicle. The effective concentration is sufficient to ameliorate the target condition and can be determined empirically using methods known to those of skill in the art. To formulate a composition, the polypeptide weight fraction is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier at an effective concentration so that the target condition is alleviated or ameliorated.

In cases where sHASEGP polypeptides or a soluble human hyaluronidase domain thereof exhibit insufficient solubility, methods to solubilize polypeptides can be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of cosolvents, such as dimethylsulfoxide (DMSO), the use of surfactants, such as TWEEN^® and Pluronic^® F68; or dissolving in aqueous sodium bicarbonate. Derivatives of the polypeptides, such as prodrugs of the polypeptides can also be used in the formulation of effective pharmaceutical compositions. For ophthalmic indications, the compositions are formulated in an ophthalmically acceptable vehicle. For the ophthalmic uses herein, local administration, by topical administration or by injection is contemplated. Time-release formulations are also desirable. Typically, the compositions are formulated for single dosage administration, such that a single dose delivers an effective amount.

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After mixing or adding the polypeptide with the excipient, the resulting mixture can be a solution, suspension, emulsion or other composition and can be formulated as an aqueous mixture, creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures. , pastes, foams, aerosols, irrigations, sprays, suppositories, bandages or any other formulation suitable for systemic, topical or local administration.

The form of the resulting mixture depends on several factors, including the desired mode of administration and the solubility of the compound in the selected carrier or excipient. If necessary, pharmaceutically acceptable salts or other derivatives of the compounds are prepared. For local internal administration, such as intramuscular, parenteral, or intra-articular administration, the compounds are preferably formulated as a solution or suspension in an aqueous-based medium such as isotonically buffered saline, or combined with a biocompatible or bioadhesive carrier intended for administration. internal.

The soluble human sHASEGP polypeptide or hyaluronidase domain is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects in the treated patient. The number and degree of side effects is understood to depend on the condition for which the compounds are administered. For example, certain toxic and undesirable side effects are tolerated when treating life-threatening conditions that would not be tolerated when treating disorders of less serious consequences. Amounts effective for therapeutic use will, of course, depend on the severity of the disease and the weight and general condition of the subject as well as the route of administration. Local administration of the therapeutic agent will typically require a lower dosage than any mode of systemic administration, although the local concentration of the therapeutic agent may in some cases be higher after local administration than can be safely achieved after systemic administration.

Since individual subjects can exhibit wide variation in the severity of symptoms and each therapeutic agent has its unique therapeutic characteristics, it is up to the physician to determine a subject's response to treatment and to vary dosages accordingly. Dosages used in vitro can provide useful information on amounts useful for in situ administration of the pharmaceutical composition and animal models can be used in some cases to determine effective dosages for the treatment of particular disorders. In general, however, for local administration, it is contemplated that an effective amount of the therapeutic agent will be an amount in the range of about 0.1 picograms (pg) to about 1 ng per kg of body weight. Various considerations for arriving at an effective amount are known to those skilled in the art and are described (see, for example, Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; Remington's Pharmaceutical Sciences, 17^to ed., Mack Publishing Co., Easton, Pa., 1990; and Mantyh et al., (Science, 278: 275-79, 1997) involving intrathecal injection of a neuronal specific toxin-ligand, each being incorporated herein by reference in its entirety).

Formulations of the sHASEGP polypeptides or soluble human hyaluronidase domains thereof for use herein include those suitable for oral, rectal, topical, inhalation, buccal (eg, sublingual), parenteral (eg, subcutaneous, intramuscular, administration, intradermal or intravenous), transdermal or any route. The most suitable route in any given case depends on the nature and severity of the condition being treated and the nature of the particular active compound being used. The formulations are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions and oil-in-water emulsions containing appropriate amounts of polypeptides and / or other pharmaceutically acceptable agents or derivatives thereof. Therapeutically active pharmaceutical polypeptides and / or other agents and derivatives thereof are typically formulated and administered in unit dosage forms or multiple dosage forms. A unit dose form, as used herein, refers to physically separate units suitable for human and animal subjects and individually packaged as is known in the art.

The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions and oral solutions or suspensions, and oil-in-water emulsions containing suitable amounts of the sHASEGP polypeptide or soluble human hyaluronidase domain thereof, and optionally, other pharmaceutically acceptable agents or derivatives thereof. Therapeutically active pharmaceutical compounds and derivatives thereof are typically formulated and administered in unit dosage forms or multiple dosage forms. A unit dose form, as used herein, refers to physically separate units suitable for human and animal subjects and individually packaged as is known in the art. Each unit dose contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect in association with the necessary pharmaceutical carrier, excipient or diluent. Examples of unit dose forms include, but are not limited to, ampoules, syringes, and individually packaged tablets or capsules. For example, a small volume formulation containing a solution stabilized with 1 to 5000 units of sHASEGP in a small volume, such as 5 to 50 µL, can be prepackaged in a syringe for use, such as after injection. injection of viscoelastic compound. Unit dose forms can be administered in fractions or multiples thereof. A multidose form is a plurality of identical unit dosage forms packaged in a single container to be administered in separate unit dose form. Examples of multidose forms include vials, bottles of tablets or capsules, or pint or gallon bottles. Therefore, the multidose form is a multiple of unit doses that are not separated on the packaging.

ES 2 335 005 T3

The composition may contain together with the active ingredient, such as a sHASEGP polypeptide: a diluent, such as lactose, sucrose, dicalcium phosphate or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate, and talc; and a binder such as starch, natural gums, such as acacia, gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone, crospovidones, and other such binders known to those skilled in the art. Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose , glycerol, glycols, ethanol and the like to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying or solubilizing agents, pH buffering agents and the like, for example acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, sodium triethanolamine acetate, triethanolamine oleate, and other such agents. Methods for preparing such dosage forms are known or will be apparent to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15^to Edition, 1975). The composition or formulation to be administered contains an amount of the active compound in an amount sufficient to alleviate the symptoms of the treated subject. For example, a conventional stabilized formulation of sHASEGP or a soluble human hyaluronidase domain thereof as provided herein includes 150 units / ml of the soluble glycoprotein formulated in EDTA, NaCl, and CaCl2. Additionally, an antibacterial or antifungal agent including, but not limited to, thiomersal may be present in the formulation. Another formulation provided herein is a stabilized solution or lyophilized form of sHASEGP or a soluble human hyaluronidase domain thereof in EDTA, NaCl, and CaCl2, containing an effective active amount of the soluble glycoprotein, such as 150 units / ml, with the addition of lactose, such as 13 mg / ml. Also provided herein is a formulation containing a stabilized solution or lyophilized form of sHASEGP or a soluble human hyaluronidase domain thereof in EDTA, NaCl and CaCl2 containing an effective active amount of the soluble glycoprotein, such as 150 units / ml, with the addition of lactose, such as 13 mg / ml, and Albumin, Pluronic® f68, TWEEN® and / or another detergent. Another formulation provided herein, lyophilized or as a stabilized solution, contains an effective amount of sHASEGP or a soluble human hyaluronidase domain thereof, such as 1 to 300 units / ml, in EDTA, NaCl, and CaCl2.

Dosage forms or compositions containing active ingredient in the range of 0.005% to 100% can be prepared, with the remainder being composed of non-toxic carrier. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents, (for example, pregelatinized cornstarch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (eg, lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (eg, magnesium stearate, talc, or silica); disintegrants (eg, potato starch or sodium starch glycolate); or wetting agents (eg sodium lauryl sulfate). Tablets can be coated by methods well known in the art.

The sHASEGPs or a soluble human hyaluronidase domain thereof or pharmaceutically acceptable derivatives can be prepared with vehicles that protect the soluble glycoprotein against rapid clearance from the body, such as time-release formulations or coatings. The compositions may include other pharmaceutically effective agents known in the general art to be of value in the treatment of one or more of the diseases or medical conditions, including, but not limited to, a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial, an amebicidal agent, a trichomonicidal agent, an antiparkinsonian agent, an antimalarial agent, an anticonvulsant agent, an antidepressant agent, an antiarthritic agent, an antifungal agent, an antihypertensive agent, an antipyretic agent, an antiparasitic agent, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha-blocking agent, an anesthetic agent, a bronchodilator agent, a biocidal agent, a bactericidal agent, a bacteriostatic agent, a beta-adrenergic blocking agent, a calcium channel blocking agent, a cardiovascular pharmacological agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, an electrolytic agent, a hypnotic agent, a hormonal agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contraction agent, an ophthalmic agent, an agent parasympathomimetic, psychic energizing agent, ophthalmic agent, parasympathomimetic agent, psychic energizing agent, sedative agent, sympathomimetic agent, tranquilizing agent, a urinary agent, a vaginal agent, a viricidal agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent, a polypeptide, a protein, a nucleic acid, a drug, a prodrug, a molecule organic and a sleep inducer to obtain desired combinations of properties. It should be understood that such combination therapy constitutes a further aspect of the compositions and methods of treatment provided herein.

1. Compositions for oral administration

Oral pharmaceutical dosage forms are solid, gel, or liquid. Solid dosage forms are tablets, capsules, granules, and bulk powders. Oral tablet types include lozenges and compression chewable tablets which may be enteric coated, sugar coated or film coated. Capsules can be hard or soft gelatin capsules, while granules and powders can be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.

ES 2 335 005 T3

Pharmaceutical compositions containing a sHASEGP or a soluble human hyaluronidase domain thereof may be in liquid form, eg, solutions, syrups, or suspensions or may be presented as a drug product for reconstitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (eg, lecithin or acacia); non-aqueous vehicles (eg, almond oil, oily esters, or fractionated vegetable oils); and preservatives (eg, methyl- or propyl-p-hydroxybenzoates or sorbic acid).

In certain embodiments, the formulations are solid dosage forms, preferably capsules or tablets. Tablets, pills, capsules, troches, and the like may contain any of the following ingredients or compounds of a similar nature: a binder; a diluent; a disintegrating agent; a lubricant; an emollient; a sweetening agent; and a flavoring agent.

Examples of binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, sucrose, and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, club moss, and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Emollients include, but are not limited to, colloidal silicon dioxide. Disintegrating agents include croscarmellose sodium, sodium starch glycolate, alginic acid, cornstarch, potato starch, bentonite, methyl cellulose, agar, and carboxymethyl cellulose. Coloring agents include, for example, any of the licensed certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended in alumina hydrate. Sweetening agents include sucrose, lactose, mannitol, and artificial sweetening agents such as saccharin, and any number of spray-dried flavors. Flavoring agents include natural plant-derived flavors such as fruits and synthetic mixtures of palatable compounds such as, but not limited to, peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Emetic coatings include fatty acids, fats, waxes, shellac, ammonia-treated shellac, and cellulose acetate phthalates. Film coatings include hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

If oral administration is desired, the sHASEGP or a soluble human hyaluronidase domain thereof could be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with an antacid or other such ingredient.

When the dosage unit form is a capsule, it may contain, in addition to the material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms may contain various other materials that modify the physical form of the dosage unit, for example coatings of sugars and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, spray, chewing gum, or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The sHASEGP or a soluble human hyaluronidase domain thereof can also be mixed with other active materials that do not alter the desired action or with materials that complement the desired action, such as antacids, H2 blockers, and diuretics. The active ingredient is a pharmaceutically acceptable compound or derivative thereof as described herein. Higher concentrations of up to 98% by weight of the active ingredient can be included.

The pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents. Enteric-coated tablets, due to the enteric coating, resist the action of stomach acid and dissolve or disintegrate in the neutral or alkaline intestine. Sugar-coated tablets are compressed tablets to which different layers of pharmaceutically acceptable substances are applied. Film-coated tablets are compression-prepared tablets that have been coated with a polymer or other suitable coating. Multiple compression tablets are compression prepared tablets generated by more than one compression cycle using the pharmaceutically acceptable substances mentioned above. Coloring agents can also be used in the above dosage forms. Flavoring and sweetening agents are used in squeeze tablets, sugar coated tablets, multiple squeeze mixes, and chewables. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are oil-in-water or water-in-oil.

Elixirs are transparent, sweetened hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for

ES 2 335 005 T3 example, sucrose, and may contain a preservative. An emulsion is a two-phase system in which a liquid is dispersed in the form of small globules throughout other liquid. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents, and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable substances used in non-effervescent granules that will be reconstituted into a liquid oral dosage form include diluents, sweeteners, and wetting agents. Pharmaceutically acceptable substances used in effervescent granules to be reconstituted into a liquid oral dosage form include organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol, and syrup. Examples of preservatives include glycerin, methyl and propyl paraben, benzoic acid, sodium benzoate, and alcohol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethyl cellulose, pectin, tragacanth, Veegum, and acacia. Diluents include lactose and sucrose. Sweetening agents include sucrose, syrups, glycerin, and artificial sweetening agents such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monoleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organic additives include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Coloring agents include any of the licensed certified water soluble FD and C colorants and mixtures thereof. Flavoring agents include natural aromas extracted from plants such as fruits and synthetic blends of compounds that produce a pleasant flavor sensation.

For a solid dosage form, the solution or suspension in, for example, propylene carbonate, vegetable oils or triglycerides is encapsulated in a gelatin capsule. Said solutions, and their preparation and encapsulation, are described in US Patents No. 4,328,245; 4,409,239 and 4,410,545. For a liquid dosage form, the solution, eg, in a polyethylene glycol, can be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, eg, water, to be readily measured for administration.

Alternatively, liquid or semisolid oral formulations can be prepared by dissolving or dispersing the sHASEGP or a soluble human hyaluronidase domain thereof in vegetable oils, glycols, triglycerides, propylene glycol esters (eg, propylene carbonate), and other carriers thereof. type and encapsulate these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in US Patent Nos. Re 28,819 and 4,358,603.

Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the sHASEGP or a soluble human hyaluronidase domain thereof in a flavored base, usually sucrose, acacia or tragacanth; and lozenges containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.

In all embodiments, the tablet and capsule formulations can be coated as is known to those of skill in the art to modify or sustain dissolution of the active ingredient. Thus, for example, they can be coated with a conventional enteric digestible coating such as phenylsalicylate, waxes and cellulose acetate phthalate.

2. Injectables, Solutions and Emulsions

Also contemplated herein is parenteral administration of sHASEGP or a soluble human hyaluronidase domain thereof, generally characterized by injection, subcutaneously, intramuscularly, or intravenously. Injectables can be prepared in conventional forms, as liquid solutions or suspensions; solid forms suitable for solution or suspension in a liquid before injection or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. Furthermore, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and cyclodextrins. The implementation of a slow release or sustained release system, so that a constant level of dosage is maintained (see, for example, US Patent No. 3,710,795) is also contemplated herein. The percentage of the sHASEGP or soluble human hyaluronidase domain thereof contained in said parenteral compositions depends on the specific nature thereof, as well as on the activity of the compound and the needs of the subject.

Parenteral administration of the compositions includes intravenous, subcutaneous, and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, soluble dry sterile products such as lyophilized powders, ready to be combined with a solvent or sterile solution just before use, including hypodermic tablets, sterile suspensions ready for injection, insoluble dry sterile ready products. to be combined with a vehicle just before use and sterile emulsions. The solutions can be aqueous or non-aqueous.

ES 2 335 005 T3

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS) and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and propylene glycol, and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparations include aqueous carriers, nonaqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, Ringer's Injection, Isotonic Dextrose Injection, Sterile Water Injection, Ringer's Dextrose Injection, and Lactate. Non-aqueous parenteral vehicles include non-volatile oils of vegetable origin, cottonseed oil, corn oil, sesame oil, and peanut oil. Antimicrobial agents in bacteriostactic or fungistatic concentrations should be added to parenteral preparations packaged in multidose containers that include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl- and propyl-p-hydroxybenzoic acid esters, thiomersal, benzoalkonium chloride, and benzoalkonium chloride. benzethonium. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). A metal ion sequestering or chelating agent includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol, and propylene glycol for water miscible carriers and sodium hydroxide, hydrochloric acid, citric acid, or lactic acid for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art.

Parenteral unit dose preparations are packaged in an ampoule, vial, or syringe with a needle. All preparations for parenteral administration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intra-arterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.

Injectables are designed for local and systemic administration. Typically a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w / w to about 90% w / w or more, preferably more than 1% w / w of the active compound of the treated tissue (s). . The active ingredient, such as a sHASEGP or a soluble human hyaluronidase domain thereof, can be administered all at once or it can be divided into several smaller doses to be administered at intervals of time. It is understood that the exact dosage and duration of treatment are a function of the tissue being treated and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. It should be noted that the concentrations and dosage values ​​may also vary with the age of the treated individual. It should be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to individual need and the professional judgment of the person administering or supervising the administration of the formulations, and that the concentration ranges set forth in This document is exemplary only and is not intended to limit the scope or practice of the claimed formulations.

The compounds provided herein can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water or other solvents before use. For example, parenteral formulations are provided herein that contain an effective amount of sHASEGP or a soluble human hyaluronidase domain thereof, such as 500 to 500,000 Units, in a stabilized solution or lyophilized form.

The compound can be suspended in micronized or other suitable form or it can be derivatized to produce a more soluble active or to produce a prodrug. The form of the resulting mixture depends on several factors, including the desired mode of administration and the solubility of the compound in the selected carrier or excipient. The effective concentration is sufficient to ameliorate the symptoms of the condition and can be determined empirically.

3. Freeze Dried Powders

Also provided herein are lyophilized powders containing sHASEGP or a soluble human hyaluronidase domain thereof that can be reconstituted for administration as solutions, emulsions, and other mixtures. These formulations can also be reconstituted and formulated as solids or gels.

ES 2 335 005 T3

The sterile, lyophilized powder is prepared by dissolving a solid portion of or mixing an aliquot of a solution containing a soluble human sHASEGP or hyaluronidase domain thereof in a suitable solvent. The solvent may contain a stability enhancing excipient or other pharmacological component of the powder or reconstituted solution prepared from the powder. Excipients that can be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, lactose, or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those skilled in the art, typically at approximately neutral pH. Subsequent filtration sterilization of the solution followed by lyophilization under conventional conditions known to those skilled in the art provides the lyophilized formulation. Generally, the solution resulting from sterile filtration is distributed into vials for lyophilization. Each vial may contain a single dosage, such as 10-1000 mg or 100-500 mg or multiple dosages of the compound.

Briefly, the lyophilized powder is prepared by dissolving dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, lactose or other suitable agent, approximately 1-20% in a suitable buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those skilled in the art at approximately neutral pH. Then a selected salt, such as, for example, the sodium salt of sHASEGP (about 1 g of the salt per 10-100 g of the buffer solution, typically about 1 g / 30 g) is added to the mixture. resulting above room temperature, such as at about 30-35 ° C and stirred until dissolved. The resulting mixture is diluted by adding more buffer, to decrease the resulting concentration of the salt by about 10-50%, typically about 15-25%. The resulting mixture is sterilized by filtration or treated to remove particulates and to ensure sterility and dispensed into vials for lyophilization. The lyophilized powder can be stored under appropriate conditions, such as at about 4 ° C to room temperature.

Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, a therapeutically effective amount of the lyophilized powder containing a sHASEGP or soluble human hyaluronidase domain thereof is added per milliliter of sterile water or other suitable vehicle. The exact amount depends on the compound selected and can be determined empirically by methods known to those of skill in the art.

Four. Topical administration

Topical mixtures are prepared as described for local and systemic administration. The resulting mixture can be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, sprays, irrigations, sprays, suppositories, bandages, patches dermal or any other formulation suitable for topical administration.

Compositions of sHASEGP or a soluble human hyaluronidase domain thereof or pharmaceutically acceptable derivatives thereof can be formulated as aerosols for topical application, such as by inhalation (see, for example, US Patent Nos. 4,404,126, 4,414. 209 and 4,264,923, which describe aerosols for the delivery of a steroid useful for the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert vehicle such as lactose. In such a case, the formulation particles will typically have diameters of less than 50 microns, such as less than 10 microns.

For administration by inhalation, the compositions for use herein may be delivered in the form of an aerosol spray presentation from pressurized containers or from a nebulizer, with the use of a suitable propellant, including, but not limited to, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide and other suitable gases. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin, for use in an inhaler or insufflator may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

The compositions may be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as to the eye, in the form of gels, creams and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa or for inhalation therapies. Nasal solutions of the active compound can also be administered alone or in combination with other pharmaceutically acceptable excipients.

For example, formulations suitable for topical application to the skin or the eye are generally formulated as an ointment, cream, lotion, pastes, gel, spray, aerosol, and oil. Carriers that can be used include petrolatum, lanolin, polyethylene glycols, alcohols, and combinations of two or more thereof. Topical formulations may advantageously contain in addition to 0.05 to 15 percent by weight of thickeners, including, but not limited to, hydroxypropylmethylcellulose, methylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, poly (alkylene glycols), poly / hydroxyalkyl, (meth) acrylates, or poly (meth) acrylamides. A topical formulation is often applied by instillation or as an ointment to the conjunctival sac. It can also be used for irrigation or lubrication of the eye, sinuses

ES 2 335 005 T3 facial and external auditory meatus. Topical formulations in the liquid state can also be present in a hydrophilic three-dimensional polymeric matrix in the form of a strip, contact lens and the like from which the active components are released. It can also be injected into the anterior chamber of the eye and other places. For example, herein is provided a formulation for intraocular use after injection of a viscoelastic compound containing a stabilized solution of an effective amount of a sHASEGP or a soluble human hyaluronidase domain thereof, such as 1 to 5000 Units. of soluble glycoprotein with 30 to 150,000 Units / mg of specific activity in a small volume, such as 5 to 50 µl.

These solutions, particularly those intended for ophthalmic use, can be formulated as 0.01% -10% isotonic solutions, at a pH of about 5-7 with appropriate salts.

5. Compositions for Other Routes of Administration

Other routes of administration, such as topical application, transdermal patches, and rectal administration are also contemplated herein.

For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories used herein refer to solid bodies for insertion into the rectum that melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. The pharmaceutically acceptable substances used in rectal suppositories are bases or carriers and agents to increase the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol), and appropriate mixtures of fatty acid mono-, di-, and triglycerides. Combinations of various bases can be used. Agents for increasing the melting point of suppositories include sperm whale and wax. Rectal suppositories can be prepared by compression or molding method. The typical weight of a rectal suppository is approximately 2 to 3 g.

Tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.

Formulations suitable for transdermal administration can be presented as separate patches adapted to remain in intimate contact with the epidermis of the recipient for an extended period of time. Said patches conveniently contain the active compound as an optionally buffered aqueous solution of, for example, a concentration of 0.1 to 0.2 M with respect to the active compound. Formulations suitable for transdermal administration can also be delivered by iontophoresis (see, for example, Pharmaceutical Research 3 (6): 318 (1986)) and typically take the form of an optionally buffered aqueous solution of the active compound.

Pharmaceutical compositions can also be administered by controlled release means and / or delivery devices (see, for example, US Patent Nos: 3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770 ; 3,916,899; 4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548; 5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566). Active compounds or pharmaceutically acceptable derivatives can be prepared with vehicles that protect the compound against rapid elimination from the body, such as time-release formulations or coatings.

In one embodiment of the compositions and methods provided herein, the therapeutic agent is administered locally in a slow release delivery vehicle, eg, encapsulated in a colloidal dispersion system or in polymer stabilized crystals. Useful colloidal dispersion systems include nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. For example, the colloidal dispersion system can be a liposome or a microsphere. Liposomes are artificial membrane vesicles that are useful as slow release delivery vehicles when injected or implanted. Some examples of lipid-polymer conjugates and liposomes are described in US Patent No. 5,631,018, which is incorporated herein by reference in its entirety. Other examples of slow release delivery vehicles are biodegradable hydrogel matrices (US Patent No. 5,041,292) conjugated dendritic polymers (US Patent No. 5,14,166), and multivesicular liposomes (Depofoam®. Depotech, San Diego, CA) (US Patent Nos. 5,723,147 and 5,766,627). One type of microspheres suitable for encapsulating therapeutic agents for local injection (eg, into subdermal tissue) are poly (D, L) lactide microspheres as described in D. Fletcher, Anesth. Analg. 84: 90-94, (1997). For example, a slow release formulation containing an effective amount of sHASEGP or a soluble human hyaluronidase domain thereof, such as 1 to 5000 Units / ml, may be employed for various uses or to treat various conditions, including, but not limited to , cosmetic formulations and treatment of spinal cord injuries.

Desirable blood levels can be maintained by continuous infusion of the active agent as determined by plasma levels. It should be noted that the attending physician would know how and when to terminate, interrupt, or adjust therapy to a lower dosage due to bone marrow, liver, or kidney toxicity or dysfunction. On the contrary, the attending physician would also know how and when to adjust the treatment to higher levels if the clinical response is not adequate (excluding toxic side effects).

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The efficacy and / or toxicity of the sHASEGP polypeptide and / or its inhibitor (s) alone or in combination with other agents, such as therapeutically effective agents, can also be evaluated by methods known in the art (see, for example, O &amp; Apos ; Reilly, Investigational New Drugs 15: 5-13 (1997)).

6. Manufacturing items

The sHASEGP polypeptides or soluble human hyaluronidase domains thereof or compositions containing any of the above agents can be packaged as articles of manufacture containing packaging material, a compound or a suitable derivative thereof provided herein, which is effective for treatment of a disease or disorder contemplated in this document, within the packaging material, and a marker indicating that the compound or a suitable derivative thereof is for treating the diseases or disorders contemplated herein. The marker can optionally include the disorders for which therapy is warranted.

The articles of manufacture provided in this document contain packaging materials. Packaging materials for use in the packaging of pharmaceuticals are well known to those of skill in the art (see, for example, US Patent Nos. 5,323,907, 5,052,558 and 5,033,352). Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, vials, tubes, inhalers, pumps, bags, vials, containers, syringes, vials, and any packaging material suitable for a selected formulation and desired mode of administration. and treatment. A wide range of formulations of the compounds and compositions provided herein are contemplated, as well as a variety of treatments for any disorder involving an HCV infection as a mediator or contributor to symptoms or cause.

Kits containing the compositions and / or combinations with instructions for administration thereof are also provided herein. The kit may further include a needle or syringe, typically packaged in sterile form, for injection of the complex and / or a packaged alcohol pad. Optionally included are instructions for administration of the active agent by a clinician or patient. For example, herein is provided a kit containing a small volume syringe with an effective amount of sHASEGP or a soluble human hyaluronidase domain thereof, such as 1 to 5000 Units of the soluble glycoprotein, in a volume of 5 at 50 µl, optionally containing a second syringe containing a viscoelastic compound. Also provided herein is a kit containing a small volume syringe containing an effective amount of sHASEGP or a soluble human hyaluronidase domain thereof, such as 1 to 500 Units of the soluble glycoprotein and a therapeutic amount of one second. active ingredient, such as a drug, small molecule, protein, or nucleic acid.

K. Animal models

Transgenic animal and animal models are provided herein, such as rodents, including mice and rats, cows, chickens, pigs, goats, sheep, monkeys, including gorillas and other primates. In particular, non-human transgenic animals are provided that contain a heterologous nucleic acid encoding a sHASEGP polypeptide or a transgenic animal in which the expression of the polypeptide has been altered, such as by substitution or modification of the promoter region or other regulatory region of the endogenous gene. Such an animal can be produced by promoting recombination between endogenous nucleic acids and an exogenous sHASEGP gene that could be overexpressed or erroneously expressed, such as by expression under a strong promoter, through a homologous or other recombination event.

Transgenic animals can be produced by introducing the nucleic acid using any known delivery method, including, but not limited to, microinjection, lipofection, and other modes of gene delivery into a germline cell or somatic cell, such as an embryonic stem cell. Typically the nucleic acid is introduced into a cell, such as an embryonic stem cell (ES), followed by injection of the ES cells into a blastocyst and implantation of the blastocyst into a foster mother, which is followed by the birth of a transgenic animal. Generally, introduction of a heterologous nucleic acid molecule into an animal chromosome occurs through recombination between the heterologous sHASEGP-encoding nucleic acid and an endogenous nucleic acid. Heterologous nucleic acid can target a specific chromosome. In some cases, knock out animals can occur. Such an animal can be produced initially by promoting homologous recombination between a sHASEGP polypeptide gene on its chromosome and an exogenous sHASEGP polypeptide gene that has been biologically inactivated (typically by insertion of a heterologous sequence, eg, an antibiotic resistance gene). In one embodiment, this homologous recombination is performed by transforming embryonic stem cells (ES) with a vector containing the insert-inactivated sHASEGP polypeptide gene, such that homologous recombination occurs, followed by injection of the ES cells. in a blastocyst and implantation of the blastocyst in a foster mother, followed by the birth of the chimeric animal ("knock out animal") in which a sHASEGP polypeptide gene has been inactivated (see Capecchi, Science 244: 1288-1292 (1989)). The chimeric animal can be reproduced to produce homozygous knock out animals which can then be used to produce additional knock out animals. Knock out animals include, but are not limited to, mice, hamsters, sheep, pigs, cattle, and other non-human mammals. For example, a knock out mouse occurs. The resulting animals can

ES 2 335 005 T3 serve as models for specific diseases, such as cancers, that exhibit under-expression of a sHASEGP polypeptide. Said knock out animals can be used as animal models of said diseases, for example, to screen or test molecules to determine the ability to treat or prevent said diseases or disorders.

Other types of transgenic animals can also be produced including those that overexpress the sHASEGP polypeptide. Such animals include "knock in" animals which are animals in which the normal gene is replaced by a variant, such as a mutant, an overexpressed form, or another form. For example, that of one species, such as an endogenous rodent gene, can be substituted for the gene of another species, such as a human. Animals can also be produced by non-homologous recombination at other sites on a chromosome; including animals that have a plurality of integration events.

After the production of the first generation of transgenic animals, a chimeric animal can be reproduced to produce additional animals that overexpress or mis-express sHASEGP polypeptides. Such animals include, but are not limited to, mice, hamsters, sheep, pigs, cattle, and other non-human mammals. The resulting animals can serve as models for specific diseases, such as cancers, that exhibit overexpression or mis-expression of a sHASEGP polypeptide. Such animals can be used as animal models of such diseases, for example, to screen or test molecules for the ability to treat or prevent said diseases or disorders. In a specific embodiment, a mouse is produced that overexpress or mis-express a sHASEGP polypeptide.

The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

L. Therapeutic uses of sHASEGP

Hyaluronidase enzymes of natural origin from slaughterhouses have been the main source of clinical enzyme preparations for more than forty years. Bovine and ovine testes are the main source of this material. However, these clinical enzyme preparations are very crude, they are marketed in preparations ranging from 0.5-5% purity based on known specific activities between 30-100,000 Units / mg. Therefore, their lack of purity combined with their abattoir origin, leaves them both immunogenic to humans and a potential source of Creutzfeld Jacob disease and other bovine and ovine pathogens. Anaphylactic reactions are known to occur against bovine and ovine hyaluronidase preparations.

Hyaluronidase obtained from cattle or bacteria has been used in the treatment of diseases associated with excess hyaluronic acid and to increase the circulation of physiological fluids and / or therapeutic agents. For example, bovine hyaluronidase can be co-injected under anesthesia into the peribulbar, retrobulbar, and subtenon blocks for ophthalmic surgical procedures. In addition, increased surgical complications appear in its absence (Brown SM et al. J Cataract Refract Surg. Sep 1999; 25 (9): 1245-9). Bovine hyaluronidase is also used as an antidote for local necrosis from paravenous injection of necrotic substances such as vinca alkaloids (Few, BJ (1987) Amer. J. Matern. Child Nurs. 12,23-26) . Bovine testes hyaluronidase is also useful for the treatment of cystic ganglions (Pauletal. J Hand Surg 1997 Apr; 22 (2): 219-21). Hyaluronidase can also be used to facilitate subcutaneous fluid delivery in hypodermoclysis (Berger EY, Am Geriatr Soc 1984 Mar; 32 (3): 199-203). Hyaluronidase has also been used to reduce infraocular pressure in the eyes of glaucoma patients and cataract patients receiving viscoelastic compounds (US Patent No. 4,820,516 issued April 11, 1989).

Hyaluronidases obtained from cattle or bacteria have also been used as a "propagating agent" to increase the activity of chemotherapeutic compounds and / or the accessibility of tumors to chemotherapeutic agents (Schuller et al., 1991, Proc. Amer. Assoc. Cancer Res 32: 173, abstract # 1034; Czejka et al., 1990, Pharmazie 45: H.9). Hyaluronidase combination chemotherapy is effective in treating a variety of cancers including urinary bladder cancer (Horn et al., 1985, J. Surg. Oncol. 28: 304-307), squamous cell carcinoma (Kohno et al., 94, J. Cancer Res. Oncol. 120: 293-297), breast cancer (Beckenlehner et al., 1992, J. Cancer Res. Oncol 118: 591-596) and gastrointestinal cancer (Scheithauer et al., 1988, Anticancer Res. 8: 391-396). Hyaluronidase is effective as the sole therapeutic agent in the treatment of brain cancer (gliomas) (published PCT application No. WO88 / 02261, published April 7, 1988). Administration of hyaluronidase also induces sensitivity of previously chemotherapy-resistant tumors of the pancreas, stomach, colon, ovaries, and breast (Baumgartneretal., 1988, Reg. Cancer Treat. 1: 55-58; Zankeretal., 1986, Proc. Amer. Assoc Cancer Res. 27: 390). Unfortunately, the contaminants and the non-human nature of such hyaluronidases result in anaphylactic reactions. In addition to its indirect anticancer effects, hyaluronidase obtained from cattle has direct anticarcinogenic effects. Hyaluronidase prevents the development of transplanted tumors in mice (De Maeyer et al., 1992, Int. J. Cancer 51: 657-660) and inhibits tumor formation after carcinogenic exposure (Pawlowski et al., 1979, Int. J. Cancer 23: 105-109; Haberman et al., 1981, Proceedings of the 17th Annual Meeting of the American Society of Clinical Oncology, Washington, DC, 22: 105, abstract no.415).

Given the value of hyaluronidases obtained from cattle as a therapeutic compound, particularly in chemotherapy in conjunction with conventional chemotherapeutics or as a chemotherapeutic by itself, there is a need in the field for substantially pure preparations of hyaluronidase of human origin. There is also a need for efficient methods

It is easy and cost effective to prepare hyaluronidase to provide commercially significant amounts of the enzyme. The present invention addresses these problems.

Hyaluronic acid is an essential component of the extracellular matrix. Hyaluronic acid is found in the connective tissue of mammals and is the main constituent of the vitreous humor of the eye. In connective tissue, the hydration water associated with hyaluronic acid generates spaces between tissues, thus generating an environment that leads to cell movement and proliferation. Hyaluronic acid plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, embryonic development, wound healing, angiogenesis, and oncogenesis (Toole, 1991, Cell Biol. Extracell. Matrix, Hay ( ed), Plenum Press, New York, 1384-1386; Bertrand et al., 1992, Int. J. Cancer 52: 1-6; Knudson et al., 1993, FASEB J. 7: 1233-1241). Furthermore, hyaluronic acid levels correlate with tumor aggressiveness (Ozello et al., 1960, Cancer Res. 20: 600-604; Takeuchi et al., 1976, Cancer Res. 36: 2133-2139; Kimata et al. , 1983, Cancer Res. 43: 1347-1354).

After spinal cord injury, glial astrocyte scars occur and contain chondroitin sulfate proteoglycans (CSPG). CSPGs play a crucial role in inhibiting axon growth (Levine, 1994; Powell et al.), For example, during fetal development, CSPGs repel axons and inhibit neuronal cell adhesion. CSPGs also play an important role in boundary formation (Snow et al., 1990, 1992; Powell and Geller, 1999). Furthermore, CSPG expression increases after CNS injury (Mckeon et al., 1991; Davies et al., 1997).

Studies indicate that the inhibitory effects of CSPG are mainly due to the glycosaminoglycan (GAG) sugar chain of chondroitin sulfate (CS) (Snow et al., 1990; Cole and McCable, 1991; Geisert and Bidanset,

1993). This is confirmed by the discovery that the administration of bacterial chondroitinase does in fact promote axon regeneration when administered intrathecally. Furthermore, electrophysiological experiments determined that regenerated CST axons established functional connections (Bradbury, et al 2002). In addition to their direct inhibitory effects, CSGPs could also interact with cell adhesion molecules or neurotrophic factors to influence neurite outgrowth (Roberts et al., 1988; Ruoslahti and Yamaguchi, 1991; Milev et al.,

1994). Therefore, recombinant mammalian hyaluronidases are useful to reverse CSPG inhibition in the glial scar and to promote axon regeneration after injury.

The amount of sHASEGP required to sufficiently degrade CSPG in the glial scar will vary. In some cases, repeated administration of 10-5000 Units of sHASEGP by intrathecal delivery will be necessary to eliminate CSPGs in the scar. In other cases, sustained release of sHASEGP through the use of a slow release formulation may be preferred. Alternatively, administration of gene therapy vectors encoding sHASEGP may be effective in increasing CSPG clearance.

SHASEGP can also be used to treat herniated discs in a process known as chemonucleolysis. Chondroitinase ABC and the enzyme that cleaves substrates similar to sHASEGP can induce reduction of intradiscal pressure in the lumbar spinal cord. (Sasaki et al., 2001, Ishikawa et al., 1999). There are three types of disc injuries. A bulging disc is one that is intact but is sticking out. In an extruded disc, the fibrous shell has broken and the NP oozes out, but is still attached to the disc. In a sequestered disc, a fragment of the NP has detached from the disc and is free in the spinal canal. Chemonucleolysis is effective on bulging and extruded discs, but not on sequestered disc injuries. In the United States, chemonucleolysis is licensed for use in the lumbar (lower) cord only. In other countries, it has also been successful in treating cervical (upper medulla) hernias. Chemonucleolysis is therefore a conservative alternative to disc surgery when it is preferable to reduce disc pressure.

The exact composition and structure of the carbohydrate chain (s) in a glycoprotein can directly influence its life in serum, since cells in the liver and the reticuloendothelial system can bind and internalize circulating glycoproteins with specific carbohydrates. Hepatocytes have receptors on their surfaces that recognize oligosaccharide chains with terminal Gal residues (i.e. the outermost end (s) of glycans relative to the polypeptide), macrophages contain receptors for terminal Man or GlcNAc residues, and hepatocytes and lymphocytes have receptors. for exposed fucose debris. However, no specific sialic acid receptors have been found. Although somewhat dependent on the spatial arrangement of the oligosaccharides, as a general rule, the greater the number of exposed sugar residues recognized by cell surface receptors in the liver and the reticuloendothelial system, the faster a serum glycoprotein will clear. Due to the absence of specific sialic acid receptors, however, oligosaccharides with all their branches terminated or "end-protected" with sialic acid will not promote the clearance of the protein to which they are attached.

The presence and nature of the oligosaccharide chain or chains in a glycoprotein can also affect important biochemical properties in addition to its recognition by specific sugar receptors on hepatic and reticuloendothelial cells. Removal of carbohydrate from a glycoprotein will usually decrease its solubility and may also increase its susceptibility to proteolytic degradation by destabilizing the correct polypeptide folding pattern and / or unmasking protease sensitive sites. For similar reasons, the glycosylation state of a protein can affect its recognition by the immune system.

ES 2 335 005 T3

SHASEGPs can be used to kill cumulus cells surrounding an oocyte prior to cryopreservation and other in vitro fertilization techniques such as intracytoplasmic sperm injection (ICSI). Hyaluronidase can be added to the collected oocytes between 10-200 U / ml in buffered saline solutions. The oocytes are separated from the released cumulus cells by aspiration and washed through several washes with media lacking hyaluronidase. The eggs can then be processed for cryopreservation or IVF techniques.

SHASEGPs are also useful for more efficient penetration of chemotherapeutic agents into solid tumors. SHASEGP can be injected intratumorally with anticancer agents or intravenously for disseminated cancers or hard-to-reach tumors. The anticancer agent can be a chemotherapeutic agent, an antibody, a peptide, or a gene therapy vector, virus or DNA. Furthermore, sHASEGP can be used to recruit tumor cells in the cycling combination for priming in previously chemoresistant tumors that have acquired multidrug resistance (St Croix et al Cancer Lett 1998 Sep 11; 131 (1): 35-44). SHASEGPs are also useful for increasing the delivery of biological compounds such as monoclonal antibodies, cytokines, and other drugs to tumors that accumulate glycosaminoglycans. Many tumors delete genes involved in glycosaminoglycan catabolism so localized accumulation can prevent antineoplastic agents and the immune system from reaching the tumor mass.

SHPASEG can also be used to increase the sensitivity of tumors that are resistant to conventional chemotherapy. In one embodiment, sHASEGP is administered to a patient who has a tumor associated with a LuCa-1 deficiency in an amount effective to increase diffusion around the tumor site (eg, increase the circulation of chemotherapeutic factors (eg, to facilitate circulation and / or concentrations of chemotherapeutic agents in and around the tumor site), inhibit tumor cell motility (for example, by degradation of HA) and / or lowering the apoptosis threshold of a tumor cell or cells (i.e., bringing the tumor cell or cells to an anoikis state), a state that makes the tumor cell or cells more susceptible to action of chemotherapeutic agents or other agents that can facilitate cell death, preferably preferably facilitate programmed cell death of cells in anoikis. Chemotherapeutic agents as used herein are intended to include all synthetic (eg, cisplatin) as well as naturally-occurring (eg, tumor necrosis factor IF) molecules that facilitate inhibition of tumor cell development and preferably facilitate, more preferably they preferentially facilitate tumor cell death.

Of particular interest is the use of sHASEGP for the treatment of metastatic and non-metastatic cancers, particularly metastatic cancers having decreased to undetectable hyaluronidase activity relative to non-cancerous (normal) cells. SHASEGP can be used as a chemotherapeutic agent (alone or in combination with other chemotherapeutic agents) in the treatment of any of a variety of cancers, particularly invasive tumors. For example, sHASEGP can be used in the treatment of small cell lung carcinoma, squamous cell lung carcinoma, as well as cancers of the breast, ovaries, head and neck and any other cancer associated with decreased levels of hyaluronidase or with a LuCa-1 gene. faulty (hpHAse) (for example, a LuCa-1 gene that does not provide expression of adequate levels of hpHAse or that encodes a defective hpHAse that does not provide an adequate level of hyaluronidase activity) or other defects associated with decreased hyaluronan catabolism. SHASEGP is preferable for the treatment of malignant tumors associated with poor HA catabolism as it does not require cellular involvement for degradation to occur.

The specific dosage appropriate for administration can be readily determined by one of ordinary skill in the art in accordance with the factors discussed above (see, for example, Harrison's Principles of Internal Medicine, 11th Edition, 1987). Furthermore, estimates for appropriate dosages in humans can be extrapolated from determinations of the level of sHASEGP enzymatic activity in vitro and / or effective dosages in animal studies. For example, hyaluronidase 70-300 TRU is effective in reducing tumor burden in a scid mouse. Given this information, corresponding dosages in the average 70 kg human will range from approximately 250,000-1,200,000 TRU of hyaluronidase. The amount of sHASEGP administered to a human patient is generally in the range of 1 TRU to 5,000,000 TRU of enzyme activity, preferably between about 1,000 TRU and 2,500,000 TRU, more preferably between about 100,000 TRU and 1,500,000 TRU, typically between approximately 250,000 TRU and 1,200,000 TRU, with approximately 725,000 TRU representing the average prescribed doses.

In one embodiment, a sHASEGP is formulated in 0.15 M saline containing sHASEGP at a concentration of approximately 150,000 TRU / ml. The formulation is then injected intravenously at 15,000 TRU / kg patient body weight. Alternatively the enzyme formulation can also be injected subcutaneously to allow the hyaluronidase to perfuse around the tumor site. In a preferred embodiment, the sHASEGP is injected peritumorally or into the tumor mass. In another preferred embodiment, the sHASEGP is formulated as a liposome and delivered by injection intravenously or near the site of cancer cells associated with a deficiency in the LuCa-1 gene (hpHAse). Intravenous injection of sHASEGP results in sHASEGP at the tumor site. Furthermore, supersalted sHASEGP is preferable for parenteral administration in that terminal sialic acids in sHASEGP prevent clearance of the enzyme from the circulation by the reticuloendothelial system. Comparisons of supersialized sHASEGP with non-sialated bovine and ovine hyaluronidases show that substantially more favorable pharmacokinetics are achieved.

ES 2 335 005 T3

Gene therapy facilitation

The efficacy of most gene delivery vehicles in vivo does not match the efficacy found in vitro. Glycosaminoglycans can hinder the transfer and diffusion of DNA and viral vectors to many cell types. Levels of such extracellular matrix material can hamper the process considerably. Dubensky et al., (Proc Natl Acad Sci USA. 1984 Dec; 81 (23): 7529-33) demonstrated that when hyaluronidase is combined with collagenase it could facilitate DNA transduction in vivo. Adeno-associated virus has also been shown to be susceptible to hyaluronidase-mediated gene therapy Favre et al, (Gene Ther 2000 Aug; 7 (16): 1417-20).

It has been determined herein that channels of defined size are opened in the extracellular matrix with sHASEGP. These pores do not increase the diffusion of substances larger than about 200-500 nm in diameter. However, smaller molecules such as retroviruses, adenoviruses, adeno-associated viruses, and DNA complexes are susceptible to sHASEGP-mediated diffusion.

Alternatively, viruses can be outfitted with the sHASEGP gene to facilitate their replication and spread within a target tissue, for example. The target tissue can be a cancerous tissue whereby the virus is capable of selective replication within the tumor. The virus can also be a non-lytic virus in which the virus selectively replicates under a tissue-specific promoter. As the virus replicates, co-expression of sHASEGP with viral genes will facilitate the spread of the virus in vivo.

Alternatively, the nucleic acid of interest and a sHASEGP can be used simultaneously or consecutively or in a way that is staggered over time. Simultaneously refers to a co-administration. In this case, these two essential components can be mixed to form a composition before being administered or they can be administered at the same time to the cell or the host organism. It is also possible to administer them consecutively, that is to say one after the other, regardless of which component of the combination product according to the invention is administered first. Finally, it is possible to use a mode of administration that is time-staggered or intermittent and that is interrupted and restarted at intervals that may or may not be regular. It is noted that the routes and sites of administration of the two components may be different. According to a particularly preferred embodiment, the sHASEGP is administered prior to the nucleic acid, the route of administration of the two components preferably being similar. The time interval between injections is not critical and can be defined by the specialist. It is possible to recommend an interval of 10 min to 72 h, advantageously 30 min to 48 h, preferably 1 to 24 h, and most preferably 1 to 6 h.

Furthermore, the combination product according to the invention can also be combined with one or more molecules intended to improve nucleic acid delivery. The molecules can be molecules that have a protective effect on the nucleic acid (protection against degradation in the cell), that improves its penetration or its expression in the host cell (fusogenic peptide, nuclear localization signal, etc.) that allows it to targets a particular cell type (ligand or antibody that recognizes a cell surface protein, etc.) or that prolongs the therapeutic effect (immunosuppressive agent, etc.). The combination product can also be combined with agents that facilitate transfection (proteins, etc.).

The combination product according to the invention can be prepared with a view to local or parenteral administration or administration through the digestive route. The routes that may be mentioned in particular are the intragastric, subcutaneous, intracardiac, intravenous, intraperitoneal, intrasynovial, intratumoral, intrapulmonary, intranasal and intratracheal routes and, very particularly, the intramuscular route. Administration can be effected by any method of the art (injection, oral route, aerosol, instillation, etc.), as a single dose or as a dose that is repeated once or several times after a particular time interval. The route of administration can be adjusted to suit the gene of interest to be transferred and the disease to be treated. The formulation can include pharmaceutically acceptable carriers (excipients, adjuvants, etc.). The substance that leads to the disruption of the extracellular matrix and the nucleic acid of interest are preferably dissolved in a buffer which is suitable for pharmaceutical use and which can be hypertonic, hypotonic or isotonic. Various buffers can be provided. Those that may be mentioned by way of illustration are a physiological saline solution (0.9% NaCl), a non-physiological saline solution (1.8% NaCl), a Hepes-Ringer solution, a Ringer-Lactate solution, a buffer that is based on Tris-HCl (10 mM Tris-HCl, pH 7.5 to 8, 1 mM EDTA; 10 mM Tris-HCl, pH 7.5 to 8, MgCl₂ 1 mM), a phosphate buffer (Krebs phosphate buffer H₂O), a solution of sugars (glucose, sucrose, trehalose, etc.) or simply water.

Hypodermoclysis

Hypodermoclysis, subcutaneous fluid infusion is a useful and easy hydration technique suitable for mildly to moderately dehydrated adult patients, especially the elderly. The method is considered safe and does not represent any serious complications. The most common adverse effect is mild subcutaneous edema that can be treated by local massage or systemic diuretics. Approximately 3 L can be administered in a 24 hour period at two separate sites. Common infusion sites are the chest, abdomen, thighs, and upper arms. The preferred solution is normal saline but other solutions such as semi normal saline, glucose with saline or 5 percent glucose can also be used. Potassium chloride can be added

ES 2 335 005 T3 to solution bag if necessary. In addition, other drugs can be delivered through similar routes. Human sHASEGP can be added to increase fluid absorption and increase the overall rate of administration. Human sHASEGP is preferable for repeated hypodermoclysis over slaughterhouse-derived enzymes in the sense that it is not likely to be immunogenic as the bovine enzyme is known to be. It can be administered at home by family members or a nurse; the technique should be familiar to all family physicians.

In outpatients, hypodermoclysis sites include the abdomen, upper chest, above the breast, over an intercostal space, and the scapular area. In bedridden patients; preferred sites are the thighs, abdomen, and outer upper arm. After one to four days, the needle and tubing should be changed, although infusion sets have been left in place for much longer periods without complications. The administration of boluses of 500 ml in one or two hours three times a day can also be administered with 150 U of sHASEGP administered subcutaneously before the first infusion in the morning.

Facilitation of therapeutic injections

Many molecules injected percutaneously reach the circulation slowly or with very little efficiency. Several factors regulate the pharmacokinetics and pharmacodynamics of molecules injected subcutaneously (SC) or intramuscularly (IM). Generally, larger molecules reach the circulation more slowly and less efficiently without active transport into the circulation. Subcutaneous bioavailability is determined by calculating the ratio of area under the curve for SC versus intravenous administration (AUC_SC/ AUC_intravenous). A second factor is the charge and affinity for matrix molecules that may play a role in subcutaneous molecule sequestration. If these materials are locally degraded they may never reach their desired targets and therefore demonstrate decreased total systemic bioavailability in target organs.

Large proteins are normally given intravenously so that the drug is directly available in the bloodstream. However, it would be advantageous if a drug could be administered subcutaneously, intramuscularly or intradermally, since these forms of administration are much easier for the patient to manipulate. Especially if the drug must be taken regularly throughout life and treatment must start early, when the patient is still a child. However, a drug with a very large and labile molecule such as coagulation factor VIII of 170 to 300 kDa normally has a very low availability if administered subcutaneously, intramuscularly or intradermally, since the uptake is not sufficient and the degradation is pronounced.

In addition to the need to increase the bioavailability of many subcutaneously administered biological compounds, faster pharmacokinetics in emergency medicine are also critically important. The time required to achieve intravenous access in many patients can prevent the use of an otherwise fast-acting drug when administered systemically. In some cases failure to reach intravenous access follows after subcutaneous injection leading to a further delay in reaching the target organs. Therefore, the more rapid availability of subcutaneous drugs would be beneficial as a first line of treatment rather than risking the time required to achieve intravenous access. Examples of molecules that can be delivered subcutaneously as well as intravenously include epinephrine, atropine, narcan, linocaine, and dextrose.

Many molecules injected percutaneously reach the circulation slowly or with very little efficiency. Several factors regulate the pharmacokinetics and pharmacodynamics of molecules injected subcutaneously (SC) or intramuscularly (IM). Generally, larger molecules reach the circulation more slowly and less efficiently without active transport into the circulation. Subcutaneous bioavailability is determined by calculating the ratio of area under the curves for SC versus intravenous administration (AUC_SC/ AUCi_ntHveEOsa). A second factor is the charge and affinity for matrix molecules that may play a role in subcutaneous molecule sequestration. If these materials are locally degraded they may never reach their desired targets and therefore demonstrate decreased total systemic bioavailability in target organs.

Large proteins are normally administered intravenously so that the drug is available directly into the bloodstream. However, it would be advantageous if a drug could be administered subcutaneously, intramuscularly or intradermally since these forms of administration are much easier for the patient to manipulate. Especially if the drug must be taken regularly throughout life and treatment must start early, when the patient is still a child. However, a drug with a very large and labile molecule such as coagulation factor VIII of 170 to 300 kDa normally has a very low bioavailability if administered subcutaneously, intramuscularly or intradermally since uptake is not sufficient and degradation is pronounced. .

In addition to the need to increase the bioavailability of many subcutaneously administered biological compounds, faster pharmacokinetics in emergency medicine are also critically important. The time required to achieve intravenous access in many patients can prevent the use of an otherwise fast-acting drug when administered systemically. In some cases failure to reach intravenous access follows after subcutaneous injection leading to a further delay in reaching the target organs. Therefore, the faster availability of subcutaneous drugs would be beneficial as a first line of treatment.

ES 2 335 005 T3 treatment more than risking the time needed to get intravenous access. Examples of molecules that can be delivered subcutaneously as well as intravenously include epinephrine, atropine, narcan, lignocaine, and dextrose.

An additional benefit of the invention is based on the ability to deliver equivalent or greater volumes of SC or IM solutions without the pain and morbidity associated with the pressure and volume of the solution at the injection site.

Vitreous hemorrhage

In an effort to minimize the potential to cause additional retinal detachment or rupture during vitrectomy, it has previously been proposed in U.S. Patent No. 5,292,509 (Hageman) to inject certain protease-free glycosaminoglycanase enzymes into the vitreous body, to cause the vitreous body to disengage or "disinsert" from the retina prior to removal of the vitreous body. Such disinsertion or uncoupling of the vitreous body is intended to minimize the likelihood of further retinal breakage or detachment as the vitreous body is removed. Examples of specific protease-free glycosaminoglycanase enzymes that can be used to cause this vitreous detachment purportedly include; chondroitinase ABC, chondroitinase AC, chondroitinase B, chondroitin 4-sulfatase, chondroitin 6-sulfatase, hyaluronidase, and beta-glucuronidase.

Although the enzyme hyaluronidase is known to be used for various ophthalmic applications including the adjunct application of vitrectomy described in US Patent No. 5,292,509 (Hageman), published studies have indicated that the enzyme hyaluronidase itself may be toxic to the retina. and / or other anatomical structures of the eye. See, The Safety of Intravitreal Hyaluronidase, Gottlieb, J. L; Antoszyk, AN, Hatchell, DL and Soloupis, P., Invest Ophthalmol Vis Sci. 31: 11, 2345-52 (1990). Also, the use of impure slaughterhouse preparations of hyaluronidase can cause uveitis or inflammation of the eye. The use of human sHASEGP is therefore preferable both for its increased potency, purity and absence of animal origin which can give rise to immunogenic reactions and antibody-mediated neutralization after repeated administration. In another embodiment, a pegylated form of a sHASEGP can be injected into the eye. Such pegylated sHASEGP is not cleared from the vitreous as quickly and maintains its activity in the vitreous for a longer period of time.

The ophthalmic toxicity of some hyaluronidase preparations has been confirmed by other investigators who have proposed that such hyaluronidase preparations be used as a toxic irritant to cause experimentally induced neovascularization of the eye, in animal toxicity models (see An Experimental Model of Preretinal Neovascularization in the Rabbit; Antoszyk, AN, Gottlieb, J. L, Casey, R..C., Hatchell, DL and Machemer, R., Invest Ophthalmol Vis Sci. 32: 1.46-51 (1991). The use of a highly purified sHASEGP devoid of mercury-based contaminants and of livestock or bacterial origin is preferable for intraocular procedures. Furthermore, a recombinant human sHASEGP is preferable over slaughterhouse preparations both for purity, absence of bovine pathogens, and reduced risk of immunogenicity. More preferably a pegylated sHASEGP is provided.

Therefore an enzymatic method is provided using a human sHASEGP to treat ophthalmic disorders of the mammalian eye. In one embodiment of the invention, said sHASEGP is PEGylated to prolong its stay within the vitreous humor and prevent localized uptake. The prevention of neovascularization and the increased rate of clearance from the vitreous of materials toxic to the retina is achieved by administering an amount of hyaluronidase effective to liquefy the vitreous of the treated eye without causing toxic damage to the eye. The liquefaction of the vitreous humor increases the rate of liquid exchange of the vitreous chamber. This increase in exchange eliminates those materials and conditions whose presence causes retinal and ophthalmic damage.

Cosmetic uses of sHASEGP

Hyaluronidase is known to have the effect of depolymerizing the long mucopolysaccharide chains of the ground substance responsible for the retention of bound water and the slowing down, by capillary compression, of the diffusion of organic fluids that eliminate metabolic waste. Said retention of water and waste associated with fat overload of the lipocytes constitutes a classic "pigskin" edema or "orange peel" edema. This depolymerization will therefore cut the long chains of mucopolysaccharides into shorter chains, and consequently the elimination of bound water, waste, restoration of venous and lymphatic circulation and disappearance of local edema.

The use of sHASEGP by way of subcutaneous administration is therefore preferred for the elimination of glycosaminoglycans involved in the accumulation of so-called cellulosis and for promoting lymphatic flow. Human sHASEGP is preferred for the treatment of cellulosis in that it is capable of eliminating such glycosaminoglycans without the inflammatory components of slaughterhouse proteins and is of high purity and unlikely to be immunogenic. SHASEGP can be administered through repeated subcutaneous injections, through transdermal delivery in the form of ointments or creams, or through the use of injectable slow-release formulations to promote the continued degradation of glycosaminoglycans and prevent their recurrence.

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Organ transplant

Hyaluronan has several biological effects, which are in part related to its molecular size (West, DC, Kumar, S. Exp.Cell. Res. 183, 179-196, 1989). The hyaluronan content in an organ increases in different conditions of inflammation of that organ. Therefore, an increased concentration of hyaluronan has been demonstrated in tissue of different organs characterized by inflammatory immunological injury such as alveolitis (Nettelbladt 0 et al, Am Rev Resp Dis 1989; 139: 759-762) and myocardial infarction (Waldenstrom et al, J Clin Invest 1991; 88 (5): 16221628). Other examples are allograft rejection after kidney transplantation (Hallgren et al, J Exp Med 1990a; 171: 2063-2076; Wells et al, Transplantation 1990; 50: 240-243), small intestine (Wallander et al, Transplant Int 1993; 6: 133-137) or cardiac (Hallgren et al, J Clin Invest 1990b; 85: 668-673); or a myocardial inflammation of viral origin (Waldenstrdm et al, Eur J Clin Invest 1993; 23: 277-282).

The appearance of interstitial edema in connection with the graft of an organ constitutes a serious problem in the field of transplantation surgery. As many as 25% of the grafts will swell to such a degree that function will be temporarily lost. Also, in 2-3% of cases, swelling causes kidney rupture, resulting in massive bleeding.

SHASEGP can be used to break down glycosaminoglycans accumulated in an organ transplant. The elimination of these glycosaminoglycans promotes the elimination of water from the graft and therefore the function of the organ. A dose ranging from 500-10,000 Units / kg can be administered to reduce pore pressure as such.

Pathological Accumulations of Glycosaminoglycans in the Brain

Hyaluronan levels are elevated in various cerebrospinal pathological conditions. Cerebrospinal hyaluronan levels are normally less than 200 pg / l in adults (Laurent et al, Acta Neurol Scand 1996 Sep; 94 (3): 194-206). These levels can rise above 8,000 pg / l in diseases such as meningitis, spinal stenosis, head injury, and stroke. Therefore, administration of sHASEGP by intrathecal delivery or systemic injection of supersialated sHASEGP can be used to degrade critically high levels of substrate.

The absence of an effective lymphatic system in the brain can also lead to life-threatening edema followed by head trauma. Hyaluronan accumulation is a result of increased synthesis by HA synthases and decreased degradation. The accumulation of hyaluronan serves the purpose of increasing the water content in damaged tissue to facilitate leukocyte extravasation but can be fatal. Administration of human sHASEGP to a patient suffering from a head injury can therefore eliminate the accumulation of tissue hyaluronan and the water associated therewith. Human sHASEGP can be administered intrathecally through a shunt or, alternatively, supersialated sHASEGP can be administered intravenously to reach brain tissue.

Following ischemia of the brain as occurs in stroke, the hyaluronan content increases dramatically due to increased expression of HA synthases and decreased catabolism. Ion pump failure and plasma leakage into the interstitium results in fluid retention that is not properly removed by the lymphatic vessels resulting in tissue necrosis. Some groups have tried to prevent the accumulation of interstitial fluid after ischemia-reperfusion by blocking vascular permeability. However, once the fluid has been extravasated, preventing vascular permeability can prevent resolution of the edema and worsen the conditions.

Human sHASEGP can also be used in the treatment of edema associated with brain tumors, particularly those associated with glioblastoma multiforme. Edema associated with brain tumors results in the accumulation of hyaluronan in the non-cancerous portions of the brain adjacent to the tumor. Administration of hyaluronidase at sites of hyaluronan accumulation (eg, by intravenous injection or through a shunt) can alleviate the edema associated with such malignant tumors by degradation of excess hyaluronan at these sites. Therefore, hyaluronidase is successful in treating brain tumors not only in reducing tumor mass and inhibiting tumor growth and / or metastasis but is also useful in alleviating edema associated with malignant tumor. Human sHASEGP can be administered for the treatment of edema in a manner similar to that of the administration of bovine testicular hyaluronidase to treat edema (see, for example, Sa Earp Arq. Braz. Med. 44: 217-20).

Treatment of glycosaminoglycan accumulation in cardiovascular diseases

Administration of hyaluronidase in animal models after an experimental myocardial infarction has been shown to reduce the size of the infarct (Maclean, et al Science 8 Oct 1976; 194 (4261): 199-200). The proposed mechanism by which bovine hyaluronidase reduces infarct size in animals is by reducing the accumulation of hyaluronan that occurs after ischemia-reperfusion. The reduction in the size of the infarct is thought to occur from increased lymphatic drainage and increased tissue oxygenation and reduced myocardial content56

ES 2 335 005 T3 of water. Although reduced infarct size could be obtained in animal models, benefits were not seen in larger clinical studies in humans. Bovine testes hyaluronidase has a remarkably short serum half-life of approximately 3 minutes in animals and humans Wolf, et al., J Pharmacol ExpTher Aug 1982; 222 (2): 331-7. This short half-life is due to terminal mannose residues that are easily recognized by phagocyte receptors of the reticuloendothelial system. Although small animals may benefit from hyaluronidase due to a smaller vascular bed, an enzyme with an increased half-life is necessary. Supersalted sHASEGP has more favorable pharmacokinetics due to sialation for which there is no phagocyte receptor. Supersialated sHASEGP at doses ranging from 100-200,000 Units / kg can be used to facilitate resolution of excess hyaluronan after ischemia-reperfusion and to reduce infarct size.

Supersalted sHASEGP can also be used to limit coronary arteriesclerosis plaques. Such plaques accumulate glycosaminoglycans and mediate macrophage and foam cell adhesion Kolodgie et al, Arterioscler Thromb Vase Biol. 1 Oct 2002; 22 (10): 1642-8. Administration of supersialated sHASEGP can be used to reduce plaque formation As repeated administration of hyaluronidase is contemplated at doses of 100,000 100,000 U / kg, the need to use a recombinant human protein with low risk of immunogenicity and an increased half-life will result in a superior plate reduction.

Peripheral tissue necrosis treatment

Tissue necrosis occurs in many diseases due to venous insufficiency. Lack of sufficient oxygenation is one of the main obstacles to tissue regrowth. Intra-arterial hyaluronidase treatment has been shown to significantly improve the clinical picture in patients with peripheral arterial occlusive disease (Elder et al, Lancet (1980) 648-649). SHASEGP can be injected intra-arterially 3-5 times a week at doses of 10-200,000 Units.

Enhancement of anesthesia

Slaughterhouse hyaluronidase is commonly used for peribulbar block in local anesthesia prior to ophthalmic surgery. The presence of the enzyme avoids the need for additional blockages and speeds up the time to the onset of akinesia (loss of eye movement). Peribulbar and subtenon block are the most common applications of hyaluronidase for ophthalmic procedures. Since Wydase® discontinuation, there have been reports of increased diplopia and ptosis with peribulbar block (Brown et al J Cataract Refract Surg 1999; 25: 1245-9).

With the Wydase® Wyeth suspension, hyaluronidase material obtained from bovine testes is currently supplied by compounding pharmacies. However, there are several concerns about the use of a sterile product prepared extemporaneously http://www.ashp.org/shortage/hyaluronidase.cfm?cfid= 11944667 &amp; CFToken = 94 2 6953 - ref # ref. Prepared compounding preparations are not FDA authorized products. As such, the FDA has no control over the quality or consistency of the manufacturing process.

The 10-500 Unit sHASEGP can be mixed directly with 5 ml of 2% lidocaine (Xylocaine), 5 ml of 0.5% bupivacaine (Marcaine) and optionally with 1: 200,000 epinephrine. SHASEGP can be used to increase the appearance of akinesia and to eliminate the need for additional blocks. The sHASEGP is also ideal for akinesis for cosmetic surgery in blepharoplasties and facelifts. SHASEGP can also be used after such surgical procedures to diffuse anti-inflammatories and to reduce tissue swelling.

SHASEGP can also be mixed with a buffer solution such as bicarbonate to prevent discomfort during the injection procedure. SHASEGP can also be mixed with laceration anesthesia to both reduce the total volume of material needed for injection and to reduce the pain of tissue swelling.

Reduction of intraocular pressure

A common side effect that occurs postoperatively in cataract patients is a significantly early and occasionally prolonged increase in intraocular pressure. This condition is sometimes serious, especially in patients with glucomatous changes in the optic disc. Although the pressure rise tends to be more severe when viscoelastic agents such as hyaluronic acid are injected into the eye during surgery, intraocular pressure may rise postoperatively even when such agents are not used. Furthermore, such an increase in pressure can occur even when no additional medications are used during the surgical procedure. In some cases, it is advantageous to leave a viscoelastic agent in the eye, which often requires large doses of carbonic anhydrase inhibitors to be administered to patients. These inhibitors lower intraocular pressure by reducing the formation of aqueous humor, a fluid that is normally secreted in the eye, by the ciliary body. Current methods of alleviating postoperative pressure increases in the eye include various types of eye drops such as beta-adrenergic blocking agents, sympathomimetic agents, miotics, alpha II selective agents, carbonic anhydrase inhibitors, and prostaglandin agents.

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A preferred method of removing viscoelastic such as hyaluronic acid is by injection of sHASEGP during or immediately after anterior segment or posterior segment surgical procedures, although other methods of administration known in the art are also possible. It is preferred if hyaluronic acid and sHASEGP are administered by injection into the anterior chamber during anterior segment ocular surgical procedures to allow the hyaluronic acid to act as a spacer during the start of the surgical procedure. In some corneal transplant cases, the combination of hyaluronic acid and sHASEGP can be placed on the surface of the intraocular structures prior to suturing the corneal transplant in place. This combination can also be used in posterior segment surgery, such as vitreous or retina surgery.

In some cases, it may be advisable to leave a viscoelastic agent such as Healon.TM., Viscoat.TM., Or other space-occupying substances in the anterior chamber of the eye at the completion of surgery. This is especially true of an increase in positive pressure when the intraocular contents tend to come forward and press against the posterior surface of the cornea. If this occurs in an eye with a synthetic intraocular lens in place, pressure on the corneal endothelium can cause significant cell damage and posterior corneal swelling and clouding can occur that is associated with decreased vision. Typically, if a patient's intraocular pressure rises significantly at the completion of the operative procedure, higher doses of carbonic anhydrase inhibitors, as well as topical eye drops such as beta blockers and alpha II agonists, need to be administered to the patient to decrease formation of aqueous humor and / or to increase the output of aqueous humor. These agents all have significant side effects and are in some cases contraindicated in patients with various types of medical conditions, such as respiratory problems, heart disease, or high blood pressure. However, the use of sHASEGP in these situations will eliminate the need to administer large doses of these drugs to these patients.

In addition, there is a significant amount of hyaluronic acid in the trabecular meshwork. SHASEGP will break it down and thus enhance aqueous outflow through the trabecular meshwork. The patient's intraocular pressure will therefore decrease. The combination of sHASEGP with other anterior chamber agents, such as a methylcellulose (Ocucoat.RTM, for example, commercially available from Storz Instrument Co.) used as spacers and / or protective agents in cataract surgery, It will also be effective in preventing significant increases in pressure because they will in effect open the trabecular meshwork and allow greater aqueous humor drainage by degradation of a significant amount of the hyaluronic acid present in the trabecular meshwork.

The removal of glycosaminoglycans from the trabecular meshwork is also useful for the reduction of intraocular pressure in individuals suffering from open-angle glaucoma. Human sHASEGP can be administered by subconjunctival injection or injection directly into the anterior chamber.

Cystic ganglions

The cystic ganglion (also known as the wrist cystic ganglion, Biblical cystic ganglion, or dorsal tendon cystic ganglion) is the most common soft tissue mass in the hand. It is a fluid-filled sac that can be felt under the skin. It is usually attached to a tendon sheath (lining that lubricates the tendon) in the hand or wrist or connected to an underlying joint; however, some have no obvious relationship to any structure. These can also appear on the feet. They often appear when there is a tear in the ligaments underlying the lining of tendons or joints and the lining herniates out of the ligament defect causing a bulge under the skin. Because it is often associated with inflammation, the inflamed tissue produces a jelly-like fluid that fills the protruding sac. They can be very hard due to the high pressure of the mucus-like fluid contained within the cyst and are often mistaken for a bony prominence.

SHASEGP can be used to enhance cystic ganglions. Intralesional injection of sHASEGP of 51,000 Units followed by fine needle aspiration will remove the cyst without the need for surgery. Corticosteroids can also optionally be injected with sHASEGP. An additional injection may be necessary for some patients.

Myxedema

Glycosaminoglycan (GAG) infiltration of the skin is a feature of hyperthyroidism, hypothyroidism, pretibial myxedema, scleromyxedema, and sclerodema. Hyaluronic acid is the main GAG in all conditions and in normal skin. There is minimal histological variability in the dermal distribution of GAG. Acquired cutaneous mucinosis have a similar distribution and biochemical composition of cutaneous GAGs. Morphological differences in fibroblast activity suggest that sclerodema and scleromyxedema mucinosis represent a local process while GAG ​​infiltration of thyroid diseases may have a systemic origin. These disorders can be ameliorated with sHASEGP from both a local and a systemic route of administration. For chronic therapy, a PEGylated sHASEGP may be envisaged.

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Pulmonary uses of sHASEGP

Hyaluronan levels in bronchoalveolar lavage (BAL) from normal individuals are generally below 15 ng / ml. However, BAL levels increase dramatically in conditions of respiratory distress (Bjermer Br Med J (Clin Res Ed) 3 Oct 1987; 295 (6602): 803-6). In ARDS, for example, hyaluronan levels can rise up to 500 ng / ml while in farmer's lung, BAL levels can exceed 1000 ng / ml (Hallgren et al Am Rev Respir Dis. Mar 1989; 139 (3): 682-7), (Larrson et al Chest. In 1992; 101 (1): 109-14). Increased hyaluronan in the lung can impede oxygen diffusion and gas exchange, as well as the activation of netrophil and macrophage responses.

Bovine hyaluronidase preparations are not preferable for the treatment of such conditions for a number of reasons. First, hyaluronidase slaughterhouse testicle preparations are known to be contaminated with serine proteases such as acrosin. Second, the strange nature of bovine enzymes increases the likelihood of an anaphylactic reaction, which could result in the death of the patient. Therefore, a highly purified preparation of recombinant human sHASEGP can be delivered by pulmonary or intravenous delivery. Human sHASEGP can also be administered to patients suffering from other pulmonary complications that are associated with elevated glycosaminoglycans or to increase delivery of other co-delivered molecules to the lung.

The invention will now be described in greater detail in connection with the following non-limiting examples.

Example 1

Microtiter plate-based hyaluronidase assays

The following example provides a rapid assay for the measurement of hyaluronidase activity of sHASEGP. This assay can be related to TRU, UI or NFU through the use of a WHO standard preparation of hyaluronidase.

Biotinylated Hyaluronan Microtiter Assay

Free carboxyl groups on hyaluronan glucuronic acid residues are biotinylated in a one-step reaction using biotin-hydrazide (Pierce), Sulfo NHS (Pierce), and 1-Ethyldimethylaminopropyl-carbodiimide (Sigma). This biotinylated HA substrate is covalently coupled to a 96-well microtiter plate in a second reaction. At the completion of the enzymatic reaction, residual substrate is detected with an avidin-peroxidase reaction that can be read on a standard ELISA plate reader. As the substrate covalently binds to the microtiter plate, artifacts such as pH-dependent shift of the biotinylated substrate do not occur. The sensitivity allows a rapid measurement of the hyaluronidase activity of cultured cells and biological samples with an inter-assay variation of less than 10%.

to. Protocol

Preparation of biotinylated HA substrate

One hundred mg HA (Sigma Chemicals) was dissolved in 0.1 M MES, pH 5.0, at a final concentration of 1 mg / ml and allowed to dissolve for at least 24 h at 4 ° C before coupling biotin . Sulfo-NHS (Pierce; Rockford IL) was added to the MES CS04 solution to a final concentration of 0.184 mg / ml. Biotinehydrazide (Pierce) was dissolved in DMSO as a 100 mM stock solution and added to the CS04 solution to a final concentration of 1 mM. A stock solution of 1-ethyl-3- (3-dimethylaminopropyl) carbidodiimide (EDAC) was prepared as a 100 mM stock solution in distilled water and added to the HA-biotin solution to a final concentration of 30 mM. This solution was allowed to stir overnight at 4 ° C. Unbound biotin and EDAC were removed by dialysis against water with 3 volume changes 1000x of water. The dialyzed, biotinylated HA (bHA) was aliquoted and stored at -20 ° C for up to several months.

Sulfo-NHS was diluted to 0.184 mg / ml in water with the bHA at a concentration of 0.2 mg / ml and pipetted into COVALINK-NH 96-well plates (NUNC; Placerville NJ) at 50 µl per well. The EDAC was diluted to 0.123 mg / ml in water and pipetted onto the COVALINK-NH plates with the bHA solution, resulting in a final concentration of bHA 10 pg / well and EDAC 6.15 pg / well. The plates were incubated overnight at 4 ° C or for 2 h at 23 ° C, which gave comparable results. After covalent immobilization of bCSO4 on the microtiter plates, the coupling solution was removed by shaking and the plates were washed 3 times in PBS containing 2M NaCl and MgSO₄ 50 mM (Buffer A). Plates could be stored at 4 ° C for up to one week.

COVALINK-NH plates with immobilized bHA were equilibrated with assay buffer 100 μl / well-0.1 M format, pH 3.7, 0.1 M NaCl, 1% TRITON X-100 detergent, 5 mM sucrolactone for hyaluronidase lysosomal; or 10 mM Hepes pH 7.4 with CaCl₂ 1 mM and 1 mg / ml human serum albumin (ICN) for enzymes active at neutral pH.

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A set of standards for the calibration of the enzymatic activity against "Relative Turbidity Reducing Units" (rTRU) was generated by dilution of bovine testicular hyaluronidase (Sigma Type VI-S) in neutral enzymatic buffer from 1.0 to 1 x 10 ⁶ rTRU / well and 100pl / well assay in triplicate. Acidic pH active hyaluronidase samples were diluted in 1:10 to 1: 130,000 lysosomal assay buffer and pipetted in triplicate at 100 µl / well. For most tests of tissue extracts and human plasma, a 30 min incubation at 37 ° C was sufficient. Positive and negative control wells (without enzyme and ABC (see below), respectively) were included in triplicate.

The reaction was stopped by adding 200 µl / well 6 M Guanidine HCl followed by three 300 µl / well washes with PBS, 2 M NaCl, MgSO₄ 50 mM, 0.05% TWEEN 20 detergent (Buffer B). An avidin-biotin complex (ABC) kit (Vector Labs; Burlingame CA) was prepared in 10 ml of PBS containing 0.1% TWEEN 20 detergent, which was pre-incubated for 30 min at room temperature during incubation. The ABC solution (100 µl / well) was added and incubated for 30 min at room temperature. The plate was washed five times with Buffer B, then an o-phenylenediamine (OPC) substrate was added at 100 µl / well by dissolving a 10 mg OPD tablet in 10 ml citrate-PO buffer.₄ 0.1 M, pH 5.3 and addition of 7.5 µl of H₂OR₂ 30%. The plate was incubated in the dark for 10-15 min, then read using a 492 nM filter on a computer controlled ELISA plate reader (TitertekMultiskan PLUS; ICN) using the Delta Soft II plate reader software from Biometallics. (Princeton NJ). A standard curve was generated using bovine testicular hyaluronidase by a four parameter curve fit of the commercial hyaluronidase preparation and unknown samples were interpolated for their absorbance at 492 nm.

To analyze the pH dependence of hyaluronidases, purified recombinant sHASEGP and bovine testicular hyaluronidase are used. The pH dependence of enzyme activity is measured by diluting purified sHASEGP or partially purified bovine testicular hyaluronidase to 0.1 rTRU in the following buffers: 50 mM format, pH 3-4.5; 50 mM acetate, pH 5-6; 50 mM MES, pH 6-7; or 50 mM HEPES, pH 7-8. The samples were tested for 30 min at 37 ° C and the activity was expressed as a percentage of the maximum activity. NaCl was not used in the buffers as it can alter the optimum pH of testicular hyaluronidase preparations (Gold, Biochem. J. 205: 69-74, 1982; Gacesa et al. Biochem. Soc. Trans. 7: 1287-1289, 1979); physiological salt concentrations (0.15 M) decreased the apparent pH optimum, an effect that was more pronounced in purified testicular enzyme preparations than in the original crude sample.

b. Results

The hyaluronan was biotinylated in a one-step reaction using biotin-hydrazide and EDAC. By limiting EDAC, which couples the free carboxyl groups in HA with biotin-hydrazide, only a small fraction of the total glucuronic acid residues in HA were labeled. This amount of EDAC (3 x 10^-5 M) added to HA (2.8 x 10 ³ M) results in a maximum of one biotin-hydrazide molecule coupled by 93 disaccharide units of HA.

A four parameter curve fit of standard bovine testicular hyaluronidase reactions measured at pH 3.7 and diluted from 1.0 to 1 x 10 was prepared. ⁶ TRU / well. Four-parameter curve fits were established from the equation y = ((A - D) / (1 + (conc / C)^TOB)) + D), in which log_Item y = In (y '/ 1-y'), y '= (y - D) / (A - D), B = -b / ln 10 and C = EXP (a / B). The four parameters (A, B, C, D) were calculated with a computer program using the 2 + 2 algorithm with linear regression (Rodbard et al., Clin. Chem. 22: 350, 1976). This curve fit incorporates the sigmoidal aspects of the standard curve. Optimal precision for measurement of a sample is typically 0.001 to 0.01 TRU / well during a 30 min incubation. During a 60 min incubation, 1/1000 of a TRU is detectable. A logarithmic standard curve over a shorter range of values ​​can also be used to establish a standard curve fit. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be excessively limited to such specific embodiments.

Example 2

SHASEGP cDNA cloning

Nucleic acid encoding human sHASEGP can be obtained by one of ordinary skill in the art through various procedures including, but not limited to, artificial gene synthesis, RT-PCR, and hybridization of cDNA libraries (for example, see Gmachl et al FEBS 336 ( 3) 1993, Kimmel et al., Proc. Natl. Acad. Sci. USA 90 1993 10071-10075). Alternatively, clones encoding human sHASEGP can be obtained from IMAGE or other providers of human gene sequences (Invitrogen Clone ID IOH10647).

The full length human PH20 cDNA was calculated to be 2009 nucleotides in length and contained an open reading frame of 1530 nucleotides. The 5 'UTR is extraordinarily large, may indicate a retained intron and can inhibit translation by preventing the ribosome from binding to the correct start methionine codon due to 9 non-coding start codons in the 5' UTR. The protein (Genbank Accession number NP_003108) is predicted to comprise SEQ ID NO: 1 of 509 amino acids with a calculated molecular mass of 58 kDa.

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For clone sequencing, PCR amplified bands were excised and eluted with the Gel Extraction Kit (Qiagen) and cloned into appropriate vectors with compatible ends after restriction digestion. All sequencing reactions were performed on double-stranded DNA with the Taq dye-labeled deoxy terminator cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions and processed on an ABI Prism automated sequencer.^TM (Applied Biosystems).

The open reading frame of human PH-20 was obtained by amplification of a human testis cDNA library (Clontech, Palo Alto CA) by Polymerase Chain Reaction using the primers of SEQ ID NO: 14 and SEQ ID NO. N °: 47. PCR products were digested with NheI and BamHI and cloned into the NheI and BamHI sites of the IRESpuro2 vector (Clontech).

Example 4

Isolation of sHASEGP from human pH20 cDNA

A catalytically active secreted recombinant human sHASEGP expression vector capable of efficient glycosylation in mammalian cells was generated as described below. Other expression constructs with promoters and selection genes are contemplated for different species such as yeast and insect cells that are also capable of generating sHASEGP. Positive selection genes such as Glutamine Synthase or Dihydrofolate Reductase (DHFR) can also be used. The examples provided below are not intended to limit but rather to provide an example of various plasmid expression systems that can be used.

To construct secreted forms of sHASEGP, truncation mutants lacking the hydrophobic C-terminus were constructed. Using a GPI cleavage prediction program, the GPI anchor cleavage site was located around amino acid position N 483 in the full length GPI anchored protein. A set of seven nested 3 'primers was used to construct a set of seven truncated deletion mutants lacking the predicted GPI anchor, starting at position Y 482 and progressively deleting one amino acid. These primers were designed to have compatible Nhe1 (5 ') and BamH1 (3') sites to clone the truncation mutants into the lrespuro2 vector unlabeled with a stop codon in the 3 'primer or as a C- His-tagged protein. terminal to facilitate purification and detection. For example, reverse primers of SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 were used to generate deletion mutants ending at position Y 482, F 481 and I 480 without a marker. 6 His. Other mutant primers with the same base pattern were generated with appropriate modifications to include and exclude particular amino acids. To generate His-tagged variants, the same set of primers was used as for non-tagged variants except that the primers lack the stop codon in the respective reverse primers, the forward primer still being the same (for a His-tagged construction see primers with SEQ ID NO: 19, 20, 21, 22, 23, 24 and 25 which are reverse primers with no stop codon corresponding to unlabeled reverse primers for their respective constructs). Overlapping primers were used to construct a six amino acid spacer followed by hexahistidine within BamH1 and Not1 sites in a lrespuro2 vector, such that His-tagged mutants were generated by ligation of the restriction enzyme-digested and PCR amplified products in the Nhe1 and BamH1 sites in the lrespuro2 vector containing the His marker.

To identify whether human sHASEGP could be modified at its carboxy terminal end to generate a secreted and active enzyme at neutral pH, a series of truncations of the GPI anchor binding site in the predicted "catalytic domain" were performed based on homology to the bee venom enzyme.

DNA encoding the full-length human sHASEGP GPI-anchored clone in IRESPuro2 was used as a template to generate the various truncated deletion mutants. Computer modeling programs provided several predicted cleavage sites for the full-length polypeptide. One such predicted site was at amino acid position N483 (SEQ ID NO: 1). PCR primers were designed to successively truncate the protein from N483 to generate six deletion mutants starting at Y 482 (lacking N) and ending at E 477 (lacking P).

to. Protocol

Generation of truncation mutant lacking N483

The full length GPI-anchored sHASEGP clone between the Nhe1 and BamH1 sites in pRESPuro2 was used as a template. This template was amplified with a 5 'primer containing a NheI site starting at the native signal peptide starting Methionine at M 1 (SEQ ID NO: 14) and a 3' primer containing a Y-ending BamHI site 482 (SEQ ID NO: 8). The PCR product was processed on a 1% agarose gel to resolve it and confirm the amplified band of the correct size, it was gel purified and digested with the restriction enzymes NheI and BamHI and the vector pRESPuro2 (Clontech) was cloned between the NheI and BamHI sites generating an expression vector to express this truncation mutant of sHASEGP that ends at the amino acid position N482 and lacks the GPI anchor with the amino acid sequence (SEQ ID NO: 5 for the resulting polypeptide sequence from sHASEGP up to Y 482) and the nucleotide sequence (SEQ ID NO: 48 - polypeptide encoding nucleotides in SEQ ID NO: 5) as indicated.

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Generation of the other truncation mutants lacking Y 482, F 481, I480, Q 479 and P 478, respectively.

The same strategy was used with the only difference being the use of the appropriate 3 'primer for each mutant. The respective 3 'primers are as follows:

3 'primer for sHASEGP mutant lacking Y 482 - SEQ ID NO: 9 3' primer for mutant lacking F 481 - SEQ ID NO: 10 3 'primer for mutant lacking 1480 - SEQ ID N #: 11 3 'primer for mutant lacking Q 479 - SEQ ID NO: 12 3' primer for mutant lacking P 478 - SEQ ID NO: 13

Generation of additional deletion mutants to determine the minimally active domain of sHASEGP

Additional deletions in blocks of ten to twenty amino acids were generated from the 3 'end of the innermost neutral pH active truncation mutant of sHASEGP, which is sHASEGP up to E 477. The NheI forward primer of SEQ ID NO: 14 is used with an appropriately placed 3 'primer to PCR amplify a sHASEGP deletion mutant of the desired length from a carboxy terminal end. For example, PCR with the primers described in SEQ ID NO: 14 and SEQ ID NO: 26 was used as the 5 'and 3' primers respectively to generate the polypeptide of SEQ ID NO: 49 when expressed at starting from a lresPuro2 vector expression construct. Similarly, PCR with the reverse 3 'primers described in SEQ ID NOS: 27, 28, 29, 30, 31 and 32 was used to generate deletion mutants that terminate at amino acid positions A 447, S 430, G 413, S 394, A 372 and S 347, respectively, of mature sHASEGP. The PCR products in each case were digested with the enzymes NheI and BamHI and the digested product was cloned in vector plresPuro2 between the NheI and BamHI sites. A few independent clones were tested in the final expression construct from each group for active secreted sHASEGP activity at neutral pH by transient transfection into CHO cells in CDCHO serum-free media (Invitrogen, CA) and samples were withdrawn from the cells. time points indicated for testing. DNA minipreps prepared from overnight cultures were used for transfection with Genejuice transfection reagent (Novagen, CA), following manufacturer's recommended protocols. Hyaluronidase activity was measured by microtiter assay as described above.

b. Results

Hyaluronidase activity was measured in sHASEGP truncation mutants to identify the minimally active domain for secreted hyaluronidase activity active at neutral pH.

<td>AMINO ACID 1 A:</td><td>U / ML / 24 H PH 7.4</td>

<td> 347</td><td> 0,000</td>

<td> 372</td><td> 0,000</td>

<td> 394</td><td> 0,000</td>

<td> 413</td><td> 0,000</td>

<td> 430</td><td> 0,000</td>

<td> 447</td><td> 0,000</td>

<td> 467</td><td> 0,089</td>

<td> 477</td><td> 0,567</td>

<td> 478</td><td> 0,692</td>

<td> 479</td><td> 0,750</td>

<td> 480</td><td> 0,575</td>

<td> 481</td><td> 0,740</td>

<td> 482</td><td> 0,329</td>

ES 2 335 005 T3

<td> 483</td><td> 0,800</td>

<td> 509</td><td> 0,044</td>

The results showed that the six one amino acid deletion mutants terminating at the indicated amino acids Y 482 to E 477 provided higher secreted activity than GPI-anchored sHASEGP.

The results also showed that deletions beyond A 467 eliminated any secreted activity. The secreted activity at neutral pH of clones A 467 decreased by approximately 10% from that found in clones P478 or N 483. Therefore, it was concluded that more of the carboxy terminal domain of human sHASEGP was required to generate the domain. hyaluronidase active at neutral pH than previously assumed from bee venom enzyme. Cysteines in the carboxy terminal domain are therefore necessary for activity at neutral pH. Thus, a very narrow range spanning approximately 10 amino acids before the GPI cleavage site at N 483 defined the minimally active domain.

Example 5

Effects of signal peptide modification on sHASEGP secretory activity

Human sHASEGP possesses an extraordinarily long predicted native leader peptide. Furthermore, the existence of two adjacent cysteine ​​residues in the leader peptide can lead to the aggregation of polypeptide multimers within the endoplasmic reticulum during high-level expression and thus prevent high-level expression of a sHASEGP. Therefore, a series of more efficient secretory leader peptides were tested to examine their ability to increase the targeting of sHASEGP for secretion.

to. Protocol

The Kappa leader peptide was constructed by overlapping primer hybridization and extension PCR with primers corresponding to the sequences of SEQ ID NOs: 37, 38, 39, and 40. The resulting PCR-amplified kappa sequence was amplified with flanking primers containing a NheI site at the 5 'end (as described in SEQ ID NO: 41) and an EcoRI site at the 3' end (as described in SEQ ID NO: 42). This allowed the cloning of the Kappa leader peptide (the polypeptide sequence is as described in SEQ ID NO: 43) into the Litmus 39 (NEB) vector between the NheI and EcoRI sites. SHASEGP has an internal EcoRI site; therefore, this kappa construct between the NheI site and EcoRI site was further amplified with a 5 'SpeI primer (as described in SEQ ID NO: 44) and a 3' MluI primer (as described in SEQ ID NO: N °: 45). The non-GPI anchor sHASEGP ending at P 478 was removed by cleavage of plresPuro2 with NheI and BamHI and cloned into a Litmus 39 vector (NEB) at the NheI and BamHI sites of the Litmus39 vector. This resulting sHASEGP-containing Litmus vector was digested with the restriction enzymes SpeI and MluI and the kappa leader construct amplified with SpeI and MluI was cloned therein. Site-directed mutagenesis was performed on this Litmus 39 vector containing both Kappa and sHASEGP sequences to generate in-frame fusion of the Kappa leader sequence with the mature sHASEGP polypeptide. Primer pairs corresponding to SEQ ID NOs: 34 and 35 were used to generate the kappa leader with the native Asp at the terminal amino acid fused to the 38 F of sHASEGP (up to P 478) (as described in SEQ ID No: 46 for the polypeptide sequence of the fusion protein). Other primer pair combinations such as those included in SEQ ID NO: 33 with SEQ ID NO: 35 were used to generate a Kappa leader terminating at the L 36 fused Asp (D) of sHASEGP, the of SEQ ID N °: 33 with SEQ ID N °: 36 were used to generate Kappa leader ending in the Gly (G) (before the terminal Asp (D)) fused with L 36 of sHASEGP and those of SEQ ID N °: 34 with SEQ ID N °: 36 they were used to generate a Kappa ending at the Gly (G) (before the Asp (D) terminal) fused with F 38 of sHASEGP. Kappa-sHASEGP fusions obtained by targeted mutagenesis were gel purified, digested with DpnI enzyme to digest any excess parental DNA, and then digested with NheI and BamHI and cloned into the NheI / BamHI digested HislresPuro2 backbone, which has the His tag (six amino acid spacer followed by six histidines) cloned between the BamH1 and Not1 sites in the vector plRESPuro2. Therefore, after ligation a construct is obtained which is NheI-kappa-sHASEGP-BamHI-His in plresPuro2. Four sets of said construct were obtained that would correspond to the combinations of G or D at the end of the Kappa leader and L36 or F38 at the beginning of mature sHASEGP. A few independent clones of each type of construct were used to transfect CHO cells in CD-CHO medium (Invitrogen, CA) to test whether the sequence of the kappa secretion leader would promote increased levels of secreted protein compared to the native secretion leader. DNA minipreps prepared from overnight cultures were used for transfection with Genejuice transfection reagent (Novagen, CA) following the manufacturer's recommended protocol and samples were removed for assay by microtiter assay at the indicated time points. Hyaluronidase activity was measured by microtiter assay as described above.

Mouse IgG Kappa chain leader peptide-sHASEGP fusion constructs were tested to test for higher levels of secreted sHASEGP activity active at neutral pH.

ES 2 335 005 T3

b. Results

<td>GENE CONSTRUCTION OF HUMAN sHASEGP</td><td>U / ML / 24 HOURS PH 7.4</td>

<td>Kappa Leader of IgG-sHASEGP AA 38-478HIS6</td><td> 3,0257</td>

<td>Native Leader-sHASEGP AA 1-478 HIS6</td><td> 0,4857</td>

Enzyme assay results indicated that the IgG Kappa leader was able to increase sHASEGP secretion approximately 7 to 8 times more than the native secretory leader when compared to clones P478, Y 482 or N 483 lacking said leader. . Other kappa leader constructs with variations of the Asp or Gly leader fusion site of the Kappa leader to sHASEGP L36 or F38 also produced increased levels of secreted hyaluronidase activity active at neutral pH. These examples are intended to broaden rather than limit the scope of the invention, as other efficient secretory leader sequences can be used with the same technology.

Example 6

Generation of a human sHASEGP expression vector

A sHASEGP without an epitopic marker was generated by cloning into a bicistronic expression cassette, HZ24 (SEQ ID NO: 47). The plasmid vector HZ24 for the expression of sHASEGP comprises a pCI vector backbone (Promega), a DNA sequence encoding amino acids 1-482 of human PH20 hyaluronidase, an internal ribosome entry site (IRES) of the ECMW virus ( Clontech), and the mouse dihydrofolate reductase (DHFR) gene. The pCI vector backbone also includes a DNA encoding the Beta-lactamase resistance (AmpR) gene, an f1 origin of replication, a cytomegalovirus (CMV) immediate early enhancer / promoter region, a chimeric intron, and a signal from late polyadenylation of SV40 (SV40). The DNA encoding the sHASEGP construct contained a Kozak consensus sequence on the native signal leader Methionine and a stop codon on Tyrosine 482. The resulting construct pCI-PH20-IRES-DHFR-SV40pa (HZ-24) results in a single mRNA species driven by the CMV promoter encoding amino acids 1-482 of PH20 and amino acids 1-187 of dihydrofolate reductase. separated by the internal site of entry to the ribosome.

The human PH20 open reading frame was amplified from an Invitrogen ORF clone (IOH10647, Invitrogen, Carlsbad CA) with a 5 'primer introducing a NheI site and a Kozack consensus sequence before the PH20 Methionine and a reverse primer that introduced a stop codon after Tyrosine 482 and introduced a BamH1 restriction site. The resulting PCR product was ligated into plasmid plRESpuro2 (Clontech, Palo Alto, CA) after digestion of the PH20 PCR fragment with NheI and BamHI.

Example 7

Generation of a cell line expressing sHASEGP

Non-transfected CHO DG44 cells growing in GIBCO modified CD-CHO medium for DHFR (-) cells supplemented with 4 mM Glutamine and 18 ml of Plurionic F68 / L (Gibco) were seeded, at 0.5 x 10⁶ cells / ml in a shaker flask in preparation for transfection. Cells were grown at 37 ° C in a humidified CO incubator.₂ 5% with 120 rpm for agitation. Non-transfected CHO DG44 cells were tested in exponential growth for viability prior to transfection.

60,000,000 viable cells from the non-transfected CHO DG44 cell culture were pelleted and resuspended at a density of 20,000,000 cells in 0.7 ml of 2x transfection buffer (2X HeBS = 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mM KCl, Na₂HPO₄ 1.4 mM, 12 mM dextrose). To each aliquot of resuspended cells, 0.09 ml of the linear HZ24 plasmid (250 pg) was added and the cell / DNA solutions were transferred to 0.4 cm gap BTX electroporation cuvettes (Gentronics) at room temperature. Negative control electroporation was performed without mixed plasmid DNA with the cells. The cell / plasmid mixtures were used for electroporation with a capacitor discharge of 330 V and 960 pF or to 350 V and 960 pF.

The cells were removed from the cuvettes after electroporation and transferred to 5 ml of modified CD-CHO media for DHFR (-) cells supplemented with 4 mM Glutamine and 18 ml of Plurionic F68 / L (Gibco) and allowed to grow in one well of a 6-well tissue culture plate without selection for 2 days at 37 ° C in a humidified CO incubator₂ at 5%.

Two days after electroporation, 0.5 ml of tissue culture medium was removed from each well and assayed for the presence of hyaluronidase activity.

ES 2 335 005 T3

Initial Hyaluronidase Activity of CHO DG44 Cells Transfected with HZ24 at 40 Hours Post-Transfection

<td></td><td>Dilution</td><td>Activity Units / ml</td>

<td>Transfection 1 to 330V</td><td>1 to 10</td><td> 0,25</td>

<td>Transfection 2 at 350V</td><td>1 to 10</td><td> 0,52</td>

<td>Negative Control</td><td>1 to 10</td><td> 0,015</td>

Cells from transfection 2 (350V) were harvested from the tissue culture well, counted, and diluted to 10,000 to 20,000 viable cells per ml. A 0.1 ml aliquot of the cell suspension was transferred to each well of five 96-well round bottom tissue culture plates. 0.1 ml of CD-CHO media (GIBCO) containing 4 mM Glutamax-1 and without hypoxanthine and thymidine supplements was added to the wells containing cells (0.2 ml final volume).

Ten clones were identified from the 5 plates grown without methotrexate.

<td>Plate / Well ID</td><td>Relative Hyaluronidase Activity</td>

<td>1C3</td><td> 261</td>

<td>2C2</td><td> 261</td>

<td>3D3</td><td> 261</td>

<td>3E5</td><td> 243</td>

<td>3C6</td><td> 174</td>

<td>2G8</td><td> 103</td>

<td>1B9</td><td> 304</td>

<td>2D9</td><td> 273</td>

<td>4D10</td><td> 302</td>

<td>1 on 1</td><td> 242</td>

<td>control (+) A1</td><td> 333</td>

<td>control (-) H12</td><td> 0</td>

Six HZ24 clones were expanded in culture and transferred to shaker flasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9, 1E11, and 4D10 were seeded in 96-well round bottom tissue culture plates using a two-dimensional infinite dilution strategy. The diluted clones were cultured in a background of 500 non-transfected CHO DG44 cells per well to provide necessary growth factors for the initial days in culture. Ten plates were prepared per subclone.

Clone 3D3 produced 24 visual subclones. Significant hyaluronidase activity was measured in the supernatants of 8 of the 24 subclones (&gt; 50 Units / ml) and these 8 subclones were expanded in T25 tissue culture flasks in the presence of 50 nM methotrexate. The 50 nM 3D3 clone was further expanded in 500 nM methotrexate giving rise to clones that produced an excess of 1,000 Units / ml in shaker flasks (5M clone 3D3).

Example 8

SHASEGP production

A vial of 5 M 3D3 was thawed and expanded from T flasks to 1 L spinner flasks in CHO CDM (Invitrogen, Carslbad CA) supplemented with 100 nM Methotrexate and Glutamax (Invitrogen). Cells were transferred from roller flasks to a 5 L bioreactor (Braun) at an inoculation density of 4.0 x 10E5 viable cells per ml. Parameters were temperature set point, 37 ° C, pH 7.2 (starting set point), with 25% dissolved oxygen set point and 0-100 cc / min air cover. At 168 h, 250 ml of Culture Medium No. 1 (CD CHO + Glucose 50 g / l) were added. At 216 hours, 250 ml of Culture Medium No. 2 were added

ES 2 335 005 T3 (CD CHO + Glucose 50 g / l + Sodium Butyrate 10 mM) and at 264 hours 250 ml of Culture Medium No. 2 were added. This process resulted in a final productivity of 1600 Units per ml with a maximum cell density of 6 million cells / ml. The addition of sodium butyrate was found to dramatically increase sHASEGP production in the final stages of production.

3D3-5 M Culture and sHASEGP Production, 5 L Bioreactor

<td>Hours of Prosecution</td><td>Cells Viable x 10E5</td><td>% Viable</td><td>Units / ml</td><td>Vol (ml)</td><td>[Glucose]</td><td>Culture</td>

<td> 0</td><td> 4,4</td><td> 100</td><td> 0</td><td> 4500</td><td> 547</td><td></td>

<td> 24</td><td> 5,7</td><td> 100</td><td> 0</td><td> 4500</td><td> 536</td><td></td>

<td> 48</td><td> 10,1</td><td> 100</td><td> 37</td><td> 4500</td><td> 501</td><td></td>

<td> 72</td><td> 17,1</td><td> 99</td><td> 62</td><td> 4500</td><td> 421</td><td></td>

<td> 96</td><td> 28,6</td><td> 99</td><td> 118</td><td> 4500</td><td> 325</td><td></td>

<td> 120</td><td> 28,8</td><td> 99</td><td> 240</td><td> 4500</td><td> 274</td><td></td>

<td> 144</td><td> 60,2</td><td> 100</td><td> 423</td><td> 4500</td><td> 161</td><td></td>

<td> 168</td><td> 55</td><td> 100</td><td> 478</td><td> 4500</td><td> 92</td><td>250 ml Crop N ° 1</td>

<td> 192</td><td> 66,6</td><td> 98</td><td> 512</td><td> 4750</td><td> 370</td><td></td>

<td> 216</td><td> 55,2</td><td> 92</td><td> 610</td><td> 4750</td><td> 573</td><td>250 ml Crop N ° 2</td>

<td> 240</td><td> 53</td><td> 88</td><td> 710</td><td> 5000</td><td> 573</td><td></td>

<td> 264</td><td> 49,8</td><td> 84</td><td> 852</td><td> 5000</td><td> 474</td><td>250 ml Crop N ° 2</td>

<td> 288</td><td> 40</td><td> 70</td><td> 985</td><td> 5250</td><td> 770</td><td></td>

<td> 312</td><td> 31</td><td> 61</td><td> 1467</td><td> 5250</td><td> 773</td><td></td>

<td> 336</td><td> 25,4</td><td> 52</td><td> 1676</td><td> 5250</td><td> 690</td><td></td>

Example 9

SHASEGP purification

Conditioned media from clone 3D3 was clarified by depth filtration and tangential flow diafiltration in 10 mM Hepes at pH 7.0. Soluble HASeGp was then purified by sequential ion exchange chromatography on Q Sepharose (Pharmacia), hydrophobic interaction chromatography on Phenyl Sepharose (Pharmacia), phenyl boronate chromatography (Prometics) and Hydroxyapatite (Biorad, Richmond, CA).

SHASEGP bound to Q Sepharose and eluted at 400 mM NaCl in the same buffer. The eluate was diluted with 2M ammonium sulfate to a final concentration of 500 mM ASO4 and passed through a Phenyl Sepharose (low sub) column, followed by binding under the same conditions to a phenyl boronate resin. The sHASEGP was eluted from the phenyl sepharose resin in Hepes pH 6.9 after washing at pH 9.0 in 50 mm bicine without ASO4. The eluate was loaded onto a ceramic hydroxyapatite resin at pH 6.9 in 5 mM PO4, 1 mM CaCl2 and eluted with 80 mM PO4 pH 7.4 with 0.1 mM CaCl2.

The resulting purified sHASEGP possessed a specific activity in excess of 65,000 USP Units / mg protein by microturbity assay using the USP reference standard. The purified sHASEGP eluted

ES 2 335 005 T3 as a single 24 to 26 minute peak from a Pharmacia 5RPC styrene divinylbenzene column with a gradient between 0.1% TFA / H₂O and 0.1% TFA / 90% acetonitrile / H₂O at 10% and resolved as a single broad band of 61 kDa by SDS electrophoresis which was reduced to a narrow band of 51 kDa after treatment with PNGASA-F. N-terminal amino acid sequencing revealed that the leader peptide had been efficiently removed.

Biochemically purified sHASEGP N-terminal Amino Acid Sequence

<td>Position</td><td> 1</td><td> 2</td><td> 3</td><td> 4</td><td> 5</td><td> 6</td><td> 7</td><td> 8</td><td> 9</td><td> 10</td><td> 11</td>

<td>Theoretical</td><td>Leu</td><td>Asn</td><td>Phe</td><td>Arg</td><td>To</td><td>Pro</td><td>Pro</td><td>Val</td><td>lie</td><td>Pro</td><td>Asn</td>

<td>Observed</td><td> -</td><td>Asn</td><td>Phe</td><td>Arg</td><td>To</td><td>Pro</td><td>Pro</td><td>Val</td><td>lie</td><td>Pro</td><td>Asn</td>

Example 10

Glycosylation analysis of sHASEGP obtained from CHO DG44

There are conflicting data as to whether sHASEGP from different species require glycosylation for their catalytic activity. For example, it is disclosed that enzymatically active bee venom hyaluronidase can be synthesized in cells lacking the glycosylation machinery, ie, such as E. coli. Furthermore, PNGase treatment of purified bovine testes hyaluronidase did not inactivate enzymatic activity (Yamagata et al 1997). Other studies describe loss of activity after deglycosylation and that disulfide bonds are additionally required.

Since all of these previous tests were performed using crude or partially purified preparations, it was not evident however whether the loss of activity was the result of exposure of deglycosylated enzyme to contaminating proteases in the crude preparations or a direct functional relationship between glycosylation and activity. catalytic.

to. Protocol

To determine whether functional N-linked glycosylation could be introduced into human sHASEGP using a CHO-based expression system under protein-free conditions, a cDNA encoding human sHASEGP-HIS6 was expressed in CHO cells using a pure IRES bicistronic cassette in chemically defined media. Cells were grown for 72 hours in CHO CDM (Invitrogen / Gibco) followed by concentration and tangential flow diafiltration on a Pellicon TFF unit (Millipore) with 30 kDa cut-off membranes. The concentrate was exchanged with 10 mM Hepes pH 7.4 50 mM NaCl. The diafiltrate was then loaded onto DEAE non-turbulent stream sepharose resin and eluted with a gradient of NaCl from 0-1 M NaCl on FPLC resin from Pharmacia. Human sHASEGP was eluted between 10-30% NaCl. The sHASEGP levels in column fractions determined that the majority of the enzyme was recovered in the 10-30% NaCl gradient. The 10-30% NaCl gradient enzyme was then further purified by affinity chromatography on a Ni-loaded IMAC resin. Human sHASEGP was eluted from IMAC resin after washing with 10 mM Imidizole with 50 mM Acetate pH 5.0. The protein was concentrated and dialyzed against 10 mM Hepes at pH 7.4. The highly purified enzyme was determined to possess a specific activity of 97,000 Units / mg protein in the presence of 1 mM Calcium and 1 mg / ml HSA in the ELISA-based biotinylated substrate microtiter assay.

To detect changes in the relative molecular mass of protein, purified human sHASEGP was treated with PNGASA or Neuraminidase overnight followed by gel electrophoresis, electroblotting and western blot analysis with an anti-His6 monoclonal antibody bound to HRP (Qiagen) and detection with ECL.

b. Results

Western blot analysis determined that human sHASEGP produced in CHO cells was sensitive to PNGASA treatment. The relative molecular mass of human sHASEGP revealed that the protein was highly glycosylated. After a complete digestion overnight with PNGASA, human sHASEGP was reduced to a single species, confirming that a slight heterogeneity of the undigested band could be attributed to trace amounts of N-linked sugars. Partial PNGaseF digestion showed a series of intermediates that shifted from no treatment and progressively shifted with longer treatment. Although the bands were somewhat diffuse on a 7% gel, at least 6 different intermediate isoforms could be visualized.

Treatment of sHASEGP with Neuraminidase revealed that CHO cells were indeed capable of synthesizing sialated human sHASEGP. After treatment with neuraminidase and Western blot analysis

ES 2 335 005 T3 of sHASEGP on 7% Gels, recombinant human sHASEGP derived from CHO exhibited an approximately 1-3 kDa shift in motility compared to untreated sHASEGP. This is therefore the first generation report of a substantially sialated human sHASEGP. This is very valuable both for the stability and for increasing the serum half-life of a human sHASEGP, since the native sperm sHASEGP of many species lacks sialation and does not react with specific sialic acid lectins.

SHASEGP FACE Analysis

Oligosaccharide analysis of active sHASEGP by FACE analysis allows rapid determination of catalytically active sHASEGP profiles.

Protocol

Purified hyaluronidase from clone 3D3 5M was evaluated using N-FACE®-Linked Oligosaccharide Profiling (Prozyme). The oligosaccharides were cleaved from 128.7 pg of glycoproteins by enzymatic digestion with N-Glycanase (also known as PNGase), labeled using the ANTS fluorophore, and separated by electrophoresis. The relative positions of the oligosaccharide bands were determined by running the sample and sample dilutions alongside an oligosaccharide standard ladder designating the migration distance in Polymerization Degree (DP) units.

Results

Profile N for the hyaluronidase sample consists of ten bands of which six (which are processed at the same time as the G5-G12 oligosaccharide pattern bands) have intensities greater than 9%. Furthermore, the band that runs next to the G9 pattern was the most intense with intensities of 35% -46%.

sHAS

EGP oligosaccharide analysis

<td>SHASEGP oligosaccharide</td><td>Degree of Polymerization</td><td>Total Percentage</td>

<td> 1</td><td> 15,64</td><td> 1,2</td>

<td> 2</td><td> 13,68</td><td> 3,4</td>

<td> 3</td><td> 11,61</td><td> 10,0</td>

<td> 4</td><td> 10,04</td><td> 10,4</td>

<td> 5</td><td> 8,37</td><td> 35,4</td>

<td> 6</td><td> 7,32</td><td> 9,7</td>

<td> 7</td><td> 6,14</td><td> 9,0</td>

<td> 8</td><td> 5,57</td><td> 12,4</td>

<td> 9</td><td> 3,84</td><td> 2,3</td>

<td> 10</td><td> 3,26</td><td> 0,5</td>

Example 11

Dependence of N-linked glycosylation of sHASEGP for enzymatic activity

to. Protocol

Samples of purified HIS6-sHASEGP were mixed with buffer containing Neuraminidase and PNGASA with and without 50 mM Octylglucoside overnight at 37 ° C. Oligosaccharides were verified to have been removed by gel shift from Western blot analysis.

ES 2 335 005 T3

b. Results

<td>SAMPLE</td><td>U / ML</td>

<td>Without reaction</td><td> 22,01</td>

<td>Neuraminidase overnight (O / N) OG 50 mM</td><td> 23,57</td>

<td>PNGaseF with OG 50 mM</td><td> 0,0</td>

<td>PNGaseF without OG 50 mM overnight (o / n)</td><td> 10,74</td>

Example 12

SHASEGP activity towards sulfated and unsulfated glycosaminoglycans

In addition to the microtiter-based assay using HA, the substrate specificity of sHASEGP towards other glycosaminoglycans or proteoglycans can be assayed using a gel shift assay with purified substrates to determine the activity of sHASEGP towards other glycosaminoglycans. Many hyaluronidase assays have been based on the measurement of the generation of new N-acetylamino reducing groups (Bonner and Cantey, Clin. Chim. Acta 13: 746-752, 1966) or loss of viscosity (De Salegui et al., Arch. Biochem. Biophys. 121: 548-554, 1967) or turbidity (Dorfman and Ott, J. Biol. Chem. 172: 367 , 1948). With purified substrates, all these methods are sufficient for the determination of the presence or absence of endoglucosamide activity.

to. Protocol

Gel shift assay - Purified substrates are mixed with recombinant sHASEGP to assay for endoglucosidase activity that results in increased substrate motility within the gel. Chondroitin Sulfate A, Agrecano and D were from Calbiochem. Hyaluronan (Human Umbilical Cord), Chondroitin Sulfate C, Dermatan Sulfate and Heparan Sulfate were obtained from Calbiochem. Human umbilical cord hyaluronan was obtained from ICN. Each test substrate is diluted to 0.1 mg / ml. 10 µl samples of purified sHASEGP or conditioned media from cells expressing sHASEGP are also mixed with 90 µl of assay substrate in desired buffer and incubated for 3 hours at 37 ° C. After incubation the samples are neutralized with sample buffer (Tris EDTA pH 8.0, Bromophenol Blue and glycerol) followed by electrophoresis in 15% polyacrylamide gels. Glycosaminoglycans were detected by staining the gels in 0.5% Alcian Blue in 3% Glacial Acetic Acid overnight followed by fading in 7% Glacial Acetic Acid. Degradation is determined by comparison of substrate motility in the presence or absence of enzyme.

b. Results

10 Units of sHASEGP were incubated_His6 in 10 µl with 90 µl of 10 mM Hepes Buffer with 50 jug / ml Human Serum Albumin for 2 hours at 37 ° C containing 10 µg of various glycosaminoglycans and proteoglycans. Electrophoretic analysis followed by Alcian blue staining revealed increased motility shifts for a single species in Chondritin Sulfate A, C and D, Agrecan and Hyaluronan but not Heparan Sulfate or Chondritin B Sulfate. While the undigested glycosaminoglycans ran as a spot in the middle of the gel, the digested products showed that the majority of the alcian blue staining ran in front of the dye, running off a small amount of material as a gradual slate.

Example 13

Effects of metal ions on sHASEGP activation

In addition to the need for glycosylation for optimal enzyme activity, human sHASEGP was found to be activated with cations for optimal enzyme activity. In the purification process, sHASEGP was found to have low specific activity after successive chromatography steps. HIS6-labeled sHASEGP was found to have very low specific activity when purified to homogeneity from DEAE followed by successive Ni-IMAC purifications. Since IMAC resins can chelate metal ions, various metals were added back to sHASEGP to determine relative enzyme activity.

ES 2 335 005 T3

to. Protocol

Purified sHASEGP was assayed after incubation with Nickel (Ni), Cobalt (Co), Zinc (Zn) Calcium (Ca) and Magnesium (Mg) 0.1 mM for 2 hours at room temperature followed by determination of hyaluronidase activity in a microtiter-based assay.

b. Results

<td>Metal Salt Additive</td><td>Neutral Activity U / ml</td>

<td>WITHOUT ADDITIVES</td><td> 11, 909</td>

<td>Ni 100 μΜ</td><td> 6,0306</td>

<td>Co100 μΜ</td><td> 8,972</td>

<td>Zn100 μΜ</td><td> 3,7476</td>

<td>Ca100 μΜ</td><td> 101,9892</td>

A significant increase in hyaluronidase activity was found after incubation of sHASEGP with 0.1 mM Calcium or 0.1 mM Magnesium. No such activation was discovered after incubation with other metals. The addition of Calcium to sHASEGP increased the specific activity of the enzyme to approximately 97,000 units per milligram of protein based on measurement at A280. A Calcium and Magnesium metal dose response curve was then tested to determine the optimal concentration of metal ions relative to enzyme.

<td>mM Divalent Metal</td><td>[Ca ++]</td><td>[Mg ++]</td>

<td> 100</td><td> 1</td><td> 1,3</td>

<td> 10</td><td> 108</td><td> 104</td>

<td> 1</td><td> 169</td><td> 164</td>

<td> 0,1</td><td> 123</td><td> 78</td>

<td> 0,01</td><td> 59</td><td> 18</td>

<td> 0,001</td><td> 47</td><td> 13</td>

<td> 0,0001</td><td> 39</td><td> 13</td>

<td> 0,00001</td><td> 55</td><td> 15</td>

Activation of sHASEGP was found to occur in the micromolar range. Concentrations above 10 mM were inhibitory for both Calcium and Magnesium. To rule out nonspecific substrate rather than enzyme activation, Calcium Chloride was incubated in 10 mM Hepes buffer with the biotinylated substrate immobilized on the microtiter plate followed by washing. No activation was found when the enzyme was added to the calcium preincubated plate that had been washed. Activation was also tested on native sHASEGP released by phospholipase C which revealed similar activation with Calcium, ruling out a carboxy terminal HIS6 epitopic marker artifact.

Example 14

Effects of albumin on sHASEGP activity

Recombinant rHUPH20 dilution and other hyaluronidase preparations obtained from slaughterhouse testes were found to require albumin in addition to Calcium for optimal activity.

ES 2 335 005 T3

to. Protocol

Human Serum Albumin (ICN) was diluted in 10 mM Hepes buffer with Calcium to determine the effects of the protein albumin on enzyme activity. Enzyme assays with sHASEGP and commercial preparations using both 1 mM CaCl2 and 1 mg / ml Human Serum Albumin were examined.

b. Results

Activation of hyaluronidase activity was discovered at high dilutions in the presence of albumin. It was not clear whether this activity was the result of preventing denaturation or whether albumin affected substrate availability. A preferable formulation of human sHASEGP could therefore include Albumin and a metal salt consisting of Calcium or Magnesium.

Example 15

In vivo purified sHASEGP propagation activity

to. Protocol

Purified sHASEGP was diluted in 10 mM Hepes pH 7.4, 150 mM NaCl, 0.1% Pluronic to 0.5 U / µl in pyrogen-free water with 0.15 M NaCl. A series of final 20 µl dilutions were made of saline to give a total of 0.01, 0.05, 0.1 Units per injection. 20 µl of Trypan Blue solution was added to a final volume of 40 µl and injected subcutaneously into the lateral skin on each side of balb mice.^{Nu / Nu} who had previously been anesthetized

ip by administration of ketamine / xylazine. The dye areas were measured in 2 dimensions with a microcalibrator at t = 0 at t = 45 min. The area was represented as mm². Recombinant human HYAL1 was included as a control which lacks activity at neutral pH but is secreted.

b. Results

<td>ESSAY ITEM</td><td>AREA WITH COLORANT AT 45 MIN</td>

<td>A. Saline Solution Control</td><td>51.5 mm²</td>

<td>B. sHASEGP 0.01 U</td><td>76.8 mm²</td>

<td>C. sHASEGP 0.05 U</td><td>98.22 mm²</td>

<td>D. sHASEGP 0.10 U</td><td>180.4 mm²</td>

<td>E. HYAL1 100 U</td><td>67.48 mm²</td>

Example 16

Kinetics of diffusion activity of sHASEGP

to. Protocol

SHASEGP separated_His6 purified recombinant in 2 aliquots. One was heated at 95 ° C for 15 minutes in a thermal cycler with a heated lid. The other remained at room temperature. Thermal inactivation of enzyme activity was verified in the microtiter-based enzyme assay. For kinetic analysis, heat inactivated material was tested against native. 4 Units of purified sHASEGP or equivalent heat inactivated material were injected subcutaneously with trypan blue dye. Areas were tested at various time points up to 15 minutes.

ES 2 335 005 T3

b. Results

<td>4 UNITS minutes after injection</td><td>4 UNITS INACTIVATED BY HEAT minutes after injection</td>

<td>t₀ = 52,38</td><td>to = 50.58</td>

<td>t₃ = 116,51</td><td>T₃ =65,48</td>

<td>t₆,5= 181,93</td><td>T₆,5 =63,87</td>

<td>t_w =216,96</td><td>Tw = 65.80</td>

<td>tie = 279.99</td><td>Tie = 74.3</td>

Example 17

Restoration of the dermal barrier decomposed by sHASEGP

to. Protocol

To establish the regeneration time of open pores with sHASEGP after subcutaneous administration, 2 Units of purified sHASEGP or saline control were injected into two opposite lateral sites subcutaneously in animals at t = 0, followed by injection with trypan blue in the same place at 30 min, 60 min and 24 hours. Dye diffusion area was recorded at t = 15 minutes post-injection for each time point compared to the control.

b. Results

<td>2 UNITS Thoras post injection of sHASEGP</td><td>SHASEGP post injection thoras SALINE SOLUTION CONTROL</td>

<td>to, 5 h = 1 83</td><td>to, 5 h = 54</td>

<td>ti h = 167</td><td>ti h = 50</td>

<td>Í22h = 61</td><td>Í22 h = 48</td>

The two results show that the dermal barrier is reconstituted 24 hours after the administration of 2 Units of enzyme.

Example 18

Determination of the size of the channels opened by sHASEGP

The channels opened by human sHASEGP in the interstitial space were shown to be sufficient to allow diffusion of a small molecule, ie, trypan blue dye. However, the upper limits on the size of particles that could diffuse in the presence of sHASEGP were unknown.

to. Protocol

Fluorescent molecules of varying sizes were used to determine the size of the channels opened by human sHASEGP. Fluorescein-treated dextrans of Average Molecular Weight of 4,400 and 2 million Da (Sigma) as well as fluorescein-labeled beads with defined diameters of 20 nanometers to 500 nanometers (Molecular Probes), were administered subcutaneously in a volume of 40 pl , following injection of sHASEGP or saline control at the same sites. The area of ​​the dye front was then measured in two dimensions at 15 minutes post-injection.

ES 2 335 005 T3

b. Results

<td>Broadcast Agent</td><td>Particle Size Diffusion Assay</td><td>Area at 15 min</td><td>Dev. Typ.</td>

<td>sHASEGP</td><td>4400 Da</td><td> 84,2</td><td> 25,7</td>

<td>Control</td><td>4400 Da</td><td> 38,0</td><td> 5,8</td>

<td>sHASEGP</td><td>2x 10E6Da</td><td> 141,2</td><td> 4,5</td>

<td>Control</td><td>2x 10E6Da</td><td> 51,7</td><td> 8,1</td>

<td>sHASEGP</td><td>20nm Diameter</td><td> 92,3</td><td> 20,6</td>

<td>Control</td><td>20nm Diameter</td><td> 51,6</td><td> 3,0</td>

<td>sHASEGP</td><td>100nm Diameter</td><td> 61,0</td><td> 5,7</td>

<td>Control</td><td>100nm Diameter</td><td> 40,0</td><td> 7,0</td>

<td>sHASEGP</td><td>200nm Diameter</td><td> 35,5</td><td> 1,6</td>

<td>Control</td><td>200nm Diameter</td><td> 27,9</td><td> 8,2</td>

<td>sHASEGP</td><td>500nm Diameter</td><td> 44,8</td><td> 13,6</td>

<td>Control</td><td>500nm Diameter</td><td> 41,2</td><td> 9,8</td>

The results demonstrated that molecules of approximately 1 kDa (Trypan Blue) at 50 nm in diameter (Latex Beads) showed increased diffusion after administration of sHASEGP. While bovine serum albumin (66 kDa) exhibited similar diffusion kinetics to trypan blue, 50 nm latex beads required significantly longer to diffuse. The 500 nm beads did not show diffusion until 480 minutes.

Example 19

Serum pharmacokinetic profiles of biotinylated antibodies after subcutaneous co-injection of human sHASEGP

to. Protocol

Female Balb / c mice were anesthetized with a ketamine / xylazine mixture. The mice were then injected subcutaneously with 20 µl of a 0.5 mg / ml solution of biotinylated mouse IgG mixed with 20 µl of saline or 20 µl of sHASEGP containing 4 Activity Units.

b. Results

<td>POST-INJECTION TIME</td><td>CONTROL</td><td>sHASEGP (4U)</td>

<td>Serum IgG t = 0 h</td><td>0 ng / ml</td><td>0 ng / ml</td>

<td>Serum IgG t = 2 h</td><td>0 ng / ml</td><td>360 ng / ml</td>

<td>Serum IgG t = 51 h</td><td>4152 ng / ml</td><td>4176 ng / ml</td>

The results demonstrate that sHASEGP increases the serum distribution kinetics of circulating large molecules. When no biotinylated IgG could be detected in the control group at 2 hours, 360 ng / ml was evident at 2 hours in the sHASEGP group.

ES 2 335 005 T3

Example 20

Spreading Activity of Subcutaneously Injected Molecules Following Intravenous Injection of Human sHASEGP

to. Protocol

Four dye injection sites were used per dose of each vehicle control and test article. The dye injection was 45 minutes after the iv injection. Each dose of test or control article was injected iv into 2 animals. Dye front area measurement after 45 minutes of enzyme administration was calculated at 2.5, 5, 10 and 15 minutes for each dose or vehicle control.

b. Results

The results demonstrated that highly purified sHASEGP was available systemically to distal tissues after intravenous administration. The spreading activity of sHASEGP administered systemically was dose dependent, with a 10 unit injection being indistinguishable from vehicle control.

<td>Guy</td><td>IV dose</td><td>Time Minutes</td><td>Average Area (mm²)</td><td>DT</td>

<td>PH20</td><td> 1000</td><td> 2,5</td><td> 86,417</td><td> 2,834193</td>

<td>PH20</td><td> 1000</td><td> 5</td><td> 102,17</td><td> 2,221146</td>

<td>PH20</td><td> 1000</td><td> 10</td><td> 124,53</td><td> 6,304944</td>

<td>PH20</td><td> 1000</td><td> 15</td><td> 129,81</td><td> 1,434319</td>

<td>PH20</td><td> 300</td><td> 2,5</td><td> 59,137</td><td> 7,218615</td>

<td>PH20</td><td> 300</td><td> 5</td><td> 73,638</td><td> 7,51197</td>

<td>PH20</td><td> 300</td><td> 10</td><td> 87,092</td><td> 8,686008</td>

<td>PH20</td><td> 300</td><td> 15</td><td> 92,337</td><td> 10,66466</td>

<td>PH20</td><td> 100</td><td> 2,5</td><td> 56,308</td><td> 7,741934</td>

<td>PH20</td><td> 100</td><td> 5</td><td> 63,156</td><td> 11,42052</td>

<td>PH20</td><td> 100</td><td> 10</td><td> 76,519</td><td> 16,18449</td>

<td>PH20</td><td> 100</td><td> 15</td><td> 77,432</td><td> 17,32264</td>

<td>PH20</td><td> 30</td><td> 2,5</td><td> 50,534</td><td> 10,64287</td>

<td>PH20</td><td> 30</td><td> 5</td><td> 59,493</td><td> 5,163971</td>

<td>PH20</td><td> 30</td><td> 10</td><td> 68,102</td><td> 11,00071</td>

<td>PH20</td><td> 30</td><td> 15</td><td> 71,118</td><td> 9,934212</td>

<td>PH20</td><td> 10</td><td> 2,5</td><td> 36,4</td><td> 3,807072</td>

<td>PH20</td><td> 10</td><td> 5</td><td> 39,859</td><td> 6,680932</td>

<td>PH20</td><td> 10</td><td> 10</td><td> 45,649</td><td> 4,44936</td>

<td>PH20</td><td> 10</td><td> 15</td><td> 48,41</td><td> 6,546835</td>

<td colspan="2">Control 0</td><td> 2,5</td><td> 34,652</td><td> 5,935037</td>

<td colspan="2">Control 0</td><td> 5</td><td> 36,279</td><td> 3,614544</td>

<td colspan="2">Control 0</td><td> 10</td><td> 44,687</td><td> 5,821216</td>

<td colspan="2">Control 0</td><td> 15</td><td> 53,002</td><td> 2,812439</td>