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

[0001] This application claims priority to the application filed March 5, 2003, United States, serial number 60 / 452,360 in accordance with USC§119 (e).

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION [0002] The present invention generally relates to neutral pH, soluble glycoproteins with hyaluronidase activity (sHASEGP), parts thereof, in particular domains with hyaluronidase activity. More specifically, the invention relates to chemical modifications, pharmaceutical compositions, expression plasmids, production methods and therapeutic methods using hyaluronidase glycoproteins and their domains and nucleic acid molecules encoding them to modify glycosaminoglycans for therapeutic use in disease treatment as well as their use to increase the diffusion of injected animals particles with a diameter smaller than 200 nm.

BACKGROUND OF THE INVENTION [0003] Glycosaminoglycans (GAGs) are complex linear extracellular matrix polysaccharides (ECMs). A characteristic feature of GAG are repetitive disaccharide subunits consisting of N-substituted D-hexosamine and uronic acid [hyaluronan (HA), chondroitin sulfate (CS), chondroitin (C), dermatan sulfate (DS), heparan sulfate (HS), heparin ( H)] or galactose [keratan sulfate (KS)]. With the exception of HA, all GAGs are in a form covalently bound to core proteins. Due to its structure, GAG in combination with core proteins are structurally classified as proteoglycans.

[0004] In mammals, hyaluronan (HA) occurs mainly in connective tissue, skin, cartilage and synovial fluid. Hyaluronan is also the main component of the vitreous humor of the eye. In connective tissue, water molecules derived from hydrated hyaluronan shape the space between the tissues, thus creating an environment conducive to the movement of cells and their proliferation. Hyaluronan plays a key role in biological phenomena associated with cell motility, including phenomena such as rapid development, regeneration, repair process, embryogenesis, embryonic development, wound healing process, angiogenesis, tumorogenesis (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, the level of hyaluronan correlates with the degree of cancer 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).

[0005] HA is found in the extracellular matrix of many tissues, in particular in soft connective tissues. He is credited with various physiological functions, such as maintaining protein homeostasis in water and plasma (Laurent TC et al. (1992) FASEB J 6: 2397-2404). HA production increases in proliferating cells and may play a role in mitosis. It is also involved in cell motility and migration and appears to play an important role in regulation, development and differentiation (Laurent et al., Supra).

[0006] HA has found use in clinical medicine. Its protective and rheological properties have proved useful in eye surgery to protect the corneal endothelium during cataract removal surgery. The presence of HA in serum is a diagnostic marker of liver diseases and various inflammations such as rheumatoid arthritis. Interstitial edema due to the accumulation of HA can lead to a disturbance in the work of many different organs (Laurent et al., Supra).

[0007] Interactions between hyaluronan and proteins are also involved in maintaining the structure of the extracellular matrix or base substance.

[0008] Hyaluronidases are enzymes found in animals and operating at neutral or acidic pH. They differ in substrate specificity and mechanism of action.

[0009] There are three main classes of hyaluronidases:

[0010] 1. Mammalian hyaluronidases (EC 3.2.1.35) are endo-beta-N-acetylhexosaminidase, and their main end product is tetra- or hexasaccharides. These enzymes possess both hydrolytic and transglycosidase activity and may degrade hyaluronan and chondroitin (CS) sulfate groups, especially C4-S and C6-S chondroitins.

[0011] 2. Bacterial hyaluronidases (EC 4.2.99.1) degrading hyaluronan and to a different degree CS and DS. They are endo-beta-N-acetylhexosaminidases acting through a beta-elimination reaction, whose final product is mainly disaccharides.

[0012] 3. Hyaluronidases (EC 3.2.1.36) found in leeches, other parasites and crustaceans are endo-beta-glucuronidases whose end products, tetra- and hexasaccharide, are formed by hydrolysis of β (1,3) binding.

[0013] Mammalian hyaluronidases can be divided into two subgroups: enzymes operating at neutral and acidic pH. Six genes coding for hyaluronidase enzymes have been identified in the human genome: HYAL1, HYAL2, HYAL3, HYAL4, HYALP1 and PH20 / SPAM1. HYALP1 is a pseudogen, and HYAL3 showed no activity of this enzyme on any known substrate. HYAL4 is chondroitinase and does not hydrolyze hyaluronan. HYAL1 is a prototype enzyme representative active at acidic pH, and PH20 is a prototype enzyme active at neutral pH. Acid-active hyaluronidases such as HYAL1 and HYAL2 lose their activity in a neutral pH environment, e.g. HYAL1 does not have in vitro catalytic capacity in an environment above pH 4.5 (Frost et al. Anal Biochemistry, 1997). HYAL2 is an enzyme operating at acidic pH, with very low specificity in vitro.

[0014] Enzymes from the hyaluronidase group also include those that are attached to the cell membrane by a phosphatidylinositol anchor, such as human HYAL2 and PH20 (Danilkovitch-Miagkova, et al. Proc Natl Acad Sci USA. 2003 Apr 15; 100 (8): 4580 -5, Phelps et al., Science 1988) and those that are soluble, e.g., human HYAL1 (Frost et al, Biochem Biophys Res Commun. 1997 Jul 9; 236 (1): 10-5). However, there is interspecies variation between them. Beef PH20 is very weakly bound to the membrane and is not anchored by a phospholipase-sensitive anchor (Lalancette et al., Biol Reprod. 2001 Aug; 65 (2): 628-36.). This unique feature of bovine hyaluronidase allows the use of hyaluronidases obtained from nuclear extracts for clinical purposes (Wydase®, Hyalase®). Other types of PH20 are anchored to the membrane through lipids and pass into a soluble form only after using detergents or lipases. For example, human PH20 is anchored in the cytoplasmic membrane with a GPI anchor. Attempts to obtain DNA constructs that encode the protein part of PH20 without a lipid anchor have resulted in a catalytically inactive or insoluble enzyme (Arming et al Eur J Biochem. 1997 Aug 1; 247 (3): 8104). Hyaluronidase naturally present in macaque sperm occurs in both soluble and membrane form. The 64 kDa membrane form is enzymatically active at pH 7.0, while the 54 kDa form is active only in an environment at pH 4.0 (Cherr et al., Dev Biol. 1996 Apr 10; 175 (1): 142-53.) . For this reason, PH20 soluble forms often do not show enzymatic activity in a neutral pH environment.

[0015] Chondroitinases are enzymes commonly found in the animal world. These enzymes degrade glycosaminoglycans in an endoglycosidase reaction. Specific examples of known chondroitinases are: ABC chondroitinase (isolated from Proteus vulgaris; Japanese Patent Application Laidopen No 6-153947, 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. Fururu, J. Biol. Chem., 243, 1543 (1968)), AC chondroitinase (isolated 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)), ACIII hyaluronidase (isolated from Flavobacterium sp. HP102; Hirofumi Miyazono, Hiroshi Kikuchi, Keiichi Yoshida, Kiyoshi Morikawa, and Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1989)), chondroitinase B (isolated from Flavobacterium heparinum; YM Michelacci and CP Dietrich, Biochem. Comm. Biophys. Res. , 973 (1974), YM Michelacci and CP Dietrich, Biochem. J., 151, 121 (1975), Kenichi Maeyama, Akira Tawada, Akiko Ueno, and Keiichi Yoshida, Seikagaku, 57, 1189 (1985), chondroitinase C ( isolated from Flavobacterium sp. HP102; Hirofumi Miyazono, Hiroshi Kikuchi, Kelichi Yoshida, Kiyoshi Morikawa, and Kiyochika Tokuyasu, Seikagaku, 61, 1023 (1939)) and the like.

[0016] Glycoproteins are composed of a polypeptide chain to which one or more carbohydrate molecules are covalently bound. There are two main categories of glycoproteins in which the carbohydrate chain is attached to the protein part by an N- or O-linked glycosidic linkage. N- and O-glycans are attached to the polypeptides via the bond between asparagine and N-acetyl-D-glucosamine and between serine (threonine) and N-acetyl-D-galactosamine, respectively. Complex N-glycans do not contain terminal mannose residues. They contain only N-acetylglucosamine terminal residue, galactose and / or sialic acid residues. Hybrid N-glycans contain in their structure both terminal mannose and terminal N-acetylglucosamine, galactose and / or sialic acid residues.

[0017] During protein synthesis occurring in the endoplasmic reticulum with N-linked glycoproteins, oligosaccharide precursors are bound to the amino group of asparagine. The oligosaccharide residue is sequentially processed by a series of specific enzymes that cleave or add sugar residues. This treatment begins in the endoplasmic reticulum and continues while passing through the yew, middle and trans compartments of the Golgi Apparatus. SUMMARY OF THE INVENTION [0018] The present invention relates to members of the family of soluble, pH neutral glycoproteins with hyaluronidase activity, and particularly soluble glycoproteins with human hyaluronidase PH-20 activity (also referred to as sHASEGP). The present invention also relates to members of the sHASEGP family, referred to herein as sHASEGP. The invention also relates to a soluble domain with hyaluronidase activity and its uses.

[0019] The invention is based on the discovery that soluble hyaluronidases, active at neutral pH, can be obtained in high yield in a eukaryotic expression system based on mammalian cells by introducing nucleic acids in the form of cDNA, lacking the sequence of a short coding region of amino acids at the C-terminus of a human PH-20. The invention also provides additional modifications of sHASEGP glycoproteins that enhance secretion using an artificial leader peptide. The invention further relates to methods of sHASEGP modification that allow extending the half-life by masking the protein with polyethylene glycol and post-translational modifications of native glycans. Earlier attempts to produce secreted human sHASEGP, active at neutral pH, have been unsuccessful. It was therefore concluded that the shortening of the human sHASEGP polypeptide resulted in both loss of enzymatic activity at neutral pH and the inability of cells to secret recombinant proteins in eukaryotic expression cells based on mammalian cells (Arming, et al Eur J Biochem 1997 Aug1; 247 (3): 810-4). From the point of view of commercial production and therapeutic use, it is important to obtain the secreted form of sHASEGP glycoprotein with hyaluronidase activity. The invention disclosed herein overcomes these difficulties. [0020] In a first aspect, the present invention provides a substantially purified glycoprotein, comprising a neutral active soluble hyaluronidase polypeptide comprising at least one sugar moiety linked by an N atom which is covalently bound to asparagine as part of that polypeptide; The substantially purified glycoprotein contains the amino acid sequence encompassed by the ID sequence. SEQ. No. 1 or a sequence that is 91% identical to the amino acid sequence covered by the ID sequence. SEQ. No. 1; and this Substantially purified glycoprotein is soluble. The research results below show that human PH-20 requires N-glycosylation for its catalytic activity, whereas bovine and bee venom hyaluronidases are active in the absence of N-glycans. The domain of human hyaluronidase without N-linked moieties is catalytically inactive. Thus, classical technology based on recombinant DNA does not allow the production of the catalytically active form of human sHASEGP, unlike HASEGP from bee venom, which can be produced in E. coli cells.

[0021] The invention includes methods and cells for producing an N-linked sHASEGP glycoprotein polypeptide using cells capable of placing said N-linked sugar moieties on the sHASEGP polypeptide. Methods for identifying correctly glycosylated sHASEPGs are disclosed below.

[0022] The invention provides sHASEGP modifications that prolong its half-life, and chemical modifications of sHASEGP using polymers such as polyethylene glycol and dextran. Such modifications protect sHASEGP from being removed from the circulation and recognized by the immune system or glycan receptors, e.g. receptors for mannose or asialoglycoproteins. The invention further provides methods of attachment to specific functional groups such as glycosylation sites, positively charged amino acids and cysteine.

[0023] The invention also provides assays for identifying effectors such as chemical compounds, including small molecules, conditions such as pH, temperature and ionic strength, which modulate sHASEGP activation, expression or / and activity. The exemplary tests evaluated the effects of the effects of test chemicals on the ability of the domain with sHASEGP hyaluronidase activity to digest a known substrate, usually glycosaminoglycans or proteoglycans. Factors modulating the activity of said domain with hyaluronidase activity, generally chemical compounds, especially small molecules are potential chemical compounds that modulate glycoprotein activity with sHASEGP hyaluronidase activity. Domains with hyaluronidase activity can also be used to produce hyaluronidase-specific antibodies with a function that interferes with its activity. The hyaluranidase domains of the present invention include, but are not limited to, an N-terminal glycosylated domain having glycosylase and hydrolase activity, lacking the C-terminal fragment and having in vitro catalytic activity.

[0024] The present invention also includes nucleic acid molecules encoding hyaluronidase activity proteins and domains, as well as nucleic acid molecules that encode hyaluronidase activity soluble domain or catalytically active fragments thereof, and such nucleic acids that encode the full sHASEGP molecule. The nucleic acid encoding the domain with hyaluronidase activity and the nucleic acid downstream of the nucleic acid sequence encoding the domain with hyaluronidase activity is shown in the ID sequence. SEQ. No. 6. The domain with hyaluronidase activity from sHASEGP is shown in the ID sequence. SEQ. No. 1 (amino acids 35-464). The protein sequence and coding for the DNA sequence of the full sHASEGP molecule is shown in ID. SEQ. No. 1 and 6.

[0025] The invention also provides nucleic acid molecules that hybridize with sHASEGP-encoding nucleic acid along their entire length or at least 70%, 80% or 90% of the entire DNA molecule encoding a full domain with hyaluronidase activity or a fragment thereof. Generally, the hybridization specificity varies under at least low conditions, generally at least moderately and often under high stringency hybridization conditions.

[0026] Such an isolated nucleic acid fragment is DNA, including genomic DNA, RNA, or other components such as peptide nucleic acid or other nucleotide analogues. An isolated nucleic acid may contain additional components, such as heterologous or native promoters or other transcription and translation regulating sequences. These genes can be combined with other genes, such as reporter genes, marker genes, or genes encoding markers. [0027] The invention also relates to an isolated nucleic acid comprising a sequence that is complementary to the nucleotide sequence encoding sHASEGP or a fragment thereof. [0028] The invention also relates to oligonucleotides or fragments thereof that can be used as probes or primers and which contain at least about 10, 14, 16 nucleotides, generally less than 1000 or less than / or equal to 100, shown in the ID sequence. SEQ. No. 6 (or complementary sequences); or contain at least about 30 (or complementary to) nucleotides or contain oligonucleotides that hybridize along their entire length (or at least about 70, 80 or 90% of their length) with any fragment or oligonucleotide. The length of these fragments depends on their use and / or the complexity of the genome of interest. Usually probes and primers contain less than 50, 150 or 500 nucleotides.

[0029] The present invention relates to plasmids containing any DNA molecules of the present invention. The invention also relates to cells containing these plasmids. Plasmid-containing cells can be, but are not limited to, bacterial, yeast, fungal, plant, insect and animal cells.

[0030] The invention also relates to improved expression systems based on mammalian cells using signal sequences enabling efficient secretion of sHASEGP. An example of such an efficient amino acid signal sequence and such a sHASEGP fusion protein is the ID sequence. SEQ. No. 43 and 46.

[0031] The invention furthermore relates to a method for 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 N-linking sugar moieties to said polypeptide; a cell culture in which the encoded polypeptide is expressed by the cell; recovering the expressed polypeptide (s).

[0032] The present invention relates to cells, generally eukaryotic cells such as mammalian and yeast cells, in which the sHASEGP polypeptide is expressed on the surface. Such cells are used in drug screening tests to identify chemical compounds that modulate sHASEGP polypeptide activity. In such assays, including in vitro binding assays and transcription based assays, direct or indirect signaling by sHASEGP is assessed, e.g. by activating growth factors.

[0033] The present invention also relates to peptides encoded by such nucleic acid molecules. Among these polypeptides is a domain with sHASEGP hyaluronidase activity or a polypeptide with amino acids substituted in such a way that the specificity and / or activity of hyaluronidase remains largely unchanged. In particular, the invention provides a mammalian, substantially purified, secretory sHASEGP glycoprotein that contains a catalytically active domain operating at neutral pH.

[0034] The invention also includes a catalytic domain with hyaluronidase activity and may further include other domains. sHASEGP can form both homodimers and heterodimers with some other proteins such as membrane protein. The invention also relates to a substantially purified glycoprotein comprising an amino acid sequence which is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to sHASEGP, and the percent identity is determined using standard algorithms and a sequence interruption cost parameter that increases the percent identity.

[0035] The present invention covers sHASEGP splice variants, especially those with a catalytically active domain having hyaluronidase activity.

[0036] In another embodiment, the invention relates to substantially purified polypeptides that comprise a domain with hyaluronidase activity from sHASEGP or a catalytically active part thereof, but do not contain the complete amino acid sequence shown in the ID sequence. SEQ. No. 1. This group includes polypeptides containing an amino acid sequence that is at least 95% or 100% identical to the ID sequence. SEQ. No. 1 and 3.

[0037] In a particular embodiment, the present invention provides a nucleic acid encoding a eukaryotic glycoprotein with hyaluronidase activity termed sHASEGP. In particular, this nucleic acid comprises the nucleotide sequence set forth in the sequence ID. SEQ. No. 6, in particular represented as nucleotides 106-1446 in the sequence ID. SEQ. No. 6 or a portion thereof that encodes a catalytically active polypeptide.

[0038] The invention also relates to nucleic acids that hybridize to the ID sequence. SEQ. No. 6 or its degenerated form in at least low stringent conditions, most often moderate, and usually under highly stringent conditions.

[0039] In one embodiment, the fragment of isolated nucleic acid hybridizes under highly stringent conditions to the nucleic acid containing the sequence set forth in ID. SEQ. No. 6 (or its degenerated form). The full sHASEGP sequence is shown in ID. SEQ. No. 1 and is encoded by DNA with an ID sequence. SEQ. No. 6 or its degenerated form.

The invention also relates to muteins of the domain with hyaluranidase activity from sHASEGP, in particular muteins in which the Cys residue in the free form of the domain with hyaluronidase activity i.e. non-disulfide bridging with any other Cys residue within the domain with hyaluronidase activity is substituted with a different amino acid , usually, though not necessarily, a conservative amino acid or such that activity is preserved, and muteins in which the glycosylation site (s) are removed.

[0041] The invention relates to sHASEGP polypeptides, including, but not limited to, splice variants and nucleic acids encoding sHASEGP glycoproteins, and domains, derivatives and analogs thereof. The invention relates to a single secretory glycoprotein chain with hyaluronidase activity that has an N-terminus functionally corresponding to the N-terminus of the sHASEGP form produced by the action of sHASEGP-forming signal peptidase. Within the sHASEGP molecule, there are seven potential N-glycosylation sites at positions N82, N166, N235, N254, N368, N393, N490, as shown in ID. SEQ. No. 1. Disulphide bridges formed between residues Cys C60-C351 and residues C224-C238 form a core domain with hyaluronidase activity. However, to maintain the catalytic activity of the sHASEGP enzyme in a neutral environment, additional cysteine ​​residues at the C-terminus in the region from amino acid position 36 to Cys464 in the ID sequence are necessary. SEQ. No. 1 containing the minimally active region of the human sHASEGP domain. Therefore, the N-glycosylation site of N490 is not necessary for adequate activity.

[0042] N-glycosylation of sHASEGP glycoproteins is critical for the stability and catalytic activity of these molecules. While changing the sHASEGP-modifying glycan type can strongly affect the antigenic properties of the protein, folding its structure, solubility and stability, it is believed that most enzymes do not require glycosylation to achieve optimal activity. Thus, sHASEGP glycoproteins are unique in that the removal of N-glycans can result in almost complete loss of hyaluronidase activity. The presence of N-linked glycans is critical to obtaining active sHASEGP. The invention also relates to protein expression systems suitable for introducing essential N-glycans into the sHASEGP structure. The present invention also relates to the introduction of a deglycosylated sHASEGP polypeptide in the presence of extracts that allow N-glycosylation of said polypeptides. In one aspect of the invention, complex-type glycosylation crowned with a sialylation step is described, and other types of glycosylation terminated by the attachment of terminal mannose residues are not excluded. Preferably, sialic acid residues are located at the N-glycan termini of the sHASEGP molecule.

[0043] N-linked oligosaccharides are divided into several main types (high mannose, complex, hybrid and sulfated), all of which have a (Man) 3-GlcNAc-GlcNAc core connected through an amide nitrogen asparagine residue that is part of Asn-X-Thr / Ser sequence (where X is not Pro). Glycosylation within the -Asn-X-Cys sequence has been described for C-reactive protein. N-glycosylation sites are often identified indirectly through an "empty" cycle in the sequencing process. Identification of the presence of a glycosylation site can be performed after the oligosaccharide is released by digestion with the PNGase F enzyme, which converts glycosylated Asn into Asp. The combined oligosaccharides released as a result of PNGase FN treatment can be purified using Bio-Gel P-6 chromatography and then separated using preparative high-performance ion exchange chromatography (HPAEC) (Townsend et al., (1989) Anal. Biochem. 182 , 1-8). Certain oligosaccharide isomers can be separated by HPAEC. The presence of fucose residues in the structure will shift the peak of the molecule to an earlier position in the HPAEC chromatogram, while additional sialic acid residues will increase the retention time. Parallel treatment of glycoproteins with known oligosaccharide structures (such as bovine fetuin, α-1 acid glycoprotein, ovalbumin, RNAse B, transferrin) can help describe the corresponding oligosaccharide peaks. Harvested oligosaccharides can be characterized using a combination of methods such as qualitative analysis and methylation analysis indicating the composition and position of glycosidic bonds (Waeghe et al., (1983) Carbohydr Res. 123, 281-304.), And NMR spectroscopy to determine the anomeric configuration glycosidic linkages (Van Halbeek (1993) in Methods Enzymol 230). [0044] The invention also provides the formulation of sHASEGP molecules. SHASEGP glycoproteins can be prepared in lyophilized form and in the form of stable solutions. Formulations containing specific metal ions such as calcium, magnesium and sodium are useful in obtaining optimal enzyme activity at neutral pH. In addition to formulations in the form of a stable solution, the present invention also relates to the form of slow-release preparations serving as a prolonged agent for the removal of glycosaminoglycans. The invention hereby provides ready-to-use kits containing packaged syringes filled with sHASEGP for administration during ocular surgery and other low volume surgery. The invention also provides balanced salt solution formulations for ex vivo use in artificial insemination techniques. [0045] The invention also relates to the use of glycoproteins with sHASEGP hyaluronidase activity for the removal of glycosaminoglycans. By degrading sHASEGP glycosaminoglycans, they open channels in the interstitial space, which allows the diffusion of particles smaller than 500 nm. Depending on the dose and formulation used, these channels remain open for 24 - 48 hours. These channels may facilitate the penetration of exogenous externally administered fluids, small molecules, proteins, nucleic acids and vectors used in gene therapy, as well as other molecules smaller than 500 nm.

[0046] sHASEGP glycoproteins can also be used to remove excess glycosaminoglycans accumulated, e.g., as a result of ischemia reperfusion, inflammation, atherosclerosis, edema, cancer, spinal cord injury and other forms of tissue scarring. In some cases, sHASEGP glycoproteins may be administered regularly via intravenous infusion. This route of administration may prove useful in situations where local access is not possible, such as in the case of the heart, brain or cancer spread throughout the body. In terms of serum half-life and distribution in the body, strongly sialated sHASEGP glycoprotein has an advantage over the non-formalized form of hyaluronidase. [0047] In certain circumstances, such as spinal cord injury, glaucoma and cosmetic procedures, prolonged administration of sHASEGP is preferred.

[0048] In another indication, one short-acting dose is more preferable. Periodic removal of glycosaminoglycans can be used to facilitate the penetration of solutions and drugs into the interstitial spaces, which can be useful in cases of diffusion of anesthetics and when administering therapeutic fluids, molecules and proteins. Subcutaneous and intramuscular administration of molecules in the presence of sHASEGP glycoproteins facilitates their faster systemic distribution. Such methods are extremely useful in situations where intravenous administration is not possible or if there is a need for faster systemic administration. Such large molecules as factor VIII, poorly available after subcutaneous administration, can be injected together with sHASEGP glycoproteins to increase their bioavailability.

[0049] The invention also provides the use of sHASEGP glycoproteins for the enzymatic removal of the cumulus matrix surrounding oocytes. Removal of the cumulus matrix using purified sHASEGP, free of toxic impurities that are present in animal extracts of hyaluronidases, is a milder method and increases oocyte survival. In addition, sHASEGP glycoproteins can be produced without the need for beef extract or derived from the cells of other organisms, which may be a potential source of viruses and other pathogens as well as spongiform encephalopathy.

[0050] Injection of small volumes of sHASEGP into the eyeball can also be used for other small spaces. SHASEGP glycoproteins can be injected into the anterior chamber of the eye to remove excess viscoelastic agent administered during surgery. Intraocular injection of glycoproteins with sHASEGP hyaluronidase activity can also be used to reduce intraocular pressure in glaucoma, to dissolve aggregates in the vitreous of the eye or mud in the vitreous, to remove the effects of vitreous hemorrhage, to treat macular degeneration, to facilitate the separation of vitreous from retina in diabetic retinopathy and in a mixture with other enzymes while helping to match the shape of the cornea with contact lenses. In some cases it may be desirable to use a stable form of sHASEGP as it is pegylated.

[0051] It may also be envisaged to use mixtures of sHASEGP in autosyringes for the administration of small volumes or rapid subcutaneous injections. Forms of such drugs may be based on examples such as Epipen®, insulin and other fluids. The methods of the present invention include administering an sHASEGP polypeptide or pharmaceutical compositions containing sHASEGP before, in parallel, or after administration of other therapeutic molecules. sHASEGP can be administered to another site or the same as said therapeutic molecule.

[0052] Thus, the present invention provides eukaryotic secretory families, active at neutral pH of glycoproteins with hyaluronidase activity referred to herein as sHASEGP and their functional domains, and in particular their catalytic domains with hyaluronidase activity, their muteins and their other derivatives and analogues. The invention also provides nucleic acids encoding sHASEGP. In addition, the invention provides the formulations and uses of said sHASEGP for the treatment of diseases and as tissue modifying enzymes.

BRIEF DESCRIPTION OF THE DRAWINGS [0053] Figure 1 is a map of the HZ24-sHASEGP vector

DETAILED DESCRIPTION OF THE INVENTION [0054] A. DEFINITIONS: Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. All patents, patent applications, published patent applications and publications, sequences from the GenBank website, websites and other published materials, which are referenced throughout this disclosure, unless otherwise stated, are an integral part of the document as a whole. In the event that there are many definitions of terms used in this document, consider those given in this section of the application.

[0055] If the reference is to a URL or other identifier of websites or an address, it is understood that such identifier may be subject to change, and the individual information to which it refers may be posted and removed, however, searching the Internet network resources allows finding equivalent information. Links to equivalent information are evidence of the disclosure and dissemination of that information.

[0056] For the purposes of this document, the abbreviations for any protecting groups, amino acids and other chemical compounds, unless otherwise indicated, are in accordance with the commonly used and recognized abbreviations or nomenclature of the TUPAC-IUB Commission on Biochemical Nomenclature (see (1972) Biochem. 11: 942-944).

[0057] As used herein, the term "eukaryotic hyaluronidase" means a diverse family of endoglucosaminidases of glycosaminoglycans in which the glutamate residue within the hyaluronidase molecule hydrolyses the β (1,4) hyaluronan and chondroitin sulfate binding through an acid-base catalysis mechanism.

[0058] Particular interest is sHASEGP mammalian, including human origin. It is well known to those skilled in the art that, in general, substitution of a single amino acid in a minor region of a polypeptide does not significantly affect biological activity (see e.g., Watson et al., (1987) Molecular Biology of the Gene, 4th Edition, The Benjamin / Cummings Pub. co., p. 224).

[0059] As used herein, the term "membrane-anchored sHASEGP" means a family of membrane-anchored hyaluronidases with a structure similar to that described in the present invention.

[0060] As used herein, the term "soluble hyaluronidase" means a polypeptide characterized by solubility under physiological conditions. An example is soluble sHASEGP, which distinguishes it from other hyaluronidases, e.g. the ability to enter the aqueous phase in a solution of Triton-X114 at 37 ° C (Bordier et al. J Biol Chem. 1981 Feb 25; 256 (4): 1604-7) in contrast to sidase-mediated lipid-mediated sHASEGP, which usually goes into a detergent-rich phase, but only after phospholipase C treatment - to a low detergent phase or to the aqueous phase.

[0061] Hence, reference to, for example, "sHASEGP" includes all glycoproteins encoded by the gene for the sHASEGP family, including but not limited to human sHASEGP, mouse sHASEGP, equivalent molecules obtained from any other source, obtained synthetically, or having the same activity . The sequences of the coding nucleic acid molecules and the encoded exemplary amino acids sHASEGP and / or their domains are shown, e.g., in ID sequence. SEQ. No. 4. The term also includes sHASEGP, in which amino acid substitution, which does not significantly change the activity, has been made, as well as sHASEGP splice variants. Suitable substitutions, including or omitting conservative amino acids, are known to those skilled in the art and can be carried out without altering the activity of the resulting molecule.

[0062] As used herein, each reference to the term "sHASEGP" in the body of this document includes a polypeptide having the amino acid sequence contained in the ID sequence. SEQ. No. 1 or a sequence 91% homologous to the amino acid sequence shown in sequence ID. SEQ. No. 1.

[0063] In particular, a sHASEGP polypeptide with domains having hyaluronidase activity is disclosed as shown in the ID sequence. SEQ. No. 4. This polypeptide is in single or double stranded form. The invention also relates to its smaller parts still having hyaluronidase activity. Domains with hyaluronidase activity derived from sHASEGP differ in size and chemical composition, including insertions and deletions in the surface domain loop. Therefore, for the purposes of this document, the catalytic domain is part of sHASEGP as defined herein and is homologous to the domain of other hyaluronidase-like sequences such as HYAL1, HYAL2, HYAL3 previously identified. However, it has not been found that an isolated single-stranded domain form with hyaluronidase activity can be functionally active in in vitro assays. Aspartate and glutamate residues necessary for activity are present in conserved motifs.

[0064] As used herein, the term "domain of soluble sHASEGP with hyaluronidase activity" means a domain derived from sHASEGP with β (1,4) endoglucosaminidase activity that exhibits hyaluronidase activity at neutral pH, is soluble under the conditions described, homologous and structurally similar to the hyaluronidase family domains with glycosyl hydrolase activity, however, it contains additional carboxy-terminal sequences necessary for activity at neutral pH. It is therefore the smallest possible part of the domain that exhibits hyaluronidase activity, as determined by standard in vitro tests and remains in soluble form. The invention includes said domains with hyaluronidase activity and their catalytically active parts. The invention also includes the truncated portion of the domain with hyaluronidase activity, containing the smallest possible fragment thereof, which has a catalytic activity as a single chain.

[0065] The term "domain with sHASEGP hyaluronidase activity" whenever referred to herein includes at least one, all or any combination thereof, or the catalytically active portion of the N-glycosylated polypeptide that comprises the amino acid sequence shown in the ID sequence. SEQ. No. 1, as well as all or any combination or catalytically active portion of the polypeptide encoded by the nucleotide sequence hybridizing under low, moderate or highly stringent conditions to the nucleotide sequence shown in ID. SEQ. No. 6, all or any combination or catalytically active portion of the polypeptide that comprises the amino acid sequence set forth in the ID sequence. SEQ. No. 1, all or any combination or catalytically active part of the polypeptide that contains an amino acid sequence showing 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% identity to the amino acid sequence shown in the ID sequence. SEQ. No. 1, and / or domains with hyaluronidase activity of the polypeptide encoded by sHASEGP splicing variants. [0066] Hence, for the purposes of this document, the term "domain with hyaluronidase activity" as defined herein is part of sHASEGP and is homologous to the domain of other sHASEGP glycoproteins. Within the broadly understood family of hyaluronidases, the catalytic domains of sHASEGP are largely similar in terms of amino acid sequence. Asp and Glu residues, necessary for activity, are present in conserved motifs.

[0067] By the term "active form" is meant the active form in vivo and / or in vitro. As described herein, a domain with hyaluronidase activity may also exist as a soluble secretion protein. This document shows that at least in vitro sHASEGP single chain forms and catalytic domains or enzymatically active parts thereof (usually C-terminal shortened fragments) exhibit hyaluronidase activity. Therefore, the invention provides isolated forms of domains with sHASEGP hyaluronidase activity and their use in in vitro drug screening assays to identify factors modulating the activity of said domains.

[0068] As used herein, the term "catalytically active sHASEGP domain" means the neutral pH endoglucosaminidase domain as defined in vitro using glycosaminoglycan substrates for this purpose.

[0069] The group of sHASEGP molecules of interest includes hyaluronidases, which in vivo and in vitro exhibit activity on chondroitin sulfates and proteoglycans (CSPG's), which include chondroitin sulfate molecules, as well as those hyaluronidases that exhibit hyaluronan activity . As used herein, human sHASEGP is encoded by a nucleic acid, such as DNA present in the human genome, including all allelic and conservative variants, provided they are not found in other mammals.

[0070] As used herein, the term "nucleic acid encoding a domain with hyaluronidase activity or its catalytically active sHASEGP domain" is understood to mean a nucleic acid encoding only said single domain chain with hyaluronidase activity or a catalytically active part thereof, but not adjacent parts within sHASEGP as contiguous sequences.

[0071] As used herein, the term "disease" or "disorder" means a pathological condition in the body resulting, e.g., from an infection or genetic defect, described by recognizable symptoms.

[0072] As used herein, the term "splice variants" means a variant obtained as a result of the differential treatment of a primary genomic nucleic acid transcript, such as DNA, resulting in more than one type of mRNA. The invention hereby provides sHASEGP splice variants.

[0073] As used herein, the term "domain with sHASEGP protein hyaluronidase activity" means a domain with sHASEGP protein hyaluronidase activity that has endoglucosaminidase activity at neutral pH. Thus, it is a minimal protein fragment that has endoglucosaminidase activity as determined by standard in vitro tests. An exemplary domain with hyaluronidase activity includes at least a fragment of the amino acid sequence set forth in the ID sequence. SEQ. No. 4, which is at least long enough to have endoglucosaminidase activity.

[0074] The invention includes nucleic acid molecules that encode a polypeptide having endoglucosaminidase activity in in vitro hyaluronidase assays and which exhibits 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% homology to full-length domain polypeptide with hyaluronidase activity sHASEGP polypeptide, or which hybridizes 100% or at least 70%, 80% or 90% to nucleic acids, which encode a domain with hyaluronidase activity, in particular under moderate or generally highly stringent conditions.

[0075] For a domain with hyaluronidase activity, residues at the N-terminus may be critical but not sufficient for the activity. This document shows that a domain with hyaluronidase activity from sHASEGP is catalytically active. Thus, a domain with hyaluronidase activity generally requires the presence of N-terminal amino acids to maintain activity. The C-terminus may be shortened to the last Cys, but the presence of several additional amino acids is required for optimal activity. The number of amino acids that can be removed is determined experimentally by testing polypeptides with hyaluronidase activity in vitro in tests that measure catalytic activity. [0076] Thus, the invention relates to smaller fragments of hyaluronidase domains, in particular single chain domains that have retained hyaluronidase activity. Such smaller versions are generally truncated by the C-terminus domain with hyaluronidase activity. Domains with hyaluronidase activity differ in size and chemical composition, including insertions and deletions in the surface loop. These domains exhibit features of a conservative structure, including at least one structural feature, such as a proton donor and / or other features of the endoglucosaminidase domain with hyaluronidase activity. Therefore, for the purposes of this document, a hyaluronidase domain is defined as a single-chain sHASEGP fragment as defined herein, is homologous and structurally similar, and has a similar retention time to a domain sequence with hyaluronidase activity or other similar sequences. This glycoprotein, as a single chain, exhibits hyaluronidase activity.

[0077] As used herein, the term "homology" means nucleic acid sequence identity greater than about 25%, such as 25% 40%, 60%, 70%, 80%, 90% or 95%. If necessary, the homology percentage will be determined. The terms "homology" and "identity" are often used interchangeably. In general, the sequences are aligned in such a way as to obtain the highest degree of alignment (see, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: 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). [0078] The number of conservative amino acids is determined by comparing sequences in standard programs according to standard algorithms using a so-called parameter. breaks set individually by the program producer. Significantly homologous nucleic acid molecules would normally hybridize under 100% or 70%, moderately stringent or high stringency conditions, 80% 90% of the full length molecule of interest. The invention also encompasses nucleic acid molecules with degenerate codons at the codon site located within the hybridizing nucleic acid molecule.

[0079] Whether any nucleic acid molecules have nucleotide sequences that are at least e.g. 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% "identical" can be determined using computer program algorithms such as the "FASTA" program using default parameters as in Pearson et al (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444), other programs include the GCG package (Devereux, J., et al, Nucleic Acids Research 12: 387 (1984)), 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 [CARILLO ETA /.] (1988) SIAM J Applied Math 48 : 1073). For example, BLAST, a database program from the National Center for Biotechnology Information, can be used to determine identity. Other commercial or publicly available programs are the DNASTAR "MEGALIGN" PROGRAM (Madison, WI) and the GAP program (University of Wisconsin Genetics Computer Group (UWG) (Madison WI)). The percentage of homology or identity of proteins and / or nucleic acids can be determined e.g. by comparing the sequences in the GAP program as in Needleman et al. (1970), J Mol Biol. 48: 443, corrected by Smith and Waterman Adv. Appl. Math (1981) 2: 482). In short, the GAP program defines similarity as the number of similar symbols stacked together (e.g., nucleotides or amino acids) divided by the total number of symbols in the shorter of the compared sequences. The default GAP program parameters include: (1) the unit matrix (result 1 is identity, result 0 is no identity) and weighted average matrix in Gribskov et al (1986) Nucl. Acids Res. 14: 6745, as described in Schwartz and Dayhoff, eds., Atlas Of Protein Sequence And Structure, National Biomedical Research Foundation, pp. 353-358 (1979); (2) the cost of interrupting sequence 3.0 for each break and the additional cost of 0.1 sequence break for each symbol in each break; (3) no interruption costs for sequence breaks at the end of the sequence. Therefore, as used herein, the term "identity" means a comparison of an analyzed and reference polypeptide or polynucleotide. [0080] As used herein, at least "90% identical to" means a percentage of identity in the range of 99 to 99.99 with respect to reference polypeptides. An identity of 90% or higher indicates that e.g. the analyzed and reference polynucleotide molecule with a length of 100 amino acids were compared and no more than 10% (i.e. 10 out of 100) of the amino acids in the analyzed polypeptide differs from the amino acids in the reference polypeptide. Similar comparisons can be made between analyzed and reference polynucleotides. The differences may mean the presence of point mutations or clusters of mutations of variable length up to the maximum permissible length, e.g. 10/100 amino acids other than the reference molecule randomly dispersed along the entire length of the molecule (about 90% identity). Differences are defined as substitutions or deletions of nucleic acids or amino acids. At a homology or identity level above about 85% -90%, the result should not depend on the software used and the set of parameters regarding the cost of breaking sequence continuity. Such a high degree of identity can also be easily estimated without using a program.

[0081] As used herein, the term "primer" means an oligonucleotide containing two or more deoxyribonucleotides or ribonucleotides, usually more than three, from which the synthesis of the primer extension product is initiated. Experimental conditions conducive to synthesis include the presence of triphosphate nucleosides and factors responsible for polymerization and extension, such as DNA polymerase, appropriate buffer, temperature and pH.

[0082] As used herein, the term "animals" includes any animals such as, but not limited to, goats, cows, deer, sheep, rodents, pigs and humans. The term "non-human animals" means the animal group of the invention excluding humans. The sHASEGP molecules of the present invention are derived from any animal, plant, prokaryotic or fungal source. Most sHASEGP comes from the animal world, including mammalian organisms.

[0083] In the meaning of the present invention, gene therapy involves the transfer of a heterologous nucleic acid, such as DNA, to specific cells - target cells, i.e. mammalian cells, and in particular human ones with abnormalities in functioning or in a condition that requires such therapy. Nucleic acid, like DNA, is expressed, which produces a therapeutic agent.

[0084] Alternatively, a heterologous nucleic acid, such as DNA, may somehow affect the expression of DNA that encodes the therapeutic agent, or may also encode a product, such as a protein or RNA, which in some way directly or indirectly affects the expression of the therapeutic agent . Gene therapy can also be used to provide a nucleic acid encoding a gene product that is intended to replace a defective gene or supplement a deficiency in a gene product produced by mammalian cells or cells into which it has been introduced. The introduced nucleic acid may encode a therapeutic substance such as a growth factor inhibitor, TNF or its inhibitor, such as its receptor, which is usually not produced in mammalian host cells or is produced, but in quantities or at a time that is inappropriate for the therapeutic effect. A heterologous nucleic acid, such as DNA, encoding a therapeutic product, may be modified prior to introduction into diseased host cells to enhance expression or other type of product change or expression. Gene therapy may also include the delivery of inhibitors, repressors or modulators of gene expression.

[0085] Within the meaning of the present invention, the term "heterologous nucleic acid" is a nucleic acid (if DNA encodes RNA and proteins) that is not naturally produced in vivo by cells in which it is expressed or influenced or encodes mediators , altering the expression of an endogenous nucleic acid, such as DNA, affecting transcription, translation or other biochemical processes. Heterologic nucleic acid may also be a foreign nucleic acid, such as DNA. Any nucleic acid, such as DNA, which would be recognized by a person skilled in the art as heterologous or foreign to the cell in which it is expressed is encompassed herein by the term "heterologous nucleic acid." Heterologous nucleic acid includes exogenously introduced DNA that is expressed endogenously. Examples of heterologous DNA are, but are not limited to, nucleic acids that encode detectable marker proteins, such as those responsible for drug resistance, nucleic acids encoding active substances such as anti-cancer agents, enzymes, hormones and nucleic acids, such as DNA that encode other types of proteins, such as antibodies. Antibodies encoded by heterologous nucleic acids can be secreted or expressed on the surface of the cell into which said heterologous nucleic acid has been introduced.

[0086] Generally, a heterologous nucleic acid obtained by artificial means or from another cell is not endogenous for the cell into which it has been introduced.

[0087] Generally, but not necessarily, such nucleic acid codes for RNA and proteins, usually not produced by the cell that expresses them.

[0088] As used herein, the term "therapeutically effective product" is a product encoded by a heterologous nucleic acid, usually DNA, which, when introduced into a host cell, is expressed and as a result alleviates, eliminates and treats the symptoms of congenital and acquired diseases.

[0089] As used herein, the term "glycoprotein mainly consists of a domain with hyaluronidase activity" means that the only part of the sHASEGP polypeptide is a domain with hyaluronidase activity or a catalytically active fragment thereof. Optionally and generally, the polypeptide contains additional amino acid sequences not derived from the sHASEGP molecule.

[0090] As used herein, the term "domain" means a portion of a molecule, e.g., glycoprotein or coding nucleic acid, which structurally and / or functionally differentiates from other parts of the molecule.

[0091] As used herein, the term "hyaluronidases" are enzymes that catalyze the hydrolysis of glycosaminoglycans.

[0092] For the sake of clarification, the term "hyaluronidase" means all forms of hyaluronidase, and its specific forms specially designated. For the purposes of this document, a domain with hyaluronidase activity contains both the sHASEGP membrane form and its soluble form.

[0093] As used herein, the term "nucleic acids" includes DNA, RNA and analogs thereof, including peptide nucleic acid (PNA) and a mixture of said elements. The nucleic acid can be single or double stranded. With respect to probes and primers, optionally labeled with a detectable label, the invention includes fluorescently or radioactively labeled single-stranded molecules. These molecules are usually of such a length that the target sequence they recognize in the library being searched for or during hybridization is statistically unique or occurs in a small number of copies (usually less than 5, generally less than 3). Generally, a probe or primer contains at least 14, 16 or 30 contiguous of sequence complementary to the gene of interest or identical to said gene. Probes and primers can be 10, 20, 30, 50, 100 or more nucleotides in length.

[0094] As used herein, the term "nucleic acid encoding a fragment or part of sHASEGP" means a nucleic acid encoding only said fragment or part of sHASEGP, but not encoding other adjacent parts of sHASEGP.

[0095] As used herein, the term "functional binding of a heterologous nucleic acid to regulatory or effector nucleotide sequences such as promoters, enhancers, translation and transcription arrest sites and other signal sequences" means the relationship between a nucleic acid, such as DNA, and said nucleotide sequence . An example would be a functional combination of heterologous DNA with a promoter, meaning a physical relationship between DNA and the promoter. As a result of this relationship, transcription of such DNA is initiated by RNA polymerase from a promoter that specifically recognizes, binds and transcribes DNA. In order to optimize expression and / or transcription in vitro, it may be necessary to remove, add or change the 5 'non-transcribed portion of the clones DNA to eliminate potentially abnormal translation initiation (i.e. start codons) or other sequences that interrupt or reduce expression, both at the level of transcription as well as translation. Alternatively, ribosome binding sites can be introduced 5 'to the start codon (see, e.g., Kozak J. Biol. Chem. 266: 19867- 19870 (1991) and thus enhance expression. The need for such modification can be determined experimentally.

[0096] As used herein, the term "complementary sequence" to at least a portion of RNA, with respect to antisense oligonucleotides, means a sequence with such complementarity that is sufficient to hybridize with said RNA, generally under moderately stringent hybridization conditions or under high stringency conditions, forming stable double-stranded complexes. In the case of double-stranded sHASEGP antisense nucleotides, both a single strand of DNA duplex (or dsRNA) and the formation of triple complexes can be tested. The ability to hybridize depends on the degree of complementarity and length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid is, the more it may contain bases that do not match the RNA encoding sHASEGP and may still form a stable double-stranded (or three-stranded complex, which may occur). The skilled person can determine the acceptable number of mismatches in the hybridized complex using standard melting point procedures.

[0097] For the purposes of this application, amino acid substitution can be carried out in any sHASEGP and domain with hyaluronidase activity, provided that it does not affect the activity of the resulting protein. The amino acid substitution to which the invention relates includes a conservative substitution that does not exclude proteolytic activity as shown in Table 1. As described herein, substitutions that alter the properties of proteins such as the removal of digestion sites and other sites are also an object of the invention. Such substitutions are generally non-conservative, but they can be quickly and successfully carried out by a person skilled in the art.

[0098] Suitable conservative amino acid substitutions are known to those skilled in the art and can be carried out without changing the biological activity, e.g. the enzymatic activity obtained by substitution of the molecule. Experts understand that, in general, a single amino acid substitution in a minor region of a polypeptide does not significantly alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., P.224) . This definition also includes the catalytically active sHASEGP fragment, and in particular a single chain part of hyaluronidase. Exemplary conservative amino acid substitutions are made according to Table 1 as follows:

[0099] Table 1 Original amino acid residue Substitution of the Ala (A) Gly conserved residue; Ser, Abu Arg (R) Lys, om Asn (N) Gln; His Cys (C) Ser Gin (Q) Asn Glu (E) ASP Gly (G) Ala; Pro His (H) Asn; Gin Ile (I) Leu; Val; Underworld; Nle; Nva Leu (L); Val; Underworld; Nle; Nv Lys (K) Arg; Gin; Glu Met (M) Leu; Tyr; How much ; 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; Underworld; Nle, Nv. Other substitutions are also allowed and may be determined by experimental method or carried out according to already known conservative substitutions.

[0100] As used herein, Abu is 2-aminobutanoic acid; Orn is ornithine. As used herein, amino acids found in the various sequences herein are identified according to the well-known three-letter or one-letter abbreviations. Nucleotides found in various DNA fragments are labeled using a standard one-letter assay routinely used in the art.

[0101] As used herein, a probe or primer designed based on the sequences disclosed herein comprise at least 10, 14, typically at least 16 adjacent nucleotides of the ID sequence. SEQ. No. 6, and probes at least 30, 50 or 100 adjacent without nucleotide ID sequence. SEQ. No. 6. The length of probe or primer to achieve specific hybridization depends on the complexity of the genome structure of interest.

[0102] As used herein, "ameliorating the symptoms of a particular disorder by administering a particular pharmaceutical composition" means any reduction, persisting or temporary, lasting or transient that can be attributed to or associated with administration of said composition.

[0103] As used herein, the term "antisense polynucleotides" means sequences from synthetic nucleotide bases complementary to mRNA or a sense strand of double-stranded DNA. Mixing sense and antisense polynucleotides under the right conditions leads to the binding of these two molecules or, in other words, their hybridization. When said polynucleotides bind (hybridize) to mRNA, protein synthesis (translation) is inhibited. When said polynucleotides bind to double-stranded DNA, RNA synthesis (transcription) is inhibited.

[0104] As a result, inhibition of translation / transcription leads to inhibition of the synthesis of proteins encoded by the antisense strand. An antisense nucleic acid molecule typically contains a sufficient number of nucleotides to specifically bind to target DNA, generally at least 5 adjacent nucleotides, often at least 15 or 16 or 30 adjacent nucleotides or modified nucleotides complementary to the coding portion of the nucleic acid molecule that encodes the gene of interest interests, e.g., a nucleic acid encoding a single hyaluronidase domain chain from sHASEGP.

[0105] As used herein, the term "matrix" means a palette of elements, such as antibodies, containing three or more components. An addressable array is characterized by the fact that each of its components is identifiable, usually by position on a solid support. Generally speaking, the matrix components are immobilized in separate identifiable positions on the solid phase surface.

[0106] As used herein, the term "antibodies" means immunoglobulins, natural or partly or completely synthetic, including any derivatives thereof, the binding specificity of which is such as the specificity of the antibodies. Thus, antibodies include any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. Antibodies include any class of immunoglobulins including IgG, IgM, IgA, IgD and IgE.

[0107] As used herein, the term "antibody fragments" means any derivative of an antibody less than the length of the full antibody molecule, retaining at least some of the binding capacity of the full antibody molecule. Examples of antibody fragments include, but are not limited to, Fab, Fab ', F (AB) 2, single chain FVS (SCFV), FV, dsFV bispecific antibody (diabody) and Fd fragments. A fragment may contain several chains connected with each other, e.g. with a disulfide bridge. Generally, the antibody fragment contains at least about 50 amino acids, and usually at least 200 amino acids.

[0108] As used herein, the Fv fragment consists of one heavy chain and one light chain variable part connected by non-covalent interactions.

[0109] As used herein, the term "dsFV" means Fv with an artificially introduced disulfide bridge.

[0110] As used herein, the term "F (AB) 2 fragment" means the antibody fragment resulting from the digestion of immunoglobulins with pepsin at pH 4.0-4.5. This fragment can be expressed as a recombinant protein to produce a corresponding fragment. [0111] As used herein, a Fab fragment is an antibody fragment resulting from the digestion of immunoglobulins with papain; can be expressed as a recombinant protein to generate the corresponding fragment.

[0112] As used herein, the term "scFVs" means an antibody fragment that contains a variable V light chain and a variable heavy chain (VH) covalently bound in any order via a polypeptide linker. The link is of such length that the two variable domains are connected by a bridge without interfering with each other. Linkers are (Gly-Ser) n residues scattered within the Glu or Lys sequence to increase solubility.

[0113] As used herein, the term "humanized antibodies" means antibodies modified to contain amino acid sequences of human origin. Administration of such antibodies to humans does not elicit an immune response. Methods for preparing such antibodies are known. For example, to produce said antibodies, hybridoma cells, prokaryotic or eukaryotic cells such as E. coli or CHO, which express monoclonal antibodies, are changed by recombinant DNA techniques to express the antibody with the constant region amino acid composition as in a human antibody. Computer programs are used to identify such regions.

[0114] As used herein, the term "bispecific antibodies" means scFV dimer, usually having a shorter peptide linker than ScFVs and, in general, dimerizing.

[0115] As used herein, the term "recombinant production" means the use of recombinant DNA methods, i.e. the use of well-known molecular biology methods, for the expression of proteins encoded by cloned DNA.

[0116] As used herein, the term "estimate" implies qualitative and quantitative determination in the sense of obtaining an absolute value for sHASEGP activity or its domain present in the sample as well as in terms of obtaining an indicator, ratio, percentage, effect indicating the level of activity. The estimation can take place directly or indirectly, and the chemical compounds in fact need not be detected for obvious reasons by proteolysis products, but can be, for example, derivatives or other substances.

[0117] As used herein, the term "biological activity" means an in vivo activity of a chemical compound or physiological response obtained after the in vivo administration of a chemical compound, composition or other mixture. Therefore, biological activity includes the therapeutic effect and pharmacological activity of such chemical compounds, composition or other mixture. Biological activity can be observed using in vitro designed systems. Thus, for the purposes of this document, the biological activity of luciferase reflects oxygenase activity in such a way that the oxidation of the substrate emits light.

[0118] As used herein, the term "functional activity" means a polypeptide or portion thereof that exhibits one or more activities associated with a full-length protein molecule.

[0119] Functional activities include, but are not limited to, biological, catalytic and enzymatic activity, antigenicity (ability to bind or compete with a polypeptide for binding to an antibody directed against this polypeptide), immunogenicity, ability to form multimers, and ability to specifically bind to the receptor or a polypeptide ligand.

[0120] As used herein, the term "conjugate" means the chemical compounds of the present invention that contain one or more sHASEGP molecules, including sHASEGP, and in particular a single domain chain with hyaluronidase activity and one or more targeting factors. These conjugates include those molecules that have been produced by recombinant fusion protein and those that have been produced by chemical methods such as by chemical linkage through e.g. sulfhydryl groups and such molecules which have been prepared by any other method where at least one sHASEGP or its domain is directly or indirectly linked via a linker (s) to the targeting agent.

[0121] As used herein, the term "targeting agent" is any protein molecule or active part thereof that provides specific binding of a conjugate to a cell surface receptor that can internalize the conjugate of the sHASEGP portion. The targeting agent may also assist or facilitate, e.g., isolation or affinity purification of the conjugate, attachment of the conjugate to the surface, or detection of the conjugate or conjugate-containing complex. [0122] As used herein, the term "antibody conjugate" means a conjugate in which the targeting agent is the antibody.

[0123] As used herein, the term "derivative" or "analog" of a molecule means a portion derived from the molecule or a modified version thereof.

[0124] As used herein, the expression "effective amount of a chemical for treating a particular disorder" is an amount sufficient to alleviate or in some way reduce the symptoms associated with the disorder. Such an amount can be administered as a single dose or according to an established procedure by which it is effective. This amount can cure the disease, but is usually given to relieve the symptoms. Often, repeated administration of a chemical is needed to achieve the desired symptom relief.

[0125] As used herein, the term "equivalent" with respect to two nucleic acid sequences means that the indicated sequences encode the same amino acid sequence or equivalent protein. If the term "equivalent" is used to refer to two proteins or peptides, it means that the two proteins or peptides have substantially the same amino acid sequence, differing only in amino acid substitutions (conservative but not only, conservative changes as shown in Table 1 above), which do not significantly affect the activity or function of proteins or peptides. If the term "equivalent" refers to a property, said property need not exist to the same extent (e.g., two peptides may have different levels of the same enzymatic activity), but the activity is usually largely the same. The term "complementary" with respect to two nucleotide sequences means that said two nucleotide sequences are capable of hybridizing, usually at less than 25%, 15%, 5% or 0% of mismatched nucleotide pairs. The percentage of complementarity will be marked if necessary. Usually, under highly stringent conditions, the two most complementary molecules hybridize.

[0126] As used herein, a factor modulating protein activity, gene or nucleic acid expression raises, lowers or changes protein activity or in some way increases or decreases or changes the expression of nucleic acid in a cell.

[0127] As used herein, the expression "inhibitor of sHASEGP activity" refers to any substance that prevents or reduces the production, post-translational (e) modification (s), maturation and localization of sHASEGP in the membrane, or any substance that interferes with or reduces proteolytic performance , especially in the case of the single chain form tested in in vitro screening.

[0128] As used herein, the expression "a method of treating or preventing a cancer" means that the method allows to reduce, alleviate, prevent, transition into remission or maintain in remission any symptoms such as a tumor, its metastasis, tumor vasculature and other characteristics of the disease. It also means that cancer and metastasis can be eliminated, reduced or prevented by treatment. Examples of disease signs include, but are not limited to, uncontrolled degradation of the basement membrane and adjacent extracellular matrix, migration, division and organization of endothelial cells into functional capillary structures with capillary stability.

[0129] As used herein, pharmaceutically acceptable salts, esters and other conjugate derivatives include any salts, esters and derivatives that can be readily prepared by a person skilled in the art using methods for such derivatization and which result in the production of chemical compounds that can be administered to animals or humans without significant toxic effects and which are in prodrug or pharmacologically active form.

[0130] As used herein, the term "prodrug" means a compound that, when administered in vivo, is metabolized or otherwise converted to a biologically, pharmacologically or therapeutically active form of a chemical compound. To obtain a prodrug, a pharmacologically active chemical compound is modified to become fully activated only when it undergoes metabolic changes. It is possible to design a prodrug that allows changing metabolic stability or drug transport parameters, neutralizing toxic side effects, and improving the taste of the drug. It also allows you to influence its other features or properties. The specialist, due to his knowledge of pharmacodynamic processes and drug metabolism in vivo, is able to design a prodrug if he is familiar with a pharmacologically active chemical compound (see Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388 -392).

[0131] As used herein, the expression "drug identified by screening" refers to any chemical compound that could potentially be used as a therapeutic or as a lead compound to design a therapeutic. These types of compounds include small molecules, including organic small molecule compounds, peptides, peptidomimetics, antisense or dsRNA molecules such as RNAi, antibodies, antibody fragments, recombinant antibodies and other compounds that can serve as potential drugs or leading starting substances.

[0132] As used herein, the term peptidomimetic "means a compound that mimics the conformation and certain stereochemical features of the biologically active form of a particular peptide. Generally, peptidomimetics are designed to mimic certain desired characteristics of a compound and eliminate undesirable features such as flexibility, which leads to loss of biologically active conformation and disruption of binding. Peptidomimetics can be prepared based on biologically active compounds, replacing certain groups or bonds with bioisosterols that contribute to the occurrence of undesirable properties. Bioisosteres are known to those skilled in the art. An example is the methylene bioisoster CH2S, which replaces the amide group in enkephalin analogues (see e.g. 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 peptidomimetic endorphin peptide. For the purposes of this application, cyclic peptides are included in the group of peptidomimetics.

[0133] As used herein, the expression "promoter region or promoter portion" means a stretch of DNA or RNA that controls transcription of DNA or RNA and with which it is operably linked. The promoter region contains a specific sequence that is recognized and bound by RNA polymerase from which transcription is initiated.

[0134] Said portion of the promoter region is referred to as the "promoter". In addition, the promoter region contains a sequence that modulates the recognition, binding and initiation of transcriptional activity of RNA polymerase. Such sequences can regulate gene expression on a cis (cis-acting element) or trans (trans-acting element) basis. Depending on the method of regulation, the promoter may be a constitutive or regulated promoter. An exemplary promoter for Procaryot in this invention is the bacteriophage T7 and T3 promoter.

[0135] As used herein, the term "receptor" means a molecule that has an affinity for a given ligand. Receptors can occur naturally or in a synthetic form. In the art, they can be anti-ligands. As used herein, the terms "receptor" and "anti-ligand" are used interchangeably. Receptors can be used unchanged or as aggregates with other types of receptors. They can be attached covalently, non-covalently in direct or indirect physical contact to the binding agent via a specific binder or linker. Examples of receptors include, but are not limited to, antibodies, internalizing membrane receptors, monoclonal antibodies, antisera directed against antigenic determinants (located on the surface of viruses, cells or other surfaces), drugs, polynucleotides, nucleic acids, peptides, factors, lectins, sugars, polysaccharides, cells, cell membranes and cell organelles.

[0136] Examples of receptors and their uses include, but are not limited to:

a) enyyms: e^EWN4 gruaa of tranpportion proteins or enyymes necessary for the release of microorganisms that could serve as a "target" in the selection of antibiotics (ligand);

b) anti-specificity: identification of iigand binding sites within the molecules in combination with the epitope of the antigen of interest; determining the mimic sequence of the antigenic epitope may lead to the creation of a vaccine in which the immunogen is based on the said sequence or leads to similar diagnostic agents or chemical compounds useful in the treatment of e.g. autoimmune diseases

c) nucleic acid: identifying k ligand, such as protein or ubiquitous RNA, binding;

d) poiipeptides with activity: cataiitic: poiimey including poiipeptides: which favor the reaction: chemical type conversion of one or more reagents into one or more products; generally, such polypeptides contain a binding site specific for at least one reagent or reaction intermediate and an active site closest to the binding site at which chemically modifying the reagent associated therewith is possible (see, e.g., US Patent No. 5,215,899);

e) hormone receptors: the designation of ligands that bind the receptor with high affinity finds application in hormone replacement therapies; for example. ligand identification: the designation of ligands that bind to these types of receptors can lead to the development of new blood pressure control drugs; f) opiate receptors: the designation of ligands that bind to opiate receptors in the brain finds application in the development of less addictive substitutes for morphine and similar drugs.

[0137] As used herein, the term "sample" means any object containing an analyte for which an analytical test will be performed. The sample may be biological, as in the case of physiological fluid or tissue. Examples of physiological fluid are urine, blood, plasma, serum, saliva, semen, stool, phlegm, cerebrospinal fluid, tears, mucus, sperm, fetal water and others. Tissues are sets of cells, usually of a particular type, found together with the intercellular substance that forms one of the building materials in the human, animal, plant, bacterial, fungal and viral structure, including connective, epithelial, muscular and nerve tissues. Examples of biological tissues are also organs, cancerous tumors, lymph nodes, arteries and single cells.

[0138] For the purposes of this document, the stringency of hybridization conditions is determined by the degree of mismatch as follows: 1) high stringency: 0.1 x SSPE, 0.1% SDS, 65 ° C 2) moderate stringency: 0.2 x SSPE, 0.1% SDS, 50 ° C 3) Low stringency: 1.0 x SSPE, 0.1% SDS, 50 ° C. It is known to those skilled in the art that the washing step selects stable hybrids and the composition of SSPE is known (see, e.g., Sambrook, EF Fritsch, T. Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold spring Harbor Laboratory Press 1989 Vol 3, p. B. 13, see also many guides describing commonly used solutions in the laboratory). SSPE is a phosphate buffer at pH 7.4 and containing 0.18 NaCl. Furthermore, it is known to those skilled in the art that hybrid stability is dependent on TmT, which is a function of sodium ion concentration and temperature (Tm = 81.5 ° C-16.6 + 0.41 (% G + C) 600 / L)), therefore the only critical parameters for hybrid stability during the washing step there are sodium ion concentration in SSPE (or SSC) and temperature.

[0139] It is understood that equivalent stringent conditions can be obtained using replacement buffers, salts or temperatures. Examples include, but are not limited to, methods in which low stringent conditions are used as follows (see also Shilo and Weinberg, Proc. Natl. Acad Sci USA 78: 6789-6792 (1981)): DNA membranes are pre-incubated 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 500ug / ml denatured salmon sperm DNA (10x) SSC, 1.5 M sodium chloride, 0.15 M sodium citrate pH 7).

[0140] Hybridization is carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll 0.2% BSA, 100VG / M sperm DNA, 10% (wt / vol) dextran sulfate and 520 X 106 cpm labeled probe ³²P. Membranes are incubated in the hybridization mixture 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 washing solution is replaced with a fresh portion and incubation is continued for an additional 1.5 hours at 60 ° C. The membranes are dried and exposed on a photographic film (autoradiography). If necessary, membranes are washed a third time at 65-68 ° C and exposed again on photographic film. Other low stringent conditions that can be used are commonly known (e.g., the use of interspecific hybridization).

[0141] Examples include, but are not limited to, methods using moderate stringent conditions including, but not limited to, methods using moderate stringent conditions as follows: DNA membranes are pre-incubated for 6 hours at 55 hours. ° C in 6x SSC solution, 5x Denhart solution, 0.5% SDS and 100ug / ml denatured salmon sperm DNA. Hybridization is carried out in the same solution and 5-20 x 10⁶ labeled probe ³²P. Membranes are incubated in a hybridization mixture 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. The membranes are washed and exposed on a photographic film. Other moderately stringent conditions are well known in the art. Membrane washing is carried out at 37 ° C for 1 hour in a solution containing 2x SSC, 0.1% SDS.

[0142] An example not limiting the scope of the present invention may be procedures using the following highly stringent conditions: initial hybridization of membranes with DNA is carried out over a time interval of 8 hours overnight at 65 ° C in a buffer composed of 6x SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 ug / ml denatured salmon sperm DNA. Membranes are hybridized for 48 hours at 65 ° C in a prehybridization mixture containing 100 ug / ml denatured salmon sperm DNA and labeled probe ³²P (5-20 x 106 cpm). Membrane washing is carried out at 37 ° C for 1 hour in a solution containing 2x SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, and in the next stage, before autoradiography, at 50 ° C for 45 min. in 0.1x SSC. Other highly stringent hybridization conditions that can be used are well known in the art.

[0143] The term "substantially similar" or "significantly homologous" or the term "similar" is interpreted by those skilled in the art in different ways depending on the context. Generally, the term means at least 60% or 70%, preferably means at least 80%, 85%, more preferably at least 90%, and most preferably at least 95% identity.

[0144] As used herein, the term "substantially identical to the product" means "sufficiently similar", so that the property of interest is sufficiently unchanged, and a substantially identical product can replace the original product.

[0145] As used herein, the term "substantially purified" means "homogeneous enough" that it appears to be free of easily detectable impurities by standard methods such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC) used by specialists to estimate such contaminants or cleanses sufficiently and sufficiently, that further purification would not detectably change the physical and chemical properties such as the enzymatic and biological activity of the substance. The methods used to purify compounds are known to those skilled in the art. The chemically substantially purified compound may still be a mixture of stereoisomers or isomers. In this case, further purification could increase the specific activity of said compound.

[0146] As used herein, the term "target cell" means a cell that expresses sHASEGP in vivo.

[0147] As used herein, the term "test substance (test chemical)" means a chemically defined compound (e.g., organic compound, inorganic compound, organic / inorganic compound, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides or hybrids of said molecules such as glycoproteins, etc.) or mixtures of compounds (e.g. test compound libraries, natural extracts or media from cell culture, etc.) that affect sHASEGP, especially a single chain form that contains a domain with hyaluronidase activity or sufficient part of that domain to maintain activity, as demonstrated by in vitro methods e.g. test subject of the invention.

[0148] As used herein, the term "therapeutic agent (agent)", "treatment regimen", "radiation protective agent" or "chemotherapeutic" means conventional drugs and therapies including vaccines known to those skilled in the art. Factors used in radiation therapy are also well known in the art.

[0149] As used herein, the term "treatment" means any method of alleviating the symptoms of a disease, abnormality and condition, or any change that is beneficial to the patient in this regard.

[0150] Treatment also includes any use of the pharmaceutical subject of the present invention.

[0151] As used herein, the term "vector (or plasmid)" refers to the individual elements used to introduce a heterologous nucleic acid into a cell for its expression and replication. Vectors usually remain in episomal form, but can be designed to integrate the gene or part of it into the chromosome in the genome. The invention also includes vectors that are artificial chromosomes, such as yeast and mammalian chromosome. The selection and use of such carriers is well known to those skilled in the art. The expression vector includes a vector capable of expressing DNA that is operably linked to regulatory sequences, such as a promoter region capable of efficiently expressing DNA fragments. Therefore, the term "expression vector" means recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses and other such vectors, which when introduced into the appropriate host cell have the ability to express cloned DNA. Suitable vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and / or prokaryotic cells and those that remain in episomal form or that integrate into the host genome.

[0152] As used herein, the expression "protein binding sequence" means a protein or peptide sequence capable of specifically binding to another protein or peptide sequence, and generally, to a set of sequences or to a specific protein or peptide sequence.

[0153] As used herein, the term "epitope markers" means a short amino acid segment corresponding to an epitope to facilitate subsequent biochemical and immunological tests of a protein or peptide with an epitope tag, the so-called tag. Epitope labeling is obtained by incorporating the tag sequence into the protein coding sequence in the appropriate expression vector. Epitope-tagged proteins can be purified by affinity using highly specific antibodies directed against said tags.

[0154] As used herein, the term "metal binding sequence" generally means a protein or peptide sequence capable of specifically binding metal ions to a set of metal ions or to one particular metal ion.

[0155] As used herein, the term "combination" means any relationship between two or more elements.

[0156] As used herein, the term "composition" means any mixture. The mixture may be in the form of an aqueous or other type of solution, suspension, liquid, powder and paste.

[0157] As used herein, the term "liquid" means any composition that has the ability to flow. Hence, liquids have a semi-liquid structure, pastes, solutions, aqueous mixtures, gels, emulsions, creams and other structures.

[0158] As used herein, the term "cell extract" means the fraction prepared from the lysate obtained by cell lysis or other method of cell disintegration. [0159] As used herein, the term "randomly selected" means that the agent is selected without taking into account the specific sequence involved in the binding of the protein or protein itself along with its associated substrates, binding agents, etc. An example of random selection of factors is the use of a chemical library or peptide combinatorial libraries or culture in a nutrient broth with a selective agent or conditioned medium for this purpose.

[0160] For the purposes of this document, the term "rationally selected" or "rationally designed" means that the factor is selected based on non-accidental criteria that take into account the sequence of the specific destination for the factor and / or the conformation of that place taking into account the action of the given agent. As described in the experimental section, the proposed hyaluronidase binding sites and (catalytic) sites are found in glycoproteins with ID sequences. SEQ. No. 1 or ID. SEQ. Nr 4. Factors can be rationally selected or rationally designed by using peptide sequences that create such sites. An example of such a rationally selected peptide factor may be a peptide whose amino acid sequence is identical to the amino acid sequence of the binding sites or domains of ATP or calmodulin.

[0161] The oligosaccharides are presumed to have a reducing end and a non-reducing end, regardless of whether the sugar residue at the reducing end is or is not in fact a reducing sugar. In accordance with the adopted nomenclature, the graphical oligosaccharide structures shown here have a non-reducing end on the left and a reducing end on the right. All oligosaccharides presented herein are described using the name or abbreviation of a given non-reducing sugar (e.g. Gal) followed by a glycosidic bond configuration (a or b), the position of the carbon atom in the ring of the non-reducing sugar involved in the glycosidic bond, the position of the carbon atom in the ring of the reducing sugar involved in glycosidic binding followed by the name or abbreviation for reducing sugar (e.g. GlcNAc). For example, the bond between two sugar residues can be represented as follows: 2,3, 2> 3 or (2,3). Each monosaccharide is pyranose.

[0162] As used herein, the term "N-linked sugar" ("N-glycan") refers to an oligosaccharide bonded through an amide nitrogen atom of an Asn residue in the sHASEPG molecule. There are several main types of N-linked oligosaccharides (N-glycans) (high mannose, complex, hybrid, sulphated), and all of them have a (Man) 3-GlcNAc-GlcNAc core connected with an amide bond as the amino group Asn enters sequence composition -Asn-Xaa-Thr / Ser- (where Xaa is not Pro). N-glycosylation sites are often indirectly identified during sequencing by the appearance of so-called "Empty" cycles. Direct identification can be performed after releasing oligosaccharides by digestion with PNG F, which converts glycosylated Asn into Asp. N-linked oligosaccharides released as a result of PNG F treatment can be purified using gel chromatography on a Bio-Gel P-6 bed and then separated using preparative (high pH) high performance ion exchange chromatography (HPAEC) (Townsend) et al., (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide isomers can be separated by HPAEC. The presence in the structure of fucose residues will shift the elution position of a given molecule on the HPAEC chromatogram to an earlier position, while additional sialic acid residues will increase the retention time. Parallel treatment of glycoproteins with known oligosaccharide structures (such as bovine fetuin, α-1 acid glycoprotein, ovalbumin, RNAse B, transferrin) in this method may facilitate the description of the corresponding oligosaccharide peaks. Harvested oligosaccharides can be characterized using a combination of methods such as qualitative and methylation analysis to determine the composition and position of glycosidic linkages (Waeghe et al., (1983) Carbohydr Res. 123, 281-304.), And NMR spectroscopy to determine the anomeric configuration of glycosidic bonds (Van Halbeek (1993) in Methods Enzymol 230). [0163] Alternatively, oligosaccharides can be identified by fluorescence assisted sugar electrophoresis (Callewaert et al. (2001) Glycobiology 11,275-281).

[0164] As used herein, the term "sialic acid" refers to each member of the 9-carbon sugars family having a carboxyl group. The most common representative of the sialic acid family is N-acetylneuraminic acid (2-keto-5acetamido-3,5-dideoxy-Dg // ce / OD-ga / acto-nonulosonic acid (often referred to as Neu5Ac, NeuAc or NANA). The other member of this family is N-glycolyl neuraminic acid (Neu5Gc or NeuGc), in which the N-acetyl NeuAc group is hydroxylated. The third member of the sialic acid family is 2-keto-3-deoxy-nonulosonic acid (KDN) (Nadano et a /. (1986) J. Biol. Chem. 261: 11550-11557; Kanamori et a /. (1990) J. Biol. Chem. 265: 21811-21819). This group also includes 9-substituted sialic acids such as 9-O-C1-C6 acyl-Neu5Ac: 9-O-lactyl-Neu5Ac or 9-O-acetylNeu5Ac, 9-deoxy-9-fluoro-Neu5Ac and 9-azido -9-deoxy-Neu5Ac (See, e.g., 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 acids in the sialylation process has been disclosed in International Patent Application WO 92/16640, published October 1, 1992.

[00165] As used herein, the term "PNGase" refers to N-glycosidase F specific for asparagine-containing peptides, such as N-glycosidase-peptide found in F / avobactenum maningoseptum. The specificity of PNGase relates to N-linked rather than O-linked oligosaccharides. Performance characteristics of PNGase can be performed using either SDS PAGE or fluorescence assisted sugar electrophoresis.

[0166] As used herein, the term "heavily substituted with terminal sialic acid residues" refers to N-linked oligosaccharides terminated with a terminal sialic acid residue. Terminal sialic acid can be identified using FACE analysis of carbohydrates released following neuraminidase treatment. [0167] Blood glycoprotein half-life is highly dependent on the composition and structure of the N-linked carbohydrate moieties. This fact is important for therapeutic glycoproteins intended for parenteral administration. Generally, to ensure maximum glycoprotein half-life, it is required that the N-linked carbohydrate group be terminated with a NeuAc-Gal-GlcNAc sequence. The glycoprotein lacking in the structure of N-glycans terminal sialic acid (NeuAc) is rapidly removed from the circulation as a result of mechanisms based on the recognition of discovered residues of N-acetyl-galactosamine (GalNAc) or galactose (Gal) (Goochee et a /. (1991) Biol / Technology 9: 1347-1355). For this reason, ensuring the presence of terminal sialic acid in N-linked structures of therapeutic glycoprotein carbohydrates is an important issue from the point of view of their commercialization.

[0168] Circulating glycoproteins are exposed to sialidase (s) (or neuraminidase), which remove terminal sialic acid residues. Typically, sialic acid removal reveals galactose residues that are then recognized and bound by galactose-specific receptors present on the surface of hepatocytes (discussed in Ashwell and Harford (1982) Ann. Rev. Biochem. 51: 531). Other sugar-specific receptors involved in the removal of glycoproteins from the circulation are also found in the liver. Such receptors also recognize N-acetylglucosamine, mannose, fucose and mannose phosphate. Glycoproteins, removed as a result of galactose receptors, are significantly degraded and then enter the bile. In contrast, glycoproteins, removed as a result of the action of mannose receptors present on Kupffer cells, reach the reticuloendothelial system (discussed in Ashwell and Harford (1982) Ann. Rev. Biochem. 51: 53).

[0169] As used herein, the term "active at neutral pH" refers to sHASEGP glycoprotein with in vitro catalytic activity against glycosaminoglycans in an environment with a pH of 5.0 - 8.0, under conditions where the salt concentration is less than 150 mM and the ionic strength is less than 50 mM [0170] As used herein, the term "stabilized solution" refers to a sHASEGP solution in which glycoprotein, stored for 30 days at room temperature, retains more than 60% of its initial activity.

[0171] For the purposes of this document, the term "unit", unless otherwise stated, is expressed in turbidity reduction units (Turbidity Reducing Unit). One TRU unit is defined as the number of hyaluronidase activity units required to reduce the turbidity of the acidified hyaluronic acid solution and is equivalent to the units described in USP-NF [(United States Pharmacopeia - NF Receptor (National Folmulary, NF XIII)]. The results of the standard curve for a hyaluronidase sample prepared based on the ELISA type test described herein can be expressed in TRU units, units contained in NF and USP (e.g. USP or WHO standard) according to American Pharmacopoeia standards (USP). Therefore, enzymatic activities measured on the basis of said ELISA type test actually refer to TRU, unless this activity is not actually measured by measuring turbidity (Dorfman et al., 1948, J. Biol. Chem. 172: 367).

[0172] As used herein, the term "potency" refers to the amount of sHASEGP required for substrate degradation in vitro. Power is expressed in TRU units or relative TRU units.

[0173] As used herein, the term "specific activity" refers to units of activity per milligram of protein. The amount of sHASEGP is determined spectrophotometrically by measuring the absorption for the sHASEGP solution at a wavelength of 280 nm, taking into account a molar extinction coefficient of about 1.7 M^-1 cm^-1.

[0174] Polyethylene glycol (PEG) is widely used in biomaterials, biotechnology and medicine, primarily because it is a biocompatible, non-toxic, non-immunogenic and water-soluble polymer (Zhao and Harris, ACS Symposium Series 680: 458-72, 1997) . In the field of drug delivery, PEG derivatives are widely used as components covalently bound to proteins (so-called "Pegylation"), which reduces the immunogenicity of proteins, their proteolysis and renal clearance and increases their solubility (Zalipsky, Adv. Drug Del. Rev. 16: 157-82, 1995). Similarly, PEG is attached to small molecules or hydrophobic drugs to increase their solubility, reduce toxicity and vary bioavailability. Pegylated medicines are usually injected as solutions.

[0175] Since most of the chemical reactions used to design degradable, soluble drug carriers are the same, they can also be used in the design of degradable gels, e.g. synthesis of crosslinked degradable PEG derivatives or formulations for use in drug delivery (Sawhney et al., Macromolecules 26 : 581-87, 1993). It is also known that intermolecular complexes can be formed by mixing solutions of two complementary polymers. Such complexes are generally stabilized by electrostatic interactions (polyanion-polycation) and / or hydrogen bonds (polyacid-polysase) between the polymers involved and / or hydrophobic interactions between polymers found in the aqueous environment (Krupers et al., Eur. Polym J. 32 : 785-790, 1996). For example, under certain conditions, mixing solutions of polyacrylic acid (PAAc) and polyethylene oxide (PEO) leads to the formation of complexes stabilized by hydrogen bonds. The phenomenon of dissociation of these complexes under physiological conditions was used to administer drugs in the free state (such as unpegilated drugs). In addition, complementary polymer complexes were prepared from both homopolymers and copolymers.

[0176] In one aspect of the invention, the molecular weight of the polyethylene glycol ranges from about 3 kDa to about 50 kDa, and preferably from about 5 kDa to about 30 kDa. Attachment of a PEG molecule to a drug via covalent bond (known as "PEGylation") can be accomplished using known chemical synthesis techniques. For example, in one embodiment of the invention, pegylation of the protein can be carried out under appropriate conditions as a result of the reaction of PEG activated with NHS with the protein.

[0177] Although numerous types of pegylation reactions have been described, those that provide directionality of the reaction, run under mild conditions and do not require further complex procedures to remove toxic catalysts or by-products are most widely used. For example, monomethoxy-PEG (mPEG) has only one reactive hydroxyl terminal group and therefore the use of this compound somewhat limits the heterogeneity of the resulting PEG-protein product mixture. In general, the activation of the hydroxyl group located at the end of the polymer, on the other side relative to the terminal methoxy group, is necessary for efficient pegylation of the protein, and its goal is to obtain a PEG derivative that will be more susceptible to nucleophilic attack. The attacking nucleophile is usually the e-NH2-lysine residue group, however, under favorable conditions it can also be other amines (e.g. α-NH2- at the N-terminus of the protein or amino groups of the histidine ring). For proteins having one lysine or cysteine ​​residue, a more targeted attachment is possible. The PEG-maleimide reagent can be used to conjugate PEG to the thiol group of said cysteine. Alternatively, PEG hydrazide may be reacted with oxidized sHASEGP periodate, followed by reduction in the presence of NaCNBH3. More specifically, pegylated sugar compounds from CMP may be reacted with sHASEGP in the presence of the corresponding glycosyltransferases. One method is the "pegylation" method in which many polymers are attached to the polypeptide of the invention. The use of this technique weakens the immune response, causing the immune system to have difficulty recognizing protein epitopes in the structure of a pegylated polypeptide responsible for inducing antibodies. To polypeptides (i.e. pharmaceutical preparations) administered directly to the human circulatory system induced a specific physiological effect, the typical possible response of the immune system in this case is the production of IgG and / or IgM type antibodies, whereas inhaled polypeptides (i.e. industrial peptides) can potentially induce IgE type production ( i.e. IgE-dependent response to an allergen). One theory explaining the described phenomenon of weakening the immune response is that the polymer (s) mask the epitope (s) on the surface of the polypeptide responsible for inducing the mechanisms of the immune system leading to the production of antibodies. Another theory or at least one of the reasons for this phenomenon is the relationship in that the higher the molecular weight of a given conjugate, the greater the effect of weakening the immune response.

[0178] Such polypeptide-bound polymers can be any polymers with a molecular weight as defined in accordance with the present invention, including natural and synthetic homopolymers such as polyols (i.e. poly-OH), polyamines (i.e. poly-NH2) and acids polycarboxylic and other heteropolymers, i.e. polymers having one or more different binding groups, for example a hydroxyl group and an amino group.

[0179] Examples of suitable polymers include polymers selected from the group consisting of polyalkylene oxides (PAOs) such as polyalkylene glycols (PAGs) (including polypropylene glycols (PEG), methoxy polyethylene glycols (mPEG) and polypropylene glycols), PEG glycidyl ethers (Epox-PEG), PEG-oxycarbonyl diimidazole (CDI-PEG), branched polyethylene glycols, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyaminyl acids, polyethylene and hydroxybutanedioic anhydride copolymer, polystyrene and hydroxybutanedioic anhydride copolymer, dextrans (including carboxymethyldextran), heparin, albumin homologues, cellulose (including methylcellulose, carboxymethylcellulose, carboxymethylcellulose, hydroxy cellulose, hydroxy cellulose, ethylcellulose), such as hydroxyethylated starch, hydroxypropylated starch), glycogen, agaroses and their derivatives, guar gum, pullulan, inulin, xanthan gum, carrageenan, pectin, alginic acid hydrolysates and biopolymers.

[0180] Preferred polymers are non-toxic polymers such as the methoxy form of polyethylene glycol (mPEG), which can be covalently bound to enzymes by relatively simple chemical reactions.

[0181] Preferred polymers in the general sense are polyalkylene oxides (PAO), such as polyethylene oxides, such as PEG, and especially mPEG. This is due to the fact that, compared to polysaccharides such as dextran, pullan and similar compounds, said preferred polymers have few reactive groups that cause undesirable crosslinking of polymers in this case.

B. TISSUE EXPRESSION PROFILES sHASEGP.

[0182] By using more sensitive techniques such as RT-PCR, it turned out that sHASEGP, initially thought to be specific for human nuclei, is expressed in many human tissues. The sHASEGP transcript is found in the spinal cord (brain), capillary endothelium, prostate, breast tissue, retina, human melanocyte fraction, fetal heart and pregnant uterus. The sHASEGP glycoprotein is also expressed in germ cell tumors. Generally, RT-PCR is required to determine the level of sHASEGP transcripts in non-testicular tissues.

C. SHASEGP ENZYMATIC ACTIVITY TESTS [0183] HYALURONIDASE ACTIVITY TEST BASED ON TURBIDIMETRIC MICROMRIMETRICATION.

[0184] Hyaluronidase activity can be determined in acidified serum solution by a modified turbidimetric test.

The required reagents are shown below.

<td>Twice deionized, UV sterilized water or sterile irrigation water</td><td>Braun</td><td>R5000-01</td>

<td>Hylumed Medical - Hyaluronan</td><td>Genzyme Advanced</td><td> 4876</td>

<td>sodium, high molecular weight HA</td><td>Biomaterials</td><td></td>

<td>Hyaluronidase reference standard</td><td>USP</td><td> 31200</td>

<td>Potassium acetate, granules, USP, ACS</td><td>JTBaker</td><td> 2914-01</td>

<td>Acetic acid, glacial,&gt; 99%</td><td>Sigma</td><td>A-6283</td>

<td>Monobasic sodium phosphate, monohydrate, USP, granules</td><td>Mallinkrodt</td><td> 7774</td>

<td>Sodium phosphate dibasic, anhydrous</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, pooled, cell-tested, hybridoma-cell tested, origin: USA</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, double hydrated, granules, USP, -FCC</td><td>JTBaker</td><td> 1336-01</td>

[0185] The following reagents are prepared: acetate buffer: 14.0 g potassium acetate and 25.0 ml glacial acetic acid are dissolved in water to a volume of 1000 ml; phosphate buffer: 2.5 g monobasic sodium phosphate, 1.0 g anhydrous dibasic sodium phosphate and 8.2 g sodium chloride are dissolved in water to a volume of 1000 ml; Stock enzyme dilution solution: 500 ml phosphate buffer is added to 500 ml water. Working solution for enzyme dilution: 33 mg of gelatin hydrolyzate in 50 ml of enzyme dilution stock solution, prepared for 2 hours. before use; Sample stabilizing buffer ("SSB" solution): 125 ml 20% human serum albumin and 50 ml 1M calcium chloride in 50 ml enzyme working solution, well mixed; Serum stock solution: horse serum is diluted 1: 9 in acetate buffer, the pH is adjusted to 3.1 with 4 M hydrochloric acid, the solution is left at room temperature for 18 to 24 hours. The solution is stored at 4 ° C and used for 30 days. Serum working solution: 10 ml of stock serum solution in 30 ml of acetate buffer, solution temperature brought to ambient temperature. Hyaluronic acid stock solution: 5.0 mg / ml hyaluronic acid in water. Hyaluronic acid working solution: 0.75 ml stock solution of hyaluronic acid in 4.25 ml phosphate buffer. Standard stock solution: the contents of one package of hyaluronidase standard (USP, standard compliant with the requirements of the American Pharmacopoeia) are dissolved in water so that the concentration is 1000 U / ml, divided into 50 portions and stored at 20 ° C. Standard working solution: 40 ml of standard stock solution in 960 ml of chilled working solution for enzyme dilution, dilution of the enzyme to obtain a concentration of 40 U / ml, solution prepared immediately before use.

[0186] All enzyme samples were diluted in a 96-well plate with a surface that provides poor protein binding as follows:

[0187] a) the maximum sensitivity range of this test is within 10-30 U / ml. To minimize the number of test replicates necessary to determine the dilutions for results within the range given, first determine the approximate value of the enzyme concentration in the sample (U / ml), and then select such a dilution (integer) so that the final enzyme concentration is about 20 U / ml.

[0188] b) minimum sample volumes needed to perform the test: FPLC fractions: 50 ml, tissue culture supernatants: 1 ml, purified and concentrated final formulation: 10 ml.

[0189] c) for samples obtained as a result of serial dilutions: 1:10 dilutions prepared in triplicate in a 96-well plate, with a surface ensuring poor protein binding, by mixing 360 ml SSB solution and 40 ml sample solution in each well. [0190] To prepare the USP standard, in accordance with the requirements of the American Pharmacopoeia (USP), a standard curve should be made on a 96-well plate with a surface ensuring poor protein binding:

<td></td><td></td><td></td><td>Standard USP curve:</td><td></td>

<td>Holes:</td><td>Standard:</td><td>Stock solution dilution [ml]:</td><td>to Working solution up to enzyme dilution [ml]:</td><td>Final concentration [U / ml]</td>

<td>A1-A3</td><td>ST01</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>

[0191] To prepare hyaluronic acid control samples in columns 1-3, the HA control should be prepared as follows in a 96 well flat bottom plate:

[0192]

HA checks:

<td>Holes:</td><td>Control:</td><td>Acid working solution hyaluronic acid [ml]</td><td>Working solution for dilution enzyme [ml]</td>

<td>H1-H3</td><td>Ko01</td><td> 0</td><td> 60</td>

[0193] Reaction plate: 30 ml of hyaluronic acid working solution is applied, using a 50 ml 8-channel pipette, to each well of a 96-well flat-bottomed plate, leaving wells H1-H3 empty. Then 60 ml / well of enzyme dilution working solution is added to wells H1-H3 on the same plate, which will serve as a control.

[0194] Serum working solution: 40 ml of the serum working solution is poured into a suitable reagent container and then placed in a heating block.

[0195] Pre-heating step: after preparing both plates, a 96-well plate with poor protein binding surface, containing diluted samples, standards and controls, and a flat-bottomed 96-well plate containing hyaluronic acid working solution, is placed in the heating block and left for 5 min. at 37 ° C.

[0196] The reaction is started by adding the enzyme to the substrate: 30 μΙ of the solution from the plate containing the enzyme solutions are added, using an 8-channel pipette with a volume of 5 - 50 μΙ, to the wells in column 1 on a flat-bottom 96-well plate (containing substrate ). To obtain a homogeneous solution, the enzyme / substrate reaction mixture is stirred for the first 15 s by aspirating 5 times and dispensing the sample again. After mixing the enzyme and substrate, remove the used pipette tips and replace with new ones before adding solutions to the wells in the next column. You must restart the stopwatch and at (t) = 0: 30, repeat the described procedure for the wells in column 2. After another 30 s, at (t) = 1: 00, the procedure is repeated for column 3. In this way, moving along the plate from left to right, every 30 s we repeat the described process for all wells containing the enzyme / substrate mixture.

[0197] Stopping the reaction: As soon as the stopwatch shows (t) = 6: 00, using an 8-channel pipette with a volume of 50-300 ml, add to each well of the column 1 flat-bottom 96-well plate a 240 ml working solution of serum from 50 ml reagent container. In order to obtain a homogeneous solution, the mixture is mixed for the first 10 s for 3 times aspiration and re-dosing of the sample. The procedure is repeated every 30 s for subsequent columns from 1 to 12. After the last column (twelfth column) has been mixed, remove the reaction plate from the heating block and place it in a reader set at a wavelength of 640 nm. The linear curve fitting function generated based on the standard curve allows extrapolation of test sample results.

[0198] ALTERNATIVE HYALURONIDASE TESTS [0199] MICROMEDIARATION TEST BASED ON BIOTINYLATED HYALURONANATE [0200] The free carboxyl groups of glucuronic acid hyaluronan are biotinylated in a one-step reaction using biotin-NH1 (Pierce) sulfuride (NH1) (3dimethylaminopropyl) carbodiimide (Sigma). Such a biotinylated HA substrate is covalently bound to the surface of a 96-well plate in a second reaction. The substrate remaining after the end of the enzymatic reaction is detected by avidin-peroxidase reaction by reading the reaction result using a standard ELISA plate reader. Because the substrate is covalently bound to the plate in this assay, there are no artifacts such as pH-dependent movement of the biotinylated substrate. The sensitivity of the test allows rapid measurement of hyaluronidase activity from cell cultures and biological samples with within 10% variation within the test.

[0201] Hyaluronidase specificity is expressed in turbidity reducing unit (TRU). One TRU unit is defined as the number of hyaluronidase activity units required to reduce the turbidity of the acidified hyaluronic acid solution and is equivalent to the units described in USP-NF [(US Pharmacopoeia of the United States Receptor NF (National Folmulary, NF XIII)]. The results of the standard curve for a hyaluronidase sample prepared based on the ELISA type test described herein can be expressed in TRU units, units contained in NF and USP (e.g. USP or WHO standard) according to American Pharmacopoeia standards (USP). Therefore, the enzymatic activities measured on the basis of said ELISA type test actually refer to TRU, unless this activity is not actually measured by measuring turbidity (Dorfman et al., 1948, J. Biol. Chem. 172: 367).

[0202] Many tests for hyaluronidase activity are based on measuring the presence of newly formed reducing N-acetylamino groups (Bonner and Cantey, Clin. Chim. Acta 13: 746-752, 1966) or on measuring the decrease in viscosity (De Salegui et al., Arch. Biochem. Biophys. 121: 548-554, 1967) or turbidity (Dorfinan and Ott, J. Biol. Chem. 172: 367, 1948). All these methods are effective when determining the presence or absence of endoglucosaminidase activity of an enzyme on purified substrates.

[0203] Significantly purified glycosaminoglycan substrates can be used in the gel migration slowdown test. Glycosaminoglycans are mixed with recombinant sHASEGP to study its endoglucosaminidase activity, which is manifested by a slowdown in the mobility of the substrate in the gel. Chondroitin-4 and 6 sulfate, dermatan sulfate, heparan sulfate can be obtained from Sigma Chemical. Hyaluronan isolated from human umbilical cord can be obtained from ICN. Each test substrate is diluted to a concentration of 0.1 mg / ml in buffer with a pH in the range 3.5 - 7.5. Samples of purified sHASEGP or conditioned media of cells expressing sHASEGP, with a volume of 10 ml, are mixed with 90 ml of substrate in the appropriate buffer and incubated for 3 hours. at 37 ° C. Then the samples are neutralized with sample buffer (Tris-EDTA, pH 8.0, bromophenol blue and glycerol) and subjected to electrophoresis separation. Glycosaminoglycans are identified by staining the gel overnight with a 0.5% solution of Alcian Blue dye in 3% glacial acetic acid, followed by staining the gel with 7% glacial acetic acid. The level of degradation is determined by comparing the mobility of the substrate in the presence and absence of enzyme.

[0204] Hyaluronidase activity can also be determined using zymography (Guentenhoner et al., 1992, Matrix 388-396). In this test, the sample is applied to a polyacrylamide gel containing SDS and hyaluronic acid, and then the proteins in this sample are electrophoretically separated (SDSPAGE). After separation, the gel is incubated in a buffer suitable for the test and then stained for identification in a hyaluronic acid gel. Proteins with hyaluronidase activity are identified by visualization as colorless bands in the gel containing the substrate.

[0205] D. IDENTIFICATION AND ISOLATION OF sHASEGP POLYPEPTIDE GENES.

[0206] Domains and / or whole genes of the sHASEGP polypeptide can be obtained by DNA isolation methods well known in the art. Any method known to those skilled in the art can be used to identify nucleic acids encoding the desired gene. Any method known in the art can be used to obtain full-length cDNA, i.e., the entire coding region or a clone of genomic DNA encoding the sHASEGP polypeptide. An example of a method is a chain polymerization reaction that can be used to amplify sequences from a cDNA or genomic DNA library expressed in normal tissue, e.g., nucleic acids encoding the sHASEGP polypeptide (SEQ. No. 1 and 2). Oligonucleotide primers that hybridize to sequences at the 3 'and 5' ends of identified sequences can be used as primers for PCR amplification of the sequence based on nucleic acid template samples (RNA or DNA, generally cDNA libraries, from a suitable source (e.g. nucleus, prostate , breasts).

[0207] PCR may be carried out, e.g. using a Cetus thermal cycler from Perkin Elmer and Taq polymerase (GeneAmp). The amplified DNA may contain mRNA or cDNA or genomic DNA from different Eucariota species. Several different degenerate primers can be selected for PCR.

[0208] The stringent hybridization conditions used in the PCR reaction in the primer annealing phase to multiply nucleic acid homologues (e.g., to obtain a sHASEGP polypeptide sequence from non-human species / organisms, or to obtain a nucleotide sequence with homology to a sHASEGP polypeptide) can be modified by allowing for flexibility in nucleotide sequence similarity between known nucleotide sequences and isolated nucleic acid homologues. Low or moderately stringent conditions apply for interspecific hybridization. For some species, hybridization is carried out under moderate to highly stringent conditions. These conditions can be determined experimentally.

[0209] After successful amplification of a nucleic acid containing all or part of the sequence of the identified sHASEGP polypeptide or all or part of the sequence encoding all or part of the homologous sHASEGP polypeptide, a fragment thereof can be cloned and sequenced and used as a probe to isolate a complete cDNA molecule or genomic clone GOUT. This, in turn, allows the determination of the complete nucleotide sequence of the gene, analysis of its expression and production of its expression product for functional analysis. Once the sequence has been determined, an open reading frame (ORF) of the protein product can be located using any suitable method known in the art, e.g. using publicly available computer programs for nucleotide sequence analysis. If an open reading frame is determined, determining the amino acid sequence of the encoded protein from said reading frame is standard operation. In the above manner, both the nucleotide sequence of the entire genes of the sHASEGP polypeptide and the amino acid sequence of the sHASEGP protein and its analogs can be identified.

[0210] The source of nucleic acid for cloning the sHASEGP polypeptide gene may be any eukaryotic cell. Nucleic acids can be isolated from cells of vertebrates, mammals, human, pig, beef, cat, bird, horse, canine as well as from primate, insect, plant and other cells. DNA can be obtained according to standard procedures known in the art, starting from DNA cloning (e.g. DNA libraries), by chemical synthesis, cloning cDNA or genomic DNA or fragments thereof, purification from given cells (see e.g. Sambrook et al. 1989, Molecular Cloning, A Laboratory Manual, 2d 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 contain, in addition to coding regions, intron and regulatory DNA regions. Clones derived from cDNA would contain only exon sequences. Regardless of the source of DNA, said gene for cloning is cloned into the appropriate vector.

[0211] In the process of molecular cloning of a gene from genomic DNA, DNA fragments are produced, some of which will encode the gene of interest.

[0212] DNA can be digested at various specific cleavage sites using various types of restriction enzymes.

[0213] Alternatively, DNAse may be used for DNA fragmentation in the presence of magnesium ions or physical methods e.g. sonication. Linear DNA fragments can be separated according to size by standard techniques including, but not limited to, agarose or polyacrylamide gel electrophoresis or by column chromatography.

[0214] Identification of generated DNA fragments can be accomplished in a number of ways.

[0215] An example is part of the sHASEGP polypeptide gene (any type) (e.g., PCR amplification product obtained as described above or an oligonucleotide with a sequence that is part of a known nucleotide sequence) or sHASEGP-specific RNA or a purified and labeled fragment thereof and produced DNA fragments can be screened by hybridization with labeled probes (Benton and Davis, Science 196: 180 (1977); Grunstein and Hogness, Proc. Natl. Acad. Sci. USA 72: 3961 (1975)). DNA fragments with significant homology to the probe will hybridize. The appropriate fragment can also be identified by digestion with restriction enzymes and comparison of the size of the fragments to the size of the expected fragments according to the restriction map, if available. Identification can also be accomplished by DNA sequence analysis and comparison to the known nucleotide sequence of the sHASEGP polypeptide. Further selection can be made based on the properties of the gene. Alternatively, the presence of the gene can be determined in a test based on the physical, chemical or immunological properties of the expressed product. Examples are cDNA clones or DNA clones that select by hybridizing the appropriate RNA based on which a protein with similar or identical gel migration is produced, with similar or identical parameters in isoelectric focusing, proteolytic digestion map, antigenic properties and hyaluronidase activity. The above-mentioned protein can be identified by binding labeled antibodies directed against the sHASEGP polypeptide (if available) to clones potentially synthesizing the sHASEGP polypeptide by ELISA (enzyme-linked immunosorbent assay).

[0216] An alternative to isolating genomic DNA for a sHASEGP polypeptide, but not limited to, is chemical synthesis of a gene sequence according to a known sequence or the transcription of cDNA into RNA that encodes the sHASEGP protein.

[0217] For example, RNA that is transcribed into cDNA intended to clone the sHASEGP protein gene can be isolated from cells expressing said protein. The isolated and identified nucleic acids can then be introduced into the appropriate vector. Many vector / host cell systems are known in the art. Potential vectors include, but are not limited to, plasmids or modified viruses, which, as tools for introducing DNA, must be compatible with the host cells used. Such vectors include, but are not limited to, bacteriophages such as lambda phage derivatives, plasmids pBR322, pUC or modified derivatives thereof, the Bluscript vector (Stratagene, La Jolla, CA). Cloning into the vector may be completed by the step of ligation of the DNA fragment into the sticky ends of said vector. [0218] If the complementary restriction sites used to cut the DNA into fragments are not present in the cloning vector, the DNA ends may be subjected to enzymatic modification. Alternatively, any needed site can be generated by ligation of the nucleotide sequence (linker) into DNA ends. The ligated linkers may contain specific chemically synthesized oligonucleotides encoding endonuclease recognition restriction sites. In an alternative method, the digested vector and sHASEGP protein gene can be modified by homopolymeric attachment of a homopolymeric linker.

[0219] Recombinant molecules can be introduced into host cells by transformation, transfection, transduction, electroporation, calcium phosphate method and other methods that result in the generation of multiple copies of the sequence of the introduced gene.

[0220] In a specific embodiment of the invention, transformation of host cells with a recombinant DNA molecule that incorporates the isolated gene for the sHASEGP protein, cDNA or synthesized DNA sequence allows the production of multiple copies of said gene.

[0221] Thus, a gene in large numbers of copies can be obtained by growing transformants and then isolating recombinant DNA from them and, when necessary, recovering the insert gene from isolated recombinant DNA.

[0222] VECTORS, PLASMIDS AND CELLS, CONTAINING NUCLEIC ACID ENCODING sHASEGP PROTEIN OR ITS DOMAIN WITH HYALURONIDASE ACTIVITY AND EXPRESSION OF sHASEGP POLYPEPTIDE IN THESE VECTORS AND CELLS.

[0223] For the expression of a recombinant one or more sHASEGP proteins, a nucleic acid containing all or part of the nucleotide sequences encoding sHASEGP can be inserted into a suitable expression vector, e.g. into a vector containing the components necessary for transcription and translation of the introduced sequence encoding said protein. The required transcription and translation signals can be complemented by a native promoter for the sHASEGP genes and / or flanking sequences.

[0224] The invention also includes vectors containing nucleic acid encoding glycoproteins with sHASEGP hyaluronidase activity that can be introduced into an expression system in which it is possible to produce a soluble, pH-active sHASEGP glycoprotein with hyaluronidase activity.

[0225] The invention also includes vectors. The term "cells" includes both eukaryotic and prokaryotic cells and vectors suitable for these cells.

[0226] The invention relates to eukaryotic cells, including Chinese Hamster Ovary Cells, lacking dihydrofolate reductase (DG44) activity. The term "suitable cells" includes yeast, fungal, plant, insect and animal cells. The listed cells are used for the production of sHASEGP glycoprotein or a domain with hyaluronidase activity of sHASEGP glycoprotein in subsequent stages: hyaluronidase activity from the medium. In an exemplary embodiment, the domain with hyaluronidase activity is secreted into the medium.

[0227] In one embodiment, the invention relates to a vector comprising a nucleotide sequence encoding a protein derived from sHASEGP that exhibits hyaluronidase activity and comprises a domain with hyaluronidase activity or only a portion thereof or multiple of a copy of the domain with hyaluronidase activity or a portion of that domain. The invention also relates to vectors that contain a nucleotide sequence encoding a domain with hyaluronidase activity and, in addition to this domain, an additional portion of sHASEGP protein that can be of varying length and even so long that together with the domain with hyaluronidase activity it forms a complete sHASEGP molecule. The invention also includes multiple copies of the nucleotide sequence encoding the domain with hyaluronidase activity and, in addition to this domain, an additional portion of sHASEGP protein of varying length, up to one that together with the domain with hyaluronidase activity forms a complete sHASEGP molecule. For expression of the sHASEGP glycoprotein or its domain with hyaluronidase activity, expression vectors may be chosen that will allow the secreted form of the expressed sHASEGP glycoprotein to be obtained. Alternatively, the signal sequence necessary for secretion of the encoded proteins may be present in the vectors. During expression of a domain with hyaluronidase activity, the nucleic acid is combined with a nucleic acid encoding a signal sequence, such as a signal sequence, a conjugation factor in Saccharomyces cerevisiae, a portion thereof, or a native signal sequence.

[0228] To produce a soluble, pH-active glycoprotein with hyaluronidase activity, cells capable of N-glycolization are needed. In a preferred embodiment, mammalian CHO cells with dihydrofolate reductase deficiency, i.e. DG44 cells, are electroporated in the presence of the coding plasmid in the following order: strong mammalian promoter, i.e. CMV, nucleic acid encoding sHASEGP followed by the IRES sequence Internal Ribosome Entry Site) and mouse gene for dihydrofolate reductase, SV40 polyadenylation sequence as shown in ID sequence. SEQ. Nr 51. Electroporated cells are initially cultured in a cell medium with strictly defined chemical composition that does not contain hypoxanthine and thymidine, and then in a medium with the addition of methotrexate in a gradually increasing concentration.

[0229] A variety of vector / host cell expression systems can be used to express protein coding sequences. Such systems include, but are not limited to, mammalian cell-based expression systems transduced with e.g. vaccinia virus or adenovirus, etc., expression systems based on insect-infected virus cells (e.g. baculovirus), expression systems based on microorganisms such as yeast containing vectors or expression systems based on bacteriophage, DNA, plasmid DNA or cosmid transformed bacteria. The vector elements responsible for expression differ in their "potency" and specificity. Depending on the vector / host cell system used, any of these elements may be used. It should be noted that by expressing DNA in bacteria for sHASEGP, no catalytically active sHASEGP molecule is obtained. Only the joint action of the bacterial expression system first, and in the next stage of the mechanism of proper glycosylation leads to obtaining an artificially glycosylated molecule.

[0230] For insertion into the vector of the insert in the form of nucleic acid fragments, any methods known to those skilled in the art may be used, including a chimeric gene containing the appropriate signal coding sequences for transcription and translation as well as a protein. These methods may include in vitro artificial recombination techniques and in vivo recombination (genetic recombination) techniques. Expression of the nucleic acid sequence encoding sHASEGP glycoprotein or its domains, derivatives, fragments or homologues can be regulated by a second sequence, so said genes or fragments thereof are expressed in host cells transformed with recombinant DNA molecule (s). For example, protein expression can be controlled by any promoter / enhancer known in the art. In a specific embodiment, the promoter is not native to the sHASEGP glycoprotein genes. The otory ferry that can be used includes, but is not limited to, the SV40 early promoter (Bernoist and Chambon, Nature 290: 304-310 (1981)), the promoter located at the 3 'end of the long repeating Rous Sarcoma virus sequence (Yamamoto et al ., Ce // 22: 787-797 (1980)), promoter for herpes virus thymidine kinase (Wagner et al. Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981)), regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39-42 (1982)), promoters of prokaryotic expression vectors such as the β-lactamase promoter (Villa-Kamaroff 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)); promoters of expression vectors used in plant cells, such as the opalin synthetase promoter (Herrar-Estrella et al., Nature 303: 209-213 (1984)) or the 35S RNA promoter for cauliflower mosaic virus (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)), elements of the yeast and other fungi such as the Gal4 promoter, alcohol dehydrogenase promoter, kinase promoter phosphoglycerol, alkaline phosphatase promoter and the following transcription control regions in animal cells that exhibit tissue specificity and have been used in transgenic animals: elastase gene control region active in acinar cells of the pancreas (Swift Et et al., Cell 38: 639-646 (1984); Omitz Et et al., Cold Spring Harbor Symp. Quant. Biol. 50: 399-409 (1986); Macdonald, Hepatology 7: 425-515 (1987)); active insulin control gene region in pancreatic beta cells (Hanahan et al., Nature 315: 115-122 (1985)), active immunoglobulin gene control region in lymphoid cells (Grosschedl et et al., Cell 38: 647-658 (1984) ; Adams et al., Nature 318: 533-538 (1985); Alexander et et al., Mol. Cell Biol. 7: 1436-1444 (1987)), murine breast cancer virus active region in testicular, breast, lymphoid and mast cells (Leder et et al., Cell 45: 485-495 (1986)), albumin gene controlling region in liver (Pickert et et al., Genes and Devel. 1: 268-276 (1987)), the region controlling the α-fetoprotein gene active in the liver (Krumlauf et et al., Mol. Cell. Biol. 5: 1639-1648 (1985); Hammer et et al., Science 235: 53-58 1987)), the liver-controlling α1-antitrypsin gene controlling region (Kelsey et al., Genes And Devel. 1: 161171 (1987)), a region controlling the β-globin gene active in myeloid cells (Mogram et al, Nature 315: 338-340 (1985); Kollias et et al., CE // 46: 89-94 (1986)) gene control region for myelin basal protein active in brain oligodendritic cells (Readhead et al., Cell 48: 703-712 (1987)), gene control region 2 myosin light chain active in skeletal muscle cells (Sani, Nature 314: 283-286 (1985)), gonadotropin release gene controlling region activated in hypothalamic gonadotropes (Mason et al., Science 234: 1372-1378 (1986)).

[0231] In a specific embodiment, the vector used comprises a promoter operably linked to nucleic acids encoding the sHASEGP glycoprotein or domain, fragment, derivative or homolog, one or more replication initiation sites, and optionally one or more selectable markers (e.g., an antibiotic resistance gene).

[0232] Specific initiation signals may be required for efficient translation of sHASEGP sequences. These signals contain the ATG initiation codon and adjacent sequences. In the event that the initiation code and the 5 'upstream sequences are inserted into the appropriate expression vector, no additional control translation signals are needed. However, in the case where only the coding sequence or part thereof is introduced, exogenous transcription initiation control signals containing the ATG initiation codon must be provided. Furthermore, for transcription to cover the entire insert, the initiation codon must be in the correct reading frame. Exogenous transcription elements and initiation codons can be of various origins, both natural and synthetic. Expression efficiency can be increased by including in the transcription elements appropriate enhancers for 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) [0233 ] Furthermore, host cells can be selected for their ability to modulate the expression of the introduced sequences or for the desired mode of expression. Such protein modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation and acylation. Posttranslational processing during which the protein is digested may also be important for correct insertion, folding and / or function. Various host cells such as CHO (DG44, DXB 11 CHO-K1), HeLa, MDCK, 293, WI38), etc. they have specific cellular machinery and characteristic mechanisms for such post-translational activities and can therefore be selected to ensure the correct modification and processing of the foreign protein introduced into them.

[0234] For long-term, high-yield production of recombinant protein, stable expression is preferred. For example, cell lines that stably express sHASEGP can be transformed using expression vectors containing viral origin of replication origin or endogenous expression elements and a marker gene. After introducing the vector, the cells grow in enriched medium and 1-2 days later in selective medium. The selectable marker provides resistance to the selection factor, so that cells that successfully express the introduced sequence can survive the selection and divide. Aggregates of stably transformed resistant cells can be propagated using cell culture techniques adapted to the cell type.

[0235] Any number of selection systems can be used to obtain transformed cell lines. Selection systems include, but are not limited to, herpes virus thymidine kinase genes (Wigler M. et al. (1977) Cell 11: 223-32) and adenine phosphoribosyl transferase (Lowy I. et al. (1980) Cell 22: 817-23) used in TK or APRT cells, respectively. Resistance to antimetabolites, antibiotics and herbicides can also be used as a basis for selection, e.g. DHFR is responsible for methotrexate resistance (Wigler M. et al. (1980) Proc Natl Acad Sci 77: 3567-70); e.g. responsible for resistance to aminoglycoside antibiotics such as neomycin or G418 (Colbere-Garapin F. et al. (1981) J Mol Biol 150: 1-14), as well as als or pat responsible for resistance to chlorsulfuron and phosphinothricin / phosphinothricin acetyltransferase, respectively (Murry, supra). Other genes responsible for selection have also been described, e.g. trpB, which allows cells to use indole instead of tryptophan or the hisD gene, which allows cells to use histinol instead of histidine (Hartman SC and RC Mulligan (1988) Proc Natl Acad Sci 85: 8047-51). Recently, it has become popular to use marker genes expressing a visible product, such as anthocyanins, β-glucuronidase together with the substrate, GUS, luciferase together with the luciferin substrate. Visible markers have found application not only in identifying transformants, but also in quantifying the transient or stable expression efficiency associated with a specific vector system (Rhodes CA et al. (1995) Methods Mol Biol 55: 121-131).

[0236] IDENTIFICATION OF TRANSFORMANTS CONTAINING POLINUCLEOTIDE SEQUENCE.

[0237] Although the presence / absence of the expression marker gene suggests that the gene of interest is also present, the presence of DNA and expression of active sHASEGP should be confirmed. For example, when the DNA for sHASEGP is an insert within the marker gene sequence, recombinant cells having sHASEGP can be recognized by stating that the gene does not function.

[0238] PURIFICATION of sHASEGP [0239] Host cells transformed with the sHASEGP nucleotide sequence can be cultured under conditions suitable for expression and recovery of the encoded protein from cell culture. The protein produced by the recombinant cell is preferably secreted, but can also be expressed into the cell depending on the sequence and / or vector used. As is understood by those skilled in the art, sHASEGP expression vectors can be designed to contain a signal sequence that facilitates the direct secretion of sHASEGP across a Procariota or Eucariot cell membrane. In other constructs, the sHASEGP coding sequence may be attached to the nucleotide sequence encoding the protein domain, facilitating purification of soluble protein (Kroll DJ et al. (1993) DNA Cell Biol 12: 441-53; cf. discussion on vectors containing fusion protein).

[0240] sHASEGP can also be expressed as a recombinant protein with one or more additional polypeptide domains added to the isolated protein to facilitate its purification. These types of domains contain, but are not limited to, metal chelating peptides, such as histidine-tryptophan motifs, allowing purification on beds with immobilized transition metal ions, protein A domains that allow purification on a bed with immobilized immunoglobulins, and FLAG tag domains. which allows purification by affinity chromatography (Immunex Corp, Seattle Wash.). The purification process facilitates the introduction of a linker sequence that can be cleaved, e.g., with factoractor XA protease or enterokinase (Invitrogen, San Diego Calif.) Between the purification facilitating domain and sHASEGP. One of these expression vectors provides a fusion protein that consists of sHASEGP and a nucleic acid encoding the 6 histidine residue motif, as well as a cleavage site for thioredoxin and enterokinase. The histidine motif facilitates purification on IMIAC (immobilized metal ion affinity chromatography as described by Porath et al. (1992) in Protein Expression and Purification 3: 263-281) while the cleavage site for enterokinase provides tools for purifying chemokines from the fusion element.

[0241] In addition to recombinant production, the sHASEGP fragment can be produced by direct peptide synthesis using solid carrier synthesis 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 either manually or automatically. You can automatically synthesize the Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer, Foster City Calif.) According to the manufacturer's instructions. Individual sHASEGP fragments can be chemically synthesized separately and combined by chemical methods until a complete molecule is obtained.

[0242] Expression vectors containing sHASEGP coding sequences or portions thereof are obtained, for example, by cloning the coding portion in place of the EcoRI site of each of the three pGEX vectors, vectors expressing glutathione S-transferase (Smith and Johnson, Gene 7: 31-40 (1988) ). This allows products to be expressed in the correct reading frame. Exemplary vectors and domain expression systems with sHASEGP protein hyaluronidase activity are well-known Pichia cell based vectors (available for example from Invitrogen, San Diego, CA), in particular those that have been designed for secretion of encoded proteins. The protein can also be expressed into the cytoplasm or inclusion bodies. One such type of vector has been described in the examples.

[0242] Vectors for transforming E. coli include, e.g., the pET expression vector (see U.S. Patent 4,952,496; available from Novagen, Madison, WI; also references published by Novagen describing the system).

[0244] Such plasmids include pET11a, which contains the T71 ac promoter, T7 terminator, inducible E. coli lac operon and the lac gene repressor; pET12A-C, which contains the T7 promoter, T7 terminator and E. coli OMPT signal sequence; pET15B and pET19B (Novagen, Madison, Wi), which contains the His-tag sequence used for purification on His column and thrombin digestion site which is used after the column purification step; T7 promoter region and T7 terminator.

[0245] The vectors are introduced into host cells such as Pichia cells and bacterial cells such as E. coli and expressed in them. Exemplary Pichia strains include, e.g., GS115. Exemplary bacterial host cell strains contain chromosomal copies of DNA encoding T7 RNA polymerase operably linked to an inducible promoter, such as the LACUV promoter (see US Patent No. 4,952,496). Such host cells include, but are not limited to, lysogenic E. cola BL21 (DE3).

[0246] sHASEGP domains, derivatives and analogs can be produced by various methods known in the art. For example, when a sHASEGP protein, domain, fragment or derivative is found, the gene product can be isolated and analyzed. The analysis can be performed using tests based on the physical and / or functional properties of the protein, including, but not limited to, analysis of radiolabelled gel electrophoresis product, immunoassays, cross-linking of the labeled product and tests of proteolytic activity. [0247] The sHASEGP protein can be isolated and purified by standard methods known in the art (also from natural sources and from host cells expressing protein complexes), including, but not limited to, a chromatographic column (e.g. ion exchange chromatography, affinity, molecular filtration, high-pressure reverse phase chromatography (HPLC), rapid protein liquid chromatography (FPLC), differential centrifugation, differential solubility and other standard techniques used for protein purification, [0248] In one embodiment, sHASEGP can be purified to a homogeneous state from chemically defined culture media, intended for culture of DG44 cells transfected with the HZ24 vector and propagated in the presence of methotrexate, by the technique of: 1) diafiltration with artificial flow, 2) binding and elution from anionic affinity chromatography, 3) flow through a column with Sefarose with phenyl groups, 4) binding and elution from a chromatographic bed based on phenyl boronate, 5) binding and elution from a hydroxyapatite chromatographic bed.

[0249] Functional properties can be evaluated using an appropriate test known in the art. [0250] Alternatively, once the sHASEGP protein, domain, fragment or derivative thereof is found to be present, the amino acid sequence can be deduced from the nucleotide sequence of the gene that encodes it. Consequently, the protein or its domain can be synthesized by standard chemical methods known in the art (e.g. see Hunkapiller et al., Nature 310: 105-111 (1984)) and then subjected to in vitro glycosylation.

[0251] Modifications of the sHASEGP protein may be carried out at the protein level. The invention includes sHASEGP proteins, its domains, derivatives and analogs that are modified differently during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, pegylation, derivatization with known protective / blocking groups, proteolytic digestion, linking with the antibody molecule and other cell ligands.

[0252] The modification can be carried out by one of many known chemical modification techniques that include, but are not limited to, specific chemical digestion with cyanogen bromide, trypsin, chymotrypsin, papain, V8, NaBH₄, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin and other factors.

[0253] In addition, domains, analogs and derivatives of sHASEGP may be chemically synthesized. For example, a peptide corresponding to a portion of the sHASEGP protein that contains the desired domain or that affects in vitro activity can be synthesized using a peptide synthesizer.

[0254] Moreover, if necessary, sHASEGP protein sequences can be introduced by substitution or addition of non-classical amino acids or amino acid analogs including, but not limited to, D isomers of ordinary amino acids, [0255] In cases where natural products are suspected are mutants or are isolated from new species, the amino acid sequence of sHASEGP from a natural source, and also that expressed in vitro or synthesized from expression vectors in vivo or in vitro, can be determined by DNA sequence analysis or alternatively, by direct sequencing of the isolated protein. This type of analysis can be performed manually or using an automatic amino acid sequencer.

[0256] Modifications - The invention includes various modifications of sHASEGP proteins and domains. The sHASEGP encoding nucleic acid can be modified according to various strategies known in the art (Sambrook et al. (1990), Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). Sequences can be digested at appropriate sites with endonuclease / restriction endonucleases, subsequently subjected to further enzymatic modifications, if necessary, isolated and ligated in vitro. In the process of producing a gene encoding a sHASEGP domain, derivative or analog, make sure that the modified gene has the original reading frame and that translation will not be interrupted by a stop codon in the gene region where the desired active region is encoded.

[0257] In addition, the coding nucleic acid molecules may be mutated in vitro and in vivo to allow and / or prevent translation, initiation, and / or termination of the sequence or to create changes in the coding region and / or to create new restriction sites or destruction already existing to facilitate further in vitro modification. As also described herein, the invention also includes muteins with alterations in the original sequence, such as replacement of cysteine ​​residues, removal or addition of glycosylation sites. In the ID sequence. SEQ. No. 1 SHASEGP has seven potential glycosylation sites. Such mutations can be carried out by any of the mutagenesis techniques known in the art, including, but not limited to, chemical mutagenesis and in vitro point mutagenesis (Hutchinson et al., J. Biol. Chem. 253: 6551-6558 (1978), TABE Linkers (Pharmacia)) In one embodiment, e.g. the sHASEGP protein or domain thereof is modified by attaching a fluorescent label. In another embodiment, the sHASEGP protein or domain thereof is modified by attaching a heterobifunctional reagent that can be used to cross-link protein complexes in the membrane. [0258] In addition, sHASEGP domains, analogs and derivatives can be chemically modified. For example, a peptide corresponding to a portion of sHASEGP that contains the desired domain or exerts an effect on the desired activity can be synthesized using a peptide synthesizer. Furthermore, if desired, non-classical amino acids or chemical amino acid analogs can be introduced as a substitution or addition to the sHASEGP sequence. Non-classical amino acids include, but are not limited to, D isomers of common amino acids, 4-aminobutanoic acid, Abu, 2-amino butanoic acid, S-ABU, e-Ahx, 6-amino caproic acid, Aib, 2-amino isobutanoic acid, 3-amino propionic acid, ornithine , norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro amino acids, synthetic amino acids such as thi-methyl amino acids, ca-methyl amino acids namethyl amino acids and generally amino acid analogues. In addition, the amino acid can be d (right-handed) or 1 (left-handed).

[0259] F. PRODUCTION OF A FUNCTIONALLY ACTIVE GLYCOSYLATED sHASEGP WITH NGLICANES.

[0259] F. RECEPTION OF FUNCTIONALLY ACTIVE GLYCOSYLATED sHASEGP WITH ATOM N-SUGAR GROUP.

[0260] To produce a catalytically stable protein it is required to obtain properly N-glycosylated human sHASEGP. N-glycosylation of sHASEGP can be carried out using a variety of techniques. Glycosylation can be carried out by introducing into cells of eukaryotic origin nucleic acids encoding such sHASEGP that ensure normal Nglycosylation, or alternatively, by placing the sHASEGP polypeptide in contact with cell extracts or purified enzymes ensuring the introduction of the N-linked sugar group into the polypeptide structure.

CHOICE OF EXPRESSION SYSTEM [0261] Mammalian cell based expression systems differ in the degree and type of glycosylation introduced into ectopically expressed peptides, for example CHO cells are highly efficient in N-glycosylation of the active sHASEGP polypeptide.

[0262] Additional expression systems based on eukaryotic cells that allow Nglycosylation and production of functional sHASEGP can be tested by introducing the sHASEGP expression plasmid into said cells and testing for activity at neutral pH. Identification of whether N-glycosylation has occurred correctly can be performed using FACE analysis of glycans released as a result of PGNase glycoprotein treatment. Glycosylation profiles for catalytically active sHASEGP glycoproteins are presented later in this document. Glycosylation verification can also be performed by treatment with PGNase F of sHASEGP glycoprotein obtained from said cells or by cloning nucleic acids encoding sHASEGP into said cells and culturing these cells in a medium containing tunicamycin.

[0263] N-glycosylation of sHASEGP polypeptide in vitro. The sHASEGP polypeptide can be Nglycosylated by contact with cell extracts with activity that allows the N-linked sugars to be introduced into the sHASEGP polypeptide structure, such as canine microsomes, or through a commercially available combined transcription and translation system (Promega Madison WI).

[0264] It is assumed that the oligosaccharides have a reducing end and a non-reducing end, regardless of whether the sugar residue at the reducing end is or is not actually a reducing sugar. In accordance with the adopted nomenclature, the graphical oligosaccharide structures shown here have a non-reducing end on the left and a reducing end on the right. All oligosaccharides presented herein are described using the name or abbreviation of a given non-reducing sugar (e.g. Gal) followed by a glycosidic configuration (alpha or beta), the position of the carbon atom in the ring of the non-reducing sugar involved in the glycosidic bond, the position of the carbon atom in the ring of the reducing sugar involved in glycosidic binding followed by the name or abbreviation for reducing sugar (e.g. GlcNAc). The bond between two sugar residues can for example be expressed as 2.3, 2&gt; 3 or (2.3). Each monosaccharide is pyranose.

[0265] As used herein, the term N-linked sugar moiety ("N-glycan") refers to an oligosaccharide bound through an amide nitrogen atom of an Asn sHASEPG residue. There are several main types of oligosaccharide-linked oligosaccharides (high-mannose, complex, hybrid, sulfated), and all of them have a (Man) 3-GlcNAc-GlcNAc core connected with an amide bond to the Asn amino group, which is part of the -Asn- sequence Xaa-Thr / Ser- (where Xaa is not Pro). N-glycosylation sites are often indirectly identified during sequencing by the appearance of so-called "Empty" cycles. Direct identification can be performed after releasing oligosaccharides by digestion with PNG F, which converts glycosylated Asn into Asp. N-linked oligosaccharides released as a result of the PGN F treatment can be purified using gel chromatography on a Bio-Gel P-6 bed and then separated using prepartative (high pH) high performance ion exchange chromatography (HPAEC) (Townsend et al ., (1989) Anal. Biochem. 182, 1-8). Certain oligosaccharide isomers can be separated by HPAEC. The presence in the structure of fucose residues will shift the elution position of a given molecule on the HPAEC chromatogram to an earlier position, while additional sialic acid residues will increase the retention time. Parallel treatment with this method of glycoproteins with known oligosaccharide structures (such as: bovine fetuin, α-1 acid glycoprotein, ovoalbumin, RNAza B, transferrin) can help describe the corresponding oligosaccharide peaks. Harvested oligosaccharides can be characterized using a combination of methods such as qualitative and methylation analysis indicating the composition and position of glycosidic bonds (Waegheet al., (1983) Carbohydr Res. 123, 281-304.), And NMR spectroscopy to determine the anomeric configuration of glycosidic bonds (Van Halbeek (1993) in Methods Enzymol 230). [0266] Alternatively, oligosaccharides can be identified in sugar electrophoresis using fluorescent labels (FACE) (Callewaert et al. (2001) Glycobiology 11,275-281).

[0267] G. DETECTION AND CHARACTERISTICS OF SHASEGP SUGAR CONNECTED BY ATOM.

[0268] The first step in glycoprotein glycan analysis is to check that the resulting protein is indeed glycosylated. The method of choice is the use of polyacrylamide gel electrophoresis under denaturing conditions in the presence of sodium dodecyl sulfate (SDSPAGE) as the last step before protein sequencing. Glycosylated proteins often migrate in the form of diffused bands during SDS-PAGE analysis. An indication of the presence of N-glycans is a significant reduction in the band width and a change in its migration position as a result of treatment with glycoprotein amidase with the peptide-N4- (N-acetyl-D-glucosaminyl) asparagine (PNGase F) molecule. If other types of glycosylation are present, other methods should be used. Immunobloting using lectins is a method that does not allow to distinguish the type of glycolization (N from O). Carbohydrate-binding protein lectins have both high affinity and narrow specificity for a wide range of defined sugar epitopes present in glycoprotein glycans (Cummings, RD (1994) Methods in Enzymol. 230, 6686.). They can easily be identified, when conjugated with biotin or dioxygenin, on a membrane obtained by western blotting by a colorimetric reaction using avidin or anti-dioxygenin conjugated alkaline phosphatase conjugates (Haselbeck, et al. (1993) Methods in Mol. Biol. 14, 161-173.), Secondary analogues conjugated with alkaline phosphatase. Glycoprotein binding tests using a panel of lectins with well-defined specificity allow for obtaining relevant information about the glycans present on its surface. It is important that the enhancement of color development in this method is high enough that one can easily see 10 to 15 ng of membrane glycoprotein transferred by western-blot. Although lectins have very high affinity for their ligands, some of them exhibit significant avidity for similar in structure epitopes. Therefore, when choosing a lectin panel for the test, remember about the possibility of cross-reactions and choose those lectins that are likely to distinguish between complex and high-mannose N-glycans from O-glycans. [0269] To determine if sHASEGP is glycosylated, sugar composition analysis can be used, which provides additional information on glycan structure for lectin analysis. Qualitative analysis of the sugar composition i) allows to identify glycosylated proteins, ii) allows to determine the molar ratio of individual sugars to protein molecules, iii) allows to identify, in some cases, glycan types, iv) is the first stage in the design of structural analysis strategies and vi) allows to assess repeatability the process of producing recombinant glycoproteins with therapeutic properties. In recent years, high performance ion exchange chromatography (under high pH conditions) with pulsed amperometric detection (HPAEC-PAD) (Townsend, et al. (1995) in Carbohydrate Analysis: High-performance liquid chromatography and capillary electrophoresis ( Z. E1 Rassi ed.) Pp. 181-209.). Methods based on fluorophore labeling have recently been introduced and many ready-made kits based on such methods are now available. The advantage of fluorescent methods is their higher sensitivity (50 times). However, one potential disadvantage is that different monosaccharides may exhibit differentiated fluorophore selectivity during the binding reaction, both in the hydrolyzate and in a mixture of external standards. Despite this, this method is attractive due to the increased sensitivity and the ability to determine the sugar composition in a small portion of the available glycoprotein sample, as well as the possibility of increasing the sensitivity using laser-labeled fluorescence.

[0270] The best way to analyze the sugar composition of small amounts of sHASEGP is preceded by electrophoretic separation of electrobloting using PVDF (PSQ) membranes (Weitzhandleret al, (1993) J. Biol. Chem. 268, 5121-5130.), And in even smaller amounts -blot. PVDF is an ideal matrix for the analysis of sugars because it does not bind monosaccharides or oligosaccharides when they are released by acid or enzymatic hydrolysis.

[0271] FACE analysis is an efficient method for determining the glycosylation profiles of sHASEGP glycoproteins. Analysis of glycosylation profiles by the FACE method (Prozyme), using gels containing 30% oligosaccharides, is one such technique. For the detection of sHASEGP glycosylation profiles, electrophoretically separated glycans released enzymatically released as a result of Nglikanase treatment (also known as PNGase) from the surface of 100 pg glycoprotein and labeled with ANTS fluorophore can be used. The relative position of the glycan-corresponding bands is determined by simultaneously analyzing the sample, its dilutions and glycan standards, and determining the migration distance in units of degree of polymerization (DP).

[0272] SCRINING METHODS FOR IDENTIFICATION OF CHEMICAL COMPOUNDS MODULATING MODULES FOR sHASEGP ACTIVITY [0273] Several examples of such tests have been presented and described herein. It is understood that hyaluronidase domains can also be used in other tests. However, as shown here, the hyaluronidase domains possess catalytic activity.

[0274] And as such they are excellent for in vitro screening assays.

[0275] said domains can also be used in binding assays.

[0276] The use of sHASEGP full glycoprotein zymogens, activated enzymes and hyaluronidase domains is contemplated in any screening assay known to those skilled in the art, including the tests presented herein. Hence, the following description, if it relates to a hyaluronidase activity test, relates to the use of one domain chain with hyaluronidase activity or the catalytically active part of each hyaluronidase, including sHASEGP. The present invention relates to other tests, such as binding assays, in particular for use with sHASEGP, including any variant thereof, e.g. splice variants.

[0277] 1. Catalytic activity tests for the identification of factors modulating the hyaluronidase activity of the sHASEGP protein.

The present invention relates to methods for identifying the modulator of sHASEGP catalytic activity, particularly one domain chain with hyaluronidase activity or a catalytically active part thereof. These methods can include: sHASEGP contact, i.e. a full zymogen molecule or active form of sHASEGP, in particular one chain of its catalytic domain, with a substrate for sHASEGP in the presence of a test substance, detection of the proteolysis of that substrate against which sHASEGP activity is tested, and then comparing this activity with a control sample. For example, the control can be sHASEGP activity tested by contacting sHASEGP, including the full zymogen molecule or the active form of sHASEGP, and in particular one chain of its catalytic domain, with a substrate for sHASEGP and the detection of proteolysis of that substrate against which sHASEGP activity is tested. The results obtained are compared for samples containing and free of test chemicals. The difference in activity shows whether a given compound modulates sHASEGP activity. The present invention also provides activators of proteolytic activation of sHASEGP, and assays for their identification are described below.

[0278] In one embodiment of the invention, multiple substances are tested at once using the screening methods described above. In another embodiment, sHASEGP is isolated from target cells, which allows the identification of factors that are potentially specific to these target cells.

[0279] In yet another embodiment of the invention, the test substance is a therapeutic compound for which the difference in sHASEGP activity measured in samples containing and lacking that test substance shows that the target cells respond to the test therapeutic compound.

[0280] One method includes the steps of: (a) contacting a sHASEGP polypeptide or domain with hyaluronidase activity with one or more test chemical compounds under conditions that permit interaction between the ligand and these chemical compounds, and (b) identifying among these compounds one or more substances that specifically bind this ligand.

[0281] Another method of the invention includes the steps of: a) contacting a sHASEGP polypeptide or domain with hyaluronidase activity with a sHASEGP polypeptide substrate and detecting degradation of that substrate against which the sHASEGP polypeptide activity is tested; b) contacting a given sHASEGP polypeptide with its substrate in the presence of a test substance and detecting the degradation of that substrate against which the activity of the sHASEGP polypeptide is tested and c) comparing the activity of the sHASEGP polypeptide determined in steps a) and b), the activity measured during step a) differs from the activity measured in step b) and indicates that the test substance modulates the activity of said sHASEGP polypeptide.

[0282] In another embodiment of the invention, multiple substances are tested simultaneously. When comparing the activity of an sHASEGP polypeptide in the presence or absence of a test substance, in order to check whether this substance is a modulator of the sHASEGP polypeptide, it is not necessary to study these activities in parallel unless such parallel measurement is standard. The activity of the sHASEGP polypeptide can be measured at one time point and compared with the activity values ​​obtained in previous measurements.

[0283] For example, the activity of the sHASEGP polypeptide can be measured in the presence of a test substance and compared with the activity value measured in a previous measurement for the sHASEGP polypeptide in the absence of the test substance. The reverse order is also possible. Such measurement can be made based on the leaflet or instructions provided with the ready test kit.

[0284] Methods for selecting substrates for individual sHASEGP glycoproteins are described in the experimental section together with specific examples of hyaluronidase activity assays.

[0285] The present invention relates to combinations and ready sets comprising these combinations, potentially including instructions for performing the test. Said combinations include the sHASEGP polypeptide to be tested and its substrate, and optionally may include reagents for detecting the proteolysis of this substrate. Substrates, which may be chromoso- and fluorogenic molecules containing glycosaminoglycans and can be digested proteolytically by individual sHASEGP polypeptides, can be identified experimentally by testing the ability of sHASEGP to digest them. The most efficiently digested substrates are identified, i.e. those that are digested at the lowest concentrations and / or at the fastest rate or under the expected conditions. [0286] In addition, the present invention provides a ready-made kit comprising the combination described above. Such kit optionally includes instructions for identifying the sHASEGP polypeptide activity modulator. In the present invention, each sHASEGP polypeptide can be used to identify modulators of its activity.

[0287] 2. Binding assays. The present invention also relates to methods for identifying and isolating agents, especially chemical compounds that bind to sHASEGP glycoproteins. These tests are designed to identify factors that bind to an isolated hyaluronidase domain (or a protein other than a sHASEGP polypeptide that contains a domain with hyaluronidase activity from the sHASEGP polypeptide), an active form, including the active form of the domain obtained from a full zymogen molecule or from extended domain with hyaluronidase activity. The identified chemical compounds are potential drugs or are used to identify compounds for the treatment of disorders and diseases associated with abnormal hyaluronidase activity. The sHASEGP polypeptides used in the methods include, as defined herein, each sHASEGP polypeptide, including a single domain chain with hyaluronidase activity from sHASEGP or a proteolytically active part thereof.

[0288] The present invention provides a variety of methods. Such methods may include solution reactions or solid phase reactions in which the sHASEGP polypeptide (s) or domain (s) with hyaluronidase activity are directly or indirectly (via a linker) bound to a solid support. Screening tests were described in the experimental section and were used to identify potential chemical compounds.

[0289] For the purposes of the present invention, all binding assays described above are prepared for sHASEGP testing.

[0290] The present invention provides methods for identifying an agent, such as a chemical, that specifically binds to a single-chain domain with hyaluronidase activity from sHASEGP, a full active sHASEGP molecule, or its double-chain domain with hyaluronidase activity. The method may consist of (a) contacting sHASEGP with one or more test factors under conditions favorable for binding between sHASEGP and a given factor, and (b) identifying among those compounds one or more factors that specifically bind sHASEGP.

[0291] For example, using such methods, a given sHASEGP polypeptide is mixed with a potential binding partner or cell extract or fraction under conditions that allow the potential binding partner to associate with that polypeptide. After mixing, the peptides, proteins or other molecules that have associated with sHASEGP are separated from the mixture. The binding agent that has bound to sHASEGP can then be separated and further analyzed. For identification and isolation of the binding agent, the entire protein may be used, for example, all disclosed protein with an ID sequence. SEQ. No.: 1. In another embodiment of the invention, a fragment of this protein may be used.

[0292] A variety of methods can be used to obtain cell extracts or body fluids, serum, urine, sweat, synovial fluid, CSF and other such fluids.

[0293] For example, cells can be disintegrated using both physical and chemical methods. Examples of physical methods of cell disintegration, but in no way limited to the following list, are sonication and mechanical cutting. Examples of chemical lysis methods, but in no way limited to the following list, are lysis detergents and enzymatic digestion. A specialist in a given field will easily adapt the methods of preparing cell extracts to obtain the extracts needed in the presented methods.

[0294] After obtaining the cell extract, it is mixed with sHASEGP under conditions favorable for the association of the protein with the binding partner. A variety of conditions can be used in this case, including conditions similar to those prevailing in the cytoplasm of a human cell or in body fluids such as blood. By introducing changes in the parameters used, such as osmolarity, pH, temperature and concentration of the cell extract, the phenomenon of protein association with a binding partner can be optimized. Similarly to the described case, methods for isolating the molecules of interest from body fluids are known.

[0295] After mixing under appropriate conditions, the protein-binding partner complex is isolated from the mixture. Various methods can be used to separate such a mixture. For example, one such method is based on the immunoprecipitation of a protein-binding agent complex as a result of the reaction of the agent with sHASEGP-specific antibodies. Alternatively, standard separation techniques such as chromatography and density gradient centrifugation can be used.

[0296] After removal of the unbound components of the cell extract, the binding agent is dissociated using conventional methods from the complex. For example, dissociation of the complex can be accomplished by changing the salt concentration or the pH of the mixture.

[0297] To facilitate the isolation of the binding partner for sHASEGP from the extract, sHASEGP can be immobilized on a solid support. For example, the protein may be associated with nitrocellulose or globules. The purpose of binding a protein or fragment thereof to a solid support is to facilitate the separation of the peptide / binding agent pair from other components in the extract. Such identified binding partners can be either single proteins or complexes of two or more proteins.

[0298] Alternatively, nucleic acid molecules encoding a single hyaluronidase chain can be used in two-hybrid yeast systems. Two-hybrid yeast systems have been used to identify other protein complexes and can successfully be adapted to use the nucleic acid molecules described herein.

[0299] Yet another in vitro binding assay, particularly useful for sHASEGP glycoprotein testing, involves using a mixture of a polypeptide that contains at least one catalytic domain of one of these proteins, and one or more potential binding agents or substrates. After incubating such a mixture under appropriate conditions, the ability of a given sHASEGP or a polypeptide fragment thereof containing the catalytic domain to bind or interact with a potential substrate is tested. In binding assays based on cell extracts, one of the components contains or is associated with a detectable label. The tag allows direct detection based on the phenomenon of radioactivity, luminescence, measurement of optical or electron density, etc. or indirect detection through the use of a label, enzyme, etc. A variety of methods can be used to detect the label, which are selected depending on the nature of the label and other test components. The label can be detected, for example, as bound to a solid substrate, or a part of the complex containing the label can be separated from the substrate that exists in solid form, followed by detection of the label.

[0300] 3. The detection of signal transduction by sHASEGP, which is a protein anchored in the cell membrane, can be performed directly and based on the cell surface receptor or indirectly using activating proteins such as growth factors that can initiate signal transduction.

[0301] In addition, a secretory sHASEGP, such as the soluble sHASEGP domain described by sequence ID. SEQ. No. 4 can be involved in signal transduction either directly, by binding to or interacting with the cell surface receptor, or indirectly through activating proteins, such as growth factors that initiate signal transduction. Transduction signal evaluation assays are well known to those skilled in the art and can easily be adapted for use with an sHASEGP polypeptide.

[0302] The present invention provides assays for identifying agents that modulate or affect the transduction signal transmitted directly or indirectly by the activation of growth factors by sHASEGP, especially all or part of the protein that is sufficient to anchor its extracellular domain on the cell surface. Such assays include, for example, transcription based assays in which signal transduction modulation is tested by measuring the effect of the reporter gene on expression (see US Patent No. 5,436,128).

[0303] 4. Methods for identifying agents that modulate expression of sHASEGP encoding nucleic acid.

In another embodiment, the invention provides a means of identifying factors that modulate the expression of sHASEGP encoding nucleic acid. Such assays can use any available method for monitoring changes in the expression level of sHASEGP encoding nucleic acids.

[0304] Monitoring of the agent's ability to modulate expression of sHASEGP encoding nucleic acid can be performed in the form of an assay. For example, mRNA expression can be monitored directly by hybridization to nucleic acids. Enzyme tests for the detection of factors modulating sHASEGP expression have also been described.

[0305] Under appropriate conditions, cell lines are exposed to the tested factors for a specified time and total RNA or mRNA is isolated using standard procedures (see, e.g., Sambrook et al (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. Cold Spring Harbor Laboratory Press). Probes for detecting differences between RNA expression levels in cells treated with a given agent and RNA expression levels in control cells can be made of nucleic acids. Usually, but not necessarily, probes are designed that hybridize only to the target nucleic acid under highly stringent conditions. Only highly complementary nucleic acid forms hybrids under highly stringent conditions. Therefore, the stringency of the test conditions determines the level of complementarity that should characterize two strands of nucleic acids so that they can form a hybrid. To maximize the differences in stability of the probe-target hybrid complexes and potential probe-non-target hybrids, stringent conditions should be used.

[0306] For example, the N- and C-terminal fragments of a given sHASEGP may be expressed in bacteria and used to search for binding proteins to them. The substrates can be fusion proteins such as His tagged or GTS tagged proteins attached to the N- or C-terminus of sHASEGP. Such fusion proteins can for example be combined with glutathione-sepharose beads and used as probes for testing from cell lysates or body fluids. Prior to the lysis stage, cells or body fluids can be exposed to potential sHASEGP-modulating factors or proteins interacting with sHASEGP domains. Proteins associated with fusion proteins present in lysates can be separated by SDS-PAGE, isolated and identified by sequencing or mass spectrometry, i.e. methods known in the art.

[0307] Antibody probes are obtained by immunizing suitable mammals according to appropriate immunization protocols, polypeptides or proteins of appropriate length (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 20, 25, 30, 35, 40 or more constitutive amino acids of the sHASEGP polypeptide) or, if there is a need to increase immunogenicity, polypeptides or proteins of suitable length conjugated to suitable carriers. Methods for preparation of immunogenic conjugates containing carriers such as bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH) or other carrier proteins. Under certain circumstances, direct conjugation using carbodiimide can be effective. In other cases, it may be advisable to use other coupling compounds such as reagents manufactured by Pierce Chemical Co. to provide access to hapten. (Rockford, IL). To facilitate carrier binding, peptide haptens may be extended at the N- or C-terminus by one cysteine ​​residue or several Cys residues may be incorporated into the structure of such haptens.

[0308] Generally, such immunogens are administered by their injection over a defined period of time together with appropriate adjuvants, as is well described in the art. To monitor the quality of the humoral immune response, antibody titers are measured during the immunization process. [0309] Antibodies directed against peptides can be produced using synthetic polypeptides including, for example, N-terminal amino acids sHASEGP.

[0310] Synthetic peptides may have a length of 1 to 3 amino acids, generally a length corresponding to at least four or more amino acids. Peptides can be conjugated to KLH using standard methods and then used to immunize animals such as rabbits or ungulates. The polyclonal antibodies are then purified, for example, using Actigel beads containing a covalently bound peptide.

[0311] For some applications, such polyclonal antibodies may meet the requirements set for them, but when it comes to pharmaceutical compositions, monoclonal antibodies are generally used. Immortalized cell lines that secrete the desired monoclonal antibodies are prepared by the method of Kohler et al. (Nature 256: 495-7 (1975) or using generally known modifications affecting the immortalization process of lymphocytes or spleen cells. The immortalized cell lines secreting the desired antibodies are screened by immunochemical tests in which the antigen is a peptide hapten, polypeptide or protein.

[0312] Once the appropriate immortalized cell line secreting the desired antibody has been identified, such cells are cultured either by in vitro methods or by their in vivo propagation in ascites fluid. Monoclonal antibodies that recognize the catalytic domain or proteolytic activation site of sHASEGP are of particular interest.

[0313] Antibodies or fragments thereof, as well as chimera-derived species including regions specifically binding to desired receptor regions may also be produced. [0314] The factors tested by the method described above can be selected randomly or rationally selected or designed.

[0315] Examples of such factors are peptides, small molecules and carbohydrates. A specialist in the field will quickly assess that there are no limits to the structure of such factors.

[0316] Peptide factors can be prepared using standard methods of peptide synthesis known in the art (solid state or solution synthesis). In addition, DNA encoding these peptides can be synthesized using commercially available oligonucleotide synthesis protocols and produced by a recombination technique using standard recombinant expression systems.

[0317] I. METHODS OF TREATMENT.

[0318] The sHASEGP glycoproteins designated by the methods described herein are used to treat or prevent the abnormal accumulation of sHASEGP substrates in animals, especially mammals, including man. In one embodiment, said method comprises administering to the mammal an effective amount of sHASEGP glycoprotein that is being treated or prevented from disease or disorder.

[0319] In another embodiment, the sHASEGP inhibitor may be used to treat excessive hyaluronidase activity. The mammalian representative can be a human. Inhibitors of the invention are inhibitors identified in screening. In addition, the invention includes antibodies, antisense nucleic acids, double-stranded RNA (dsRNA) and RNAi.

[0320] 1. Nucleic antisense acid treatment: In a specific embodiment as described above to prevent excessive chondroitinase activity, sHASEGP protein functions are reduced or inhibited by specific antisense nucleic acids. The invention relates to the therapeutic or prophylactic use of nucleic acids consisting of at least 6 nucleotides, generally up to about 150 nucleotides, which are antisense to the sequence of a gene or cDNA encoding a protein or part of sHASEGP. The term "antisense" sHASEGP nucleic acid ", as used herein, means a nucleic acid capable of hybridizing to the RNA portion (generally mRNA) of the sHASEGP protein as a result of some sequence complementarity and generally under high stringency conditions. The antisense nucleic acid may be complementary to the coding and / or non-coding region of the mRNA of the sHASEGP protein. This type of antisense nucleic acid finds application in therapies that reduce or inhibit the function of the sHASEGP protein and can be used to treat or prevent disorders as described above.

[0321] Antisense nucleic acids of the sHASEGP protein consist of at least 6 nucleotides and are generally oligonucleotides (covering a range of 6 to 150 nucleotides including a range of 6 to 50 nucleotides). The antisense molecule can be complementary to the entire hyaluronidase domain or part thereof. For example, an oligonucleotide is composed of at least 10 nucleotides, at least 15 nucleotides, at least 100 nucleotides and at least 125 nucleotides. Oligonucleotides can be like DNA or RNA, they can be chimera, derivatives or modified versions, single-stranded or double-stranded. The oligonucleotide may be modified at the base, sugar moiety and phosphate backbone site. . The oligonucleotide may contain other attached groups such as peptides or agents that facilitate transport across the cell membrane (see, e.g., 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 No. WO 88/09810, published December 15, 1988) or blood-brain barrier (see, e.g., PCT No. WO 89/10134, published on April 25, 1988), initiators of hybridization (see, e.g., Krol et al., BioTechniques 6: 958 -976 (1988)) or intercalar factors (see, e.g., Zon. Pharm. Res. 5: 539-549 (1988)).

[0322] The antisense nucleic acid of the sHASEGP protein is generally an oligonucleotide, usually single-stranded DNA or RNA, an analogue thereof, or a mixture thereof. For example, said nucleotide contains an antisense sequence to a portion of the nucleic acid encoding the human sHASEGP protein. The oligonucleotide can be modified at any position in the structure with usually single-stranded substituents generally known in the art.

[0323] The antisense oligonucleotide of the sHASEGP protein may contain at least one modified base selected from the group consisting of, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcysteine, 5 (carboxyhydroxymethyl) , 5-carboxymethylaminomethyl-2-thioury &lt;duna, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylcouosine, inosine, N6isopentenyloadenine, 1-methylguanine, 1-methylolysine, 2,2-dimethylguanine 2-methyloadenine, 2-methylguanine, 3-methylcytosine, 5-methylcysteine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methylolaminomethyluracil, 5-methoxyaminomethyl-2-thiouracyl, b-D-mannosylcenoozymethoxy-5-methacacoroxymethyl 2-methylthio-N6-isopentenyl adenine, uracil-5-oxoacetic acid (v), wyutoxosine, pseudouracil, cuenosine, 2-thiocytosine, 5-methyl-2-thiouracyl, 2-thiouracil, 4-thiouracyl, 5-methylouracyl, methyl ester, methylester (v) uracyl-5-oxoacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3-n-2 carboxypropyl) -uracil, (ACP3) and 2,6-diaminopurine.

[0324] In a further embodiment, said nucleotide comprises at least one modified sugar residue selected from the group consisting of, but not limited to, arabinose, 2-fluoroarabinose, xylulose and hexose. The oligonucleotide may contain at least a modified phosphate backbone selected from phosphorothioates, dithiophosphates, phosphorothioates, phosphorothioates, diamidophosphates, methylphosphonates, alkylated phosphotriesters and formacetals or analogues thereof.

[0325] The oligonucleotide may be an α-anomeric nucleotide. The α-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which the strands run parallel to each other.

[0326] The oligonucleotide may be conjugated to another molecule such as, but not limited to, a peptide, hybridization initiating cross-linking agent, transport agent, and hybridization initiating cutting agent. Oligonucleotides can be synthesized by standard methods known in the art, e.g. using an automated DNA synthesizer (such as commercially available from

Biosearch, Applied Biosystems, etc.). For example, oligonucleotide phosphorothioates can be synthesized by the method described by Stein et al., Nucl. Acids Res. 16: 3209 (1988)), oligonucleotide methylphosphonates can be prepared using a polymeric glass support with controlled porosity (Sarin et al., Proc. Natl. Acad. Sci. USA 85: 7448-7451 (1988)), etc. In a specific embodiment, the antisense oligonucleotide of the sHASEGP protein comprises catalytic RNA or ribozyme (see e.g. PCT No. WO 90/11364, published October 4, 1990; Sarver et al., Science 247: 1222-1225 (1990)). In another embodiment, the oligonucleotide is a 2'-Omethyl carbonyl nucleotide (Inoue et al., Nucl. Acids Res. 15: 6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215: 327-330 (1987)).

[0327] Alternatively, the oligonucleotide may be double-stranded RNA (dsRNA) or as RNAi.

[0328] In an alternative embodiment, the antisense nucleic acid of the sHASEGP protein is produced by intracellular transcription and based on an exogenous sequence.

[0329] For example, the vector may be introduced in vivo in such a way that it is absorbed by the cell and transcribed in that cell to produce antisense nucleic acids (RNA). Such a vector would contain the antisense nucleic acid sequence of the sHASEGP protein. This type of vector may remain episomal or may integrate into the genome as long as it can be transcribed and produce the desired antisense RNA. Such a vector can be constructed by recombinant DNA technology standard in the art. The vectors may be viral plasmids or other known in the art used for replication and expression in mammalian cells. Expression of the sHASEGP antisense RNA coding sequence may be from beneath any known mammalian cell promoter known in the art. Such promoters can be constitutive or inducible. Such promoters include, but are not limited to, the SV40 early promoter (Bernoist and Chambon, Nature 290: 304-310 (1981), the promoter located at the 3 'end of the long repeating sequence of Rous Sarcoma virus (Yamamoto et al., Cell 22: 787 -797 (1980), promoter for herpes simplex thymidine kinase (Wagner et al. Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981), regulatory sequences of the metallothionein gene (Brinster et al., Nature 296: 39- 42 (1982)), etc.

[0330] Antisense nucleic acids contain sequences complementary to at least a portion of the RNA transcript of the sHASEGP protein gene, including the human sHASEGP protein gene. Absolute complementarity is not required. The amount of sHASEGP antisense nucleic acid effective in treating and preventing cancer depends on the nature of the disease and can be determined experimentally by standard clinical techniques.

[0331] If possible, before the antisense nucleic acid is tested and used in humans, it is desirable to determine the cellular cytotoxicity of the antisense nucleic acid in vitro and then in an appropriate animal model.

[0332] 2. RNA interference (RNAi) (see, e.g., Chuang et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4985) can be used to inhibit the expression of the gene encoding sHASEGP. Interfering RNA (RNAi) fragments, especially double-stranded (ds) RNAi, can be used to generate so-called loss of function in the absence of sHASEGP. Methods associated with the use of RNAi to silence genes in organisms including mammals, C. elegans, Drosophila, plant and human are known (see e.g. Fire et al. (1998) Nature 391: 806-811; 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; International PCT application No. WO 01/29058; International PCT application No. WO 99/32619).

[0333] Double-stranded RNA (dsRNA) expressing constructs are introduced into a host, such as an animal or plant, using a replicable vector that remains episomal or integrates into the genome. Choosing the appropriate sequences, dsRNA expression may interfere with the accumulation of endogenous mRNA encoding sHASEGP. RNAi can also be used to inhibit expression in vitro.

[0334] Regions contain at least about 21 (or 21) nucleotides that are selective (i.e. unique) for sHASEGP are used to prepare RNAi. Smaller fragments of about 21 nucleotides can be transformed directly (i.e. in vitro or in vivo) into cells; larger RNAi ds RNA molecules are generally introduced into vectors that encode them. The dsRNA molecules are at least about 21 bp long 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 skilled in the art.

[0335] 3. Gene therapy in an exemplary embodiment, nucleic acids that contain the nucleotide sequence encoding the sHASEGP protein or functional functional domains or derivatives thereof are administered for function by gene therapy. The term "gene therapy" means therapy carried out by administering nucleic acid to the subject. In this embodiment, the nucleic acid produces an encoded protein that affects the therapeutic effect by supporting the action of the sHASEGP protein. Any method of gene therapy known in the art can be used (see Goldspiel et al., Clinical Pharmacy 12: 488-505 (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 11 5: 155- 215 (1993). For example, one gene therapy therapeutic composition contains a nucleic acid encoding an sHASEGP protein that is part of an expression vector that expresses the sHASEGP protein or domain, a fragment thereof, or a chimeric protein in appropriate host cells. In particular, such a nucleic acid has a promoter, constitutive or inductive, operably linked to the sHASEGP protein coding region and optionally tissue-specific. In another embodiment, a nucleic acid molecule is used in which the sequence encoding the sHASEGP protein and other desired sequences are flanked by regions that support homologous recombination at a desired location in the genome, thereby enabling intra-chromosomal expression of the sHASEGP protein nucleic acid (Koller and Smithies, Proc. Natl Acad. Sci. USA 86: 8932-8935 (1989); Zijlstra et al., Nature 342: 435-438 (1989)).

[0336] Administration of a nucleic acid to a patient may be carried out in a direct manner in which the patient is in direct contact with the nucleic acid or vector that carries that acid, or in an indirect manner in which the patient is transplanted with cells previously transformed with nucleic acid in vitro.

[0337] In a particular embodiment, the nucleic acid is administered directly in vivo, where it is expressed and produces the encoded product. Such an effect can be achieved by a variety of methods known in the art, e.g. by introducing it into a suitable expression vector and administering in such a way that it becomes inside the cell e.g. as a result of infection using defective attenuated retroviral vectors or other viral vectors (see US Patent No. 4980286), by direct injection of bare DNA, micro-shooting (e.g. 'gene cannon'; Biolistic, Dupont), coating with lipids, surface, receptors or transfection agents, encapsulation in liposomes, microparticles or microcapsules, or administration together with a peptide, which is known to get into the venom, or by co-administration with a ligand for the receptor mediating endocytosis (see, e.g., Wu and Wu, J. Biol. Chem, 262: 4429-4432 (1987)) (which can be used to initiate expression of receptors in target cells), etc. In another embodiment, a nucleic acid-ligand complex can be formed in which the ligand is a fusogenic viral protein that causes the breakup of endosomes , thus protecting the nucleic acid against lysosomal degradation. In yet another embodiment, the nucleic acid may be targeted in vivo to specific cells through which it is taken up and expressed through a specific receptor (see PCT No. WO 92/06180, April 16, 1992; (Wu et al.); WO 92 / 22635, December 23, 1992 (Wilson et al.); WO 92/20316, October 26, 1992; (Findeis et al.); WO 93/14188, July 22, 1993 (Clarke et al.), WO 93/2022, October 14, 1993 (Young)). Alternatively, the nucleic acid may be introduced into the cell and incorporated into the DNA of the host cell 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)).

[0338] A specific embodiment of the invention utilizes a viral vector that contains sHASEGP nucleic acid. For example, a retroviral vector may be used (see Miller et al., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors have been modified to remove the retroviral sequence necessary for viral genome packaging and integration into host cell DNA . The sHASEGP nucleic acid protein for gene therapy is cloned into a vector, which facilitates the administration of this gene to a patient. More details on retroviral vectors can be found in Boesen et al., Biotherapy

6: 291-302 (1994), describing the use of a retroviral vector to introduce the MDR1 gene into hematopoietic stem cells to increase their resistance to chemotherapy. [0339] Other references describing 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: 129-141 (1993); and Grossman and Wilson, Curr. Opin. In Genetics And Devel. 3: 110-114 (1993).

[0340] Adenoviruses are other viral carriers that can be used in gene therapy. Adenoviruses are particularly attractive carriers for the delivery of genes to respiratory epithelial cells. Adenoviruses naturally infect the respiratory epithelium where they cause mild diseases. The other target site for adenoviruses is the liver, central nervous system, endothelial cells and muscles. The advantage of adenoviruses is the ability to infect non-dividing cells. Kozarski and Wilson in Current Opinion in Genetics and Development 3: 499503 (1993) published a review article on adenovirus-based gene therapy. Bout et al. in an article published in Human Gene Therapy 5: 3-10 (1994) demonstrated the use of adenoviral vectors in gene transfer to the respiratory endothelium of Rhesus monkeys. Other examples of the use of adenoviruses 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).

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

[0342] Another approach to gene therapy involves gene transfer to cells in cell culture by methods such as electroporation, lipofection, calcium phosphate transfection or a virus infection. Typically, the transfer involves transfer to the cells of a selectable marker. These cells are then selected to isolate those that have taken up the transferred gene and expressed it. Such cells are administered to the patient in the next step.

[0343] In this embodiment, the nucleic acid is introduced into the cell prior to administration to the patient of recombinant cells obtained in vivo. Such introduction may be carried out 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 sequence, cell fusion, gene transfer on a chromosomal carrier, gene transfer via microcells, spheroplast fusion , etc. Many methods for introducing foreign genes into cells are known in the art (see, e.g., Loeffler and Behr, Meth. Enzymol. 217: 599-618 (1993); Cohen et al., Meth. Enzymol. 217: 618-644 (1993); Cline, Pharmac. Ther. 29: 69-92 (1985)), which can be used as long as the necessary developmental and physiological functions of the target cell are not disturbed. The technique should ensure stable transfer of the nucleic acid into a cell in which it can be expressed, inherited and expressed by daughter cells. [0344] The resulting recombinant cells can be administered to a patient by various methods known in the art. In implementation, epithelial cells are injected, e.g., subcutaneously. In another embodiment, the recombinant skin cells can be used as a skin graft onto a patient's skin. Recombinant blood cells (e.g. hematopoietic stem or progenitor cells) can be administered intravenously. The number of cells to be used depends on the desired effect, patient condition, etc., and can be determined by a person skilled in the art. [0345] Cells into which the nucleic acid introduced for gene therapy include any desired and available cell type such as, but 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 types of stem and progenitor cells, in particular hematopoietic or progenitor stem cells, e.g. stem cells obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver and other sources.

[0346] For example, the cell used in gene therapy is autologous to the patient. In an embodiment in which the recombinant cells are used in gene therapy, the nucleic acid encoding the sHASEGP protein is introduced into the cells in such a way that it can be expressed by daughter cells and the recombinant cells are administered in vivo for therapeutic effect. In a specific embodiment, stem or progenitor cells were used. Any stem and / or progenitor cells that are possible to isolate and keep alive in vivo can potentially be used in accordance with this embodiment.

[0347] Such stem cells include, but are not limited to, hematopetic stem cells (HSC), stem epithelial cells of tissues such as skin, intestinal epithelium, embryonic myocardial cells, liver stem cells (PCT No. WO 94/08598, April 28, 1994), and nerve cells (Stemple and Anderson, Cell 71: 973-985 (1992)).

[0348] Epithelial stem cells (ESCs) or keratinocytes can be obtained from tissue such as skin and intestinal lining by known procedures (Rheinwald, Meth. Cell Bio. 21A: 229 (1980)). In tissue tissue, such as skin, renewal occurs by stem cell mitosis within the germ leaf, the leaf closest to the basal lamina. Stem cells in the lining of the intestine ensure rapid renewal of this tissue. ESC or keranocytes obtained from the skin or epithelium of the patient or donor can be cultured in cell cultures ((Rheinwald, Meth. Cell Bio. 21A: 229 (1980); Pittelkow and Scott, Cano. Clinic Proc. 61: 771 (1986)) If the ESCs are from a donor, suppression / suppression of host anti-transplant immune response methods (e.g. irradiation, administration of drugs or antibodies promoting moderate immunosuppression) may be used.

[0349] With respect to hematopoietic stem cells (HSCs), any technique for isolation, propagation and maintenance of HSCs in vitro can be used in this embodiment, which includes (a) isolation and derivation of a cell culture from bone marrow cells isolated from a future host or donor, or (b) the use of previously derived long-term culture that can cause allergies or be xenogenic.

[0350] Generally, non-autologous HSCs can be utilized using a method of suppressing future host / patient immune responses in response to a transplant. In a particular embodiment, the human bone pin cells can be obtained from the iliac crest of the needle by aspiration through the needle (see, e.g., Kodo et al., J. Clin. Invest. 73: 1377-1384 (1984)). For example, HSCs can be in highly enriched or significantly purified form. Said enrichment can be obtained earlier during or after prolonged cultivation by any techniques known in the art. Long-term bone marrow cultures can be established and maintained using modified cell culture techniques according to Dexter ((Dexter et al., J. Cell Physiol. 91: 335 (1977)) or Witlock-Witte (Witlock and Witte, Proc. Natl Acad. Sci. USA 79: 36083612 (1982)).

[0351] In a specific embodiment, the nucleic acid to be introduced into the cell as part of gene therapy contains an inducible promoter operably linked to the coding region, whereby the expression of the nucleic acid is under the control of the appropriate transcription inducer and depends on its presence or absence.

[0352] 3. Prodrugs - The invention includes a method of treating cancer. The method involves administering a prodrug that is digested by sHASEGP at a specific site, resulting in an active drug or precursor that can be converted to the active drug in vivo. On contact with a cell expressing active sHASEGP, the prodrug is converted into an active drug. The prodrug may be a conjugate that contains an active drug such as an anti-cancer drug, cytotoxic agent or other therapeutic agent (TA) associated with the substrate for the target sHASEGP. In a conjugate, said drug or agent is inactive or unable to enter the cell, but it gains activity as a result of digestion. A prodrug, for example, may contain chondroitin sulfate, usually relatively short because it has less than about 20 disaccharide subunits that is digested by specific sHASEGP. Cytotoxic agents include, but are not limited to, alkylating agents, antiproliferative agents, and tubulin binding agents. Other cytotoxic agents include drugs such as vincristine, mitomycin, bleomycin and paclitaxel.

[0353] J. PHARMACEUTICAL COMPOSITIONS AND METHODS OF ADMINISTRATION.

[0354] 1. Components of pharmaceutical compositions. The invention includes pharmaceutical compositions containing active sHASEGP, as well as substance variants that modulate the activity of the sHASEGP polypeptide. The invention also relates to another method of treatment or a substance for the treatment of hyaluronidase-related disorders, e.g. an antibody substance.

[0355] The sHASEGP polypeptide and the second substance may be packaged as separate pharmaceutical compositions for administration in a mixture, sequentially or irregularly. Alternatively, they may be provided for administration as a single composition or two compositions for administration as a single composition. Variants can be packaged as ready-to-use kits.

[0356] 2. Formulations and route of administration.

[0357] Human-derived sHASEGP polypeptides and their soluble domains with hyaluronidase activity of the present invention can be formulated as pharmaceutical compositions intended for single dose administration. The concentration of polypeptides used in the formulation allows effective delivery at the time of administration of the amount that is effective in the planned therapy. Usually, the pharmaceutical compositions are formulated for single administration. For formulation purposes, the weighed sHASEGP polypeptide fraction, its soluble domains with hyaluronidase activity of human origin, or a mixture of both of these components is dissolved, suspended, dispersed or otherwise mixed with the selected carriers in an effective concentration that allows reducing or alleviating the condition being treated.

[0359] In addition, the polypeptides may be formulated as the only active pharmaceutical ingredient in the composition, or may be mixed with other active ingredients. Liposomal suspension, also liposomes directed to a given tissue, can be a suitable and pharmaceutically acceptable carrier that can be prepared by methods known to those skilled in the art. An example may be the method for preparing liposome formulations described in U.S. Patent No. 4,522,811.

[0360] The active sHASEGP or its soluble domain with human hyaluronidase activity is contained in a pharmaceutically acceptable carrier in an amount sufficient to produce a therapeutically useful effect without causing the patient being treated to have undesirable side effects. The therapeutically effective concentration can be determined empirically by testing polypeptides in known in vitro and in vivo systems, such as the inventive assay or see, e.g., 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, Steinkuhler 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 e.g., 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: 822-826, 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 extrapolate to human dosing.

[0361] Typically, the invention includes therapeutically effective dosing. The amount administered can be in the order of 0.001 to 1 mg / ml blood, including a range of about 0.005-0.05 mg / ml blood and about 0.01 mg / ml blood. Pharmaceutical unit doses are formulated to deliver from about 1 mg to about 1000 mg, including from about 10 to about 500 mg, and including about 25-75 mg of the primary active ingredient or combination of active ingredients per unit dose. The exact dose can be determined empirically.

[0362] In some cases, a high unit dose of sHASEGP is preferred. For example, for intravenous administration, a concentration of 500 to 100,000 units per milliliter of sHASEGP is preferred. Freeze-dried sHASEGP formulations are ideal for storing large amounts of sHASEGP unit doses. The invention encompasses 200,000 units of freeze-dried vials with sHASGEP for intravenous administration.

[0363] The invention also includes high concentration doses for the administration of sHASEGP in small volumes. The invention includes the administration of 5000 units / ml sHASEGP in a volume of 10-100 μ, which is injected into the anterior chamber of the eye to dissolve the viscoelastic substance administered during cataract surgery and implanting an additional lens. Small injection volumes of 50-200U / ml used in intraocular space procedures such as treatment of vitreous hemorrhage or separation of vitreous in diabetic retinopathy are also included in the invention.

[0364] The active ingredient may be administered in one dose or divided into a number of smaller doses administered at intervals. It is understood that the exact dose and duration of treatment depend on the condition being treated and can be determined experimentally using known test protocols or by extrapolating data obtained in in vitro or in vivo experiments. It should be noted that concentration and dose values ​​may vary depending on how severe the patient's condition is to be alleviated. It is understood that a specific dosage regimen should be established for each individual case, depending on the individual needs and the expert opinion of the person administering the drug or supervising the administration of the pharmaceutical composition. It is also understood that the concentration range provided herein is by way of example only and is not intended to narrow the scope of the invention, to the use of the claimed composition or a combination containing this drug.

[0365] Pharmaceutically acceptable derivatives contain acids, salts, esters, hydrates, solvents and prodrug forms. Usually, a derivative whose pharmacokinetic properties are better than the corresponding sHASEGP or its soluble domain with hyaluronidase activity is selected.

[0366] Thus, to form a pharmaceutical composition, the effective concentrations or amounts of one or more polypeptides of the present invention or a pharmaceutically acceptable derivative thereof are mixed with a suitable pharmaceutical carrier or carrier for systemic, external local or local administration. SHASEGP polypeptides or its soluble domains with hyaluronidase activity are included in the effective therapeutic dose used to alleviate or treat the disease for which treatment is encompassed by the present invention. The concentration of the active polypeptide in the composition depends on the absorption, inactivation, rate of excretion of the active polypeptide, schedule and amount of administration, particular formulation, and other factors known to those skilled in the art.

[0367] Therapeutic agents used in said methods may be administered by any route known to those of ordinary skill in the art, but are not limited to the route of administration externally topically, intrathecally, intracapsular, intraocular, intraventricular, intrathecal, intravenous, intramuscular, intraperitoneal, intradermal, intratracheal, as also a route of administration being a combination of any two or more of these modes of administration. A pulmonary dry powder formulation is also provided in the present invention.

[0368] The most optimal route of administration will vary depending on the proposed application, eg use as a factor that facilitates subcutaneous fluid administration, use to reduce intraocular pressure in the eye of glaucoma patients receiving viscoelastics, as a "spreading agent" for enhancing chemotherapeutic activity. The injection sites will also be different, such as a specific internal organ, cancer, intraocular space and epidermis. Methods of administration include, but are not limited to, topical, local, intraocular, intrathecal, intraocular, intraventricular, intrathecal, intravenous, intramuscular, intratracheal, intraperitoneal, intradermal and intra-cutaneous administration and a combination of two or more of these methods. For example, for the treatment of various types of cancer, such as squamous cell carcinoma of the skin ( squamous cell carcinoma), breast cancer, bladder cancer and gastrointestinal cancer, local administration, including intratumoral (e.g. intrathecal, intraventricular or intracapsular) has the advantage that the therapeutic agent can be administered in a high concentration without the risk of complications. which usually accompany systemic administration of the therapeutic agent.

[0369] Pharmaceutical or cosmetic carriers or vehicles suitable for administration of sHASEGP polypeptides or a soluble form of the human-derived hyaluronidase domain of the present invention include any carrier known to those of skill in the art that is suitable for the particular mode of administration. Furthermore, the polypeptides can be formulated as the only pharmaceutically active ingredient in the composition, they can be combined with other active ingredients that do not interfere with the desired effect, or with materials that complement the desired effect and are known to those skilled in the art. The sHASEGP polypeptides of the present invention serve as examples, which can be used as "delivery" or "spreading" agents in combination with a second active substance as a therapeutically effective agent including, but not limited to, a drug or prodrug. The purpose of such a combination is to more easily deliver or enhance the activity of the second active agent. In a particular embodiment, to inhibit or reduce blood supply during ophthalmic surgery, the sHASEGP polypeptide or its soluble domain with human hyaluronidase activity may be formulated together with an anesthetic agent such as lignocaine or bupivacaine or a mixture thereof and optionally with a hormone agent such as epinephrine. The sHASEGP polypeptide or its soluble domain with hyaluronidase activity of human origin can also be formulated with various types of chemotherapeutic agents, such as toxin and TNF, to enhance the effect of the chemotherapeutic agent and / or its availability to target tumor cells. The active ingredient is contained in the carrier in an amount sufficient to produce a therapeutically useful effect without the serious toxic effects of the subject being treated. The effective concentration can be determined empirically by testing chemicals using in vivo and in vitro systems including the animal models described herein.

[0370] Solutions or suspensions used for parenteral, intradermal or topical external administration may contain the following components: sterile diluent such as water for injections, saline, solid oil, polyethylene glycol, glycerin, propylene glycol and other synthetic solvent, antibacterial substances such as benzyl alcohol, methyl paraben, antioxidants such as ascorbic acid and sodium bisulfite, chelating agents , such as ethylenediaminetetraacetic acid (EDTA), buffers such as acetates, citrates and phosphates, and osmotic pressure regulating agents such as sodium chloride, calcium chloride, magnesium chloride, dextrose, glycerol and boric acid. Preparations for parenteral administration may be in ampoules, disposable syringes, glass, plastic or other material and in single or multiple dose vials.

[0371] sHASEGP polypeptides or their soluble domains with hyaluronidase activity of human origin may be suspended in macromolecular form or other suitable form, or derivatized with a more soluble and more active product or prodrug. The form of the final mixture depends on many factors including the intended mode of administration and the solubility of the polypeptide in the selected carrier or vehicle. The effective concentration is sufficient to alleviate the disease state targeted by the therapy and can be determined empirically using methods known to those skilled in the art. The weight fraction of the polypeptide for formulation of the composition is dissolved, suspended, dispersed or mixed in an effective manner in a selected bed in an effective concentration, i.e. one that causes the amelioration or resolution of the disease state being targeted by the therapy.

[0372] In the case where sHASEGP polypeptides or a soluble domain with human hyaluronidase activity exhibit insufficient solubility, methods for dissolving polypeptides known to those skilled in the art may be used that include, but are not limited to, the use of cosolvents such as dimethyl sulfoxide (DMSO), the use of surfactants such TWEEN® and Pluronic® F68 or dissolution in sodium bicarbonate. Polypeptide derivatives, such as polypeptide prodrugs, can also be used in the formulation of effective pharmaceutical compositions. Pharmaceutical compositions for use in ophthalmology are formulated with an ophthalmologically acceptable carrier. Local administration for ophthalmological applications by external topical administration or injection is included in the present invention. Formulations allowing sustained release of the drug are also desirable. Typically, the pharmaceutical compositions are formulated for administration in a single dose that contains a therapeutically effective amount.

[0373] After mixing or adding the polypeptide to the bed, the resulting mixture may be a solution, suspension, emulsion or other form of composition and may be present in the formulation of an aqueous mixture, cream, gel, ointment, emulsion, solution, medicine, liquid, suspension, tincture, paste , foam, aerosol, rinse aid, aerosol, spray, suppositories, dressings or any other formulation suitable for systemic, local external or local administration.

[0374] The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of the chemical in the selected carrier or bed. If necessary, said chemical compound is converted into pharmaceutically acceptable salts or other derivatives. For local internal administration, such as intramuscular, parenteral or intra-articular, the chemical compounds are preferably formulated in the form of an aqueous suspension-suspension in the form of an isotonic buffered saline or are mixed with a biocompatible, intended for internal administration, adjuvant or bioadhesive.

[0375] The sHASEGP polypeptide or its soluble domain with hyaluronidase activity of human origin is contained in a pharmaceutically acceptable carrier in an amount sufficient to produce a therapeutically useful effect in the patient being treated while avoiding undesirable side effects. It is understood that the number and severity of the side effects depend on the medical condition being the reason for administering these ingredients. An example would be some toxic and undesirable effects that are tolerated when the disease being treated is life threatening and which would not be tolerated in the treatment of disorders or less serious conditions. The effective amounts for therapeutic use will of course depend on the severity of the disease, the weight and general condition of the subject being treated, and the route of administration. Local administration of the therapeutic agent usually requires a lower dose than for any of the routes of systemic administration, although the local concentration of the therapeutic agent may, in some cases, be higher after local administration than the concentration achievable after systemic administration.

[0376] Due to the fact that different individuals undergoing treatment may experience many different symptoms of the disease with varying severity and each therapeutic agent has unique therapeutic properties, it is up to the physician to determine how strong the individual's response to therapy should be, and accordingly what dosage should be Doses determined in vitro should provide data to help determine the amount of pharmaceutical compositions administered in situ. In some cases, an animal model may be used to determine the effective dosage for the treatment of particular diseases. In general, however, for local administration, an effective amount of therapeutic agent is an amount in the range of from about 0.1 pg to about 1 ng per kilogram body weight, which includes the present invention. It is known to those skilled in the art that in determining the effective amount of a therapeutic agent, a variety of factors are taken into account, as described e.g. in Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990; and Mantyh et al., (Science, 278: 275-79, 1997) including intrathecal injection of neuronal ligand-toxin. Each of the above publications constitutes an integral part of this document in its entirety.

[0377] Formulations of sHASEGP polypeptides or soluble domains thereof with human-derived hyaluronidase activity for use as used herein include formulation methods suitable for oral, rectal, topical, inhaled, buccal (e.g. sublingual), parenteral (e.g. subcutaneously, intramuscularly, intradermally or intravenously), transdermally or by any other route of administration. The most appropriate route of administration in a given case depends on the nature and severity of the condition being treated and the nature of the particular active chemical used. For administration to humans and animals, the formulations are provided in a pharmaceutical unit dose, such as tablets, capsules, pills, powder, granules, sterile parenteral fluids or solutions, and oral fluids and solutions, oil-in-water emulsions containing appropriate amounts of polypeptides and / or other factors and their derivatives. Pharmaceutically therapeutically active polypeptides and / or other agents and their derivatives are usually formulated and administered in a pharmaceutical unit dose or multiple dose variant. The term "unit dosage form" as used herein means physically separate units of the drug individually packaged, suitable for use in humans and animals, as is known in the art.

[0378] The invention includes pharmaceutical compositions intended for human and animal use in the form of a tablet, capsule, pill, powder, granules, sterile parenteral fluids or solutions, and oral fluids and solutions, oil-in-oil emulsions containing appropriate amounts of sHASEGP or soluble form of its domain with hyaluronidase activity and optionally another agent or a pharmaceutically acceptable derivative thereof. Therapeutically active chemical pharmaceutical compounds and their derivatives are usually formulated and administered in unit or multiple doses of the drug. The term "unit dosage form" as used herein means physically discrete units suitable for use in humans and animals and individually packaged as is known in the art. Each unit dosage form contains a predetermined quantity of a pharmaceutically active compound sufficient, in association with the required carrier, vehicle or diluent, to achieve the desired therapeutic effect. Examples of unit dosage forms include, but are not limited to, ampoules, syringes and individually packaged tablets or capsules. Examples include low-volume formulations that contain a stabilized solution containing from 1 to 5000 units of sHASEGP in a low volume of 5 to 50 [mu] L and can be prepared in a single-use syringe for injection, e.g. a viscoelastic substance. Unit dosage forms may be administered in parts or in multiples of the dose. Multiple dosing is a multiple of identical unit dose forms packaged in individual containers for administration as separate unit dose forms. Examples of multiple dose forms include vials, bottles containing tablets, capsules, or bottles of about half a liter (1 pint, GB = 0.57 L, US = 0.47 L) or 4.546 L (1 gallon). Hence, the multiple dosage form is a multiple of the unit dosage form in one package.

[0379] The pharmaceutical composition may contain, in addition to the active ingredient such as the sHASEGP polypeptide, a diluent such as lactose, sucrose, dicalcium phosphate, carboxymethyl cellulose, a moisturizing agent such as magnesium stearate, calcium stearate and talc, and as a binder may contain starch, natural gums such as acacia, glucose, molasses, polyvinylpyrrolidone, celluloses and their derivatives, povidone, crospovidones and other binders known in the art. To obtain a solution or suspension, liquid pharmaceutical compositions intended for administration may be prepared, e.g., by dissolving, dispersing or mixing the active compound by other methods as defined above, as well as dissolving, dispersing or mixing by other methods of pharmaceutical adjuvants in a carrier such like water, saline, dextrose aqueous solution, glycerol, glycol, ethanol and the like. If necessary, the pharmaceutical composition intended for administration to patients may contain smaller amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents or solubilizing agents, buffering agents and the like e.g. acetate, sodium citrate, cyclodextran derivatives, sorbitol monolaurate, triethanolamine / sodium 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, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975). The pharmaceutical composition or formulation intended for administration to patients contains an active compound in an amount sufficient to alleviate the symptoms of the subject being treated. An example is the standard stabilized formulation of sHASEGP or its soluble domain with hyaluronidase activity, the subject of the invention, which contains 150 U / ml soluble glycoprotein formulated in EDTA, NaCl and CaCb. Additionally, antibacterial or antifungal agents may be present in the formulation, including but not limited to thimerosal. Another formulation of the present invention is in the form of a stabilized solution of the lyophilized form of sHASEGP or its soluble domain with hyaluronidase activity in EDTA, NaCl and CaCb, containing active soluble glycoprotein at a concentration of 150 U / ml with the addition of lactose at a concentration of 13 mg / ml. The invention also includes a formulation comprising a stabilized solution or lyophilized form of sHASEGP or a soluble domain thereof with hyaluronidase activity in EDTA, NaCl and CaCf., As well as albumin, Pluronik® F68, TWEEN® and / or other detergents. Another formulation encompassed by the present invention, both the lyophilized form and the stabilized solution, contains an effective amount of sHASEGP or a soluble domain thereof with hyaluronidase activity at a concentration of 1 to 300 U / ml in EDTA, NaCl and CaCfe.

[0380] It is possible to prepare a dosage form or pharmaceutical composition containing the active ingredients in the range of 0.005% to 100% by weight balanced with a non-toxic carrier. For oral administration, the pharmaceutical composition may take the form of, e.g., tablets or capsules prepared by methods commonly used in the art with pharmaceutically acceptable excipients such as binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, fillers (e.g. lactose, microcrystalline cellulose, calcium hydrogen phosphate), moisturizing agents (e.g. magnesium stearate, talc or silica), acceleration agents (e.g. potato starch, sodium starch), carboxylic acid, sodium starch (e.g. sodium lauryl sulfate). The tablets can be coated using methods well known in the art.

[0381] sHASEGP, its soluble domain with hyaluronidase activity, or pharmaceutically acceptable derivatives can be prepared with carriers in slow release or coated form that protect soluble glycoprotein from rapid removal from the body. Such a composition may contain, to obtain the desired combination of properties, other pharmaceutically active agents commonly known in the art and essential in the treatment of one or more diseases including, but not limited to, a chemotherapeutic agent, analgesic, anti-inflammatory agent, antibacterial agent, tacicide, agent anti-malarial, anticonvulsant, antidepressant, anti-arthritis agent, antifungal agent, antihypertensive agent, antipyretic agent, antiparasitic agent, antihistamine, alpha-adrenoceptor agonist, alpha-blocker, anesthetic, bronchodilator, biocide, bactericide, bacteriostatic agent, beta-adrenergic blocker, calcium channel blocker, cardiac drug , contraceptive, decongestant, diuretic, suppressant, diagnostic agent, electrolyte, hypnotic, hormonal, hyperglycemic, muscle relaxant, muscle relaxant, ocular, parasympathomimetic, psychic, sedative, sympathomimetic, attractant, urinary tract, vaginal, virucidal, vitamin agent, non-steroidal anti-inflammatory agent, angiotensin converting enzyme inhibitor, polypeptide, protein, nucleic acid, drug, prodrug, organic compound and tranquilizer. It is understood that such combination therapy is another aspect of the pharmaceutical composition and treatment methods of this invention.

[0382] 1. COMPOSITION USED FOR ORAL ADMINISTRATION [0383] Oral drug dosage forms are in solid, gel or liquid form. Solid dosage forms are tablets, capsules, granules and loose powders. Types of oral tablets include compressed tablets, chewable tablets and lozenges, which may be coated with an acid-resistant substance (enteric-coated tablets), a sugar coating or a polymer. Capsules can be made of hard or soft gelatin, while granules and powders can be provided in effervescent or non-effervescent form in combination with other ingredients known to those skilled in the art.

[0384] A pharmaceutical composition containing sHASEGP or its soluble domain with hyaluronidase activity may be in liquid form, e.g., in the form of solutions, syrups or suspensions, or as a drug which must be dissolved in water or another suitable excipient before use. Liquid media of this type can be prepared with pharmaceutically acceptable admixtures, such as suspending agents, using methods commonly used in the field. sorbitol syrup, cellulose derivatives or hardened edible fats), emulsifiers (e.g. lecithin or acacia), anhydrous auxiliaries (e.g. almond oil, fatty acid esters or fractionated edible oils), preservatives (methyl or propyl p-hydroxybenzoate or sorbic acid) .

[0385] In some embodiments, the formulations are in the form of solid dosage forms, preferably in the form of capsules or tablets. Tablets, pills, capsules, troches and the like may contain any of the listed ingredients or compounds of a similar nature, i.e. a binder, diluent, spreader, moisturizer, glidant, sweetener and flavor.

[0386] Examples of binders include microcrystalline cellulose, tragacanth, glucose solution, acacia, vegetable glue, gelatin solution, sucrose and starch paste. Moisturizers include talc, magnesium or calcium stearate, stellate fatty acids, and stearic acid. Examples of diluents include, for example, lactose, sucrose, starch, kaolin, mannitol, dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Spreading agents include internally cross-linked sodium carboxymethyl cellulose, corn starch, potato starch, k sodium carboxymethyl starch, alginic acid, bentonite, methyl cellulose, agar and carboxymethyl cellulose. Coloring substances include, for example any FDA-approved, water-soluble FD and C dye and mixtures of dyes, as well as water-soluble FDA-approved dyes suspended in aluminum hydrate. Sweeteners include sucrose, lactose, mannitol, artificial sweeteners such as saccharin and any number of spray dried flavors. Flavors include natural flavors extracted from plants e.g. from fruit, synthetic mixtures producing a pleasant aroma, such as, but not limited to, peppermint or methyl salicylate. Surfactants include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene sorbitan monolaurate ether. Antiemetic coatings include methylcellulose, sodium carboxymethylcellulose, polyethylene glycol and acetate phthalate.

[0387] For oral administration, if desired, sHASEGP or a soluble domain thereof with hyaluronidase activity may be provided in a pharmaceutical composition that protects it from the acidic environment of the stomach. An example would be a pharmaceutical composition formulated as an enteric-coated tablet that does not disintegrate in the stomach and releases the drug substance in the small intestine. The pharmaceutical composition may be formulated in combination with an antacid or other such substance.

[0388] If the dosage form is a capsule, it may contain, in addition to the material as described above, a liquid carrier such as liquid fat. In addition, unit dosage forms may contain a variety of other substances that modify the physical unit dosage form, e.g., sugar coating and other enteric coating forms. Chemical compounds can be administered as components of an elixir, suspension, syrup and wafer, aerosol, chewing gum or similar forms. A syrup may contain, in addition to the active ingredients, sucrose and sweeteners as well as certain preservatives, dyes, colorants and flavors.

[0389] sHASEGP or its soluble domain with hyaluronidase activity can also be mixed with other active substances that do not adversely affect the desired effect or with substances that complement the desired effect, such as antacids, H2 blockers or diuretics. The active ingredient is a compound or a pharmaceutically acceptable derivative thereof as described herein, and its concentration may be up to about 98% by weight of the active ingredient.

[0390] Pharmaceutically acceptable carriers contained in tablets are binders, moisturizers, diluents, spreaders, coloring agents, flavoring agents and surface active agents. Gastro-resistant tablets, due to their shell, are resistant to the acidic environment in the stomach and dissolve or disintegrate in the neutral or alkaline environment of the small intestine. Coated tablets are compressed tablets that have been coated with various layers of pharmaceutically acceptable substances. Polymer coated tablets are compressed tablets that have been coated with a polymer or other suitable coating material. Multiple compressed tablets are tablets resulting from more than one compression cycle using the pharmaceutically acceptable substances mentioned above. Coloring agents may be used in the above dosage forms. Flavorings and colourants can be used in pressed, coated, multiple pressed and chewable tablets. Flavoring and coloring substances are particularly useful in the formation of chewable tablets and lozenges.

[0391] 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, potions and syrups. Emulsions occur in the form of oil in water or in the form of water in oil.

[0392] Potions are clear, sweetened and hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs contain a solvent. Syrups are concentrated aqueous solutions of sugar, e.g. sucrose, and contain a preservative. The emulsion is a two-phase system in which the liquid is dispersed in another liquid in the form of small balls. Pharmaceutically acceptable carriers used in emulsions are anhydrous liquids, emulsifiers and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives.

Pharmaceutically acceptable substances used in non-effervescent granules for reconstitution in liquid oral form include diluents, sweeteners and surfactants. Pharmaceutically acceptable substances used in effervescent granules for reconstitution in liquid oral form contain organic acids and a source of carbon dioxide. Coloring and flavoring agents are used in all of the above dosage forms.

[0393] Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol. Examples of anhydrous 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, magnesium aluminum silicate and acacia. Diluents include lactose and sucrose. Sweeteners include sucrose, syrups, glycerin and artificial sweeteners such as saccharin. Surfactants include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene ether lauryl sulfate. Organic admixtures include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium bicarbonate. Coloring agents include any FDA approved and certified water soluble dye or dye mixture. Flavors include natural flavor extracts from plants, e.g. fruit, and synthetic mixtures of compounds that produce a pleasant taste sensation.

[0394] In the form of a solid dosage form, the solution or suspension, e.g. in propylene carbonate, vegetable oils or triglycerides, is enclosed in gelatin capsules. Such a solution and its preparation and encapsulation are disclosed in US Patent No. 4,328,245; 4,409,239; and 4,410,545. The liquid dosage form, i.e. the solution, e.g. in polyethylene glycol, may be diluted in a sufficient amount of a pharmaceutically acceptable carrier (e.g. in water), which makes it easier to measure the right dose before giving you medicine. [0395] Alternatively, oral liquid or semi-solid formulations can be prepared by diluting or dispersing sHASEGP or its soluble domain with hyaluronidase activity in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g. in propylene carbonate) and other carriers. Oral liquid or semi-solid formulations can also be prepared by encapsulating said solutions or suspensions in hard or soft shells of gelatin capsules. Another useful formulation is the US Patent Nos. Re 28.819 and 4.358.603.

[0396] Formulations suitable for buccal (sublingual) administration include, for example, lozenges comprising sHASEGP or a soluble domain thereof with hyaluronidase activity in an aromatized drug base, usually sucrose, acacia or tragacanth. These formulations also include lozenges containing a chemical compound in a non-reactive base such as gelatin, glycerin, sucrose or acacia.

[0397] As known to those of ordinary skill in the art, tablet and capsule formulations may be coated in all implementations to modify or extend the disintegration time of the active ingredient, e.g., a conventional, gastroresistant coating such as phenyl salicylate, waxes and cellulose acetate phthalate.

[0398] 2. INJECTION LIQUIDS, SOLUTIONS AND EMULSIONS.

[0399] Included in the present invention is parenteral administration of sHASEGP or its soluble domain with hyaluronidase activity, generally by subcutaneous, intramuscular or intravenous injection. Injectable liquids may be prepared in a form commonly known as a liquid solution or suspension, in solid form, suitable for reconstitution prior to injection of a solution or suspension in a liquid or as an emulsion. A suitable adjuvant is e.g. water, saline, dextrose, glycerol or ethanol. In addition, if desired, pharmaceutical compositions intended for administration to a patient may contain smaller amounts of non-toxic auxiliary substances such as surfactants or emulsifiers, buffering agents, stabilizers, solubilizing agents and other agents such as sodium acetate, sorbitan monolaurate, oleate triethanolamine and cyclodextrin. The invention also includes implantation of slow or delayed drug release systems that allow a constant dosage level to be maintained (see, e.g., US Patent No. 3,710,795). The percentage of sHASEGP or its soluble domain with hyaluronidase activity contained in this type of parenteral composition depends on its specific nature as well as the activity of the chemical compound and the treatment requirements.

[0400] Parenteral administration of the pharmaceutical composition includes intravenous, subcutaneous and intramuscular administration. Preparations for parenteral administration include sterile injectable solutions, sterile dry soluble products such as lyophilized powders ready to mix with solvent or sterile solution, subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to mix with excipient just before use and sterile emulsions. The solution may be aqueous or anhydrous.

[0401] For intravenous administration, suitable carriers include saline solution or saline solution in PBS, and solutions containing thickening agents such as glucose, polyethylene glycol, polypropylene glycol and mixtures thereof.

[0402] Pharmaceutically acceptable carriers, used in preparations intended for parenteral administration, contain an aqueous vehicle, anhydrous vehicle, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifiers, capture or chelating agents and other pharmaceutically acceptable substances.

[0403] Examples of aqueous media include sodium chloride, Ringer's solution, isotonic dextrose, sterile water, dextrose and lactated Ringer's. Anhydrous vehicles for parenteral administration contain vegetable-based solid oils, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in concentrations that inhibit bacterial or fungal growth must be added to parenteral preparations packaged in multi-dose containers that contain phenols or cresols, mercury compounds, benzyl alcohol, chlorobutanol, methyl and propyl ester of p-hydroxybenzoic acid, thiomersal, benzalkonium chloride , benzylammonium chloride. Isotonic factors include sodium chloride and dextrose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaline hydrochloride. Suspending and dispersing agents include sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose and polyvinylpyrrolidone. Emulsifiers include polysorbate 80 (TWEEN 80). Factors that capture and chelate metal ions include EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol as a miscible vehicle and sodium hydroxide, hydrochloric acid, citric acid and lactic acid for pH adjustment.

[0404] The concentration of pharmaceutically active compounds is set at the level of effective amount that allows obtaining 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.

[0405] Unit dose preparations intended for parenteral administration are packaged in ampoules, vials and needle syringes. All preparations for parenteral administration must be sterile, as is known and practiced in the art.

[0406] For example, an effective intravenous and intraarterial infusion of a sterile aqueous solution containing the active compound is an effective method of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an active substance which requires injection to produce the desired pharmacological effect. [0407] Drugs injected into the treated tissue (s) are intended for local or systemic administration. Usually, an effective therapeutic dose is formulated with an active substance content of at least 0.1% w / w to about 90% w / w or more, preferably more than 1% w / w. The active substance, like sHASEGP or its soluble domain with hyaluronidase activity, may be administered in a single dose or in several smaller doses administered at intervals. It is understood that the precise dosage and duration of treatment depends on the type of tissue being treated and can be determined experimentally using known test protocols or by extrapolating results obtained in in vitro and in vivo tests. It should be noted that the concentration and dose may vary depending on the age of the subject being treated. It is also understood that for each particular subject being treated, a specific dosage regimen should be established during the treatment period and depending on individual needs and the expert opinion of the person administering the drug or supervising the formulation. It is also understood that the concentration ranges provided herein are exemplary only and do not limit the scope or practical use of the claimed formulation.

[0408] The chemical compounds of the present invention may be formulated for parenteral injection, e.g., a large single-dose bolus injection or intravenous infusion. Formulations for injection may be presented in unit dosage form with an added preservative, e.g. in ampoules or multiple dose containers. The pharmaceutical composition may be in the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulation components such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredients may be in powder form for reconstitution prior to use in a suitable vehicle e.g. in sterile pyrogen-free water or other solvents. For example, the invention relates to a dosage form for parenteral administration that contains an effective amount of sHASEGP or a soluble domain thereof with hyaluronidase activity, i.e. from 500 to 500,000 U in a stabilizing solution or lyophilized form.

[0409] Said chemical compound may be suspended in the microparticle or other suitable form, or may be derivatized to provide a more active product or prodrug. The form of the resulting mixture depends on many factors, including the intended route of administration and the solubility of the compound in the chosen carrier or vehicle. The effective concentration is sufficient to alleviate the symptoms of the condition being treated and can be determined empirically. [0410] 3. LYOPHILIZED POWDERS [0411] The invention also includes freeze-dried powders containing sHASEGP or a soluble domain thereof with hyaluronidase activity, which can be reconstituted as a solution, emulsion or other mixture prior to administration. Formulations of this type can be made and retested in solid or gel form.

[0412] A sterile lyophilized powder is prepared by dissolving a portion of its solid form in a suitable solvent or by mixing a portion of the solution containing sHASEGP or its soluble domain with hyaluronidase activity. The solvent may contain an excipient that increases solubility as well as other pharmaceutical components of the powder or reconstituted powder-based solution. Excipients may include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose, lactose and other appropriate types. The solvent may also contain a buffer, such as citrate buffer, sodium or potassium phosphate, or other buffers known to those skilled in the art, typically at near neutral pH. The lyophilized form of the drug is obtained as a result of subsequent sterile filtration, used alternately with lyophilization, carried out under standard conditions known to those skilled in the art. Generally, the solution obtained after sterile filtration is portioned into vials for lyophilization. Each vial may contain a single dose, such as 10-1000 mg, 100-500 mg, or a multiple of the dose of the substance.

[413] Briefly, lyophilized powder is prepared by dissolving dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, lactose and other relevant agents in an amount of about 1-20% in a suitable buffer at a pH near neutral as citrate. sodium or potassium phosphate and other such buffers known to those skilled in the art. Then selected salt, e.g. sHASEGP sodium (about 1 gm of salt per 10-100 gms buffer, usually 1 gm / 30 gms) is added to the resulting mixture at a temperature higher than room temperature, such as about 30-35 ° C and stirred until dissolved. The resulting solution is diluted by adding buffer to reduce the final concentration.

[0414] Reconstitution of said lyophilized powder in water for injection provides a dosage form for parenteral administration. To this end, the therapeutic amount of lyophilized powder containing sHASEGP or its soluble domain with hyaluronidase activity is reconstituted by the addition of sterile water or other suitable carrier per ml. The exact amount depends on the chemical chosen and can be determined empirically by methods known to those skilled in the art.

[0415] 4. EXTERNAL LOCAL ADMINISTRATION [0416] Mixtures intended for external topical administration are prepared as already described for local and systemic administration. For external topical administration, the resulting mixture may be in the form of a solution, suspension, tincture, pastes, foams, aerosols, sprays, suppositories, dressings, bandages, skin patches or other forms suitable for administration.

[0417] Pharmaceutical compositions of sHASEGP, its soluble domain with hyaluronidase activity or pharmaceutically acceptable derivatives thereof may be in the form of aerosols for topical administration such as e.g. inhalation (see e.g. US Patent Nos. 4,044,126, 4,414,209, and 4,364,923 which describes aerosols for administration steroids useful in the treatment of inflammatory diseases, especially asthma). Drug forms intended for administration to the respiratory system may be in the form of aerosols, sprayable solutions using a nebulizer or as a microparticle inhalation powder, in pure form or with the addition of a non-reactive carrier, e.g. lactose. When administered to the respiratory tract, the drug molecules, as mentioned above, will usually have a diameter less than 50 microns, for example less than 10 microns.

[0418] The pharmaceutical composition for administration by inhalation, as described in the present invention, may be provided in the form of an aerosol or spray in pressurized packs or spray with a suitable gas that includes, but is not limited to, dichloro-difluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane, carbon dioxide and other suitable gases. In the case of a pressurized aerosol, the unit dose may be determined by measurement using a valve. Capsules and cartridges of e.g. gelatin for use in inhalers or insufflators may be in the form of a mixture of a powdered drug and a suitable base powder such as lactose or starch.

[0419] The pharmaceutical composition may be in a form suitable for local, external and internal administration, such as for local external administration to the surface of the skin or mucous membranes (e.g. to the eye), in the form of gels, creams and lotions intended for ocular administration, intracapsular and intramedullary administration. Transdermal, ocular, mucosal or inhalation administration is also included in the invention. For nasal administration, a solution containing either the active substance alone or a solution variant in admixture with a pharmaceutically acceptable excipient is intended.

[0420] As an example, drug forms suitable for topical topical administration to the surface of the skin or to the eye are used, which are generally in the form of ointments, cream, lotion, paste, gel, spray, aerosol and oil. Carriers that can be used include petroleum jelly, lanolin, polyethylene glycols, alcohols, and combinations of two or more of the listed ingredients. Drug forms intended for topical administration may preferably contain from 0.05 to 15% by weight thickeners, including, but not limited to, hydroxypropyl methylcellulose, methylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, polyalkylene glycols, polymethoxyacrylates, or polymethylacrylamides. The topical topical formulation is often applied to the conjunctival sac in drops or as an ointment. It can also be used to irrigate or moisturize the eye, facial sinuses and the external auditory canal. In the liquid state, the aforementioned form of the drug may also exist as a three-dimensional matrix composed of a polymer in the form of a strip, contact lenses and similar forms from which active ingredients are released. The topical external formulation may also be injected into the anterior chamber of the eye and elsewhere. For example, the invention includes a drug form for intraocular use after injection of a viscoelastic material, comprising a stabilized solution of an effective amount of sHASEGP or a soluble domain thereof with hyaluronidase activity ranging from 1 to 5000 U soluble glycoprotein with 30-150,000 U / mg specific activity in a small volume of such like from 5 to 50 street

[0421] Such solutions, in particular intended for ophthalmological use, may be in the form of a 0.01% -10% isotonic solution at a pH of about 5-7 in a suitable salt.

[0422] 5. Pharmaceutical compositions for other routes of administration.

[0423] Other routes of administration, such as topical and external application, epidermal patches, and rectal administration are included in the present invention.

[0424] An example are pharmaceutical dosage forms for rectal administration in the form of rectal suppositories, capsules and tablets with systemic effect. The term "rectal suppositories", as used herein, means a solid form for insertion into the anus that melts or softens at body temperature releasing one or more pharmaceutically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are base substances or vehicles and agents that increase the melting point. Examples of such base substances are cocoa butter (cocoa oil), glycerol-gelatin, CARBOWAX (polyoxyethylene glycol) and suitable mixtures of mono-, di- and triglycerides of fatty acids. It is possible to use other combinations of base substances. Factors that increase the melting point of suppositories include spermaceti and wax. Rectal suppositories can be made by pressing or molding. The typical weight of a rectal suppository is about 2 to 3 gm.

[0425] Tablets and capsules for rectal administration are prepared by the same methods and using the same pharmaceutically acceptable substance as the oral dosage forms.

[0426] Forms of the drug suitable for transdermal administration may exist as separate patches that are adapted to be in close contact with the patient's epidermis for a prolonged period of time. These types of patches contain a suitable active ingredient, such as, optionally, buffered aqueous solutions containing the active ingredient in a concentration of e.g. 0.1 to 0.2 M. A suitable dosage form for transdermal administration may also be provided by iontophoresis (see e.g. Pharmaceutical Research 3 (6): 318 (1986)) and usually takes the form of an optionally buffered solution of the active ingredient.

[0427] Pharmaceutical compositions can also be administered using controlled release and / or drug delivery apparatus (see 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; 5,073,548; 5,073,543; 5,073,543; ; 5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566). The active ingredients or their pharmaceutically acceptable derivatives can be prepared together with carriers that protect the active ingredient against rapid removal from the body, as in the case of a coating or prolonged drug formulation.

[0428] In one embodiment of the pharmaceutical composition and methods encompassed by the present invention, the therapeutic agent is administered topically in a slow releasing vehicle. e.g. in the form of capsules with a colloidal dispersion system or stabilized polymer crystals. Useful colloidal dispersion systems include nanocapsules, microspheres, coating beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. An example is the colloidal scattering system, which can be in the form of a liposome or microsphere. Liposomes are artificial membranes that are used as carriers that slowly release the drug after injection or implantation. Several examples of lipid-polymer conjugates and liposomes have been disclosed in US Patent No. 5631018, which is incorporated herein by reference in its entirety. Another example of slow release drug carriers is the biodegradable matrix (US Patent No. 5041292), dendritic polymer conjugates (US Patent No. 5,714,166), multi-bubble liposomes (Depofoam®, Depotech, San Diego, CA) (US Patent No. 5,723,147 and 5,766,627). One type of microsphere suitable for encapsulating therapeutic agents intended for local injection (e.g., subcutaneous tissue) is poly (D, L) lactide, as described in D. Fletcher, Anesth. Analg. 84: 90-94, (1997) An example is a slow-release form of a drug containing a therapeutic amount of sHASEGP or its soluble domain with hyaluronidase activity in the range of 1 to 5000 U / ml, which can be used for various purposes and for the treatment of various diseases including, but not limited to, cosmetic and for the treatment of spinal cord injuries.

[0429] The desired blood level can be maintained by intravenous infusion of the active therapeutic agent, as is the case with plasma administration. It should be noted that the physician involved in the treatment would know how and when to terminate, interrupt or reduce the therapeutic dose due to bone marrow, liver or kidney toxicity or impairment. [0430] The efficacy and / or toxicity of the sHASEGP polypeptide and / or its inhibitor (s), alone or in combination with other factors such as therapeutically effective agents, can be assessed by methods known in the art (see e.g. O &amp; Apos; Reilly , Investigational New Drugs 15: 5-13 (1997)).

[0431] 6. PRODUCTS FOR PRODUCTION [0432] sHASEGP polypeptides or their soluble domains with human hyaluronidase activity or pharmaceutical compositions containing any of the previously described factors can be prepared as kits containing a package, the chemical compound contained in this package or a suitable derivative thereof which is the subject of the invention, which is effective in the treatment of diseases or disorders contemplated by the present invention and an information label, that the chemical compound or its respective derivative is intended for the treatment of the diseases or disorders covered by the present invention. Optionally, the label may describe disorders for which the described therapy is suitable.

[0433] The industrial goods of the present invention include packaging. The packaging used for packaging pharmaceutical products is well known to those skilled in the art (see US Patent Nos. 5,323907, 5052558 and 5033352). Examples of packaging used in the pharmaceutical industry include, but are not limited to, contour packaging, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles and any other packaging material suitable for the chosen formulation and intended method of administration and treatment . The wide range of chemical compound and pharmaceutical composition formulations of the present invention is contemplated herein for any disorder in which HCV infection is the mediating factor or cause.

[0434] The present invention provides ready-made kits containing said pharmaceutical compositions and / or combinations with instructions for their administration. Such a kit may still typically contain a sterile packaged needle or injection syringe for the complex, and a packaged alcohol swab. Optionally, the instructions describe the administration of the active agent by the clinician or by the patient. For example, the subject of the present invention is a ready-made kit containing a small-volume syringe containing an effective amount of sHASEGP or its soluble domain with human hyaluronidase activity, in an amount of 1-5000 U soluble glycoprotein, in a volume of 5 - 50 m. Optionally, such a kit may contain a second syringe containing a viscoelastic agent. The present invention also relates to a ready-made kit containing a small volume syringe containing an effective amount of sHASEGP or a soluble domain with human hyaluronidase activity, in an amount of 1 - 500 U soluble glycoprotein and a therapeutic amount of a second active ingredient such as a drug, small molecule, protein or acid nucleic.

[0435] K. ANIMAL MODELS [0436] The present invention relates to transgenic animal models and transgenic animals such as mice and rats, cows, chickens, pigs, goats, sheep, monkeys, including gorillas and other non-human primates. Particularly, the invention relates to transgenic non-human animals into which a nucleic acid encoding the sHASEGP polypeptide has been introduced, or transgenic animals in which the expression of that polypeptide is modified by changing or modifying a promoter or other endogenous region of the gene. Such an animal can be obtained by supporting recombination between an endogenous nucleic acid and an exogenous sHASEGP gene, which gene may be overexpressed or whose expression may be inhibited by the expression of that gene under a strong promoter by homologous recombination or other type of recombination.

[0437] Transgenic animals can be obtained by incorporation of a nucleic acid using any of the methods of introduction, including, but not limited to, microinjection, lipofection and other methods of introducing genes into reproductive or somatic cells, such as embryonic stem cells. Typically, such a nucleic acid is introduced into a cell, such as an embryonic stem cell (ES), and the next steps are: injection of ES cells into the blastocyst, implantation of the blastocyst into the uterus of the surrogate mother and subsequent birth of the transgenic animal. Generally, the introduction of a heterologous nucleic acid into an animal's chromosome occurs as a result of recombination between a heterologous nucleic acid encoding sHASEGP and an endogenous nucleic acid. Such a heterologous nucleic acid can be built into a specific chromosome. In some cases, knockout animals can be received. Such an animal can be initially obtained by initiating homologous recombination between the gene encoding the sHASEGP polypeptide found on the animal's chromosome and the exogenous gene encoding the sHASEGP polypeptide that is biologically inactive (usually as a result of insertion of a heterologous sequence, e.g. antibiotic resistance gene). In one embodiment of the invention, such homologous recombination is carried out by transforming embryonic stem (ES) cells with a vector containing, inactivated by insertion, the sHASEGP polypeptide gene in such a way that homologous recombination occurs. Then ES cells are injected into the blastocyst followed by implantation of the blastocyst (into the womb) of the surrogate mother and birth of a chimera (knockout animal) carrying the inactive gene encoding the sHASEGP polypeptide (see Capecchi, Science 244: 1288-1292 (1989)). Such a chimera can be bred to obtain knocked out homozygous progeny animals which can then be used to obtain further knocked out animals. Knockout animals include, but are not limited to, mice, hamsters, sheep, pigs, cattle and non-human mammals. An example is knockout mice. The animals thus obtained can serve as models for specific diseases, such as tumors showing reduced expression of the sHASEGP polypeptide. Knockout animals can be used as animal models for this type of disease, for the purpose of screening or testing to assess the ability of molecules to treat or prevent said diseases or disorders.

[0438] It is also possible to obtain other types of transgenic animals, including those that show high levels of sHASEGP polypeptide expression. The group of such animals includes knock-out animals obtained by knock-in (directed sequence insertion) in which the normal gene is replaced by a variant such as a mutant, overexpressed form or other form. For example, a gene from one species, such as the endogenous rat gene, may be replaced by a gene from another species, e.g., a human gene. Animals can also be obtained by non-homologous recombination at other places on the chromosome. The present invention also includes animals having many genome-integrated fragments of introduced DNA.

[0439] After receiving a first generation transgenic animal, such a chimera can be cultured to obtain further animals characterized by overexpression, diminished expression or lack of expression of the sHASEGP polypeptide. These include, but are not limited to, mice, hamsters, sheep, pigs, cattle and non-human mammals. The animals obtained in the above manner can serve as models for specific diseases, such as tumors showing overexpression or lack of expression of the sHASEGP polypeptide. For example, these animals can be used as animal models for this type of disease to screen or test for molecules for the ability of these molecules to treat or prevent such diseases or disorders. In a particular embodiment, the present invention provides mice overexpressing or not expressing the sHASEGP polypeptide.

[0440] The examples below serve only as demonstration and in no way limit the scope of the invention.

[0441] L. THERAPEUTIC APPLICATIONS of sHASEGP.

[0442] For over 40 years slaughterhouses have been the primary source of preparations used for clinical purposes containing naturally occurring hyaluronidases. The primary source of this enzyme are beef and sheep testicles. However, such enzyme preparations are very crude. They are sold as preparations with a purity in the range of 0.5% - 5% and specific activity in the range of 30 - 100,000 U / mg. The low level of purity of these preparations and the source of origin (slaughterhouse) make them immunogenic and constitute a potential source of infection with Creutzfeldt-Jacob disease and other pathogens of cattle and sheep. There are known cases of anaphylactic reactions in response to the administration of preparations containing bovine and sheep hyaluronidase.

[0443] Bovine or bacterial hyaluronidase has been used to treat diseases associated with excess hyaluronic acid and to improve the distribution of physiological fluids and / or therapeutic agents in the body. For example, during ophthalmic surgery, bovine hyaluronidase may be injected around the ear, out of the ear, or under the back of Tenon's block (sub-Tenon's block) together with anesthetics. Moreover, when no bovine hyaluronidase was used, the number of postoperative complications increased (Brown SM et al. J Cataract Refract Surg. 1999 Sep; 25 (9): 1245-9.). Bovine hyaluronidase is also used as an antidote for local necrosis resulting from peri-venous injection of necrotic substances such as Vinca alkaloids (Few, BJ (1987) Amer. J. Matern. Child Nurs. 12, 23-26). Bovine testicular hyaluronidase is also used to treat gelatinous cysts of the tendon sheath (Paul et al. J Hand Surg 1997 Apr; 22 (2): 219-21). It can also be used to facilitate subcutaneous fluid administration during subcutaneous infusions (Berger EY, Am Geriatr Soc 1984 Mar; 32 (3): 199-203). It also found use as an intraocular pressure lowering agent in patients with glaucoma and patients with cataract who are receiving viscoelastic fluids (US Patent No. 4820516, published on April 11, 1989).

[0444] Bovine or bacterial hyaluronidases have also been used as so-called "Spreading agents", enhancing the activity of chemotherapeutics and / or reaching cancer (Schuller et al., 1991, Proc. Amer. Assoc. Cancer Res. 32: 173, abstract no. 1034; Czejka et al ., 1990, Pharmazie 45: H.9). The combination of chemotherapy and hyaluronidase is effective in the treatment of various types of cancer, including 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), gastrointestinal cancer (Scheithauer et al., 1988, Anticancer Res. 8: 391-396). Hyaluronidase alone as a therapeutic agent is effective in the treatment of brain cancer (glioblastoma) (PCT Patent Application No. WO88 / 02261, published 07.04.1988). The administration of hyaluronidase also stimulates the reactivity of primary chemotherapy resistant pancreatic, stomach, colon, ovarian and breast tumors (Baumgartner et al., 1988, Reg. Cancer Treat. 1: 55-58; Zanker et al., 1986, Proc. Amer Assoc. Cancer Res. 27: 390). Unfortunately, the presence of impurities and the fact that these hyaluronidases are not of human origin causes an anaphylactic reaction.

[0445] In addition to its indirect activity, bovine hyaluronidase also has direct anti-tumor activity. It prevents the growth of transplanted mice (De Maeyer et al., 1992, Int. J. Cancer 51: 657-660) and inhibits the development of cancer caused by carcinogens (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).

[0446] Given the therapeutic significance of bovine hyaluronidases, particularly in chemotherapy, in combination with conventional chemotherapeutics or as a chemotherapeutic, there is a need in this regard for substantially purified preparations of hyaluronidase of human origin. There is also a need for efficient, low-cost methods for producing hyaluronidase to provide significant amounts of this enzyme to the market. The present invention solves these problems.

[0447] Hyaluronic acid is an essential component of the extracellular matrix. Occurs in the connective tissue of mammals and is the main component of the vitreous humor of the eye. In connective tissue, water molecules derived from hydrated hyaluronan shape the space between the tissues, thus creating an environment conducive to the movement of cells and their proliferation. Hyaluronan plays a key role in biological phenomena associated with cell motility, including phenomena such as rapid development, regeneration, repair process, embryogenesis, embryonic development, wound healing, angiogenesis, tumorogenesis (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, the level of hyaluronan correlates with the degree of aggressiveness of the cancer (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).

[0448] Following spinal cord injury, astrocytes produce glial scars containing proteoglycan sulfate (CSPG). CSPGs play an important role in inhibiting axon growth (Levine, 1994; Powell et al.). For example, during fetal development, CSPG repels axons and inhibits neural cell adhesion. These molecules also play an important role in forming connections between astrocytes (Snow et al., 1990, 1992; Powell and Geller, 1999). In addition, an increase in CSPG expression is observed following central nervous system (CNS) injury (Mckeon et al., 1991; Davies et al., 1997).

[0449] Studies show that the inhibitory effects of CSPG molecules relate essentially to chondroitin sulfate (CS) - the glycosaminoglycan sugar chain (GAG) (Snow et al., 1990; Cole and McCable, 1991; Geisert and Bidanset, 1993). This is supported by the finding that intrathecal administration of bacterial chondroitinase actually stimulates axon regeneration. In addition, as a result of electrophysiological experiments, it was found that the axons regenerated as a result of CST therapy regained their functional connections (Bradbury, et al. 2002). In addition to the direct inhibitory effects of CSPG molecules, they can also interact with cell adhesion molecules or neurotrophic factors, thereby affecting axon growth (Roberts et al., 1988; Ruoslahti and Yamaguchi, 1991; Milev et al., 1994). Therefore, recombinant mammalian hyaluronidase is useful in suppressing the inhibitory effect of CSPG molecules in the neuroglial scar and promoting axon regeneration after trauma.

[0450] Depending on the case, the amount of sHASEGP glycoproteins required for efficient degradation of CSPG molecules in the neuroglial scar will vary. In some cases, repetitive intrathecal administration of 10,000 U will be required to remove CSPG molecules in the scar. In other cases, continuous delivery of sHASEGP by using a sustained release formulation will be preferred. Alternatively, vectors used for gene therapy encoding sHASEGP may be effective to increase the effect of CSPG removal.

[0451] sHASEGP glycoproteins may also find use in the treatment of intervertebral disc hernia by a method called chemonucleolysis. Chondroitinase ABC, an enzyme that digests similar substrates as sHASEGP, can induce a reduction in intra-lumbar pressure in the lumbar spine (Sasaki et al., 2001, Ishikawa et al., 1999). There are three types of disc injuries. The protruding intervertebral disc (protrusion) is one whose continuity is intact, but there is a bulge. In the case of disc extrusion, the atherosclerotic nucleus (NP) interrupts the continuity of the fibrous ring, but remains in the disc. In the case of disk sequestration, a piece of NP escapes outside the disk into the spinal canal. Chemonucleolysis is effective for disk protrusion and extrusion, but not for disk sequestration. In the USA, chemonucleolysis is approved for use only in diseases of the lumbar spine. In other countries, it is used to effectively treat cervical (upper) spinal hernia. Chemonucleolysis is therefore a conservative method that is an alternative to disc surgery in cases where reduction of intra-disc pressure is indicated.

[0452] Since endoplasmic reticulum or liver cells can bind and internalize circulating glycoproteins containing specific sugars in their structure, the specific composition and structure of the sugar chain (s) of these glycoproteins may directly affect the serum half-life of these glycoproteins. Hepatocytes have receptors on their surface that recognize glycan chains containing terminal (i.e. located at the furthest end (s) to the polypeptide) of the Gal residue. In contrast, macrophages have receptors that recognize terminal Man or GIcNAc residues, and hepatocytes and lymphocytes - receptors that bind exposed fucose residues. No receptors specific for sialic acids have been identified. Despite this, and to some extent depending on the spatial structure of glycans, the rule is that the higher the number of exposed sugar residues recognized by surface receptors of liver cells and endoplasmic reticulum, the faster the glycoproteins will be removed from the serum. Due to the lack of receptors specifically recognizing sialic acids, glycans containing side chains terminated or crowned with sialic acid do not induce the process of removing the protein to which they are attached.

[0453] The presence and nature of glycan (s) on the glycoprotein may affect not only the recognition of glycoprotein by sugar-specific receptors that are present in the liver and endoplasmic reticulum, but also its significant biochemical properties. The removal of glycans from the glycoprotein most often causes a decrease in its solubility, but also an increase in susceptibility to proteolytic degradation, which is the result of destabilization of the correct folding of the protein and / or the discovery of sites sensitive to proteases. For similar reasons, the glycosylation status of a protein may affect the recognition of the protein by the immune system.

[0454] sHASEGP glycoproteins can be used to remove cumulus cells surrounding the oocyte before freezing the egg or before using other in vitro fertilization techniques such as intracytoplasmic sperm injection (ICSI). Hyaluronidase can be added to harvested oocytes in buffered 10-200 U / ml saline. Oocytes are separated from removed cumulus cells by aspiration and washed several times with hyaluronidase-free medium. Then oocytes can be frozen or used in in vitro fertilization techniques.

[0455] sHASEGP glycoproteins are also used to improve the penetration of chemotherapeutics into solid tumors. These glycoproteins may be injected intratumorally together with anti-tumor agents or intravenously in the case of disseminated or hard-to-reach tumors. Such an anti-tumor agent may be a chemotherapeutic, antibody, peptide or vector used in gene therapy, virus or DNA. In addition, sHASEGP glycoproteins can be used to loosen intercellular junctions in tumor mass, which makes it possible to increase the sensitivity of drug-resistant cells to chemotherapeutic agents (St Croix et al Cancer Lett 1998 Sep 11; 131 (1): 35-44). SHASEGP glycoproteins are also used to facilitate the delivery of biological preparations that cause the deposition of glycosaminoglycans such as monoclonal antibodies, cytokines and other anti-cancer drugs. Many cancers induce the deletion of genes involved in the catabolism of glycosaminoglycans, which causes the local accumulation of these molecules, which prevents anti-cancer agents and components of the immune system from entering the tumor mass.

[0456] sHASEGP can also be used to increase the sensitivity of cancer resistant to conventional chemotherapy methods. In one embodiment of the invention, sHASEGP is administered to a cancer patient associated with a LUKA-1 defect in an amount that allows increased diffusion within the tumor (e.g. to increase circulation and / or accumulation of chemotherapeutic agents within and around the tumor), inhibition of tumor cell (s) motility (e.g. by degradation of HA), and / or by lowering the threshold of susceptibility of the cancer cell (s) to apoptosis (e.g., by introducing the cancer cell (s) into the anoikis state, i.e. a condition that makes the cancer cell (s) more susceptible to chemotherapeutic agents or) other factors that can lead to cell death, particularly to facilitate programmed cell death in anoikis. As used herein, the term "chemotherapeutics" includes all synthetic (e.g., cisplatin) and naturally occurring molecules (e.g., tumor necrosis factor, (IF)) that help inhibit the growth of a cancer cell and favorably lead to cancer cell death, and very preferably selectively lead to cancer cell death. [0457] Of particular interest is the use of sHASEGP in the treatment of metastatic and non-metastatic tumors, especially metastatic tumors, characterized by reduced or undetectable levels of hyaluronidase activity on non-cancer cells (normal cells). The sHASEGP glycoprotein can be used as a chemotherapeutic agent (alone or in combination with other chemotherapeutic agents) in the treatment of any cancer, especially in the treatment of invasive cancer. For example, sHASEGP can be used to treat small cell lung cancer, squamous cell lung cancer, as well as breast, ovarian, head and neck cancer or any cancer associated with inhibited hyaluronidase levels or with a LUCA-1 (hpHAza) gene defect (e.g. with such a defect in the LUCA-1 gene that does not ensure the expression of an adequate level of hpHAase or encodes a defective hpHAase (which does not provide an adequate level of hyaluronidase activity) or other defects associated with reduced catabolism of hyaluronan. Since degradation using sHASEGP does not require cell involvement, the use of sHASEGP in the treatment of malignancies associated with deficiency in HA catabolism is preferred.

[0458] The specific dosage appropriate to the use of the drug can be readily determined by one skilled in the art taking into account the factors discussed above (see, e.g., Harrison's Principles of Internal Medicine, 11th Ed., 1987). In addition, it is possible to extrapolate the correct dosage in humans based on in vitro sHASEGP enzymatic activity measurement and / or based on knowledge of effective doses used in animal studies. For example, 70-300 TRU hyaluronidase is effective in reducing tumor mass in SCID mice. Based on this data, a suitable dose of hyaluronidase for a human, weighing about 70 kg, would be about 250,000 - 1,200,000 TRU. The amount of sHASEGP administered to humans is usually in the range of 1 - 5,000 TRU, preferably in the range 100,000 - 1,500,000 TRU, usually between 250,000 and 1,200,000 TRU and corresponds to an average prescribed dose of TRU 725,000.

[0459] In one embodiment of the invention, the sHASEGP formulation is prepared as a solution at a concentration of about 150,000 TRU / mL in 0.15 M saline. This preparation is then injected intravenously at a dose of 150,000 TRU / kg body weight of the patient. Alternatively, the enzyme formulation can be injected subcutaneously to allow hyaluronidase to penetrate into the tumor area. In a preferred embodiment of the invention, sHASEGP are injected peritumorally or intratumorally. In another preferred embodiment of the invention, the sHASEGP preparation is prepared in the form of liposomes and administered either intravenously (by injection), intratumorally or in the vicinity of tumor cells defective in the LUCA-1 gene (hpHAza). Intravenous injection of sHASEGP results in the site of sHASEGP reaching the tumor site. In addition, since terminal sialic acids prevent sHASEGP from being removed from the circulation via an endoplasmic reticulum, highly sialated sHASEGP is beneficial for parenteral administration. A comparison of high-level sHASEGP with unialized bovine and sheep-hyaluronidase showed significantly better pharmacokinetics of this glycoprotein.

[0460] SUPPORT OF GENE THERAPY [0461] The efficacy of most of the in vivo carriers used for gene introduction does not match the efficacy observed in vitro. Glycosaminoglycans can impede the transfer and diffusion of DNA and viral vectors into many cell types. A significant amount of such a substance derived from the extracellular matrix can significantly hinder these processes. Dubensky et al. (Proc Natl Acad Sci USA 1984 Dec; 81 (23): 7529-33) showed that hyaluronidase in combination with collagenase can facilitate DNA transduction in vivo. It has also been shown that adeno-associated virus can be effectively used in hyaluronidase-assisted gene therapy (Favre et al, Gene Ther 2000 Aug; 7 (16): 1417-20).

[0462] In the present invention, we have shown that sHASEGP opens the channels of the extracellular matrix of a certain size. These channels do not support the diffusion of particles larger than about 200-500 nm in diameter. The use of sHASEGP facilitates the diffusion of smaller molecules such as retroviruses, adenoviruses, adeno-associated viruses and DNA complexes.

[0463] Alternatively, to facilitate the replication and spread of viruses in target tissues, they may for example be equipped with a gene encoding sHASEGP. The target tissue may be a tumor tissue within which said virus is capable of selective replication. Such a virus can also be a virus that does not lyse target cells, which is selectively under a given promoter. At the time of virus replication, co-expression of sHASEGP with viral genes will facilitate the spread of the virus in vivo.

[0464] Alternatively, the nucleic acid of interest and sHASEGP may be used simultaneously or sequentially or in a staggered manner. The term "simultaneously" refers to simultaneous administration. In this case, both necessary ingredients may be mixed prior to administration to obtain a pharmaceutical composition, or may be administered to the host cell or organism at the same time. It is also possible to administer these substances one after the other, irrespective of which component of the invention is administered first. Finally, it is possible to use the administration regimen, with doses being spread out or administered in a manner in which dosing is withheld and resumed at intervals that may or may not be regular. It is emphasized here that the routes and places of administration of these two ingredients may be different. Thus, according to one particularly preferred embodiment, the invention provides sHASEGP administered prior to the nucleic acid in such a way that the routes of administration of the two components are preferably similar. The time interval between injections is not critical and can be determined by a person skilled in the art. A time interval of 10 min to 72 hours, preferably 30 min, may be recommended. up to 48 hours, more preferably 1 to 24 hours and very preferably from 1 to 6 hours. [0465] In addition, the combination of the invention may be linked to one or more molecules in which they are presumed to improve delivery of a given nucleic acid. Such molecules may be nucleic acid protective molecules (by the term "protective" is meant protection against degradation in the cell), molecules that improve the penetration of the nucleic acid or its expression in the host cell (fusogenic peptide, nuclear localization signal, etc.), molecules penetration into one specific cell type (ligand or antibody recognizing cell surface proteins, etc.), molecules prolonging the therapeutic effect (immunosuppressive factors, etc.). Such a combination can also be combined with factors that facilitate transfection (proteins, etc.).

[0466] In accordance with the present invention, the pharmaceutical composition may be prepared for local or parenteral administration or oral administration. In particular, routes of administration such as intragastric, subcutaneous, intracardiac, intravenous, intraperitoneal, intra-synovial, intrathecal, pulmonary, intranasal and intratracheal administration should be mentioned, especially intramuscular administration. Administration of the pharmaceutical composition may be performed using any technique known to the skilled person (injection, oral route, aerosol, instillation, etc.), in the form of a single dose or once or repeatedly at specified intervals. The route of administration may be adapted to the gene to be delivered and is of interest and the type of disease being treated. The formulation may include pharmaceutically acceptable carriers (excipients, adjuvants, etc.). The substance causing disorganization of the extracellular matrix and the nucleic acid of interest are preferably dissolved in a pharmaceutically acceptable buffer, which buffer may be hypertonic, hypotonic or isotonic. Different buffers are considered. Those that could be mentioned as examples are saline solution (0.9% NaCl), non-physiological saline solution (1.8% NaCl), Hepes-Ringer solution, Ringer's solution with the addition of lactate, buffer prepared on the basis of Tris-HCl solution (10 mM Tris-HCl, pH 7.5 -8, 1 mM EDTA; 10 mM Tris-HCl, pH 7.5 - 8, 1 mM MgCl2), phosphate buffer (Krebs buffer: phosphate monohydrate), sugar solution (e.g. glucose, sucrose, trehalose etc. .) or water alone.

[0467] SUBcutaneous infusion [0468] Subcutaneous infusion or subcutaneous fluid infusion is a useful and easy irrigation technique suitable for light to moderately dehydrated patients, especially for the elderly. This method is considered safe and does not cause any serious complications. The most common side effect is light subcutaneous edema, which can be treated with local massage or a systemic diuretic. Such a therapeutic fluid can be administered in a volume of approximately 3 liters over a 24 hour period at two separate sites. Typical infusion sites are the chest, abdomen, thigh and upper arm. Preferably, the solution is normal saline. However, other saline solutions such as semi-normal saline, glucose solution in saline or 5% glucose may also be used. If desired, sodium chloride may be added to this solution. In addition, other drugs may be provided through similar routes of administration. Human sHASEGP can be added to assist fluid absorption and increase the overall rate of administration. The use of human sHASEGP for repeated subcutaneous infusions is more preferred compared to animal-derived enzymes. The reason for this is that human sHASEGP, unlike the bovine enzyme, is most likely not immunogenic, it can be given at home by family members or a nurse, and the technique of administration is known to every family doctor.

[0469] For outpatient patients, subcutaneous infusion sites include the abdomen, upper chest, above the breast, above the intercostal space and the scapula area. For bedridden patients, the preferred injection site is the thighs, abdomen and outside of the upper arm. The needle and tubing should be changed after one to two days, although the infusion set was left at the infusion site for a long time without any complications. Subcutaneous administration of one large dose of sHASEGP (150 U) three times in one day, before the first morning infusion, in a volume of 500 ml for 1 to 2 hours is also possible.

[0470] FACILITATION OF THERAPEUTIC INJECTIONS [0471] Many transdermally injected molecules reach circulation slowly or with very low efficiency. Several factors influence the pharmacokinetics and pharmacodynamics of injected subcutaneous (SC) or intramuscular (IM) molecules. Generally, larger particles not supported by active transport reach circulation more slowly and less efficiently. Subcutaneous bioavailability is determined by calculating the ratio of the area under the curve obtained for CS administration to the area under the curve for intravenous administration (AUCSC / AUC_dabout_fromintravenous). The second factor is the charge and affinity for extracellular matrix molecules, which play a key role in subcutaneous particle sequestration. If such ingredients are locally degraded, they will never reach their desired target and will therefore show reduced total systemic bioavailability to target organs.

[0472] Because large molecules are usually administered intravenously, the medicine is immediately available in the bloodstream. However, it would be an advantage that the medicine can be administered subcutaneously, intramuscularly or intradermally, as administration by such methods is much easier for the patient to perform. This is especially true if such a medicine must be taken regularly throughout life, and treatment begins fairly early when the patient is a child. However, a large drug or labile molecule, such as coagulation factor VIII with a weight of 170-300 kDa, is usually characterized by very low bioavailability for subcutaneous, intramuscular or intradermal administration, when absorption is insufficient and degradation is significant. [0473] From the point of view of emergency medicine, in addition to the need to increase the bioavailability of many subcutaneously administered biological preparations, their faster pharmacokinetics is also very important. An unsuccessful attempt to gain venous access may, for many patients, prevent the use of another fast-acting systemic drug. In some cases, after venous access fails, subcutaneous injection is used, which further leads to delayed (drug) reaching target organs. Therefore, instead of risking the loss of time needed to gain intravenous access, first aid would be beneficial in terms of first aid to ensure faster drug availability. Examples of molecules that can be delivered subcutaneously as well as intravenously include epinephrine, atropine, narcan, lignocaine and dextrose.

[0474] Many molecules injected transdermally reach circulation slowly or with very low efficiency. Several factors influence the pharmacokinetics and pharmacodynamics of injected subcutaneous (SC) or intramuscular (IM) molecules. Generally, larger particles not supported by active transport reach circulation more slowly and less efficiently. Subcutaneous bioavailability is determined by calculating the ratio of the area under the curve obtained for CS administration to the area under the curve for intravenous administration (AUCSC / AUC_dabout_fromintravenous). The second factor is the charge and affinity for matrix particles, which play a key role in subcutaneous sequestration of molecules. If such ingredients are locally degraded, they will never reach their desired target and will therefore show reduced total systemic bioavailability relative to target organs.

[0475] Because large molecules are usually administered intravenously, the medicine is immediately available in the bloodstream. However, it would be an advantage that the medicine can be administered subcutaneously, intramuscularly or intradermally, as administration by such methods is much easier for the patient to perform. This is especially true if such a medicine must be taken regularly throughout life, and treatment begins fairly early when the patient is a child. However, a large drug or labile molecule, such as coagulation factor VIII with a weight of 170-300 kDa, is usually characterized by very low bioavailability for subcutaneous, intramuscular or intradermal administration, when absorption is insufficient and degradation is significant. [0476] From the point of view of emergency medicine, in addition to the need to increase the bioavailability of many subcutaneously administered biological preparations, their faster pharmacokinetics is also very important. The time needed for intravenous access in many patients may exclude the use of another fast-acting systemic drug. In some cases, after venous access fails, subcutaneous injection is used, which further leads to delayed (drug) reaching target organs. Therefore, instead of risking the loss of time needed to gain intravenous access, first aid would be beneficial in terms of first aid to ensure faster drug availability. Examples of molecules that can be delivered subcutaneously as well as intravenously include epinephrine, atropine, narcan, lignocaine and dextrose.

[0477] An additional benefit of the present invention is the possibility of subcutaneous (SC) or intramuscular (IM) delivery of equivalent or larger volumes in a non-painful manner and without causing disease conditions related to the pressure and volume of the solution at the injection site.

[0478] GLYALY EYE BLEEDING BLEEDING [0479] To minimize the likelihood of further detachment or tearing of the retina during vitrectomy, US Patent No. 5292509 (Hageman) was proposed to inject specific protease-free glycosaminoglycanase into the vitreous body prior to removal before removal vitreous, cause it to separate or detach from the retina. Such "detachment" or separation of the vitreous humor of the eye minimizes the likelihood of retinal tearing or detachment during vitreous removal. Examples of such specific, protease-free, glycosaminoglycanases that can be used to cause vitreous detachment appear to be chondroitinase ABC, chondroitinase AC, chondroitinase B, chondroitin-4-sulfate sulfatase, chondroitin-6-sulfate sulfate, hyaluronidase and .

[0480] Although it is known that hyaluronidases are suitable for various ophthalmic applications, including the vitrectomy support described in US Patent No. 5,292,509 (Hageman), the results of published studies have shown that hyaluronidase itself can be toxic to retina and / or other anatomical structures of the eye. See: The Safety of Intravitreal Hyaluronidase; Gottleib, JL; Antoszyk, AN, Hatchell, DL and Soloupis, P., Invest Ophthalmol Vis Sci 31:11, 2345-52 (1990). In addition, the use of contaminated hyaluronidase preparations from slaughterhouses can lead to the development of uveitis or eye inflammation. The use of human sHASEGP is therefore beneficial both because of its greater potency, purity and the fact that such an enzyme is not of animal origin, which in the opposite case may lead to the induction of immunogenic reactions and the formation of antibodies neutralizing the activity of subsequent doses of this glycoprotein. In another embodiment of the invention, pegylated sHASEGP may be injected into the eye. Such pegylated sHASEGP is not removed so quickly from the vitreous, where it maintains its activity for a long time.

[0481] The ocular toxic effect of some hyaluronidase preparations has been confirmed by researchers who proposed using such preparations as toxic irritants to induce the experimental formation of new ocular blood vessels in animal models for toxicity testing (see An Experimental Model of Preretinal Neovascularization in the Rabbit; Antoszyk, AN, Gottleib, JL, Casey, R C., Hatchell, DL and Machemer, R., Invest Ophthalmol Vis Sci 32: 1.46-51 (1991). As for intraocular treatments, the use of highly purified sHASEGP, free from impurities containing mercury or bovine or bacterial compounds, is preferred. In addition, due to the lack of bovine pathogens alone and the reduced risk of immunogenicity, recombinant human sHASEGP is more advantageous compared to preparations from slaughterhouses. Pegylated sHASEGP is expected to be most preferred.

[0482] The present invention relates to an enzymatic method using sHASEGP for the treatment of ophthalmological disorders in mammals. In one embodiment, the present invention includes said sHASEGP, which is pegylated to prolong its residence time in the vitreous and prevent its local absorption. The effect of preventing the formation of blood vessels and the increased rate of removal of particles toxic to the retina from the vitreous is achieved by administering hyaluronidase in an amount sufficient to liquefy the vitreous without causing toxic damage to the treated eye. Vitreous liquefaction increases the rate of fluid exchange in a vitreous chamber. Such an increase in the rate of fluid exchange leads to the removal of these particles and conditions that cause damage to the eye and retina.

[0483] COSMETIC APPLICATIONS of sHASEGP [0484] Hyaluronidases are known to induce the depolymerization effect of long chains of mucopolysaccharide, the primary substance responsible for retaining bound water molecules and slowing down, by closing capillaries, the diffusion of organic fluids removing the by-products of metabolism.

This retention of water and by-products of metabolism in combination with fat filling fat cells is a classic swelling of the skin called 'Pig skin' or 'orange peel'. In this case, depolymerization will therefore lead to cutting the long chains of mucopolysaccharide into smaller fragments, thereby leading to the elimination of water, by-products of metabolism, return to normal venous and lymphatic circulation, and the disappearance of local edema.

[0485] Therefore, it is preferred to use sHASEGP, by its subcutaneous administration, to remove glycosaminoglycans involved in the accumulation of so-called cellulitis and stimulation of circulation in the lymphatic system. Due to the fact that sHASEGP is able to remove these glycosaminoglycans and is characterized by a lack of immunogenic animal protein components (slaughterhouses), high purity and a probable lack of immunogenicity, the use of human sHASEGP for the treatment of cellulitis is preferred. To stimulate constant degradation of glycosaminoglycans and prevent their re-accumulation, sHASEGP can be administered subcutaneously as injections, transdermally in the form of ointments or creams or as injectable delayed-release formulations.

[0486] ORGAN TRANSPLANTATION [0487] Hyaluronan produces several biological effects that are partly due to the size of this molecule (West, DC, Kumar, S. Exp. Cell. Res. 183, 179-196, 1989). The content of hyaluronan in the body increases in various inflammations. Therefore, increased levels of hyaluronan have been found in the tissues of various inflammatory organs and damage resulting from the immune system, such as alveolitis or alveolitis (Nettelbladt et al., Am Rev Resp Dis 1989; 139: 759-762) and myocardial infarction cardiac (Waldenstrom et al., J Clin Invest 1991; 88 (5): 1622-1628). Other examples include renal allograft rejection (Ha'llgren et al., J Exp Med 1990a; 171: 2063-2076; Wells et al., Transplantation 1990; 50: 240-243), small intestine transplantation (Wallander et al., Transplant Int 1993; 6: 133-137) or heart transplant (Hallgren et al., J Clin Invest 1990; 85: 668-673) or myocarditis of viral origin (Waldenstrdm et al., Eur J Clin Invest 1993; 23: 277 -282).

[0488] The appearance of interstitial edema associated with organ transplantation is a major problem in transplant surgery. As much as 25% of transplants will develop swelling to such an extent that organ function will be temporarily lost. In addition, in 2-3% of cases, edema caused a tear in the kidney leading to extensive haemorrhage.

[0489] The sHASEGP glycoprotein can be used to degrade glycosaminoglycans accumulated in the transplanted organ. Eliminating these particles removes water from the transplanted organ and restores its function. To reduce interstitial pressure, a sHASEGP dose may be administered in the range 500 - 10,000 U / kg.

[0490] PATHOLOGICAL AGGREGATION OF GLYCOSAAMINOGLICANES IN THE BRAIN [0491] Elevated levels of hyaluronan are observed in many cerebrospinal diseases. Normally the so-called "Cerebrospinal" hyaluronan in adults is less than 200 mg / l (Laurent et al, Acta Neurol Scand 1996 Sep; 94 (3): 194-206). This level may rise to more than 8,000 mg / l for diseases such as meningitis, spinal stenosis, head trauma and stroke. Therefore, the administration of high-sialated sHASEGP by intrathecal or systemic injection can be used to degrade substrates of this enzyme at elevated concentrations.

[0492] Lack of efficiently circulating lymph in the brain can also lead to life-threatening edema resulting from head trauma. Accumulation of hyaluronan is the result of increased HA synthase synthesis and reduced hyaluronan degradation. It aims to increase the water content of damaged tissue so as to allow leukocytes to extravasate, however, in some cases it can be fatal. In this case, administration of human sHASEGP to a patient suffering from head trauma may result in the removal of hyaluronan accumulated in the tissue along with the associated water. Human sHASEGP can be administered intrathecally via a fistula. Alternatively, to deliver it to the brain tissue, high-ialized sHASEGP may be administered intravenously.

[0493] Hyaluronan levels increase dramatically in cerebral ischemia resulting from stroke, which is associated with increased expression of HA synthase and its reduced catabolism. Ion pump failure and plasma leakage into the interstitial tissue causes fluid retention, which, if not properly removed by the lymphatic system, can lead to necrosis. Fluid accumulation in interstitial tissue in cases of ischemia following reperfusion has been attempted to prevent by blocking the permeability of blood vessels. However, when fluid flows out of the blood vessel, blocking the permeability of the blood vessels can also block the absorption of edema and exacerbate the patient's condition.

[0494] Human sHASEGP can also be used to treat edema associated with a brain tumor, especially one that is associated with glioblastoma multiforme. Edema, resulting from brain tumors, occurs as a result of the accumulation of hyaluronan in the cancerous part of the brain. Hyaluronidase administration to hyaluronan accumulation sites (e.g. by intravenous injection or fistula) may alleviate edema associated with this type of cancer by degrading the hyaluronan accumulated in these areas. Therefore, hyaluronidase is effective in the treatment of brain tumors not only because its effect reduces the tumor mass and inhibits its growth and / or metastasis, but also because its use relieves edema resulting from the described neoplastic condition. During the treatment of edema, human sHASEGP may be administered in a manner analogous to that used for bovine testicular hyaluronidase (see, e.g., Sa Earp Arq. Braz. Med. 44: _217-20).

[0495] TREATMENT OF GLUCOSOAMINOGLYCAN ACCUMULATION IN CARDIOVASCULAR DISEASE [0496] Using animal models of experimental myocardial infarction, it was shown that administration of hyaluronidase reduced the area of ​​ischemic necrosis in experimental animals (Maclean, et al. 194-26 Oct 19 200). The proposed mechanism by which hyaluronidase reduces the area of ​​ischemic necrosis in animals is to reduce the accumulation of hyaluronan that occurs in cases of ischemia following reperfusion. It is believed that the reduction in ischemic necrosis is associated with an increase in lymphatic drainage, oxygenation of the tissue and reduction of myocardial water content. While the effect of reducing ischemic necrosis in animal models has been achieved, such beneficial effects have not been achieved in large-scale human studies. Bovine testicular hyaluronidase has a very short serum half-life of approximately 3 min for animals and humans. (Wolf, et al., J Pharmacol Exp Ther 1982 Aug; 222 (2): 331-7). Such a short half-life is the result of the presence of terminal mannose residues that are easily recognized by scavenger receptors of the endoplasmic reticulum. The use of hyaluronidase can have a beneficial effect in small animals due to their smaller vascular bed. There is a need for an enzyme with an increased half-life. High-ialized sHASEGP is characterized by favorable pharmacokinetics, resulting from the presence of terminal sialic acids in sHASEGP glycan structures, which is why they are not recognized by scavenger receptors. Highly sialated sHASEGP, at a dose of 100 - 200,000 U / kg, can be used to facilitate the absorption of excess hyaluronan resulting from reperfusion ischemia and to reduce the size of the ischemic necrosis.

[0497] High-ialized sHASEGP can also be used to reduce the formation of atherosclerotic plaques. Such plaques accumulate glycosaminoglycans and mediate the adhesion of macrophages and foam cells (Kolodgie et al, Arterioscler Thromb Vasc Biol. 2002 Oct 1; 22 (10): 1642-8). Administration of high-sialated sHASEGP can be used to reduce the formation of atherosclerotic plaque. The present invention includes repeated administration of hyaluronidase at doses of 100 - 100,000 U / kg, and the use of human recombinant protein for these purposes, with a low risk of immune response and increased half-life, will result in greater reduction of atherosclerotic plaque.

[0498] TREATMENT OF PERIPHERAL PERIPHERAL TISSUE [0499] In many diseases, tissue necrosis occurs as a result of venous insufficiency. Lack of efficient oxygenation is one of the main obstacles to tissue regeneration. Intraarterial hyaluronidase treatment has been shown to improve the clinical picture of a patient suffering from peripheral arterial disease (Elder et. Al, Lancet (1980) 648-649). The sHASEGP glycoprotein can be injected into the artery 3 to 5 times a week at doses of 10 - 200,000 U.

[0500] GAIN ANESTHESIA [0501] Animal-derived hyaluronidases are commonly used to induce periocular blockade under local anesthesia used in ophthalmic surgery. The presence of the enzyme eliminates the need for further blockages and accelerates the occurrence of akinesia (eye stillness). Intraocular blockade and blockade consisting in the administration of an anesthetic under the posterior part of the Tenon capsule sub-Tenon's block) are the most common methods used during ophthalmic procedures in which hyaluronidase can be used. Since the withdrawal of Wydase®, in cases of periocular blockade, an increasing number of postoperative complications associated with double vision and eyelid drooping have been reported (Brown et al J Cataract Refract Surg 1999; 25: 1245-9).

[0502] When cessation of use of Wydase®, hyaluronidase derived from bovine testicles appeared on the market. However, there are several problems associated with the use of such sterile on-site products (see:

<a href="http://www.ashp.org/shortage/hyaluronidase.cfm?cfid=11944667&amp;CFToken=942">http://www.ashp.org/shortage/hyaluronidase.cfm?cfid=11944667&amp;CFToken=942 </a>6953 - ref # ref.

Such preparations are products not approved by the FDA. Thus, the FDA has no control over their quality and compliance with the requirements of their production process.

[0503] 10-500 U sHASEGP glycoprotein can be mixed directly with 5 ml 2% lidocaine (xylocaine), 5 ml 0.5% bupivacaine (markin) and optionally with epinephrine in a ratio of 1: 200,000. SHASEGP glycoprotein can be used to accelerate the occurrence of akinesia and eliminate the need for additional blockages. This glycoprotein is also useful for causing akinesia in cosmetic surgery associated with eyelid surgery and face lifting. It can also be used to support the diffusion of anti-inflammatory agents and to reduce swelling of tissues resulting from such operations.

[0504] The sHASEGP glycoprotein may also be mixed with a buffered saline solution, such as a bicarbonate buffer, to prevent discomfort felt during injection. When making incisions, sHASEGP can be mixed with the anesthetic both to reduce the total volume of material required for injection, as well as to reduce pain due to tissue swelling.

[0505] REDUCTION OF INTRACULAR PRESSURE [0506] A common side effect in patients who have undergone cataract surgery is a significant and rarely prolonged increase in intraocular pressure. This condition can be serious in patients with optic disc changes due to glaucoma. Usually, the increase in pressure is more drastic when injecting viscoelastic agents such as hyaluronic acid into the eye, but intraocular pressure after surgery rises even when such factors have not been used. In addition, the described increase in pressure may occur even when no additional medications are used during the surgery. In some cases, it is recommended to leave the viscoelastic factor in the eye, which often requires patients to be given high doses of carbonic anhydrase inhibitors. These inhibitors reduce intraocular pressure by reducing the production of aqueous humor, a fluid normally secreted in the eye by the ciliary body. Currently known methods for alleviating postoperative increases in ocular pressure include various types of eye drops containing ingredients such as receptor blocking agents. β-adrenergic, sympathomimetic factors, miotics, alpha-2 adrenomimetics, carbonic anhydrase inhibitors and agents acting on the basis of the mechanism of prostaglandin action.

[0507] A preferred method of removing a viscoelastic substance, such as hyaluronic acid, is to inject sHASEGP during or immediately after surgery on the anterior or posterior segment of the eye. However, other methods of administration known in the art are also possible. During anterior surgery, to allow hyaluronic acid to act as a septum at the start of surgery, it is preferred that hyaluronic acid and sHASEGP are administered by injection into the anterior chamber of the eye. In some cases, during corneal transplantation, the combination of hyaluronic acid and sHASEGP may be placed on the surface of intraocular structures prior to suturing the corneal transplant. This type of combination can also be used in posterior ocular surgery, such as surgery for retinal and vitreous diseases.

[0508] In some cases it is recommended to leave a viscoelastic agent such as Healon.TM, Viscoat.TM or other fillers in the anterior chamber after surgery. This procedure works especially when the pressure increases, when the contents of the inside of the eyeball tend to approach and press on the posterior surface of the cornea. If this occurs in an eye containing a synthetic intraocular lens, corneal endothelial pressure can cause significant cell damage, resulting in corneal edema and loss of transparency, resulting in poor vision. Usually, if the patient has a markedly elevated pressure, then at the end of the operation, both to reduce the production of aqueous humor and / or to increase its outflow, such patients should be given high doses of carbonic anhydrase inhibitors, locally in the form of drops containing beta-blockers and alpha-2 receptor agonists. All these factors cause side effects and in some cases they are not recommended for patients with different types of medical conditions such as breathing problems, heart disease or high blood pressure. However, the use of sHASEGP in such cases eliminates the need for such patients to be given high doses of these drugs. [0509] In addition, a significant amount of uronic acid is present in the collagen fiber mesh in the corner of the eye's filtration. Under the influence of sHASEGP, it degrades and thereby improves the outflow of liquid through the collagen fiber mesh in the eye angle. Thus, the intraocular pressure in the patient's eye will decrease. Combinations of sHASEGP with other factors of the anterior chamber of the eye, such as methyl cellulose (e.g. Ocucoat.RTM, a preparation commercially available from Storz Instrument Co.), used as septum and / or safety devices during cataract surgery, will also be effective in preventing a significant increase in pressure. As a result, they will open the collagen fiber mesh in the angle of eye filtration and allow greater drainage of aqueous humor by degrading significant amounts of hyaluronic acid present there.

[0510] Removal of glycosaminoglycans from the collagen fiber mesh in the corner of the eye is also useful for reducing intraocular pressure in people suffering from open angle glaucoma. Human sHASEGP can be administered by subconjunctival injection or directly into the anterior chamber of the eye.

[0511] GELARETTE CELLS TENDON SCRAPS [0512] Jelly cyst of the tendon sheath (also known as carpal cyst, biblical tumor or dorsal tendon cyst) is the most common soft tissue tumor of the hand. You can feel a bubble filled with fluid under the skin. It is usually associated with the tendon sheath (tendon moisturizing sheath) of the hand or wrist or connected to the underlying joint. Some of these cysts have no visible connection to any structure. Jelly cysts of tendon sheaths may also occur in the foot. They often appear in places of injury to the ligaments surrounding the lining of the tendons or joints, where the lining protrudes through the defect of the ligament and the formation of a tumor under the skin. Inflammation is often associated with this condition. The inflamed tissue produces a jelly-like substance that fills the protruding tumor. Tumors of this type, due to the high pressure of the mucous fluid filling the cyst, can be very hard, which is why they are often mistaken for bone processes.

[0513] sHASEGP can be used to treat gelatinous cysts of the tendon sheath. Intra-articular injection of 5 - 1000 U sHASEGP followed by gentle suction with a needle allows the cyst to be removed without the need for surgery. Optionally, corticosteroids may be injected together with sHASEGP [0514] MYLUCUME Edema [0515] Cutaneous infiltrates of glycosaminoglycans are a hallmark of hyperthyroidism, hypothyroidism, mucosal edema and tissue edema. Hyaluronic acid is the main GAG in all these conditions and in normal skin. There is minimal histological variation in the distribution of GAG molecules in the skin. Acquired mucous skin degeneration (mucinosis) is characterized by a similar distribution of GAGs and their chemical composition. Some morphological differences in fibroblast activity suggest that mucous degeneration of the skin in the case of lower leg mucosa edema and tissue edema is a local process, while GAG ​​infiltrates in thyroid disease are of systemic origin. These disorders can be alleviated by sHASEGP administered both topically and systemically. For chronic treatment, the use of pegylated sHASEGP may be envisaged.

[0516] USES OF sHASEGP IN LUNG DISEASES [0517] The concentration of hyaluronan in the bronchial vesicular fluid (BAL) of normal individuals is usually less than 15 ng / ml. However, this concentration increases dramatically for respiratory distress syndrome (Bjermer Br Med J (Clin Res Ed) 1987 Oct 3; 295 (6602): 803-6). For example, in the case of ARDS, the concentration of hyaluronan may rise to 500 ng / ml, while in the case of so-called "Farmer's lungs", this concentration reaches 1000 ng / ml (Hallgren et al Am Rev Respir Dis. 1989 Mar; 139 (3): 682-7), (Larrson et al Chest. 1992

Jan; 101 (1): 109-14). An increase in the amount of hyaluronan in the lungs can interfere with oxygen diffusion and gas exchange as well as the activation of neutrophils and macrophage responses.

[0518] For many reasons, bovine hyaluronidase preparations are not recommended for the treatment of such conditions. First, it is known that hyaluronidase preparations from animal testicles are contaminated with serine proteases such as acrosine. Secondly, foreign enzymes of bovine origin increase the likelihood of an anaphylactic reaction that can lead to the patient's death. In contrast, the highly purified recombinant human sHASEGP preparation can be administered both into the lungs and intravenously. Human sHASEGP can also be given to patients suffering from other lung diseases associated with elevated glycosaminoglycans. Its administration may improve the lung penetration of other molecules administered simultaneously with sHASEGP [0519] The examples cited in the following experimental section illustrate individual embodiments of the invention without, however, limiting its content in any way.

[0520] EXAMPLE 1 [0521] MICROFILERATION BASED ACTIVITY TESTING [0522] The following example shows a quick test for measuring sHASEGP hyaluronidase activity. Activity in this test is expressed in units of TRU, IU or NFU in relation to standard hyaluronidase preparations described by WHO.

[0523] MICROMINETRICATION TEST USING BIOTINYLATED HYALURONATE [0524] The free carboxyl groups of the glucuronic acid residues of hyaluronan are biotinylated in a one-step reaction using biotin hydrazide (Pierce), Sulfo-NHP (Pierodimethimide). This biotinylated HA substrate is covalently bound in a second step to a 96-well microtiter plate. After completion of the enzymatic reaction, the remaining residue of the substrate is detected by the avidinaperoxidase reaction, the result of which can be read in a standard ELISA plate reader. In the case where the substrate is covalently attached to the surface of the plate, no artifacts such as pH-dependent leaching of the biotinylated substrate occur. The sensitivity of the test allows rapid measurement of hyaluronidase activity isolated from cell cultures and biological samples with a variation between tests of 10%.

[0525] a. PROTOCOL [0526] PREPARATION OF BIOTINYLATED HA SUBSTRATE [0527] Before binding to biotin, 100 mg HA (Sigma Chemicals) was dissolved in 0.1 M MES, pH 5.0 to a final concentration of 1 mg / ml leaving for dissolution for a period of at least 24 h at 4 ° C. Sulfo-NHS (Pierce; Rockford IL) was added to the CS04 MES solution so that the final concentration was 0.184 mg / ml. Biotin hydrazide (Pierce) was dissolved in DMSO to give a 100 mM stock solution, which was added to the CS04 solution to a final concentration of 1 mM. 1-ethyl-dimethylaminopropyl-carbodiimide stock solution (EDAC) was prepared as a 100 mM solution in water, which was then added to the biotin and HA solution so that the final EDAC concentration was 30 mM. This solution was stirred overnight at 4 ° C. Unbound biotin and EDAC were removed by dialysis against water with three changes of water volume 1000 times larger than the dialyzed sample. Dialyzed, biotinylated HA (bHA) was aliquoted and stored at 20 ° C for up to several months.

[0528] Sulfo-NHS was diluted in water with bHA (2 mg / ml) to a concentration of 0.184 mg / ml and pipetted onto a COVALINK-NH 96 well plate (NUNC, Placerville, NJ) at 50 mg per well. EDAC was diluted to 0.123 mg / ml in water and pipetted into a COVALINK-NH 96 well plate containing bHA solution, finally achieving a concentration of bHA -10 mg / well and EDAC 6.15 mg / well. Plates were incubated overnight at ° C or for 2 hours. at 23 ° C, which gave comparable results. After covalent immobilization of bCS04 in microtiter plates, the coupling solution was removed by shaking, the plates were washed 3 times in PBS containing 2 M NaCl and 50 mM MgSO4 (Buffer A). Plates can be stored at 4 ° C for up to one week.

[0529] COVALINK-NH plates with immobilized bHA were equilibrated with 100 μg / well assay buffer (0.1 M formate, pH 3.7, 0.1 M NaCl, 1 °% TRITON X-100, 5 mM saccharolactone for lysosomal hyaluronidase or 10mM Hepes pH 7.4 with 1 mM CaCf and 1 mg / ml albumin (ICN) human serum for enzymes active at neutral pH). A set of standards for calibration of enzymatic activity expressed in "relative turbidity reduction units" (rTRU) was prepared by dissolving hyaluronidase from bovine nuclei (Sigma type VI-S) in a buffer for enzymes operating at neutral pH in the amount of 1.0 to 1 x 10 -⁶ rTRU / well. Samples in a volume of 100 ml / well were tested in triplicate. Acidic active hyaluronidase samples were prepared in lysosomal hyaluronidase buffer at a dilution of 1:10 to 1: 130,000 and pipetted at 100 ml / well. Incubation for 30 min was sufficient for most tests performed on tissue extracts and human plasma. at 37 ° C. Wells containing positive and negative controls (no enzyme or no ABC respectively - see below) were prepared in triplicate.

[0530] Reactions were terminated by the addition of 6 M Guanidine-HCl in a volume of 200 ml / well, after which the plate was washed three times with 300 ml / well with a solution of PBS with 2 M NaCl, 50 mM MgSO₄, 0.05% TWEEN 20 (buffer B). A kit containing the avidin-biotin complex (ABC) (Vector Labs; Burlingame CA) was prepared in 10 ml PBS containing 0.1% TWEEN 20, which was preincubated for 30 min. in room temperature. ABC solution (100 ml / well) was added and incubated for 30 min. in room temperature. The plate was washed 5 times with buffer B followed by addition of the substrate phenylenediamine (OPD) at 100 m / well by dissolving a 10 mg OPD tablet in 10 ml 0.1 M citrate-phosphate buffer (pH 5.3) and adding 7.5 ml 30% H2O2. The plate was incubated in the dark for 10-15 minutes and then read using an ELISA plate reader at 492 nm (Titertek Multiskan PLUS; ICN) using Delta Soft II software for a plate reader from Biometallics (Princeton NJ) to monitor the reaction. The standard curve for bovine hyaluronidase was prepared using a four-parameter correlation curve for commercial hyaluronidase preparations, and in the case of unknown samples the values ​​were interpolated by measuring absorbance at 492 nm.

[0531] For the analysis of the dependence of hyaluronidase activity on pH, purified recombinant sHASEGP and hyaluronidase isolated from bovine nuclei are used. The effect of pH on enzymatic activity is measured by diluting purified sHASEGP or partially purified hyaluronidase isolated from bovine nuclei to a concentration of 0.1 rTRU in the following buffers: 50 mM formate, pH 3-4.5; 50 mM acetate, pH 5-6;

mM MES, pH 6-7; or 50 mM HEPES, pH 7-8. Samples are tested for 30 min. at 37 ° C and the activity is expressed as a percentage of the maximum activity. NaCl was not used for the preparation of buffers because it can change the optimal pH for the preparation of bovine hyaluronidase (Gold, Biochem. J. 205: 69-74, 1982; Gacesa et al. Biochem. Soc. Trans. 7: 1287-1289, 1979). The physiological salt concentration (0.15 M) lowered the actual optimal pH, and this effect was stronger for purified beef testicle enzyme preparations than for the original raw sample.

[0532] b. RESULTS [0533] Hyaluronan was biotinylated in a one step reaction using biotin hydrazide and EDAC. By limiting the amount of EDAC, which compound couples free HA carboxyl groups to biotin hydrazide, only a small portion of all uronic acid residues in HA have been labeled. EDAC (3 x 10^-5 M) added to HA (2.8 x 10^-3 M) causes one molecule of biotin hydrazide to conjugate to 93 disaccharide HA subunits.

[0534] A four-parameter correlation curve was prepared for a standard bovine testicle-isolated hyaluronidase reaction measured at pH 3.7, with an enzyme dilution of 1 to 1 x 10 "⁶TRU / well. The four-parameter correlation curve was determined from the equation y = ((AD) / (1+ (conc / C)^ANDB)) + D), where log, y = In (y '/ 1-y'), y '= (yD) / (AD), B = -b / ln 10 and C = EXP (a / B) . Four parameters (A, B, C, D) were calculated using a computer program and a 2 + 2 algorithm with linear regression (Rodbard et al., Clin. Chem. 22: 350, 1976). The correlation curve takes into account the sigmoidal nature of the standard curve. Optimal sample measurement accuracy is usually achieved in the range of 0.001 to 0.1 TRU / well in 30 min. incubation. During an incubation of 60 minutes detectable activity was 1/1000 TRU. You can also use the standard logarithmic curve to determine the standard curve for a smaller range. Although the present invention has been presented in connection with specific preferred embodiments, it should be understood that the claimed invention is in no way limited to them.

[0535] EXAMPLE 2 [0536] cDNA CLONING for sHASEGP [0537] One skilled in the art can obtain nucleic acid encoding human sHASEGP by a variety of methods including, but not limited to, artificial gene synthesis, RT-PCR, cDNA library screening by hybridization (e.g., 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 from another distributor having human genome sequences (Invitrogen Clone ID IOH10647).

[0538] The total length of human PH20 cDNA is 2009 nucleotides and contains an open reading frame of 1530 nucleotides. The 5'UTR is unusually large, which may suggest that one of the introns, containing 9 non-coding start codons, has not been removed and inhibits translation by preventing the ribosome from binding to the correct initiating methionine codon. Based on the sequence, it is predicted that the protein (GenBank Accession Number e.g. NP_003108) contains 509 amino acids of the ID sequence. SEQ. No. 1, and the calculated molecular mass is 58kDa.

[0539] For DNA sequencing of clones, the PCR-expanded bands were excised from the gel and the DNA, recovered using the ready-made Gel Extraction Kit (Qiagen) and cloned into the appropriate vector into compatible restriction sites obtained by restriction enzyme digestion. All sequencing reactions were performed using double-stranded DNA and TaqDyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems) in an automatic ABI sequencer according to the manufacturer's Prism ™ instructions (Applied Biosystems).

[0540] An open human PH-20 reading frame was obtained by amplifying a human nucleus cDNA library (Clontech, Palo Alto CA) by a Polymerase Chain Reaction using an ID primer pair. SEQ. No. 14 and ID. SEQ. Nr 47. PCR products were digested with NheI and BamHI enzymes and cloned into NheI and BamHI restriction sites of the IRESpuro2 vector (Clontech).

[0541] EXAMPLE 4 [0542] ISOLATION OF sHASEGP FROM HUMAN PH20 cDNA [0543] An expression vector for the catalytically active secretory recombinant form of human origin sHASEGP, capable of effective glycosylation in mammalian cells, was generated as described below. The invention also encompasses other expression constructs with promoters and selection genes of functional host cells from various species, such as yeast and insect cells, which are also capable of producing sHASEGP. Positive selection genes such as glutamine synthase or dihydrofolate reductase (DHFR) can also be used. The examples given below are not intended to narrow the scope of the invention, but rather to provide examples of several expression systems that could be used.

[0544] To obtain the soluble form of sHASEGP, deletion mutants were prepared that do not have a hydrophobic C-terminal end. Using the GPI program to predict cleavage sites, a GPI anchor digestion site was located that in the full length protein molecule with a GPI anchor is located near the amino acid at position N483. With a set of seven 3 'primers (i.e. nested primers), seven deletion mutants were constructed that did not have the GPI anchor envisaged in the GPI program starting at position Y482 and successively removed one amino acid in each subsequent mutant. These primers were designed with restriction sites for the NheI (5 ') and BamHI (3') enzymes to allow the cloning of deletion mutants into the IRESpuro2 vector in variants without the tag and stop codon in the 3 'primer or with the His-tag tag at the C-terminus proteins to facilitate the purification and detection step, e.g. ID primers. SEQ. No. 8, ID. SEQ. No. 9 and ID. SEQ. No. 10 was used to generate deletion mutants with terminal amino acid at positions Y482, F481 and I480 and without 6xHis-tag. Other primers were made with a similar base composition and appropriate modifications so that the amino acid is introduced into the sequence or removed. The same primers were used to generate His tag tag variants, but the untagged variants were used except that the antisense reverse primer did not contain a stop codon and the forward primer remained the same (His-tag construct amplified with primers SEQ ID NO. Nos. 19, 20, 21, 22, 23, 24 and 25, which are antisense, do not contain a stop codon and correspond to antisense primers without His-tag). Overlapping primers were used to construct a six-amino acid linker with six additional histidines between the BamHI and NotI sites of the IRESpuro2 vector. His-tag mutants were obtained by ligation of the PCR product digested with NheI and BamHI enzymes into the IRESpuro2 vector containing His-tag.

[0545] Based on homology to the bee venom enzyme, it was checked which modifications at the C-terminal lead to the production of a secretory enzyme active at neutral pH. To this end, a series of deletions were carried out from the end with the GPI anchor attachment site to the potential "catalytic domain".

[0546] DNA encoding the clone of the complete human sHASEGP molecule with GPI anchor and contained in the IRESpuro2 vector was used as a template to generate various types of truncated deletion mutants. Modeling programs provide several predicted cleavage sites for a full-length polypeptide. One of these sites was at position N483 (SEQ ID No. 1). To generate six deletion mutants, the PCR primers were designed to successively shorten the protein from position N483 starting trimming at position Y482 (no N) and ending at position E477 (no P).

[0547] a. PROTOCOL [0548] MANUFACTURE OF SHORTENED MUTANTS DEVOTED OF N483:

[0549] A full-length sHASEGP clone with a GPI anchor located between NheI and BamHI restriction sites in the pIRESpuro2 vector was used as a template. This matrix was amplified with a 5 'primer containing the NheI site that begins with the methionine of the native signal peptide at position M1 (SEQ ID NO: 14) and with a 3' primer containing the BamHI site ending at position Y482 (SEQ ID NO: 8). The PCR product was applied to a 1% agarose gel to separate and confirm the correct size of the propagated band. The band was then excised from the gel, purified, digested with NheI and BamHI enzymes and cloned into the pIRESpuro2 vector (Clontech) between Nhel and BamHI restriction sites. In this way, an expression vector was obtained for expressing the truncated sHASEGP mutant ending in position N482 and lacking the GPI anchor with the amino acid sequence (SEQ ID NO. No. 5 for the sequence of the resulting sHASEGP polypeptide up to position Y482) and the nucleotide sequence (SEQ ID NO: 48 - nucleotides encoding the polypeptide SEQ ID NO: 5) as designated.

[0550] Generation of other truncated mutants, lacking Y482, F481, I480, Q479, and P478, respectively.

[0551] The same strategy was used with the only difference being using the appropriate 3 'primer for each mutant. Suitable mutants are as follows:

[0552] 3 'primer for the sHASEGP mutant that is lacking Y482 -ID. SEQ. No. 9 [0553] A 3 'primer for a mutant that is lacking F481-ID. SEQ. No. 10 [0554] A 3 'primer for a mutant that lacks I480 -ID. SEQ. No. 11 [0555] A 3 'primer for a mutant that is lacking Q479 -ID. SEQ. No. 12 [0556] A 3 'primer for a mutant that is lacking in P478 -ID. SEQ. No. 13 [0557] Generation of further mutants to determine the minimally active sHASEGP domain. [0558] Further deletions, in blocks of ten amino acids, starting from the innermost 3 'end of the truncated sHASEGP mutant active at neutral pH, which is the mutant up to position E477. A sensible primer with a NheI ID restriction site. SEQ. No. 14 was used together with the appropriately located 3 'primer to duplicate sHASEGP deletion mutants of the desired length in PCR from the C-terminus. An example is PCR using the primers described in ID. SEQ. Nos. 14 and 26 as 5 'and 3' primers, respectively, were used to generate the polypeptide with the ID sequence. SEQ. No. 49 provided that it is expressed from the IRESpuro2 expression construct. Similarly, the 3 'antisense primers described in ID. SEQ. No. 27,28,29,30,31 and 32 were used to generate deletion mutants at position A 447, S430, G413, S394, A372 and S347 of the mature sHASEGP molecule, respectively. The PCR products were in each case digested with NheI and BamHI enzymes, and the digest was cloned into the pIRESpuro2 vector between NheI and BamHI restriction sites. Several independent clones from each group were tested for the presence of secretion, active at neutral pH, sHASEGP. The test was performed after transient transfection of CHO cells in serum-free CD-CHO medium (Invitrogen, CA). Samples for the test were taken at the appointed time. DNA from miniprep bacterial cultures grown overnight was introduced into cells by transfection using Genejuice transfection reagent (Novagen, CA) according to manufacturer's recommended protocols. Hyaluronidase activity was measured in a microplate test as described above.

[0559] b. RESULTS [0560] Hyaluronidase activity of truncated sHASEGP mutants was measured to identify the minimally active secretory domain at neutral pH.

<td>Amino acid from 1 to:</td><td>U / ML / 24HRS PH7.4</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>

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

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

[0561] The results obtained showed that all six amino acid deletion mutants with one point mutation ending in the indicated amino acid positions, i.e. at positions Y482 to E477 had a higher secretory activity than GPH anchor sHASEGP.

[0562] The results also indicated that deletions up to position A467 completely eliminated secretory activity. Secretory activity at the neutral pH of A467 clones was reduced to approximately 10% of clone P478 or N483 activity. From this it was concluded that, in order to obtain hyaluronidase activity which is active at neutral pH, a larger fragment of the human sHASEGP C-terminal domain is necessary, as previously determined from the bee venom enzyme. Cysteines in the C-terminal domain are therefore necessary for activity at neutral pH. The minimally active domain is defined by a narrow fragment of 10 amino acids before the GPI cleavage site at N483.

[0563] EXAMPLE 5 - EFFECT OF SIGNAL PEPTIDE MODIFICATION ON SECRET ACTIVITY OF sHASEGP.

[564] Human sHASEGP has the exceptionally long native signal peptide expected. In addition, the presence of two cysteine ​​residues in the signal peptide may, during intensive expression, lead to aggregation of polypeptide multimers within the endoplasmic reticulum, which will reduce the efficiency of this expression. Therefore, a series of signal peptides that were more effective in their ability to enhance sHASEGP secretion were tested.

[0565] a. PROTOCOL [566] The Kappa signal peptide was constructed by overlapping steps of attachment and extension of PCR using primers corresponding to SEQ Nos. 37, 38, 39 and 40. The resulting Kappa sequence was amplified by PCR using primers containing 5 'end of the cleavage site for NheI enzymes (as described in SEQ ID NO: 41) and EcoRI site at the 3' end (as described in SEQ ID NO: 42). This allowed for cloning of the Kappa signal peptide (the polypeptide sequence is described in SEQ ID NO: 43) into the Litmus 39 (NEB) vector between NheI and EcoRI restriction sites. sHASEGP has an EcoRI site within its sequence. Therefore, the Kappa construct located between NheI and EcoRI was amplified with the 5 'SpeI primer (as described in SEQ ID NO: 44) and the 3'MluI primer (as described in SEQ No. 45). sHASEGP without a GPI anchor, with a terminal amino acid at position P478, was excised from the pIRESpuro2 vector with NheI and BamHI enzymes and cloned into the Litmus 39 (NEB) vector at the NheI and BamHI sites. The resulting Litmus containing sHASEGP, i.e. Litmus-sHASEGP, was digested with SpeI and MluI enzymes, and then ligated with the PCR product - Kappa construct amplified with SpeI and MluI restriction sites. To create a common reading frame in the Litmus 39 vector for the Kappa signal sequence and mature sHASEGP polypeptide, point mutagenesis was performed. Primer pairs matching ID. SEQ. Nos. 34 and 35 were used to generate the Kappa sequence with native Asp as a terminal amino acid attached at position F38 of sHASEGP (to P 478) (as described in SEQ ID NO: 46 for polypeptide or fusion protein sequence). Other combinations of primer pairs as implemented in ID. SEQ. No. 33 and ID. SEQ. No. 35, was used to generate the Kappa signal sequence terminating in the Asp (D) terminal linked to the L36 sHASEGP molecule, and the ID pair. SEQ. No. 33 and ID. SEQ. No. 36 was used to generate a Kappa sequence terminated with terminal Gly (G) (upstream of terminal Asp (D)) bound to the amino acid at position L36 of sHASEGP. ID. SEQ. No. 34 with ID. SEQ. No. 36 were used to make the construct terminated in the Kappa sequence on Gly (G) (upstream of the Asp (D) terminal) attached to F38 sHASEGP. Kappa-sHASEGP constructs obtained by point mutagenesis were gel purified, subjected to restriction digestion with DpnI enzyme to remove any host DNA contamination, then digested with NheI and BamHI enzymes and cloned into the NheI / BamHI cleavage site of the HisIresPuro2 vector backbone which has the His-tag tag (six-amino acid linker with six additional histidines) cloned between the BamHI and NotI cleavage site in the pIREsPuro2 vector. Hence, after ligation, a construct was obtained that corresponds to the G or D combination at the end of the Kappa leader sequence and the L36 or F38 position at the beginning of mature sHASEGP. Several independent clones of each type of construct were transfected into CHO cells cultured in CD-CHO medium (Invitrogen, CA) to check if the presence of the Kappa leader sequence would increase the amount of secreted protein compared to the amount of native secreted protein. DNA isolated in mini-preparation from overnight cultures was introduced by transfection using the Genjuice transfection agent (Novagen, CA), following the manufacturer's instructions. Samples were then taken for microplate testing at a specified time. Hyaluronidase activity was measured in a microplate test as described above.

[0567] The Kappa chain construct of the mouse IgG leader sequence sHASEGP was tested for higher levels of secretion of active sHASGEP operating at neutral pH.

[568] b. RESULTS

<td>Human sHASEGP gene construct</td><td>U / ML / 24HOURS PH 7.4</td>

<td>Kappa IgG leader sequence sHASEGP AA 38-478 HIS6</td><td> 3,0257</td>

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

[569] The results of enzymatic tests indicate that the Kappa IgG leader sequence was able to increase the level of sHASEGP secretion about 7 to 8 times compared to the secretion of the native leader sequence. Other constructs containing this sequence with variants of the fusion site leader sequence from Asp to Gly of the Kappa chain to the L36 or F38 position of sHASEGP also gave a higher level of activity of secreted hyaluronidase at neutral pH. These examples are intended to broaden rather than narrow the scope of the invention, since other effective leader sequences may also be used in the technology described.

[0570] EXAMPLE 6 [0571] MANUFACTURE OF THE HUMAN EXPRESSION HUMAN EXPRESSION VECTOR [0572] sHASEGP without epitope tag was produced by cloning into the HZ24 bicistronic expression cassette (SEQ ID No. 47). The HZ24 plasmid vector for sHASEGP expression comprises the basic part of the pCI vector (Promega), the DNA sequence encoding amino acids 1 to 482 of human PH20 hyaluronidase, the sequence of the internal ribosome binding site (ang. internal ribosome entry site, IRES) derived from the EMCV virus (Clontech) and the mouse dihydrofolate reductase (DHFR) gene. The skeleton of the pCI vector also contains DNA encoding the β-lactamase resistance gene (AmpR), f1 origin of replication, regulatory region enhancer / cytomegaly promoter, chimeric intron and SV40-derived polyadenylation signal (SV40). The DNA encoding the sHASEGP construct contained the Kozak sequence upstream of the methionine of the native leader sequence and a stop codon at position 482. The resulting construct pCI-PH20-IRES-DHFRSV40pa (HZ-24) is formed as a single mRNA molecule under the CMV promoter that encodes the amino acid sequence from 1 to 482 PH20 and amino acid sequence 1 to 187 of dihydrofolate reductase separated by the IRES sequence.

[0573] The human PH20 open reading frame was amplified on an IOH10647 clone matrix (Invitrogen, Carlsbad CA) using a 5 'primer containing a NheI restriction site and Kozak consensus sequence before methionine PH20 and using an antisense primer which he introduced behind tyrosine 482 stop codon and BamHI cutting site. The obtained PCR product was digested with NheI and BamHI enzymes and then introduced under ligation into the plasmid pIRESpuro2 (Clontech, Palo Alto, CA).

[0574] EXAMPLE 7 [0575] RECEIVING sHASEGP EXPRESSION CELL LINE [0576] Non-transfected DG44 CHO cells growing in modified medium for CDCHO DHFR (-) cells, supplemented with 4mM glutamine and 18 ml Plurionic F68 / L preparation (Gibco) before transfection, sown in bottles intended for shaking cell cultures in an amount of 0.5 x 10⁶ cells / ml. Cells were grown at 37 ° C in a humidified atmosphere saturated with 5% CO2, in an incubator with shaking at 120 rpm. Exponentially growing, non-transfected DG44 CHO cells were tested for viability before transfection. 60,000,000 live non-transfected cells from DG44 CHO cultures were centrifuged and the pellets were suspended to obtain a density of 20,000,000 cells per 0.7 ml 2 x concentrated transfection buffer (2x HeBS = 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mM KCl, 1.4 mM Na2HPO4, 12 mM. Dextrose). 0.09 ml of the linear form of the HZ24 plasmid (250 ug) was added to each aliquot of suspended cells, and the cell / DNA solution was transferred to the electroporation cuvette (0.4 cm BTX Gentronics gap) at room temperature. Negative control was electroporated cells without DNA. The cell / DNA mixture was electroporated at 330V and 960uF or at 350V and 960uF.

[0578] After electroporation, the cells were transferred from the cuvette to a 6-well cell culture plate and cultured for 2 days at 37 ° C in a humidified atmosphere saturated with 5% CO2, using 5 ml of modified DHFR (-) cell medium supplemented with 4 mM glutamine and containing 18 ml Plurionic F68 / L (Gibco).

[579] Two days after electroporation, 0.5 ml of medium was withdrawn from each well and tested for hyaluronidase activity.

[0580] Initial hyaluronidase activity in medium of DG44 CHO cells transfected with HZ24 10 in 40 hours. after transfection.

<td></td><td>dilution</td><td>U / ml activity</td>

<td>Transfection 1 at 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>

[0581] Cells transfected under current conditions at 350V (transfection 2) were harvested from the well, counted and diluted to a concentration of 10,000-20,000 viable cells per ml. A 0.1 ml aliquot of cell suspension was transferred to each of five wells in a 96 well round bottom plate. 0.1 ml medium for CD-CHO (Gibco) containing 4 mM Glutamax-1, without hypoxanthine and thymidine, was added to each cell well (final volume 0.2 ml).

[0582] Ten clones were obtained from the culture in the presence of methotrexate carried out on 5 plates.

<td>Plate / hole 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>1E11</td><td> 242</td>

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

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

[0583] Six HZ24 transfected clones were expanded and transferred as a single-cell suspension into shaking bottles. Clones 3D3, 3E5, 2G8, 2D9, 1E11 and 4D10 were plated on a round bottom 96-well plate using a two-way dilution strategy. Diluted clones grew on untransfected DG44 CHO medium (500 cells per well) to provide the necessary growth factors during the first days of culture. 10 plates were prepared for each of the 10 clones.

[0584] Clone 3D3 formed 24 visible clones. Hyaluronidase activity was measured in the supernatant 8 of 24 subclones (&gt; 50 U / ml). Said 8 clones were expanded in T-25 culture bottles in the presence of 50 nM methotrexate. Clone 3D3, grown in the presence of 5 nM methotrexate, was then expanded in the presence of 500 nM methotrexate, thus selecting clones producing more than 1

000 U / ml in shaking bottles (3D3 clone 5M).

[0585] EXAMPLE 8 [0586] PRODUCTION of sHASEGP [0587] A tube of frozen 3D3 5M clone cells was thawed, and cells propagated in spinner flask bottles in 1L CHO CDM (Invitrogen, Carlsbad CA) supplemented with 100 nM methotrexate and Glutamax (Invitrogen). Cells were transferred from spinner flask vessels to a 5L bioreactor (Braun) in 4 x 10 innoculum⁵ live cells / ml. The values ​​of initial parameters were: 37 ° C, pH 7.2 and 25% dissolved oxygen and aeration 0-100 cc / min. After 168 hours the first portion of medium (1, 250 ml feed) (CD CHO + 50g / L glucose) was added. After 216 hours of culture, a second portion of medium (2, 250 ml feed) was added (CD CHO + 50g / L glucose + 10 mM sodium butyrate). Finally, 1600 U / ml was obtained with a maximum cell density of 6 million / ml. The addition of sodium butyrate caused a strong increase in sHASEGP production in its final stages.

3D3-5N clone growth and sHASEGP production, 5L bioreactor

<td>Hours growth</td><td>Number of live cellsx10 E5</td><td>% live cells</td><td>U / ml</td><td>Vol. (ML)</td><td>[Glucose]</td><td>Addition of nutrients</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>250ml nutrients top up 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>250ml nutrients top up 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>250ml nutrients top up 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>

[0588] EXAMPLE 9 [0589] PURIFICATION of sHASEGP [0590] The conditioned medium from clone 3D3 was clarified by deep filtration and tangential flow diafiltration in Hepes pH 7.0 buffer. Soluble sHASEGP was purified by sequential chromatography on an ion exchange Sepharose Q bed (Pharmacia), followed by hydrophobic interaction chromatography on a phenyl-Sepharose bed (Pharmacia), phenyl boronate chromatography (Prometics) on a hydroxyapatite bed chromatography (BioRad, Richmond, CA) .

[591] sHASEGP was bound to a Sepharose Q type bed and eluted with 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 substitution) bed column, then bound to a phenyl boronate bed column. After the washing step with buffer with 50mM bicine without AsO₄ at pH 9.0, sHASEGP was eluted from said column in Hepes buffer pH 6.9. The eluate was applied to a ceramic hydroxyapatite bed in 5mM PO₄ with 1 mM CaCf at pH 6.9 and eluted with 80 mM PO₄ with 1 mM CaCl2 at pH 7.4.

[0592] Purified sHASEGP had a specific activity of over 65,000 USP / mg protein as determined by a turbidimetric assay using USP standards. Purified sHASEGP was eluted from the 5RPC styrene divinyl benzene column as a single peak from 24 to 26 min. at a gradient between 0.1% TFA / H2O to 0.1% TFA / 90% acetonitrile / 10% H2O and separated as a single 61 kDa wide band by SDS electrophoresis. After PNGase-F treatment, the band weight was 52 kDa. N-terminal amino acid sequencing showed that the signal sequence was successfully deleted.

[0593] N-terminal amino acid sequence of purified sHASEGP.

<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>Theoretically</td><td>Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ala</td><td>Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>own</td>

<td>The observed</td><td> -</td><td>own</td><td>phe</td><td>Arg</td><td>ala</td><td>Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>own</td>

[0594] EXAMPLE 10 [0595] ANALYSIS OF GLYCLOSILATION OF SHHEGEGP PRODUCED IN DG44 CHO CELLS [0596] There is conflicting data on whether sHASEGP from different species requires glycosylation for its catalytic activity. For example, it has been shown that the enzymatically active bee venom hyaluronidase can be synthesized in cells that do not have the machinery necessary for glycosylation, e.g., E. coli. In addition, PGNase treatment of purified bovine testicular hyaluronidase did not cause inactivation of the enzyme (Yamagata et al 1997). Other studies report loss of activity as a result of deglycosylation and that disulfide bonds are additionally required.

[0597] However, since all these tests were, however, performed on crude or partially purified preparations, it is not entirely clear whether the loss of activity was due to the presence of the deglycosylated enzyme in the test or the crude protease preparations contained in the impurities or the direct relationship between glycosylation and enzymatic activity.

[0598] a. PROTOCOL [0599] To check if functional N-glycosylation could be introduced into human sHASEGP using an expression system based on CHO cells under protein-free conditions, the cDNA encoding human sHASEGP-HIS6 was expressed using an IRESpuro bicistronic cassette , in CHO cells cultured in chemically defined media. Cells were cultured in CHO CDM medium (Invitrogen / Gibco) for 72 h and then concentrated and filtered using a Pellicon TFF membrane (Millipore) with a 30 kDa permeability limit. The concentrate buffer was exchanged for 10 mM Hepes pH 7.4, 50 mM NaCl. The filtered concentrate was then applied to a DEAE-sepharose bed and eluted in a 0 - 1 M NaCl gradient on a Pharmacia bed intended for FPLC. Elution of human sHASEGP occurred at a concentration of 10-30% NaCl. The content of sHASEGP in the column fractions indicated that most of the enzyme was recovered at 10-30% NaCl salt concentration. The enzyme-containing fractions collected at 10-30% salt concentration were then purified by means of affinity chromatography on a Ni-IMAC ion exchanger. Human sHASEGP, after washing, was eluted from the IMAC bed with 10 mM imidazole with 50 mM acetate pH 5.0. Protein was concentrated and dialyzed in buffer against 10 mM HEPES pH 7.4. The enzymatic activity of such a highly purified enzyme, measured in an ELISA microtiter assay based on the use of biotinylated substrates, was 97,000 U / mg protein in the presence of 1 mM calcium ions and 1 mg / ml HSA.

[0600] To identify changes in relative molecular weight of the protein, purified human sHASEGP was treated overnight with PNGase or neuraminidase followed by gel, electrotransfer and western-blot electrophoresis using HRP-conjugated anti-His6 monoclonal antibody (Qiagen) and ECL detection.

[0601] b. RESULTS [0602] Western blot analysis showed that human sHASEGP produced in CHO cells was sensitive to PNGase. The relative molecular weight of such human sHASEGP indicated that this protein was previously highly glycosylated. As a result of overnight PNGase digestion, one human sHASEGP population was obtained, indicating that the low protein heterogeneity within the band containing the control preparation (not digested with PNGase) could be due to the presence of N-linked sugar moieties. Partial digestion of the glycoprotein PNGase F led to the formation of a series of intermediates migrating in the gel from the position corresponding to the protein not treated with PNGase F to further positions depending on the time of digestion. Although the 7% gel bands were diffused, at least 6 different intermediate isoforms could be seen.

[0603] Treatment with sHASEGP with neuraminidase has shown that CHO cells are in fact capable of synthesizing sialated human sHASEGP. As a result of sHASEGP treatment with neuraminidase and protein analysis using a 7% gel western blot, synthesized by CHO cells, human sHASEGP showed a slowed gel migration of approximately 1 - 3 kDa compared to control sHASEGP (enzyme untreated). Thus, this is the first report that we have received largely saluted sHASEGP. Calming is important for both stability and increasing the half-life of human sHASEGP, while the sHASEGP species found in the semen of many species are not salified and do not react with sialic acid-recognizing lectins.

[0604] FACE sHASEGP analysis [0605] FACE glycan analysis of sHASEGP allows rapid determination of catalytically active sHASEGP glycoprotein profiles.

[0606] PROTOCOL [0607] Purified expression hyaluronidase in 3D3 5M clone was analyzed by FACE® N-glycan profiling (Prozyme). Glycans were released from glycoproteins (128.7 pg) by treatment with N-glycanase (abbreviated PNGase), labeled with ANTS fluorophore and separated by electrophoresis. The relative positions of the corresponding glycan bands were determined by separating the sample and its dilutions in parallel with the separation of the standards with known migration positions expressed in units of "polymerization degree" (DP). [0608] RESULTS [0609] The released N-glycans profile of a given hyaluronidase sample comprised ten bands from six (of which migrated in positions corresponding to the G5 - G12 standards bands) had an intensity greater than 9%. In addition, the band migrating in parallel with the G9 standard was the most intense band - 35% - 46%.

[0610] sHAS [0611] EGP glycan analysis.

<td>glycan from sHASEGP</td><td>Degree of Polymerization</td><td>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>

[0612] EXAMPLE 11 [0613] DEPENDENCE OF ENZYMATIC ACTIVITY ON N-GLYCOSILATION OF sHASEGP 10 [0614] a. PROTOCOL [0615] Samples of purified His6-labeled sHASEGP were mixed with a buffer containing neuraminidase and PNGase and addition of okag and ozag incubated overnight at 37 C. Digestion of oligosaccharides was identified by delaying gel migration analyzed by western blotting.

[0616] b.

[0617] RESULTS

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

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

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

<td>PNGase F with 50mM OG</td><td> 0,0</td>

<td>PNGase F without 50mM OG o / n</td><td> 10,74</td>

[0618] EXAMPLE 12 [0619] SHASEGP ACTIVITY WITH REGARD TO SULPHATED AND NON-Sulphated

GLYCOSOSAMINOGLICANS [0620] In addition to the HA microtiter assay, the sHASEGP substrate specificity for other glycosaminoglycans or proteoglycans can be tested in a gel slowing test using purified substrates. Many tests for hyaluronidase activity are based on measuring the presence of newly formed reducing acetylamino groups (Bonner and Cantey, Clin. Chim. Acta 13: 746-752, 1966) or on measurements of a decrease in viscosity (De Salegui et al., Arch. Biochem. Biophys. 121: 548-554, 1967) or turbidity (Dorfinan and Ott, J. Biol. Chem. 172: 67, 1948). All these methods are effective for determining the presence or absence of endoglucosaminidase activity on purified substrates. [0621] a. PROTOCOL [0622] GEL MIGRATION SLOW TEST - Glycosaminoglycans are mixed with recombinant sHASEGP to test its endoglucosaminidase activity, which is manifested by an increase in increased mobility of the gel substrate. Chondroitin A sulfate, agrekan and D were from Calbiochem. Hyaluronan (human umbilical cord), chondroitin C sulfate, dermatan sulfate, heparan sulfate were from Calbiochem. Hyaluronan from human umbilical cord was obtained from ICN. Each test substrate was diluted to a concentration of 0.1 mg / ml. Samples of purified sHASEGP or conditioned media from cells expressing sHASEGP, 10 ml, were mixed with 90 ml of test substrate in the appropriate buffer and incubated for 3 hours. at 37 ° C. After incubation, the samples were neutralized with assay buffer (Tris-EDTA, pH 8.0, bromophenol blue and glycerol) and subjected to electrophoresis on a 15% polyacrylamide gel. Glycosaminoglycans were identified by staining the gel overnight with a 0.5% solution of Alcian Blue dye in 3% glacial acetic acid, followed by discoloration with 7% glacial acetic acid. The level of degradation was determined by comparing substrate mobility in the presence and absence of enzyme.

[0623] b. RESULTS [0624] sHASEGP-His6 protein at 100 U / 10 ml in 90 ml of 10 mM HEPES buffer containing 50 mg / ml human serum albumin and 10 mg of various glycosaminoglycans and proteoglycans, incubated for 2 hours. at 37 ° C. The electrophoretic separation and subsequent staining with Alcian Blue showed accelerated migration for chondroitin A, C and D, aggrecan and hyaluronan, but it did not apply to either heparan sulfate or chondroitin sulfate B. While undigested glycosaminoglycans migrated in the middle of the gel in a smudged manner, the digested products constituted the majority of Alcian blue stained bands migrating along with the dye front together with a small amount of additional migrating material as so-called "incremental ladder".

[0625] EXAMPLE 13 - INFLUENCE OF METAL IONS ON sHASEGP ACTIVATION [0626] In addition to the glycosylation required for sHASEGP enzymatic activity, it has been shown that human sHASEGP is additionally activated by cations for its optimal enzymatic activity. It was shown that after subsequent sHASEGP purification steps using chromatography it showed low activity. Labeled sHASEGP glycoprotein His6 had very low specific activity when it was purified on a DEAE bed, followed by subsequent purification steps on a Ni-IMAC bed. Because the IMAC bed chelates metal ions, various metals were added back to sHASEGP to determine its relative enzymatic activity.

[0627] a. PROTOCOL [0628] Purified sHASEGP was incubated for 2 hours. at room temperature with 0.1 mM nickel (Ni), cobalt (Co), zinc (Zn), calcium (Ca) and magnesium (Mg), and then its activity was tested by determining hyaluronidase activity using a microtiter assay. [0629] b. RESULTS

<td>Addition of metal salt</td><td>Enzyme activity at neutral pH [U / ml]</td>

<td>No extras</td><td> 11,909</td>

<td>100 mM Ni</td><td> 6,0306</td>

<td>100 mM Co</td><td> 8,972</td>

<td>100 mM Zn</td><td> 3,7476</td>

<td>100 mM Ca</td><td> 101,9892</td>

[0630] A significant increase in hyaluronidase activity was observed after incubation of sHASEGP with 0.1 mM calcium or 0.1 mM magnesium. This type of activation was not observed during the incubation of the enzyme with other metals. Addition of calcium to sHASEGP increased specific activity by approximately 97,000 U per mg of protein, as measured by absorbance at 280 nm. Then the dependence of this activity on the dose of calcium or magnesium was determined in order to determine the optimal concentration of these ions for the enzyme.

<td>Double charged metal [mM]</td><td>[Ca²⁺]</td><td>[Mg²⁺]</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>

[0631] Activation of sHASEGP has been shown to occur at a concentration at the micromolar level. A concentration of both calcium and magnesium ions above 10 mM showed inhibitory effect. To rule out the possibility that the substrate rather than the enzyme could be activated unspecifically, CaCk in 10 mM HEPES buffer was incubated in a microtiter plate with the immobilized biotinylated substrate, and then the plate was washed. When enzyme was added to such a plate, no activation was observed. Such activation was also tested using native sHASEGP released by phospholipase C, which showed similar susceptibility to calcium activation, which excluded the possibility of C-terminal His6 tag-related artifacts.

[0632] EXAMPLE 14 [0633] EFFECT OF ALBUMIN ON ACTIVITY OF sHASEGP [0634] It was found that for optimal enzyme activity, when diluting recombinant rHUPH20 and other preparations of bovine hyaluronidase, albumin is required.

[0635] a. PROTOCOL [0636] Human serum albumin (ICN) was diluted in 10 mM HEPES buffer with calcium to determine the effects of albumin on enzymatic activity. Enzymatic tests with sHASEGP and commercial preparations were performed using both 1 mM CaCk and 1 mg / ml human serum albumin.

[0637] b. Results [0638] Activation of hyaluronidase activity occurred at high dilutions of albumin. It was not clear whether this activation was the result of preventing denaturation or whether albumin affected substrate access. A preferred formulation of human sHASEGP could then contain albumin and a metal salt consisting of either calcium or magnesium.

[0639] EXAMPLE 15 - EXTENDED IN VIVO ACTIVITY OF PURIFIED sHASEGP [0640] a. PROTOCOL [0641] Purified sHASEGP in 10 mM Hepes pH 7.4, 150 mM NaCl, 0.1% Pluronic was diluted to a concentration of 0.5U / l in apyrogenic water with 0.15 M NaCl. To give total doses of 0.01, 0.05, 0.1 U per injection, a series of dilutions were made in a total volume of 20 L of saline. Then 20 l of Trypan Blue solution was added to the solutions to a total volume of 40 l and injected subcutaneously in both sides of the mice ^{Nu / nu}BALB / c previously anesthetized by intraperitoneal administration of ketamine / xylazine. The areas of color were measured in two dimensions using a micro vernier caliper during t = 0 to t = 45 min. The size of the area is expressed in mm². As a control, recombinant human HYAL1 was used, which does not have activity at neutral pH, but is a secretory protein. [0642] b. Results

<td>TESTED INGREDIENT</td><td>DYE AREA @ 45 MIN,</td>

<td>A, Control - saline</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>

[0643] EXAMPLE 16 - KINETICS OF SHASEGP DIFFUSION ACTIVITY [0644] a. PROTOCOL [0645] Recombinant purified sHASEGP-His6 was divided into two portions. One was heated at 95 ° C for 15 min in a thermocycler with heated lid. A second portion was left at room temperature. Thermal inactivation of the enzyme was checked in an enzymatic assay based on microtitration. A kinetic analysis was carried out comparing the thermally inactivated material with the native material. Purified sHASEGP or the corresponding heat inactivated material was injected subcutaneously at 4 U together with Trypan blue. The color area was measured at various time points up to 15 min.

[0646] b. RESULTS

<td>sHASEGP [4 U] minutes after absorption</td><td>sHASEGP [4 U] (THERMAL INACTIVATED ENZYME) tmmuty after wsftzytoteau</td>

<td>t0 = 52.38</td><td>t0 = 50.58</td>

<td>t3 = 116.51</td><td>ts = 65.48</td>

<td>these,₅= 181,93</td><td>t6₅ =63,87</td>

<td>t10 = 216.96</td><td>t10 = 65.80</td>

<td>t16 = 279.99</td><td>t16 = 74.3</td>

[0647] EXAMPLE 17 - REBUILDING THE SKIN BARRIER INTERRUPTED BY sHASEGP [0648] a. PROTOCOL [0649] To determine the regeneration time of open pores as a result of subcutaneous administration of sHASEGP, 2 units of purified sHASEGP or saline as a control, two subcutaneously injected animals at t = 0. Then trypan blue was administered to the same place at t = 30 min, t = 60 min. it = 24 hours Dye diffusion area was measured 15 min. after administration for each time point and compared with control values ​​obtained.

[0650] b. RESULTS

<td>2 UNITS hours after sHASEGP injection</td><td>CONTROL - PHYSIOLOGICAL SALT total hours after sHASEGP injection</td>

<td>^t0.5h = ¹⁸³</td><td>^t0.5h = ⁵⁴</td>

<td>t1h = 167</td><td>t1h = 50</td>

<td>^t22h = ⁶¹</td><td>^t22h = ⁴⁸</td>

[0651] The results obtained show that the skin barrier returns to its original state within 24 hours. after administration of 2 enzyme units.

[0652] EXAMPLE 18 - MEASUREMENT OF SIZE OF OPENED CHANNELS BY sHASEGP [0653] Human sHASEGP has been shown to open channels in the interstitial space sufficiently to allow diffusion of small molecules such as Trypan Blue. However, it was not known what are the upper limits of the size of particles that can diffuse in the presence of sHASEGP.

[0654] a. PROTOCOL [0655] Fluorescent molecules of various sizes were used to measure the size of the open channels of human sHASEGP: fluorescent dextrans with an average molecular weight of 4,400 and 2 million Da (Sigma) as well as fluorescein-labeled spheres with a defined diameter from 20 nm to 500 nm (Molecular Probes). These particles were administered subcutaneously in a volume of 40 ml, after which sHASEGP or saline control was injected at the same site. The dye front area was then measured in two dimensions in 15 min. from the injection. [0656] b. RESULTS

<td>Diffusion factor</td><td>The size of the particles used for the diffusion test</td><td>Area in 15 min,</td><td>Standard deviation</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>2 x 10⁶ da</td><td> 141,2</td><td> 4,5</td>

<td>Control</td><td> 2 ^x 10⁶ da</td><td> 51,7</td><td> 8,1</td>

<td>sHASEGP</td><td>20 nm in diameter</td><td> 92,3</td><td> 20,6</td>

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

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

<td>Control</td><td>100 nm in diameter</td><td> 40,0</td><td> 7,0</td>

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

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

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

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

[0657] The results obtained show that particles ranging in size from about 1 kDa (Trypan Blue) to 50 nm in diameter (latex beads) showed increased diffusion as a result of sHASEGP administration. While bovine serum albumin (66 kDa) had similar diffusion kinetics to Trypan Blue, latex beads with a diameter of 50 nm needed much more time for diffusion. The 500 nm beads did not diffuse up to 480 min.

[0658] EXAMPLE 19 - PHARMACOKINETIC PROFILES OF BIOTINYLATED ANTIBODIES OF ANOTHER ARIS AFTER PARALLEL HUMAN SUBJECTIVE HUMAN INJECTION sHASEGP [0659] a. PROTOCOL [0660] Female mice with Balbina ketin Mice were injected subcutaneously with 20 ml 0.5 mg / ml solution of biotinylated mouse IgG mixed with 20 ml saline or 20 ml sHASEGP in an amount corresponding to 4 U activity.

[0661] b. RESULTS

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

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

<td>IgG serum t = 2 hours</td><td>0 ng / ml</td><td>360 ng / ml</td>

<td>IgG serum t = 51 hours</td><td>4152 ng / ml</td><td>4176 ng / ml</td>

[0662] The results show that sHASEGP improves the kinetics of distribution of large molecules in serum. While in 2 hours after injection, no biotinylated IgG was identified in the control group, the antibody concentration in the sHASEGP treated group was 360 ng / ml.

[0663] EXAMPLE 20 - PARTICLE SPREAD ACTIVITY

INJECTED SUBcutaneously after PREVIOUS INTRAVENAL HUMAN INJECTION sHASEGP.

[0664] a. PROTOCOL [0665] Dye was injected at four sites, each of which was used for each dose of test ingredient and control vehicle. The dye injection took 45 min. after intravenous injection. Each dose of test or control component was administered intravenously to two animals. Measure the dye front area after 45 min. after administration of the enzyme was carried out in 2.5, 5, 10 and 15 min. for each dose or vehicle control.

[0666] b. RESULTS [0667] The results show that highly purified sHASEGP was systemically available in distal tissues after intravenous administration. The spread activity of systemically administered sHASEGP was dose-dependent. No differences were observed between the control vehicle and the 10 U injection of sHASEGP.

<td>^Typ</td><td>IV dose</td><td>Time [minutes]</td><td>Medium surface</td><td>field (mm²)</td><td>Deviation standard</td>

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

<td>Control</td><td> 0</td><td> 5</td><td> 36,279</td><td></td><td> 3,614544</td>

<td>Control</td><td> 0</td><td> 10</td><td> 44,687</td><td></td><td> 5,821216</td>

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

List of Sequences [0668] &lt;110&gt; DeliaTroph Pharmaceuticals, Inc.

Frost, Gregory Kundu, Anirban Bookbinder, Louis &lt;120&gt; Soluble glycoprotein with hyaluronidase activity (SHASEGP), the same preparation process, its uses and pharmaceutical composition &lt;130&gt; DELIA1340WO &lt;150&gt; US 60 / 452,360 &lt;151&gt; 2003-03-05 &lt;160&gt; 53 &lt;170&gt; FastSEQ for Windows 4.0 &lt;210&gt; 1 &lt;211&gt; 509 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220&gt;

&lt;221&gt; CARBOHYD &lt;222&gt; 82, 166, 235, 254, 368, 393, 490 &lt;400&gt; 1

<td>Underworld</td><td>Gly</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>lys</td><td>His</td><td>How much</td><td>phe</td><td>phe</td><td>Arg</td><td>Cheese</td><td>phe</td><td>val</td><td>lys</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Cheese</td><td>Cheese</td><td>Gly</td><td>val</td><td>Cheese</td><td>Gin</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>phe</td><td>Leu</td><td>Leu</td><td>How much</td><td>Pro</td><td>Cys</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Cys</td><td>Leu</td><td>Thr</td><td>Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ala</td><td>Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>own</td><td>val</td><td>Pro</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>phe</td><td>Leu</td><td>Trp</td><td>ala</td><td>Trp</td><td>own</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Cys</td><td>Leu</td><td>Gly</td><td>lys</td><td>phe</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>asp</td><td>Glu</td><td>Pro</td><td>Leu</td><td>asp</td><td>Underworld</td><td>Cheese</td><td>Leu</td><td>phe</td><td>Cheese</td><td>phe</td><td>How much</td><td>Gly</td><td>Cheese</td><td>Pro</td><td>Arg</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>How much</td><td>own</td><td>ala</td><td>Thr</td><td>Gly</td><td>Gin</td><td>Gly</td><td>val</td><td>Thr</td><td>How much</td><td>phe</td><td>Tyr</td><td>val</td><td>asp</td><td>Arg</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Gly</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>Tyr</td><td>How much</td><td>asp</td><td>Cheese</td><td>How much</td><td>Thr</td><td>Gly</td><td>val</td><td>Thr</td><td>val</td><td>own</td><td>Gly</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gly</td><td>How much</td><td>Pro</td><td>Gin</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>asp</td><td>His</td><td>Leu</td><td>asp</td><td>lys</td><td>ala</td><td>lys</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>lys</td><td>asp</td><td>How much</td><td>Thr</td><td>phe</td><td>Tyr</td><td>Underworld</td><td>Pro</td><td>val</td><td>asp</td><td>own</td><td>Leu</td><td>Gly</td><td>Underworld</td><td>ala</td><td>val</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>How much</td><td>asp</td><td>Trp</td><td>Glu</td><td>Glu</td><td>Trp</td><td>Arg</td><td>Pro</td><td>Thr</td><td>Trp</td><td>ala</td><td>Arg</td><td>own</td><td>Trp</td><td>lys</td><td>Pro</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>lys</td><td>asp</td><td>val</td><td>Tyr</td><td>lys</td><td>own</td><td>Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Leu</td><td>val</td><td>Gin</td><td>Gin</td><td>Gin</td><td>own</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>val</td><td>Gin</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Glu</td><td>ala</td><td>Thr</td><td>Glu</td><td>lys</td><td>ala</td><td>lys</td><td>Gin</td><td>Glu</td><td>phe</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Glu</td><td>lys</td><td>ala</td><td>Gly</td><td>lys</td><td>asp</td><td>phe</td><td>Leu</td><td>val</td><td>Glu</td><td>Thr</td><td>How much</td><td>lys</td><td>Leu</td><td>Gly</td><td>lys</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>Arg</td><td>Pro</td><td>own</td><td>His</td><td>Leu</td><td>Trp</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>phe</td><td>Pro</td><td>asp</td><td>Cys</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>own</td><td>His</td><td>His</td><td>Tyr</td><td>lys</td><td>lys</td><td>Pro</td><td>Gly</td><td>Tyr</td><td>own</td><td>Gly</td><td>Cheese</td><td>Cys</td><td>phe</td><td>own</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>val</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>own</td><td>asp</td><td>asp</td><td>Leu</td><td>Cheese</td><td>Trp</td><td>Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td rowspan="2">Thr</td><td colspan="3" rowspan="2">Ala Leu Tyr 260</td><td rowspan="2">Pro</td><td rowspan="2">Cheese</td><td colspan="2" rowspan="2">Ile Tyr</td><td colspan="8">Leu Asn Thr Gin Gin Ser Pro Val</td>

<td> 265</td><td colspan="7"> 270</td>

<td>ala</td><td>Ala</td><td>Thr</td><td>Leu</td><td>Tyr</td><td>val</td><td>Arg</td><td>own</td><td>Arg</td><td>val</td><td>Arg</td><td>Glu</td><td>ala</td><td>How much</td><td>Arg</td><td>val</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Cheese</td><td>lys</td><td>How much</td><td>Pro</td><td>asp</td><td>ala</td><td>lys</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>Pro</td><td>val</td><td>phe</td><td>ala</td><td>Tyr</td><td>Thr</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>asp</td><td>Gin</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>Leu</td><td>Cheese</td><td>Gin</td><td>asp</td><td>Glu</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Leu</td><td>val</td><td>Tyr</td><td>Thr</td><td>phe</td><td>Gly</td><td>Glu</td><td>Thr</td><td>val</td><td>ala</td><td>Leu</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Gly</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>val</td><td>How much</td><td>Trp</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>Arg</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>Cys</td><td>Leu</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>asp</td><td>own</td><td>Tyr</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>How much</td><td>Leu</td><td>own</td><td>Pro</td><td>Tyr</td><td>How much</td><td>How much</td><td>own</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>val</td><td>Thr</td><td>Leu</td><td>ala</td><td>ala</td><td>lys</td><td>Underworld</td><td>Cys</td><td>Cheese</td><td>Gin</td><td>val</td><td>Leu</td><td>Cys</td><td>Gin</td><td>Glu</td><td>Gin</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>val</td><td>Cys</td><td>How much</td><td>Arg</td><td>lys</td><td>own</td><td>Trp</td><td>own</td><td>Cheese</td><td>Cheese</td><td>asp</td><td>Tyr</td><td>Leu</td><td>His</td><td>Leu</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>own</td><td>Pro</td><td>asp</td><td>own</td><td>phe</td><td>ala</td><td>How much</td><td>Gin</td><td>Leu</td><td>Glu</td><td>lys</td><td>Gly</td><td>Gly</td><td>lys</td><td>phe</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>val</td><td>Arg</td><td>Gly</td><td>lys</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Glu</td><td>asp</td><td>Leu</td><td>Glu</td><td>Gin</td><td>phe</td><td>Cheese</td><td>Glu</td><td>lys</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>phe</td><td>Tyr</td><td>Cys</td><td>Cheese</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Glu</td><td>lys</td><td>ala</td><td>asp</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>val</td><td>lys</td><td>asp</td><td>Thr</td><td>asp</td><td>ala</td><td>val</td><td>asp</td><td>val</td><td>Cys</td><td>How much</td><td>ala</td><td>asp</td><td>Gly</td><td>val</td><td>Cys</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>How much</td><td>asp</td><td>ala</td><td>phe</td><td>Leu</td><td>lys</td><td>Pro</td><td>Pro</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Pro</td><td>Gin</td><td>How much</td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>phe</td><td>Tyr</td><td>own</td><td>ala</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>ala</td><td>Thr</td><td>Underworld</td><td>phe</td><td>How much</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td>

<td>Cheese</td><td>How much</td><td>Leu</td><td>phe</td><td>Leu</td><td>How much</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>val</td><td>ala</td><td>Cheese</td><td>Leu</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 2 &lt;211&gt; 35 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 2

<td>Underworld</td><td>Gly</td><td>Val Leu</td><td>lys</td><td>phe</td><td>lys</td><td>His</td><td>How much</td><td>phe</td><td>Phe Arg</td><td>Cheese</td><td>phe</td><td>val</td><td>lys</td>

<td> 1</td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Cheese</td><td>Cheese</td><td>Gly Val</td><td>Cheese</td><td>Gin</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>Phe Leu</td><td>Leu</td><td>How much</td><td>Pro</td><td>Cys</td>

<td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

Cys Leu Thr 35 &lt;210&gt; 3 &lt;211&gt; 474 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 3

<td>Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ala</td><td>Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>Asn Val</td><td>Pro</td><td>phe</td><td>Leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td> 15</td><td></td>

<td>ala</td><td>Trp</td><td>own</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Cys</td><td>Leu</td><td>Gly Lys</td><td>phe</td><td>asp</td><td>Glu</td><td>Pro</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>asp</td><td>Underworld</td><td>Cheese</td><td>Leu</td><td>phe</td><td>Cheese</td><td>phe</td><td>How much</td><td>Gly</td><td>Cheese Pro</td><td>Arg</td><td>How much</td><td>own</td><td>ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Thr</td><td>Gly</td><td>Gin</td><td>Gly</td><td>val</td><td>Thr</td><td>How much</td><td>phe</td><td>Tyr</td><td>val</td><td>Asp Arg</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>Tyr</td>

<td>Pro</td><td>50 Tyr</td><td>How much</td><td>asp</td><td>Cheese</td><td>How much</td><td>55 Thr</td><td>Gly</td><td>val</td><td>Thr</td><td>val</td><td>60 own</td><td>Gly</td><td>Gly</td><td>How much</td><td>Pro</td>

<td>65 Gin</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>70 Gin</td><td>asp</td><td>His</td><td>Leu</td><td>asp</td><td>75 lys</td><td>ala</td><td>lys</td><td>lys</td><td>asp</td><td>80 How much</td>

<td>Thr</td><td>phe</td><td>Tyr</td><td>Underworld</td><td>85 Pro</td><td>val</td><td>asp</td><td>own</td><td>Leu</td><td>90 Gly</td><td>Underworld</td><td>ala</td><td>val</td><td>How much</td><td>95 asp</td><td>Trp</td>

<td>Glu</td><td>Glu</td><td>Trp</td><td>100 Arg</td><td>Pro</td><td>Thr</td><td>Trp</td><td>ala</td><td>105 Arg</td><td>own</td><td>Trp</td><td>lys</td><td>Pro</td><td>110 lys</td><td>asp</td><td>val</td>

<td>Tyr</td><td>lys</td><td>115 own</td><td>Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>120 Leu</td><td>val</td><td>Gin</td><td>Gin</td><td>Gin</td><td>125 own</td><td>val</td><td>Gin</td><td>Leu</td>

<td>Cheese</td><td>13 0 Leu</td><td>Thr</td><td>Glu</td><td>ala</td><td>Thr</td><td>135 Glu</td><td>lys</td><td>ala</td><td>lys</td><td>Gin</td><td>140 Glu</td><td>phe</td><td>Glu</td><td>lys</td><td>Ala</td>

<td>145 Gly</td><td>lys</td><td>asp</td><td>phe</td><td>Leu</td><td>150 val</td><td>Glu</td><td>Thr</td><td>How much</td><td>lys</td><td>155 Leu</td><td>Gly</td><td>lys</td><td>Leu</td><td>Leu</td><td>160 Arg</td>

<td>Pro</td><td>own</td><td>His</td><td>Leu</td><td>165 Trp</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>170 phe</td><td>Pro</td><td>asp</td><td>Cys</td><td>Tyr</td><td>175 own</td><td>His</td>

<td>His</td><td>Tyr</td><td>lys</td><td>180 lys</td><td>Pro</td><td>Gly</td><td>Tyr</td><td>own</td><td>185 Gly</td><td>Cheese</td><td>Cys</td><td>phe</td><td>own</td><td>190 val</td><td>Glu</td><td>How much</td>

<td>lys</td><td>Arg</td><td>195 own</td><td>asp</td><td>asp</td><td>Leu</td><td>Cheese</td><td>200 Trp</td><td>Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>205 Cheese</td><td>Thr</td><td>ala</td><td>Leu</td>

<td>Tyr</td><td>210 Pro</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>Leu</td><td>215 own</td><td>Thr</td><td>Gin</td><td>Gin</td><td>Cheese</td><td>220 Pro</td><td>val</td><td>ala</td><td>ala</td><td>Thr</td>

<td>225 Leu</td><td>Tyr</td><td>val</td><td>Arg</td><td>own</td><td>230 Arg</td><td>val</td><td>Arg</td><td>Glu</td><td>ala</td><td>235 How much</td><td>Arg</td><td>val</td><td>Cheese</td><td>lys</td><td>240 How much</td>

<td>Pro</td><td>asp</td><td>ala</td><td>lys</td><td>245 Cheese</td><td>Pro</td><td>Leu</td><td>Pro</td><td>val</td><td>250 phe</td><td>ala</td><td>Tyr</td><td>Thr</td><td>Arg</td><td>255 How much</td><td>val</td>

<td>phe</td><td>Thr</td><td>asp</td><td>260 Gin</td><td>val</td><td>Leu</td><td>Lye</td><td>phe</td><td>265 Leu</td><td>Cheese</td><td>Gin</td><td>asp</td><td>Glu</td><td>270 Leu</td><td>val</td><td>Tyr</td>

<td>Thr</td><td>phe</td><td>275 Gly</td><td>Glu</td><td>Thr</td><td>val</td><td>ala</td><td>280 Leu</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Gly</td><td>285 How much</td><td>val</td><td>How much</td><td>Trp</td>

<td>Gly</td><td>290 Thr</td><td>Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>295 Arg</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>300 Cys</td><td>Leu</td><td>Leu</td><td>Leu</td><td>asp</td>

<td>305 own</td><td>Tyr</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>310 How much</td><td>Leu</td><td>own</td><td>Pro</td><td>Tyr</td><td>315 How much</td><td>How much</td><td>own</td><td>val</td><td>Thr</td><td>320 Leu</td>

<td>ala</td><td>ala</td><td>lys</td><td>Underworld</td><td>325 Cys</td><td>Cheese</td><td>Gin</td><td>val</td><td>Leu</td><td>330 Cys</td><td>Gin</td><td>Glu</td><td>Gin</td><td>Gly</td><td>335 val</td><td>Cys</td>

<td>How much</td><td>Arg</td><td>lys</td><td>340 own</td><td>Trp</td><td>own</td><td>Cheese</td><td>Cheese</td><td>345 asp</td><td>Tyr</td><td>Leu</td><td>His</td><td>Leu</td><td>350 own</td><td>Pro</td><td>asp</td>

<td>own</td><td>phe</td><td>355 ala</td><td>How much</td><td>Gin</td><td>Leu</td><td>Glu</td><td>360 lys</td><td>Gly</td><td>Gly</td><td>lys</td><td>phe</td><td>365 Thr</td><td>val</td><td>Arg</td><td>Gly</td>

<td>lys</td><td>370 Pro</td><td>Thr</td><td>Leu</td><td>Glu</td><td>asp</td><td>375 Leu</td><td>Glu</td><td>Gin</td><td>phe</td><td>Cheese</td><td>380 Glu</td><td>lys</td><td>phe</td><td>Tyr</td><td>Cys</td>

<td>385 Cheese</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>390 Leu</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Glu</td><td>395 lys</td><td>ala</td><td>asp</td><td>val</td><td>lys</td><td>400 asp</td>

<td>Thr</td><td>asp</td><td>ala</td><td>val</td><td>405 asp</td><td>val</td><td>Cys</td><td>How much</td><td>ala</td><td>410 asp</td><td>Gly</td><td>val</td><td>Cys</td><td>How much</td><td>415 asp</td><td>ala</td>

<td>phe</td><td>Leu</td><td>lys</td><td>420 Pro</td><td>Pro</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>425 Glu</td><td>Glu</td><td>Pro</td><td>Gin</td><td>How much</td><td>430 phe</td><td>Tyr</td><td>own</td>

<td>ala</td><td>Cheese</td><td>435 Pro</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>440 ala</td><td>Thr</td><td>Underworld</td><td>phe</td><td>How much</td><td>445 val</td><td>Cheese</td><td>How much</td><td>Leu</td>

<td>phe 465</td><td>450 Leu</td><td>How much</td><td>How much</td><td>Cheese</td><td>Cheese 470</td><td>455 val</td><td>ala</td><td>Cheese</td><td>Leu</td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 4 &lt;211&gt; 448 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 4

<td>Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ala</td><td>Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>own</td><td>val</td><td>Pro</td><td>phe</td><td>Leu</td><td>Trp</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>ala</td><td>Trp</td><td>own</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Cys</td><td>Leu</td><td>Gly</td><td>lys</td><td>phe</td><td>asp</td><td>Glu</td><td>Pro</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Leu</td><td>asp</td><td>Underworld</td><td>Cheese</td><td>Leu</td><td>phe</td><td>Cheese</td><td>phe</td><td>How much</td><td>Gly</td><td>Cheese</td><td>pro</td><td>Arg</td><td>How much</td><td>own</td><td>ala</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Thr</td><td>Gly</td><td>Gin</td><td>Gly</td><td>val</td><td>Thr</td><td>How much</td><td>phe</td><td>Tyr</td><td>val</td><td>asp</td><td>Arg</td><td>Leu</td><td>Gly</td><td>Tyr</td><td>Tyr</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Tyr</td><td>How much</td><td>asp</td><td>Cheese</td><td>How much</td><td>Thr</td><td>Gly</td><td>val</td><td>Thr</td><td>val</td><td>own</td><td>Gly</td><td>Gly</td><td>How much</td><td>Pro</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Gin</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>asp</td><td>His</td><td>Leu</td><td>asp</td><td>lys</td><td>ala</td><td>lys</td><td>lys</td><td>asp</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Thr</td><td>phe</td><td>Tyr</td><td>Underworld</td><td>Pro</td><td>val</td><td>asp</td><td>own</td><td>Leu</td><td>Gly</td><td>Underworld</td><td>ala</td><td>val</td><td>How much</td><td>asp</td><td>Trp</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Glu</td><td>Glu</td><td>Trp</td><td>Arg</td><td>Pro</td><td>Thr</td><td>Trp</td><td>ala</td><td>Arg</td><td>own</td><td>Trp</td><td>lys</td><td>Pro</td><td>lys</td><td>asp</td><td>val</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Tyr</td><td>lys</td><td>own</td><td>Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Leu</td><td>val</td><td>Gin</td><td>Gin</td><td>Gin</td><td>own</td><td>val</td><td>Gin</td><td>Leu</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td>Leu</td><td>Thr</td><td>Glu</td><td>ala</td><td>Thr</td><td>Glu</td><td>lys</td><td>ala</td><td>lys</td><td>Gin</td><td>Glu</td><td>phe</td><td>Glu</td><td>lys</td><td>ala</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Gly</td><td>lys</td><td>asp</td><td>phe</td><td>Leu</td><td>val</td><td>Glu</td><td>Thr</td><td>How much</td><td>lys</td><td>Leu</td><td>Gly</td><td>lys</td><td>Leu</td><td>Leu</td><td>Arg</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>Pro</td><td>own</td><td>His</td><td>Leu</td><td>Trp</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>phe</td><td>Pro</td><td>asp</td><td>Cys</td><td>Tyr</td><td>own</td><td>His</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>His</td><td>Tyr</td><td>lys</td><td>lys</td><td>Pro</td><td>Gly</td><td>Tyr</td><td>own</td><td>Gly</td><td>Cheese</td><td>Cys</td><td>phe</td><td>own</td><td>val</td><td>Glu</td><td>How much</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>lys</td><td>Arg</td><td>own</td><td>asp</td><td>asp</td><td>Leu</td><td>Cheese</td><td>Trp</td><td>Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>Cheese</td><td>Thr</td><td>ala</td><td>Leu</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>Pro</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>Leu</td><td>own</td><td>Thr</td><td>Gin</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>val</td><td>ala</td><td>ala</td><td>Thr</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>Leu</td><td>Tyr</td><td>val</td><td>Arg</td><td>own</td><td>Arg</td><td>val</td><td>Arg</td><td>Glu</td><td>ala</td><td>How much</td><td>Arg</td><td>val</td><td>Cheese</td><td>lys</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Pro</td><td>asp</td><td>ala</td><td>lys</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>Pro</td><td>val</td><td>phe</td><td>ala</td><td>Tyr</td><td>Thr</td><td>Arg</td><td>How much</td><td>val</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>phe</td><td>Thr</td><td>asp</td><td>Gin</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>Leu</td><td>Cheese</td><td>Gin</td><td>asp</td><td>Glu</td><td>Leu</td><td>val</td><td>Tyr</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Thr</td><td>phe</td><td>Gly</td><td>Glu</td><td>Thr</td><td>val</td><td>ala</td><td>Leu</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Gly</td><td>How much</td><td>val</td><td>How much</td><td>Trp</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>Arg</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>Cys</td><td>Leu</td><td>Leu</td><td>Leu</td><td>asp</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>own</td><td>Tyr</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>How much</td><td>Leu</td><td>own</td><td>Pro</td><td>Tyr</td><td>How much</td><td>How much</td><td>own</td><td>val</td><td>Thr</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>ala</td><td>ala</td><td>lys</td><td>Underworld</td><td>Cys</td><td>Cheese</td><td>Gin</td><td>val</td><td>Leu</td><td>Cys</td><td>Gin</td><td>Glu</td><td>Gin</td><td>Gly</td><td>VAL</td><td>Cys</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>How much</td><td>Arg</td><td>lys</td><td>own</td><td>Trp</td><td>own</td><td>Cheese</td><td>Cheese</td><td>asp</td><td>Tyr</td><td>Leu</td><td>His</td><td>Leu</td><td>own</td><td>Pro</td><td>asp</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>own</td><td>phe</td><td>ala</td><td>How much</td><td>Gin</td><td>Leu</td><td>Glu</td><td>lys</td><td>Gly</td><td>Gly</td><td>lys</td><td>phe</td><td>Thr</td><td>val</td><td>Arg</td><td>Gly</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>lys</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Glu</td><td>asp</td><td>Leu</td><td>Glu</td><td>Gin</td><td>phe</td><td>Cheese</td><td>Glu</td><td>lys</td><td>phe</td><td>Tyr</td><td>Cys</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>Cheese</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Glu</td><td>lys</td><td>ala</td><td>asp</td><td>val</td><td>lys</td><td>asp</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>Thr</td><td>asp</td><td>ala</td><td>val</td><td>asp</td><td>val</td><td>Cys</td><td>How much</td><td>ala</td><td>asp</td><td>Gly</td><td>val</td><td>Cys</td><td>How much</td><td>asp</td><td>ala</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>phe</td><td>Leu</td><td>lys</td><td>Pro</td><td>Pro</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Pro</td><td>Gin</td><td>How much</td><td>phe</td><td>Tyr</td><td>own</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

&lt;210&gt; 5 &lt;211&gt; 482 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 5

<td>Underworld</td><td>Gly</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>lys</td><td>His</td><td>How much</td><td>phe</td><td>phe</td><td>Arg</td><td>Cheese</td><td>phe</td><td>val</td><td>lys</td>

<td> 1</td><td></td><td></td><td></td><td> 5</td><td></td><td></td><td></td><td></td><td> 10</td><td></td><td></td><td></td><td></td><td> 15</td><td></td>

<td>Cheese</td><td>Cheese</td><td>Gly</td><td>val</td><td>Cheese</td><td>Gin</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>phe</td><td>Leu</td><td>Leu</td><td>How much</td><td>Pro</td><td>Cys</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>Cys</td><td>Leu</td><td>Thr</td><td>Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ala</td><td>Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>own</td><td>val</td><td>Pro</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>phe</td><td>Leu</td><td>Trp</td><td>ala</td><td>Trp</td><td>own</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Cys</td><td>Leu</td><td>Gly</td><td>lys</td><td>phe</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>asp</td><td>Glu</td><td>Pro</td><td>Leu</td><td>asp</td><td>Underworld</td><td>Cheese</td><td>Leu</td><td>phe</td><td>Cheese</td><td>phe</td><td>How much</td><td>Gly</td><td>Cheese</td><td>Pro</td><td>Arg</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>How much</td><td>own</td><td>ala</td><td>Thr</td><td>Gly</td><td>Gin</td><td>Gly</td><td>val</td><td>Thr</td><td>How much</td><td>phe</td><td>Tyr</td><td>val</td><td>asp</td><td>Arg</td><td>Leu</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Gly</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>Tyr</td><td>How much</td><td>asp</td><td>Cheese</td><td>How much</td><td>Thr</td><td>Gly</td><td>val</td><td>Thr</td><td>val</td><td>own</td><td>Gly</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Gly</td><td>How much</td><td>Pro</td><td>Gin</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>asp</td><td>His</td><td>Leu</td><td>asp</td><td>lys</td><td>ala</td><td>lys</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>lys</td><td>asp</td><td>How much</td><td>Thr</td><td>phe</td><td>Tyr</td><td>Underworld</td><td>Pro</td><td>val</td><td>asp</td><td>own</td><td>Leu</td><td>Gly</td><td>Underworld</td><td>ala</td><td>val</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>How much</td><td>asp</td><td>Trp</td><td>Glu</td><td>Glu</td><td>Trp</td><td>Arg</td><td>Pro</td><td>Thr</td><td>Trp</td><td>ala</td><td>Arg</td><td>own</td><td>Trp</td><td>lys</td><td>Pro</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>lys</td><td>asp</td><td>val</td><td>Tyr</td><td>lys</td><td>own</td><td>Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Leu</td><td>val</td><td>Gin</td><td>Gin</td><td>Gin</td><td>own</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>val</td><td>Gin</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Glu</td><td>ala</td><td>Thr</td><td>Glu</td><td>lys</td><td>ala</td><td>lys</td><td>Gin</td><td>Glu</td><td>phe</td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td>

<td>Glu</td><td>lys</td><td>ala</td><td>Gly</td><td>lys</td><td>asp</td><td>phe</td><td>Leu</td><td>val</td><td>Glu</td><td>Thr</td><td>How much</td><td>lys</td><td>Leu</td><td>Gly</td><td>lys</td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>Arg</td><td>Pro</td><td>own</td><td>His</td><td>Leu</td><td>Trp</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>phe</td><td>Pro</td><td>asp</td><td>Cys</td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td>

<td>Tyr</td><td>own</td><td>His</td><td>His</td><td>Tyr</td><td>lys</td><td>lys</td><td>Pro</td><td>Gly</td><td>Tyr</td><td>own</td><td>Gly</td><td>Cheese</td><td>Cys</td><td>phe</td><td>own</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>val</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>own</td><td>asp</td><td>asp</td><td>Leu</td><td>Cheese</td><td>Trp</td><td>Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Thr</td><td>ala</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>Leu</td><td>own</td><td>Thr</td><td>Gin</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>val</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>ala</td><td>ala</td><td>Thr</td><td>Leu</td><td>Tyr</td><td>val</td><td>Arg</td><td>own</td><td>Arg</td><td>val</td><td>Arg</td><td>Glu</td><td>ala</td><td>How much</td><td>Arg</td><td>val</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Cheese</td><td>lys</td><td>How much</td><td>Pro</td><td>asp</td><td>ala</td><td>lys</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>Pro</td><td>val</td><td>phe</td><td>ala</td><td>Tyr</td><td>Thr</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>asp</td><td>Gin</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>Leu</td><td>Cheese</td><td>Gin</td><td>asp</td><td>Glu</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Leu</td><td>val</td><td>Tyr</td><td>Thr</td><td>phe</td><td>Gly</td><td>Glu</td><td>Thr</td><td>val</td><td>ala</td><td>Leu</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Gly</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>val</td><td>How much</td><td>Trp</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>Arg</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>Cys</td><td>Leu</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>asp</td><td>own</td><td>Tyr</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>How much</td><td>Leu</td><td>own</td><td>Pro</td><td>Tyr</td><td>How much</td><td>How much</td><td>own</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>val</td><td>Thr</td><td>Leu</td><td>ala</td><td>ala</td><td>lys</td><td>Underworld</td><td>Cys</td><td>Cheese</td><td>Gin</td><td>val</td><td>Leu</td><td>Cys</td><td>Gin</td><td>Glu</td><td>Gin</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>val</td><td>Cys</td><td>How much</td><td>Arg</td><td>lys</td><td>own</td><td>Trp</td><td>own</td><td>Cheese</td><td>Cheese</td><td>asp</td><td>Tyr</td><td>Leu</td><td>His</td><td>Leu</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>own</td><td>Pro</td><td>asp</td><td>own</td><td>phe</td><td>ala</td><td>How much</td><td>Gin</td><td>Leu</td><td>Glu</td><td>lys</td><td colspan="2">Gly gly</td><td>lys</td><td>phe</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>val</td><td>Arg</td><td>Gly</td><td>lys</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Glu</td><td>asp</td><td>Leu</td><td>Glu</td><td>Gin</td><td>phe</td><td>Cheese</td><td>Glu</td><td>lys</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>phe</td><td>Tyr</td><td>Cys</td><td>Cheese</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Glu</td><td>lys</td><td>ala</td><td>asp</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>val</td><td>lys</td><td>asp</td><td>Thr</td><td>asp</td><td>ala</td><td>val</td><td>asp</td><td>val</td><td>Cys</td><td>How much</td><td>ala</td><td>asp</td><td>Gly</td><td>val</td><td>Cys</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

Ile Asp Ala Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gin Ile

<td> 465</td><td> 470</td><td> 475</td><td> 480</td>

<td>Phe Tyr</td><td></td><td></td><td></td>

&lt;210&gt; 6 &lt;211&gt; 1530 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;220&gt;

&lt;221&gt; CDS &lt;222&gt; (1) ... (1530) &lt;223&gt; ΡΗ-20 Glycoprotein ο hyaluronidase activity anchored by GPI &lt;400&gt; 6

<td rowspan="2">atg Underworld 1</td><td colspan="2" rowspan="2">gga gtg Gly Val</td><td rowspan="2">eta Leu</td><td colspan="4">aaa ttc aag cac</td><td colspan="8">atc ttt ttc aga age ttt gtt aaa</td><td rowspan="2"> 48</td>

<td>lys 5</td><td>phe</td><td>lys</td><td>His</td><td>How much</td><td>phe 10</td><td colspan="3">Phe Arg Cheese</td><td>phe</td><td>val 15</td><td>lys</td>

<td>tCA</td><td>agt</td><td>GGA</td><td>gta</td><td>tcc</td><td>cag</td><td>ata</td><td>gtt</td><td>ttc</td><td>acc</td><td>ttc</td><td>ctt</td><td>cOT</td><td>att</td><td>ca</td><td>tgt</td><td> 96</td>

<td>Cheese</td><td>Cheese</td><td>Gly</td><td>val</td><td>Cheese</td><td>Gin</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>phe</td><td>Leu</td><td>Leu</td><td>How much</td><td>Pro</td><td>Cys</td><td></td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td>

<td>TGC</td><td>TTG</td><td>act</td><td>cOT</td><td>aat</td><td>ttc</td><td>aga</td><td>GCA</td><td>Cct</td><td>Cct</td><td>gtt</td><td>att</td><td>ca</td><td>aat</td><td>gtg</td><td>Cct</td><td> 144</td>

<td>Cys</td><td>Leu</td><td>Thr</td><td>Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ala</td><td>Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>own</td><td>val</td><td>Pro</td><td></td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td>

<td>ttc</td><td>ctc</td><td>TGG</td><td>gcc</td><td>TGG</td><td>aat</td><td>gcc</td><td>ca</td><td>agt</td><td>gaa</td><td>ttt</td><td>tgt</td><td>ctt</td><td>GGA</td><td>aaa</td><td>ttt</td><td> 192</td>

<td>phe</td><td>Leu</td><td>Trp</td><td>ala</td><td>Trp</td><td>own</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Cys</td><td>Leu</td><td>Gly</td><td>lys</td><td>phe</td><td></td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td></td>

<td>gat</td><td>gag</td><td>ca</td><td>eta</td><td>gat</td><td>atg</td><td>age</td><td>ctc</td><td>ttc</td><td>tet</td><td>ttc</td><td>ata</td><td>GGA</td><td>age</td><td>ccc</td><td>ega</td><td> 240</td>

<td>asp</td><td>Glu</td><td>Pro</td><td>Leu</td><td>asp</td><td>Underworld</td><td>Cheese</td><td>Leu</td><td>phe</td><td>Cheese</td><td>phe</td><td>How much</td><td>Gly</td><td>Cheese</td><td>Pro</td><td>Arg</td><td></td>

<td> 55</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td>

<td>ata</td><td>aac</td><td>gcc</td><td>ace</td><td>ggg</td><td>caa</td><td>GGT</td><td>gtt</td><td>aca</td><td>ata</td><td>ttt</td><td>tat</td><td>gtt</td><td>gat</td><td>aga</td><td>ctt</td><td> 288</td>

<td>How much</td><td>own</td><td>ala</td><td>Thr</td><td>Gly</td><td>Gin</td><td>Gly</td><td>val</td><td>Thr</td><td>How much</td><td>phe</td><td>Tyr</td><td>val</td><td>asp</td><td>Arg</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td>

<td>GGC</td><td>trays</td><td>tat</td><td>Cct</td><td>trays</td><td>ata</td><td>gat</td><td>tCA</td><td>atc</td><td>aca</td><td>GGA</td><td>gta</td><td>act</td><td>gtg</td><td>aat</td><td>GGA</td><td> 336</td>

<td>Gly</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>Tyr</td><td>How much</td><td>asp</td><td>Cheese</td><td>How much</td><td>Thr</td><td>Gly</td><td>val</td><td>Thr</td><td>val</td><td>own</td><td>Gly</td><td></td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td>

<td>GGA</td><td>atc</td><td>ccc</td><td>eag</td><td>aag</td><td>att</td><td>tcc</td><td>tta</td><td>caa</td><td>gac</td><td>cat</td><td>cOT</td><td>gac</td><td>aaa</td><td>get</td><td>aag</td><td> 384</td>

<td>Gly</td><td>How much</td><td>Pro</td><td>Gin</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>asp</td><td>His</td><td>Leu</td><td>asp</td><td>lys</td><td>ala</td><td>lys</td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td>

<td>aaa</td><td>gac</td><td>att</td><td>aca</td><td>ttt</td><td>tat</td><td>atg</td><td>ca</td><td>gta</td><td>gac</td><td>aat</td><td>TTG</td><td>GGA</td><td>atg</td><td>get</td><td>gtt</td><td> 432</td>

<td>lys</td><td>asp</td><td>How much</td><td>Thr</td><td>phe</td><td>Tyr</td><td>Underworld</td><td>Pro</td><td>val</td><td>asp</td><td>own</td><td>Leu</td><td>Gly</td><td>Underworld</td><td>ala</td><td>val</td><td></td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td></td>

<td>att</td><td>gac</td><td>TGG</td><td>gaa</td><td>gaa</td><td>TGG</td><td>aga</td><td>ccc</td><td>act</td><td>TGG</td><td>GCA</td><td>aga</td><td>aac</td><td>TGG</td><td>aaa</td><td>Cct</td><td> 480</td>

<td>How much</td><td>asp</td><td>Trp</td><td>Glu</td><td>Glu</td><td>Trp</td><td>Arg</td><td>Pro</td><td>Thr</td><td>Trp</td><td>ala</td><td>Arg</td><td>own</td><td>Trp</td><td>lys</td><td>Pro</td><td></td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td>

<td>aaa</td><td>gat</td><td>gtt</td><td>trays</td><td>aag</td><td>aat</td><td>agg</td><td>tet</td><td>att</td><td>gaa</td><td>TTG</td><td>gtt</td><td>cag</td><td>caa</td><td>caa</td><td>aat</td><td> 528</td>

<td>lys</td><td>asp</td><td>val</td><td>Tyr</td><td>lys</td><td>own</td><td>Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Leu</td><td>val</td><td>Gin</td><td>Gin</td><td>Gin</td><td>own</td><td></td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td><td></td>

<td rowspan="2">gta val</td><td colspan="2">caa ctt</td><td colspan="3" rowspan="2">agt etc aca Ser Leu Thr 180</td><td colspan="9">gag gee act gag aaa gca aaa caa gaa</td><td rowspan="2">ttt phe</td><td rowspan="2"> 576</td>

<td>Gin</td><td>Leu</td><td colspan="2">Glu Ala</td><td>Thr 185</td><td>Glu</td><td>lys</td><td>ala</td><td>lys</td><td colspan="2">Gin Glu 190</td>

<td>gaa</td><td>aag</td><td>GCA</td><td>ggg</td><td>aag</td><td>gat</td><td>ttc</td><td>cOT</td><td>gta</td><td>gag</td><td>act</td><td>ata</td><td>aaa</td><td>TTG</td><td>GGA</td><td>aaa</td><td> 624</td>

<td>Glu</td><td>lys</td><td>ala</td><td colspan="2">Gly Lys</td><td>asp</td><td>phe</td><td>Leu</td><td>val</td><td>Glu</td><td>Thr</td><td>How much</td><td>lys</td><td>Leu</td><td>Gly</td><td>lys</td><td></td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td>

<td>tta</td><td>ctt</td><td>CGG</td><td>ca</td><td>aat</td><td>cac</td><td>TTG</td><td>TGG</td><td>GGT</td><td>tat</td><td>tat</td><td>ctt</td><td>ttt</td><td>ccg</td><td>gat</td><td>tgt</td><td> 672</td>

<td>Leu</td><td>Leu</td><td>Arg</td><td>Pro</td><td>own</td><td>His</td><td>Leu</td><td>Trp</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>phe</td><td>Pro</td><td>asp</td><td>Cys</td><td></td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td></td>

<td>trays</td><td>aac</td><td>cat</td><td>cac</td><td>tat</td><td>aag</td><td>aaa</td><td>ccc</td><td>GGT</td><td>trays</td><td>aat</td><td>GGA</td><td>agt</td><td>TGC</td><td>ttc</td><td>aat</td><td> 720</td>

<td>Tyr</td><td>own</td><td>His</td><td>His</td><td>Tyr</td><td>lys</td><td>lys</td><td>Pro</td><td colspan="2">Gly Tyr</td><td>own</td><td>Gly</td><td>Cheese</td><td>Cys</td><td>phe</td><td>own</td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td><td></td>

<td>gta</td><td>gaa</td><td>ata</td><td>aaa</td><td>aga</td><td>aat</td><td>gat</td><td>gat</td><td>ctc</td><td>age</td><td>TGG</td><td>TTG</td><td>TGG</td><td>aat</td><td>gaa</td><td>age</td><td> 768</td>

<td>val</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>own</td><td>asp</td><td>asp</td><td>Leu</td><td>Cheese</td><td>Trp</td><td>Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>Cheese</td><td></td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td><td></td>

<td>act</td><td>GCT</td><td>ctt</td><td>trays</td><td>ca</td><td>tcc</td><td>att</td><td>tat</td><td>TTG</td><td>aac</td><td>act</td><td>cag</td><td>cag</td><td>tet</td><td>Cct</td><td>gta</td><td> 816</td>

<td>Thr</td><td>ala</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>Leu</td><td>own</td><td>Thr</td><td>Gin</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>val</td><td></td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td><td></td>

<td>GCT</td><td>GCT</td><td>aca</td><td>ctc</td><td>tat</td><td>gtg</td><td>cgc</td><td>aat</td><td>ega</td><td>gtt</td><td>CGG</td><td>gaa</td><td>gee</td><td>atc</td><td>aga</td><td>gtt</td><td> 864</td>

<td>ala</td><td>ala</td><td>Thr</td><td>Leu</td><td>Tyr</td><td>val</td><td>Arg</td><td>own</td><td>Arg</td><td>val</td><td>Arg</td><td>Glu</td><td>ala</td><td>How much</td><td>Arg</td><td>val</td><td></td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td><td></td>

<td>tcc</td><td>aaa</td><td>ata</td><td>Cct</td><td>gat</td><td>GCA</td><td>aaa</td><td>agt</td><td>ca</td><td>ctt</td><td>ccg</td><td>gtt</td><td>ttt</td><td>GCA</td><td>tat</td><td>acc</td><td> 912</td>

<td>Cheese</td><td>lys</td><td>How much</td><td>Pro</td><td>asp</td><td>ala</td><td>lys</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>Pro</td><td>val</td><td>phe</td><td>ala</td><td>Tyr</td><td>Thr</td><td></td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td><td></td>

<td>cgc</td><td>ata</td><td>gtt</td><td>ttt</td><td>act</td><td>gat</td><td>caa</td><td>gtt</td><td>TTG</td><td>aaa</td><td>ttc</td><td>ctt</td><td>tet</td><td>caa</td><td>gat</td><td>gaa</td><td> 960</td>

<td>Arg</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>asp</td><td>Gin</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>Leu</td><td>Cheese</td><td>Gin</td><td>asp</td><td>Glu</td><td></td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td><td></td>

<td>ctt</td><td>gtg</td><td>tat</td><td>aca</td><td>ttt</td><td>GGC</td><td>gaa</td><td>act</td><td>gtt</td><td>GCT</td><td>cOT</td><td>GGT</td><td>GCT</td><td>tet</td><td>GGA</td><td>att</td><td> 1008</td>

<td>Leu</td><td>val</td><td>Tyr</td><td>Thr</td><td>phe</td><td>Gly</td><td>Glu</td><td>Thr</td><td>val</td><td>ala</td><td>Leu</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Gly</td><td>How much</td><td></td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td>

<td>gta</td><td>ata</td><td>TGG</td><td>GGA</td><td>acc</td><td>ctc</td><td>agt</td><td>ata</td><td>atg</td><td>ega</td><td>agt</td><td>atg</td><td>aaa</td><td>tet</td><td>TGC</td><td>TTG</td><td> 1056</td>

<td>val</td><td>How much</td><td>Trp</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>Arg</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>Cys</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td>

<td>ctc</td><td>eta</td><td>gac</td><td>aat</td><td>trays</td><td>atg</td><td>gag</td><td>act</td><td>ata</td><td>cOT</td><td>aat</td><td>Cct</td><td>trays</td><td>ata</td><td>atc</td><td>aac</td><td> 1104</td>

<td>Leu</td><td>Leu</td><td>asp</td><td>own</td><td>Tyr</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>How much</td><td>Leu</td><td>own</td><td>Pro</td><td>Tyr</td><td>How much</td><td>How much</td><td>own</td><td></td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td><td></td>

<td>gtc</td><td>aca</td><td>eta</td><td>GCA</td><td>gee</td><td>aaa</td><td>atg</td><td>tgt</td><td>age</td><td>caa</td><td>gtg</td><td>ctt</td><td>TGC</td><td>cag</td><td>gag</td><td>caa</td><td> 1152</td>

<td>val</td><td>Thr</td><td>Leu</td><td>ala</td><td>ala</td><td>lys</td><td>Underworld</td><td>Cys</td><td>Cheese</td><td>Gin</td><td>val</td><td>Leu</td><td>Cys</td><td>Gin</td><td>Glu</td><td>Gin</td><td></td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td><td></td>

<td>GGA</td><td>gtg</td><td>tgt</td><td>ata</td><td>agg</td><td>aaa</td><td>aac</td><td>TGG</td><td>aat</td><td>tea</td><td>agt</td><td>gac</td><td>tat</td><td>ctt</td><td>cac</td><td>ctc</td><td> 1200</td>

<td>Gly</td><td>val</td><td>Cys</td><td>How much</td><td>Arg</td><td>lys</td><td>own</td><td>Trp</td><td>own</td><td>Cheese</td><td>Cheese</td><td>asp</td><td>Tyr</td><td>Leu</td><td>His</td><td>Leu</td><td></td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td><td></td>

<td>aac</td><td>ca</td><td>gat</td><td>aat</td><td>ttt</td><td>GCT</td><td>att</td><td>caa</td><td>ctt</td><td>gag</td><td>aaa</td><td>GGT</td><td>GGA</td><td>aag</td><td>ttc</td><td>aca</td><td> 1248</td>

<td>own</td><td>Pro</td><td>asp</td><td>own</td><td>phe</td><td>ala</td><td>How much</td><td>Gin</td><td>Leu</td><td>Glu</td><td>lys</td><td>Gly</td><td>Gly</td><td>lys</td><td>phe</td><td>Thr</td><td></td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td><td></td>

<td>gta</td><td>cgt</td><td>GGA</td><td>aaa</td><td>ccg</td><td>aca</td><td>ctt</td><td>gaa</td><td>gac</td><td>cOT</td><td>gag</td><td>caa</td><td>ttt</td><td>tet</td><td>gaa</td><td>aaa</td><td> 1296</td>

<td>val</td><td colspan="2">Arg Gly</td><td>lys 420</td><td>Pro</td><td colspan="4">Thr Leu Glu Asp 425</td><td>Leu</td><td colspan="2">Glu Gin</td><td>phe</td><td>Cheese 430</td><td>Glu</td><td colspan="2">lys</td>

<td>ttt</td><td>tat</td><td>TGC</td><td>agc</td><td>tgt</td><td>tat</td><td>agc</td><td>acc</td><td>TTG</td><td>agt</td><td>tgt</td><td>aag</td><td>gag</td><td>aaa</td><td>GCT</td><td>gat</td><td> 1344</td>

<td>phe</td><td>Tyr</td><td>Cys</td><td>Cheese</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Glu</td><td>lys</td><td>ala</td><td>asp</td><td></td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td><td></td>

<td>gta</td><td>aaa</td><td>gac</td><td>act</td><td>gat</td><td>GCT</td><td>gtt</td><td>gat</td><td>gtg</td><td>tgt</td><td>att</td><td>GCT</td><td>gat</td><td>GGT</td><td>gtc</td><td>tgt</td><td> 1392</td>

<td>val</td><td>lys</td><td>asp</td><td>Thr</td><td>asp</td><td>ala</td><td>val</td><td>asp</td><td>val</td><td>Cys</td><td>How much</td><td>ala</td><td>asp</td><td>Gly</td><td>val</td><td>Cys</td><td></td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td><td></td>

<td>ata</td><td>gat</td><td>GCT</td><td>ttt</td><td>eta</td><td>aaa</td><td>Cct</td><td>ccc</td><td>atg</td><td>gag</td><td>aca</td><td>gaa</td><td>gaa</td><td>Cct</td><td>caa</td><td>att</td><td> 1440</td>

<td>How much</td><td>asp</td><td>ala</td><td>phe</td><td>Leu</td><td>lys</td><td>Pro</td><td>Pro</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Pro</td><td>Gin</td><td>How much</td><td></td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td><td></td>

<td>ttc</td><td>trays</td><td>aat</td><td>GCT</td><td>tea</td><td>ccc</td><td>tcc</td><td>aca</td><td>eta</td><td>tet</td><td>gee</td><td>aca</td><td>atg</td><td>ttc</td><td>att</td><td>gtt</td><td> 1488</td>

<td>phe</td><td>Tyr</td><td>own</td><td>ala</td><td>Cheese</td><td>Pro</td><td>cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>ala</td><td>Thr</td><td>Underworld</td><td>phe</td><td>How much</td><td>val</td><td></td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td><td></td>

<td>agt</td><td>att</td><td>TTG</td><td>ttt</td><td>ctt</td><td>atc</td><td>att</td><td>tet</td><td>tet</td><td>gta</td><td>GCG</td><td>agt</td><td>TTG</td><td>taa</td><td></td><td></td><td> 1530</td>

<td>Cheese</td><td>How much</td><td>Leu</td><td>phe</td><td>Leu</td><td>How much</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>val</td><td>ala</td><td>Cheese</td><td>Leu</td><td> *</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td> 500</td><td></td><td></td><td></td><td></td><td> 505</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; Ί &lt;211&gt; 509 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;220&gt;

&lt;221&gt; CARBOHYD &lt;222&gt; 82, 166, 235, 254, 368, 393, 490 &lt;400&gt; 7

<td>Underworld 1</td><td>Gly</td><td>val</td><td>Leu</td><td>lys 5</td><td>phe</td><td>lys</td><td>His</td><td>How much</td><td>phe 10</td><td>phe</td><td>Arg</td><td>Cheese</td><td>phe</td><td>val 15</td><td>lys</td>

<td>Cheese</td><td>Cheese</td><td>Gly</td><td>val twenty</td><td>Cheese</td><td>Gin</td><td>How much</td><td>val</td><td>phe 25</td><td>Thr</td><td>phe</td><td>Leu</td><td>Leu</td><td>How much thirty</td><td>Pro</td><td>Cys</td>

<td>Cys</td><td>Leu</td><td>Thr 35</td><td>Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ala 40</td><td>Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro 45</td><td>own</td><td>val</td><td>Pro</td>

<td>phe</td><td>Leu 50</td><td>Trp</td><td>ala</td><td>Trp</td><td>own</td><td>ala 55</td><td>Pro</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Cys 60</td><td>Leu</td><td>Gly</td><td>lys</td><td>phe</td>

<td>asp 65</td><td>Glu</td><td>Pro</td><td>Leu</td><td>asp</td><td>Underworld 70</td><td>Cheese</td><td>Leu</td><td>phe</td><td>Cheese</td><td>phe 75</td><td>How much</td><td>Gly</td><td>Cheese</td><td>Pro</td><td>Arg 80</td>

<td>How much</td><td>own</td><td>ala</td><td>Thr</td><td>Gly 85</td><td>Gin</td><td>Gly</td><td>val</td><td>Thr</td><td>How much 90</td><td>phe</td><td>Tyr</td><td>val</td><td>asp</td><td>Arg 95</td><td>Leu</td>

<td>Gly</td><td>Tyr</td><td>Tyr</td><td>Pro 100</td><td>Tyr</td><td>How much</td><td>asp</td><td>Cheese</td><td>How much 105</td><td>Thr</td><td>Gly</td><td>val</td><td>Thr</td><td>val 110</td><td>own</td><td>Gly</td>

<td>Gly</td><td>How much</td><td>Pro 115</td><td>Gin</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu 120</td><td>Gin</td><td>asp</td><td>His</td><td>Leu</td><td>asp 125</td><td>lys</td><td>ala</td><td>lys</td>

<td>lys</td><td>asp 130</td><td>How much</td><td>Thr</td><td>phe</td><td>Tyr</td><td>Underworld 135</td><td>Pro</td><td>val</td><td>asp</td><td>own</td><td>Leu 140</td><td>Gly</td><td>Underworld</td><td>ala</td><td>val</td>

<td>How much 145</td><td>asp</td><td>Trp</td><td>Glu</td><td>Glu</td><td>Trp 150</td><td>Arg</td><td>Pro</td><td>Thr</td><td>Trp</td><td>ala 155</td><td>Arg</td><td>own</td><td>Trp</td><td>lys</td><td>Pro 160</td>

<td>lys</td><td>asp</td><td>val</td><td>Tyr</td><td>lys 165</td><td>own</td><td>Arg</td><td>Cheese</td><td>How much</td><td>Glu 170</td><td>Leu</td><td>val</td><td>Gin</td><td>Gin</td><td>Gin 175</td><td>own</td>

<td>val</td><td>Gin</td><td>Leu</td><td>Cheese 180</td><td>Leu</td><td>Thr</td><td>Glu</td><td>ala</td><td>Thr 185</td><td>Glu</td><td>lys</td><td>ala</td><td>lys</td><td>Gin 190</td><td>Glu</td><td>phe</td>

<td>Glu</td><td>lys</td><td>ala 195</td><td>Gly</td><td>lys</td><td>asp</td><td>phe</td><td>Leu 200</td><td>val</td><td>Glu</td><td>Thr</td><td>How much</td><td>lys 205</td><td>Leu</td><td>Gly</td><td>lys</td>

<td colspan="2">Leu Leu</td><td colspan="3" rowspan="2">Arg Pro Asn</td><td rowspan="2">His</td><td rowspan="2">Leu 215</td><td colspan="2" rowspan="2">Trp Gly</td><td rowspan="2">Tyr</td><td rowspan="2">Tyr</td><td colspan="2" rowspan="2">Leu Phe 220</td><td rowspan="2">Pro</td><td rowspan="2">asp</td><td rowspan="2">Cys</td>

<td></td><td> 210</td>

<td>Tyr</td><td>own</td><td>His</td><td>His</td><td>Tyr</td><td>lys</td><td>lys</td><td>Pro</td><td>Gly</td><td>Tyr</td><td>own</td><td>Gly</td><td>Cheese</td><td>Cys</td><td>phe</td><td>own</td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td>

<td>val</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>own</td><td>asp</td><td>asp</td><td>Leu</td><td>Cheese</td><td>Trp</td><td>Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td>

<td>Thr</td><td>ala</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>Leu</td><td>own</td><td>Thr</td><td>Gin</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>val</td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td>

<td>ala</td><td>ala</td><td>Thr</td><td>Leu</td><td>Tyr</td><td>val</td><td>Arg</td><td>own</td><td>Arg</td><td>val</td><td>Arg</td><td>Glu</td><td>ala</td><td>How much</td><td>Arg</td><td>val</td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td>

<td>Cheese</td><td>lys</td><td>How much</td><td>Pro</td><td>asp</td><td>ala</td><td>lys</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>Pro</td><td>val</td><td>phe</td><td>ala</td><td>Tyr</td><td>Thr</td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td>

<td>Arg</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>asp</td><td>Gin</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>Leu</td><td>Cheese</td><td>Gin</td><td>asp</td><td>Glu</td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td>

<td>Leu</td><td>val</td><td>Tyr</td><td>Thr</td><td>phe</td><td>Gly</td><td>Glu</td><td>Thr</td><td>val</td><td>ala</td><td>Leu</td><td colspan="2">Gly Ala</td><td>Cheese</td><td>Gly</td><td>How much</td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td>

<td>val</td><td>How much</td><td>Trp</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>Arg</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>Cys</td><td>Leu</td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>asp</td><td>own</td><td>Tyr</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>How much</td><td>Leu</td><td>own</td><td>Pro</td><td>Tyr</td><td>How much</td><td>How much</td><td>own</td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td>

<td>val</td><td>Thr</td><td>Leu</td><td>ala</td><td>ala</td><td>lys</td><td>Underworld</td><td>Cys</td><td>Cheese</td><td>Gin</td><td>val</td><td>Leu</td><td>Cys</td><td>Gin</td><td>Glu</td><td>Gin</td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>val</td><td>Cys</td><td>How much</td><td>Arg</td><td>lys</td><td>own</td><td>Trp</td><td>own</td><td>Cheese</td><td>Cheese</td><td>asp</td><td>Tyr</td><td>Leu</td><td>His</td><td>Leu</td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td>

<td>own</td><td>Pro</td><td>asp</td><td>own</td><td>phe</td><td>ala</td><td>How much</td><td>Gin</td><td>Leu</td><td>Glu</td><td>lys</td><td colspan="2">Gly gly</td><td>lys</td><td>phe</td><td>Thr</td>

<td></td><td></td><td></td><td></td><td> 405</td><td></td><td></td><td></td><td></td><td> 410</td><td></td><td></td><td></td><td></td><td> 415</td><td></td>

<td>val</td><td>Arg</td><td>Gly</td><td>lys</td><td>Pro</td><td>Thr</td><td>Leu</td><td>Glu</td><td>asp</td><td>Leu</td><td>Glu</td><td>Gin</td><td>phe</td><td>Cheese</td><td>Glu</td><td>lys</td>

<td></td><td></td><td></td><td> 420</td><td></td><td></td><td></td><td></td><td> 425</td><td></td><td></td><td></td><td></td><td> 430</td><td></td><td></td>

<td>phe</td><td>Tyr</td><td>Cys</td><td>Cheese</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Glu</td><td>lys</td><td>ala</td><td>asp</td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td>

<td>val</td><td>lys</td><td>asp</td><td>Thr</td><td>asp</td><td>ala</td><td>val</td><td>asp</td><td>val</td><td>Cys</td><td>How much</td><td>ala</td><td>asp</td><td>Gly</td><td>val</td><td>Cys</td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

<td>How much</td><td>asp</td><td>ala</td><td>phe</td><td>Leu</td><td>lys</td><td>Pro</td><td>Pro</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Pro</td><td>Gin</td><td>How much</td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

<td>phe</td><td>Tyr</td><td>own</td><td>ala</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>ala</td><td>Thr</td><td>Underworld</td><td>phe</td><td>How much</td><td>val</td>

<td></td><td></td><td></td><td></td><td>4B5</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td> 495</td><td></td>

<td>Cheese</td><td>How much</td><td>Leu</td><td>phe</td><td>Leu</td><td>How much</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>val</td><td>ala</td><td>Cheese</td><td>Leu</td><td></td><td></td><td></td>

500 505 &lt;210&gt; 8 &lt;211&gt; 34 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer with a BamHI enzyme cleavage at the 5 'end, for producing a GPI anchor with a deletion from position N483, whose sequence terminates at position Y482.

&lt;400&gt; 8 aattggatcc tcagtagaaa atttgaggtt cttc 34 &lt;210&gt; 9 &lt;211&gt; 33 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer with BamHI enzyme cleavage at the 5 'end, for producing a GPI anchor deleted from position Y482, whose sequence terminates at position F481 &lt;400&gt; 9 aattggatcc tcagaaaatt tgaggttctt ctg 33 &lt;210&gt; 10 &lt;211&gt; 34 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer with BamHI enzyme cleavage at the 5 'end, for producing a GPI anchor deleted from position F481 whose sequence terminates at position 1480 &lt;400&gt; 10 aattggatcc tcaaatttga ggttcttctg tctc 34 &lt;210&gt; 11 &lt;211&gt; 32 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer with BamHI enzyme cleavage at the 5 'end, for producing a GPI anchor with a deletion from position 1480, whose sequence terminates at position Q479 &lt;400&gt; 11 aattggatcc tcattgaggt tcttctgtct cc 32 &lt;210&gt; 12 &lt;211&gt; 32 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer with BamHI enzyme cleavage at the 5 'end, for producing a GPI anchor deleted from position Q479 whose sequence terminates at position P478 &lt;400&gt; 12 aattggatcc tcaaggttct tctgtctcca tg 32 &lt;210&gt; 13 &lt;211&gt; 31 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer with BamHI enzyme cleavage at the 5 'end for producing a GPI anchor deleted from position P478 whose sequence terminates at position E477 &lt;400&gt; 13 aattggatcc tcattcttct gtctccatgg g 31 &lt;210&gt; 14 &lt;211&gt; 32 &lt; 212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Sense primer with NheI enzyme cleavage at the 5 'end &lt;400&gt; 14 aattgctagc atgggagtgc taaaattcaa gc 32 &lt;210&gt; 15 &lt;211&gt; 1473 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;220&gt;

&lt;221&gt; CDS &lt;222&gt; (1) ... (1473) &lt;223&gt; sHaSeGP up to position P478 with His-tag &lt;400&gt; 15

<td colspan="4">atg gga gtg eta</td><td colspan="4">aaa ttc aag cac</td><td rowspan="2">atc How much</td><td rowspan="2">ttt phe 10</td><td colspan="3" rowspan="2">ttc aga age Phe Arg Ser</td><td colspan="3">ttt gtt aaa</td><td rowspan="2"> 48</td>

<td>Underworld 1</td><td>Gly</td><td>val</td><td>Leu</td><td>lys 5</td><td colspan="2">Phe Lys</td><td>His</td><td>phe</td><td>val 15</td><td>lys</td>

<td>tea</td><td>agt</td><td>GGA</td><td>gta</td><td>tcc</td><td>cag</td><td>ata</td><td>gtt</td><td>ttc</td><td>acc</td><td>ttc</td><td>ctt</td><td>cOT</td><td>att</td><td>ca</td><td>tgt</td><td> 96</td>

<td>Cheese</td><td>Cheese</td><td>Gly</td><td>val</td><td>Cheese</td><td>Gin</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>phe</td><td>Leu</td><td>Leu</td><td>How much</td><td>Pro</td><td>Cys</td><td></td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td><td></td>

<td>TGC</td><td>TTG</td><td>act</td><td>cOT</td><td>aat</td><td>ttc</td><td>aga</td><td>GCA</td><td>Cct</td><td>Cct</td><td>gtt</td><td>att</td><td>ca</td><td>aat</td><td>gtg</td><td>Cct</td><td> 144</td>

<td>Cys</td><td>Leu</td><td>Thr</td><td>Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ala</td><td>Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>own</td><td>val</td><td>Pro</td><td></td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td><td></td>

<td>ttc</td><td>ctc</td><td>TGG</td><td>gee</td><td>TGG</td><td>aat</td><td>gee</td><td>ca</td><td>agt</td><td>gaa</td><td>ttt</td><td>tgt</td><td>ctt</td><td>GGA</td><td>aaa</td><td>ttt</td><td> 192</td>

<td>phe</td><td>Leu</td><td>Trp</td><td>ala</td><td>Trp</td><td>own</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Cys</td><td>Leu</td><td>Gly</td><td>lys</td><td>phe</td><td></td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td><td></td>

<td>gat</td><td>gag</td><td>ca</td><td>eta</td><td>gat</td><td>atg</td><td>age</td><td>ctc</td><td>ttc</td><td>tet</td><td>ttc</td><td>ata</td><td>GGA</td><td>age</td><td>ccc</td><td>ega</td><td> 240</td>

<td>asp</td><td>Glu</td><td>Pro</td><td>Leu</td><td>asp</td><td>Underworld</td><td>Cheese</td><td>Leu</td><td>phe</td><td>Cheese</td><td>phe</td><td>How much</td><td>Gly</td><td>Cheese</td><td>Pro</td><td>Arg</td><td></td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td><td></td>

<td>ata</td><td>aac</td><td>gee</td><td>acc</td><td>ggg</td><td>caa</td><td>GGT</td><td>gtt</td><td>aca</td><td>ata</td><td>ttt</td><td>tat</td><td>gtt</td><td>gat</td><td>aga</td><td>ctt</td><td> 288</td>

<td>How much</td><td>own</td><td>ala</td><td>Thr</td><td>Gly</td><td>Gin</td><td>Gly</td><td>val</td><td>Thr</td><td>How much</td><td>phe</td><td>Tyr</td><td>val</td><td>asp</td><td>Arg</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td><td></td>

<td>GGC</td><td>trays</td><td>tat</td><td>Cct</td><td>trays</td><td>ata</td><td>gat</td><td>tea</td><td>atc</td><td>aca</td><td>GGA</td><td>gta</td><td>act</td><td>gtg</td><td>aat</td><td>GGA</td><td> 336</td>

<td>Gly</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>Tyr</td><td>How much</td><td>asp</td><td>Cheese</td><td>How much</td><td>Thr</td><td>Gly</td><td>val</td><td>Thr</td><td>val</td><td>own</td><td>Gly</td><td></td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td><td></td>

<td>GGA</td><td>atc</td><td>ccc</td><td>cag</td><td>aag</td><td>att</td><td>tcc</td><td>tta</td><td>caa</td><td>gac</td><td>cat</td><td>cOT</td><td>gac</td><td>aaa</td><td>get</td><td>aag</td><td> 384</td>

<td>Gly</td><td>How much</td><td>Pro</td><td>Gin</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>asp</td><td>His</td><td>Leu</td><td>asp</td><td>lys</td><td>ala</td><td>lys</td><td></td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td><td></td>

<td>aaa</td><td>gac</td><td>att</td><td>aca</td><td>ttt</td><td>tat</td><td>atg</td><td>ca</td><td>gta</td><td>gac</td><td>aat</td><td>TTG</td><td>GGA</td><td>atg</td><td>get</td><td>gtt</td><td> 432</td>

<td>lys</td><td>asp</td><td>How much</td><td>Thr</td><td>phe</td><td>Tyr</td><td>Underworld</td><td>Pro</td><td>val</td><td>asp</td><td>own</td><td>Leu</td><td>Gly</td><td>Underworld</td><td>ala</td><td>val</td><td></td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td><td></td>

<td>att</td><td>gac</td><td>TGG</td><td>gaa</td><td>gaa</td><td>TGG</td><td>aga</td><td>ccc</td><td>act</td><td>TGG</td><td>GCA</td><td>aga</td><td>aac</td><td>TGG</td><td>aaa</td><td>Cct</td><td> 480</td>

<td>How much</td><td>asp</td><td>Trp</td><td>Glu</td><td>Glu</td><td>Trp</td><td>Arg</td><td>Pro</td><td>Thr</td><td>Trp</td><td>ala</td><td>Arg</td><td>own</td><td>Trp</td><td>lys</td><td>Pro</td><td></td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td><td></td>

<td>aaa</td><td>gat</td><td>gtt</td><td>trays</td><td>aag</td><td>aat</td><td>agg</td><td>tet</td><td>att</td><td>gaa</td><td>TTG</td><td>gtt</td><td>cag</td><td>caa</td><td>caa</td><td>aat</td><td> 528</td>

<td>lys</td><td>asp</td><td>val</td><td>Tyr</td><td>lys</td><td>own</td><td>Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Leu</td><td>val</td><td>Gin</td><td>Gin</td><td>Gin</td><td>own</td><td></td>

<td colspan="9"> 165</td><td> 170</td><td colspan="7"> 175</td>

<td>gta</td><td>caa</td><td>ctt</td><td>agt</td><td>ctc</td><td>aca</td><td>gag</td><td>gee</td><td>act</td><td>gag</td><td>aaa</td><td>GCA</td><td>aaa</td><td>caa</td><td>gaa</td><td>ttt</td><td> 576</td>

<td>val</td><td>Gin</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Thr</td><td>Glu</td><td>ala</td><td>Thr</td><td>Glu</td><td>lys</td><td>ala</td><td>lys</td><td>Gin</td><td>Glu</td><td>phe</td><td></td>

<td></td><td></td><td></td><td> 180</td><td></td><td></td><td></td><td></td><td> 185</td><td></td><td></td><td></td><td></td><td> 190</td><td></td><td></td><td></td>

<td>gaa</td><td>aag</td><td>GCA</td><td>ggg</td><td>aag</td><td>gat</td><td>ttc</td><td>cOT</td><td>gta</td><td>g^andg</td><td>act</td><td>ata</td><td>aaa</td><td>TTG</td><td>GGA</td><td>aaa</td><td> 624</td>

<td>Glu</td><td>lys</td><td>ala</td><td>Gly</td><td>lys</td><td>asp</td><td>phe</td><td>Leu</td><td>val</td><td>Glu</td><td>Thr</td><td>How much</td><td>lys</td><td>Leu</td><td>Gly</td><td>lys</td><td></td>

<td></td><td></td><td> 195</td><td></td><td></td><td></td><td></td><td> 200</td><td></td><td></td><td></td><td></td><td> 205</td><td></td><td></td><td></td><td></td>

<td>tta</td><td>ctt</td><td>CGG</td><td>ca</td><td>aat</td><td>cac</td><td>TTG</td><td>TGG</td><td>GGT</td><td>tat</td><td>tat</td><td>ctt</td><td>ttt</td><td>ccg</td><td>gat</td><td>tgt</td><td> 672</td>

<td>Leu</td><td>Leu</td><td>Arg</td><td>Pro</td><td>own</td><td>His</td><td>Leu</td><td>Trp</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>phe</td><td>Pro</td><td>asp</td><td>Cys</td><td></td>

<td></td><td> 210</td><td></td><td></td><td></td><td></td><td> 215</td><td></td><td></td><td></td><td></td><td> 220</td><td></td><td></td><td></td><td></td><td></td>

<td>trays</td><td>aac</td><td>cat</td><td>cac</td><td>tat</td><td>aag</td><td>aaa</td><td>ccc</td><td>GGT</td><td>trays</td><td>aat</td><td>GGA</td><td>agt</td><td>TGC</td><td>ttc</td><td>aat</td><td> 720</td>

<td>Tyr</td><td>own</td><td>His</td><td>His</td><td>Tyr</td><td>lys</td><td>lys</td><td>Pro</td><td>Gly</td><td>Tyr</td><td>own</td><td>Gly</td><td>Cheese</td><td>Cys</td><td>phe</td><td>own</td><td></td>

<td> 225</td><td></td><td></td><td></td><td></td><td> 230</td><td></td><td></td><td></td><td></td><td> 235</td><td></td><td></td><td></td><td></td><td> 240</td><td></td>

<td>gta</td><td>gaa</td><td>ata</td><td>aaa</td><td>aga</td><td>aat</td><td>gat</td><td>gat</td><td>ctc</td><td>age</td><td>TGG</td><td>TTG</td><td>TGG</td><td>aat</td><td>gaa</td><td>age</td><td> 768</td>

<td>val</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>own</td><td>asp</td><td>asp</td><td>Leu</td><td>Cheese</td><td>Trp</td><td>Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>Cheese</td><td></td>

<td></td><td></td><td></td><td></td><td> 245</td><td></td><td></td><td></td><td></td><td> 250</td><td></td><td></td><td></td><td></td><td> 255</td><td></td><td></td>

<td>act</td><td>GCT</td><td>ctt</td><td>trays</td><td>ca</td><td>tcc</td><td>att</td><td>tat</td><td>TTG</td><td>aac</td><td>act</td><td>cag</td><td>cag</td><td>tet</td><td>Cct</td><td>gta</td><td> 816</td>

<td>Thr</td><td>ala</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>Leu</td><td>own</td><td>Thr</td><td>Gin</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>val</td><td></td>

<td></td><td></td><td></td><td> 260</td><td></td><td></td><td></td><td></td><td> 265</td><td></td><td></td><td></td><td></td><td> 270</td><td></td><td></td><td></td>

<td>GCT</td><td>GCT</td><td>aca</td><td>ctc</td><td>tat</td><td>gtg</td><td>cgc</td><td>aat</td><td>ega</td><td>gtt</td><td>CGG</td><td>gaa</td><td>gee</td><td>atc</td><td>aga</td><td>gtt</td><td> 864</td>

<td>ala</td><td>ala</td><td>Thr</td><td>Leu</td><td>Tyr</td><td>val</td><td>Arg</td><td>own</td><td>Arg</td><td>val</td><td>Arg</td><td>Glu</td><td>ala</td><td>How much</td><td>Arg</td><td>val</td><td></td>

<td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td><td></td><td></td><td></td>

<td>tcc</td><td>aaa</td><td>ata</td><td>Cct</td><td>gat</td><td>GCA</td><td>aaa</td><td>agt</td><td>ca</td><td>ctt</td><td>ccg</td><td>gtt</td><td>ttt</td><td>GCA</td><td>tat</td><td>acc</td><td> 912</td>

<td>Cheese</td><td>lys</td><td>How much</td><td>Pro</td><td>asp</td><td>ala</td><td>lys</td><td>Cheese</td><td>Pro</td><td>Leu</td><td>Pro</td><td>val</td><td>phe</td><td>ala</td><td>Tyr</td><td>Thr</td><td></td>

<td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td><td></td><td></td><td></td>

<td>cgc</td><td>ata</td><td>gtt</td><td>ttt</td><td>act</td><td>gat</td><td>caa</td><td>gtt</td><td>TTG</td><td>aaa</td><td>ttc</td><td>ctt</td><td>tet</td><td>caa</td><td>gat</td><td>gaa</td><td> 960</td>

<td>Arg</td><td>How much</td><td>val</td><td>phe</td><td>Thr</td><td>asp</td><td>Gin</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>Leu</td><td>Cheese</td><td>Gin</td><td>asp</td><td>Glu</td><td></td>

<td> 305</td><td></td><td></td><td></td><td></td><td> 310</td><td></td><td></td><td></td><td></td><td> 315</td><td></td><td></td><td></td><td></td><td> 320</td><td></td>

<td>ctt</td><td>gtg</td><td>tat</td><td>aca</td><td>ttt</td><td>GGC</td><td>gaa</td><td>act</td><td>gtt</td><td>GCT</td><td>cOT</td><td>GGT</td><td>GCT</td><td>tet</td><td>GGA</td><td>att</td><td> 1008</td>

<td>Leu</td><td>val</td><td>Tyr</td><td>Thr</td><td>phe</td><td>Gly</td><td>Glu</td><td>Thr</td><td>val</td><td>ala</td><td>Leu</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Gly</td><td>How much</td><td></td>

<td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td><td> 335</td><td></td><td></td>

<td>gta</td><td>ata</td><td>TGG</td><td>GGA</td><td>acc</td><td>ctc</td><td>agt</td><td>ata</td><td>atg</td><td>ega</td><td>agt</td><td>atg</td><td>aaa</td><td>tet</td><td>TGC</td><td>TTG</td><td> 1056</td>

<td>val</td><td>How much</td><td>Trp</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>Arg</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>Cys</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td> 350</td><td></td><td></td><td></td>

<td>ctc</td><td>eta</td><td>gac</td><td>aat</td><td>trays</td><td>atg</td><td>gag</td><td>act</td><td>ata</td><td>cOT</td><td>aat</td><td>Cct</td><td>trays</td><td>ata</td><td>atc</td><td>aac</td><td> 1104</td>

<td>Leu</td><td>Leu</td><td>asp</td><td>own</td><td>Tyr</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>How much</td><td>Leu</td><td>own</td><td>Pro</td><td>Tyr</td><td>How much</td><td>How much</td><td>own</td><td></td>

<td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td><td></td><td></td><td></td>

<td>gtc</td><td>aca</td><td>eta</td><td>GCA</td><td>gee</td><td>aaa</td><td>atg</td><td>tgt</td><td>age</td><td>caa</td><td>gtg</td><td>ctt</td><td>TGC</td><td>cag</td><td>gag</td><td>caa</td><td> 1152</td>

<td>val</td><td>Thr</td><td>Leu</td><td>ala</td><td>ala</td><td>lys</td><td>Underworld</td><td>Cys</td><td>Cheese</td><td>Gin</td><td>val</td><td>Leu</td><td>Cys</td><td>Gin</td><td>Glu</td><td>Gin</td><td></td>

<td></td><td> 370</td><td></td><td></td><td></td><td></td><td> 375</td><td></td><td></td><td></td><td></td><td> 380</td><td></td><td></td><td></td><td></td><td></td>

<td>GGA</td><td>gtg</td><td>tgt</td><td>ata</td><td>agg</td><td>aaa</td><td>aac</td><td>TGG</td><td>aat</td><td>tea</td><td>agt</td><td>gac</td><td>tat</td><td>ctt</td><td>cac</td><td>ctc</td><td> 1200</td>

<td>Gly</td><td>val</td><td>Cys</td><td>How much</td><td>Arg</td><td>lys</td><td>own</td><td>Trp</td><td>own</td><td>Cheese</td><td>Cheese</td><td>asp</td><td>Tyr</td><td>Leu</td><td>His</td><td>Leu</td><td></td>

<td> 385</td><td></td><td></td><td></td><td></td><td> 390</td><td></td><td></td><td></td><td></td><td> 395</td><td></td><td></td><td></td><td></td><td> 400</td><td></td>

<td>aac</td><td>ca</td><td>gat</td><td>aat</td><td>ttt</td><td>GCT</td><td>att</td><td>caa</td><td>ctt</td><td>gag</td><td>aaa</td><td>GGT</td><td>GGA</td><td>aag</td><td>ttc</td><td>aca</td><td> 1248</td>

<td>own</td><td>Pro</td><td>asp</td><td>own</td><td>phe</td><td>ala</td><td>How much</td><td>Gin</td><td>Leu</td><td>Glu</td><td>lys</td><td>Gly</td><td>Gly</td><td>lys</td><td>phe</td><td>Thr</td><td></td>

405 410 415

<td rowspan="2">gta val</td><td colspan="3">cgt gga aaa</td><td colspan="12">ccg aca ctt gaa gac ctg gag caa ttt tet gaa aaa</td><td rowspan="2"> 1296</td>

<td>Arg</td><td>Gly</td><td>lys 420</td><td>Pro</td><td>Thr</td><td colspan="2">Leu Glu</td><td>asp 425</td><td colspan="2">Leu Glu</td><td>Gin</td><td colspan="3">Phe Cheese Glu 430</td><td>lys</td>

<td>ttt</td><td>tat</td><td>TGC</td><td>agc</td><td>tgt</td><td>Lati</td><td>agc</td><td>acc</td><td>TTG</td><td>agt</td><td>tgt</td><td>aag</td><td>gag</td><td>aaa</td><td>GCT</td><td>gat</td><td> 1344</td>

<td>phe</td><td>Tyr</td><td>Cys</td><td>Cheese</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Glu</td><td>lys</td><td>ala</td><td>asp</td><td></td>

<td></td><td></td><td> 435</td><td></td><td></td><td></td><td></td><td> 440</td><td></td><td></td><td></td><td></td><td> 445</td><td></td><td></td><td></td><td></td>

<td>gta</td><td>aaa</td><td>gac</td><td>act</td><td>gat</td><td>GCT</td><td>gtt</td><td>gat</td><td>gtg</td><td>tgt</td><td>att</td><td>GCT</td><td>gat</td><td>GGT</td><td>gtc</td><td>tgt:</td><td> 1392</td>

<td>val</td><td>lys</td><td>asp</td><td>Thr</td><td>asp</td><td>ALa</td><td>val</td><td>asp</td><td>val</td><td>Cys</td><td>He</td><td>Jaa</td><td>asp</td><td>Gly</td><td>val</td><td>Cys</td><td></td>

<td></td><td> 450</td><td></td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td><td></td>

<td>ata</td><td>gat</td><td>GCT</td><td>ttt</td><td>eta</td><td>aaa</td><td>Cct</td><td>ccc</td><td>atg</td><td>gag</td><td>aca</td><td>gaa</td><td>gaa</td><td>Cct</td><td>GGA</td><td>tcc</td><td> 1440</td>

<td>How much</td><td>asp</td><td>ala</td><td>phe</td><td>Leu</td><td>lys</td><td>Pro</td><td>Pro</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>Glu</td><td>Glu</td><td>Pro</td><td>Gly</td><td>Cheese</td><td></td>

<td> 465</td><td></td><td></td><td></td><td></td><td> 470</td><td></td><td></td><td></td><td></td><td> 475</td><td></td><td></td><td></td><td></td><td> 480</td><td></td>

<td>GGT</td><td>tet</td><td>GGT</td><td>GCT</td><td>cac</td><td>cat</td><td>cac</td><td>cat</td><td>cac</td><td>cat</td><td>taa</td><td></td><td></td><td></td><td></td><td></td><td> 1473</td>

<td>Gly</td><td>Cheese</td><td>Gly</td><td>ala</td><td>His</td><td>His</td><td>His</td><td>His</td><td>His</td><td>His</td><td> *</td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td> 485</td><td></td><td></td><td></td><td></td><td> 490</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 16 &lt;211&gt; 490 &lt;212&gt; PRT &lt;213&gt; Homo sapiens &lt;400&gt; 16

<td>Underworld</td><td>Gly</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>lys</td><td>His</td><td>How much</td><td>phe</td><td>phe</td><td>Arg</td><td>Cheese</td><td>phe</td><td>val</td><td>lys</td>

<td>1 Cheese</td><td>Cheese</td><td>Gly</td><td>val</td><td>5 Cheese</td><td>Gin</td><td>How much</td><td>val</td><td>phe</td><td>10 Thr</td><td>phe</td><td>Leu</td><td>Leu</td><td>How much</td><td>15 Pro</td><td>ss</td>

<td>Cys</td><td>Leu</td><td>Thr</td><td>twenty Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ALa</td><td>25 Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>thirty own</td><td>val</td><td>Pro</td>

<td>phe</td><td>Leu</td><td>35 Trp</td><td>ala</td><td>Trp</td><td>own</td><td>ala</td><td>40 Pro</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Cys</td><td>45 Leu</td><td>Gly</td><td>lys</td><td>phe</td>

<td>asp</td><td>50 hu</td><td>Pro</td><td>Leu</td><td>asp</td><td>Underworld</td><td>55 Cheese</td><td>Leu</td><td>phe</td><td>Cheese</td><td>phe</td><td>60 How much</td><td>Gly</td><td>Cheese</td><td>Pro</td><td>Arg</td>

<td>65 How much</td><td>own</td><td>ALa</td><td>Thr</td><td>Gly</td><td>70 Gin</td><td>Gly</td><td>val</td><td>Thr</td><td>How much</td><td>75 phe</td><td>Tyr</td><td>val</td><td>asp</td><td>Arg</td><td>80 Leu</td>

<td>Gly</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>85 Tyr</td><td>How much</td><td>asp</td><td>Cheese</td><td>How much</td><td>90 Thr</td><td>Gly</td><td>val</td><td>Thr</td><td>val</td><td>95 own</td><td>Gly</td>

<td>Gly</td><td>How much</td><td>Pro</td><td>100 Gin</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>105 GLn</td><td>asp</td><td>His</td><td>Leu</td><td>asp</td><td>110 lys</td><td>ala</td><td>lys</td>

<td>lys</td><td>asp</td><td>115 How much</td><td>Thr</td><td>phe</td><td>Tyr</td><td>Underworld</td><td>120 Pro</td><td>val</td><td>asp</td><td>own</td><td>Leu</td><td>125 Gly</td><td>Underworld</td><td>ala</td><td>val</td>

<td>How much</td><td>130 asp</td><td>Trp</td><td>Glu</td><td>Glu</td><td>Trp</td><td>135 Arg</td><td>Pro</td><td>Thr</td><td>T-rp</td><td>ala</td><td>140 Arg</td><td>own</td><td>Trp</td><td>lys</td><td>Pro</td>

<td>145 lys</td><td>asp</td><td>val</td><td>Tyr</td><td>lys</td><td>150 own</td><td>Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>155 Leu</td><td>val</td><td>GLn</td><td>Gin</td><td>Gin</td><td>160 own</td>

<td>val</td><td>Gin</td><td>Leu</td><td>Cheese</td><td>165 Leu</td><td>Thr</td><td>Glu</td><td>ala</td><td>Thr</td><td>170 Glu</td><td>lys</td><td>ALa</td><td>lys</td><td>GLn</td><td>175 Glu</td><td>phe</td>

<td>Glu</td><td>lys</td><td>ala</td><td>180 Gly</td><td>lys</td><td>asp</td><td>phe</td><td>Leu</td><td>185 val</td><td>Glu</td><td>Thr</td><td>How much</td><td>lys</td><td>190 Leu</td><td>Gly</td><td>lys</td>

<td>Leu</td><td>Leu</td><td>195 Arg</td><td>Pro</td><td>own</td><td>His</td><td>Leu</td><td>200 Trp</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>205 phe</td><td>Pro</td><td>asp</td><td>Cys</td>

<td>Tyr</td><td>210 own</td><td>His</td><td>His</td><td>Tyr</td><td>lys</td><td>215 lys</td><td>Pro</td><td>Gly</td><td>T} yr</td><td>own</td><td>220 Gly</td><td>Cheese</td><td>Cys</td><td>phe</td><td>Abb</td>

<td>225 val</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>230 ABn</td><td>Archbishop</td><td>asp</td><td>Leu</td><td>Cheese</td><td>235 Trp</td><td>Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>240 Cheese</td>

<td>Thr</td><td>ala</td><td>Leu</td><td>Tyr</td><td>245 Pro</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>Leu</td><td>250 own</td><td>Thr</td><td>Gin</td><td>GLn</td><td>Cheese</td><td>255 Pro</td><td>val</td>

<td>ala</td><td>ala</td><td>Thr</td><td>260 Leu</td><td>Tyr</td>

<td>Cheese</td><td>lys</td><td>275 How much</td><td>Pro</td><td>asp</td>

<td>Arg</td><td>290 How much</td><td>Vdl</td><td>phe</td><td>tir</td>

<td>305 Leu</td><td>val</td><td>• yyr</td><td>TLR</td><td>phe</td>

<td>val</td><td>How much</td><td>Trp</td><td>Gly</td><td>325 Thr</td>

<td>Leu</td><td>Leu</td><td>asp</td><td>340 own</td><td>Tyr</td>

<td>val</td><td>Thr</td><td>355 Leu</td><td>ala</td><td>ala</td>

<td>Gly</td><td>370 val</td><td>Qys</td><td>How much</td><td>turg</td>

<td>385 own</td><td>Pro</td><td>asp</td><td>own</td><td>phe</td>

<td>val</td><td>Arg</td><td>Gly</td><td>lys</td><td>405 Pro</td>

<td>phe</td><td>Tyr</td><td>Cys</td><td>420 Cheese</td><td>Cys</td>

<td>val</td><td>lys</td><td>435 asp</td><td>Thr</td><td>asp</td>

<td>How much</td><td>450 asp</td><td>ala</td><td>phe</td><td>Leu</td>

<td>465 Gly</td><td>Cheese</td><td>Gly</td><td>ala</td><td>His</td>

<td></td><td></td><td></td><td></td><td> 485</td>

<td>val</td><td>Arg</td><td>own</td><td>265 Arg</td><td>val</td>

<td>ala</td><td>lys</td><td>280 Cheese</td><td>Pro</td><td>Leu</td>

<td></td><td>295 Gin</td><td>val</td><td>Leu</td><td>lys</td>

<td>310 Gly</td><td>Glu</td><td>Thr</td><td>val</td><td>ALa</td>

<td>Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>330 Arg</td>

<td>Underworld</td><td>Glu</td><td>Thr</td><td>345 How much</td><td>Leu</td>

<td>lys</td><td>Underworld</td><td>360 Cys</td><td>Cheese</td><td>Gin</td>

<td>lys</td><td>375 own</td><td>Trp</td><td>own</td><td>Cheese</td>

<td>390 ala</td><td>How much</td><td>Gin</td><td>Leu</td><td>Glu</td>

<td>Thr</td><td>Leu</td><td>Glu</td><td>asp</td><td>410 Leu</td>

<td>Tyr</td><td>Cheese</td><td>Thr</td><td>425 Leu</td><td>Cheese</td>

<td>ala</td><td>val</td><td>440 asp</td><td>val</td><td>Cys</td>

<td>lys</td><td>455 Pro</td><td>Pro</td><td>Underworld</td><td>Glu</td>

<td>'470 His'</td><td>His</td><td>His</td><td>His</td><td>His</td>

490

<td>Arg</td><td>Glu</td><td>ala</td><td>270 How much</td><td>Arg</td><td>val</td>

<td>Pro</td><td>val</td><td>285 phe</td><td>ala</td><td>Tyr</td><td>Thr</td>

<td>PPe</td><td>300 Leu</td><td>Cheese</td><td>Gin</td><td>asp</td><td>Glu</td>

<td>311 Leu</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Gly</td><td>320 How much</td>

<td>Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>335 Cys</td><td>Leu</td>

<td>own</td><td>Pro</td><td>Tyr</td><td>350 How much</td><td>How much</td><td>own</td>

<td>val</td><td>Leu</td><td>365 Cys</td><td>Gin</td><td>Glu</td><td>Gin</td>

<td>Cheese</td><td>380 asp</td><td>Tyr:</td><td>Leu</td><td>His</td><td>Leu</td>

<td>339 ' lys</td><td>Gly</td><td>Gly</td><td>lys</td><td>phe</td><td>400 Thr</td>

<td>Glu</td><td>Gin</td><td>phe</td><td>Cheese</td><td>415 Glu</td><td>lys</td>

<td>Cys</td><td>lys</td><td>Glu</td><td>430 lys</td><td>ala</td><td>asp</td>

<td>How much</td><td>Ala ·</td><td>445 asp</td><td>Gly</td><td>val</td><td>Cys</td>

<td>Thr</td><td>460 Glu</td><td>Glu</td><td>Pro</td><td>Gly</td><td>Cheese</td>

<td> 475</td><td></td><td></td><td></td><td></td><td> 480</td>

&lt;210&gt; 17 &lt;211&gt; 39 &lt;212 DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Sense primer with His &lt;400&gt; linker sequence 17 ataattggat ccggttctgg tgctcaccat caccatcac 39 &lt;210&gt; 18 &lt;211&gt; 38 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer with His &lt;400&gt; linker 18 tataattgcg gccgcctaat ggtgatggtg atggtgag 38 &lt;210&gt; 19 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; 3 'Antisense primer without stop codon for production of the truncated HISsHASEGP product without GPI anchor whose sequence terminates at position N483 &lt;400&gt; 19 aatggatcca ttgtagaaaa tttgaggttc 30 &lt;210&gt; 20 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; 3 'Antisense primer without stop codon, to generate a truncated HIS-sHASEGP product without GPI anchor whose sequence terminates at position Y482 &lt;400&gt; 20 aatggatccg tagaaaattt gaggttcttc 30 &lt;210&gt; 21 &lt;211&gt; 30 &lt;212&gt; DNA &lt; 213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; 3 'Antisense primer without stop codon to generate the kkroonoo HIS-sHASEGP product without GPI anchor whose sequence terminates at position F481 &lt;400&gt; 21 aattggatcc gaaaatttga ggttcttctg 30 &lt;210&gt; 22 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213 &gt; Artificial sequence &lt;220&gt;

&lt;223&gt; 3 'Antisense primer without stop codon to generate the terminal project

HIS-sHASEGP without GPI anchor, whose sequence terminates at position I480 &lt;400&gt; 22 attggatcca atttgaggtt cttctgtctc. 30 &lt;210&gt; 23 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; 3 'Antisense primer without stop codon, to generate a truncated HIS-sHASEGP product without GPI anchor whose sequence terminates at position Q479 &lt;400&gt; 23 aattggatcc ttgaggttct tctgtctcc 29 &lt;210&gt; 24 &lt;211&gt; 29 &lt;212&gt; DNA &lt; 213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; 3 'Antisense primer without stop codon, to generate a truncated HIS-sHASEGP product without a GPI anchor whose sequence terminates at position P478 &lt;400&gt; 24 aattggatcc aggttcttct gtctccatg 29 &lt;210&gt; 25 &lt;211&gt; 28 &lt;212&gt; DNA &lt; 213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; 3 'Antisense primer without stop codon, to generate a truncated HIS-sHASEGP product without GPI anchor whose sequence terminates at position E477 &lt;400&gt; 25 aattggatcc ttcttctgtc tccatggg 28 &lt;210&gt; 26 &lt;211&gt; 36 &lt;212&gt; DNA &lt; 213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer of the sHASEGP deletion mutant whose sequence terminates at position A467 &lt;400&gt; 26 aattggatcc ctaagcatct atacagacac catcag 36 &lt;210&gt; 27 &lt;211&gt; 35 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer of the sHASEGP deletion mutant, whose sequence ends at position A447 &lt;400&gt; 27 aattggatcc ctaagctttc tccttacaac tcaag 35 &lt;210&gt; 28 &lt;211&gt; 34 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer of the sHASEGP deletion mutant, whose sequence ends at position S430 &lt;400&gt; 28 aattggatcc ctaagaaaat tgctccaggt cttc 34 &lt;210&gt; 29 &lt;211&gt; 36 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer of the sHASEGP deletion mutant, whose sequence ends at position G413 &lt;400&gt; 29 aattggatcc ctatccacct ttctcaagtt gaatag 36 &lt;210&gt; 30 &lt;211&gt; 36 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer of the sHASEGP deletion mutant whose sequence terminates at position S394 &lt;400&gt; 30 aattggatcc ctatgaattc cagtttttcc ttatac 36 &lt;210&gt; 31 &lt;211&gt; 35 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer of the sHASEGP deletion mutant, whose sequence ends at position A372 &lt;400&gt; 31 aattggatcc ctatgctagt gtgacgttga ttatg 35 &lt;210&gt; 32 &lt;211&gt; 33 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer of the sHASEGP deletion mutant, whose sequence ends at position S347 &lt;400&gt; 32 aattggatcc ctaacttcgc attatactga ggg 33 &lt;210&gt; 33 &lt;211&gt; 29 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; The sense primer LN used in site-directed mutagenesis to produce the sHASEGP fusion protein with the Kappa leader sequence. L36 is the first sHASEGP amino acid following the Kappa leader sequence.

&lt;400&gt; 33 ctgaatttca gagcacctcc tgttattcc 29 &lt;210&gt; 34 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; The sense primer FR used in site-directed mutagenesis to produce the sHASEGP fusion protein with the Kappa leader sequence. F38 is the first sHASEGP amino acid following the Kappa leader sequence &lt;400&gt; 34 ttcagagcac ctcctgttat tccaaatg 28 &lt;210&gt; 35 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Asp antisense primer used in site directed mutagenesis to produce sHASEGP fusion protein with Kappa leader sequence. Asp is the last amino acid of the Kappa leader sequence before L36 or F38 in the sequence PH-20 &lt;400&gt; 35 gtcaccagtg gaacctggaa ccc 23 &lt;210&gt; 36 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; The Gly antisense primer used in site-directed mutagenesis to produce the sHASEGP fusion protein with the Kappa leader sequence. Gly is the last amino acid of the Kappa leader sequence before L36 or F38 in the PH-20 &lt;400&gt; sequence 36 accagtggaa cctggaaccc agag 24 &lt;210&gt; 37 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Primer sensible for the first fragment of the Kappa leader sequence &lt;400&gt; 37 gagacagaca cactcctgct atgggtactg 30 &lt;210&gt; 38 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer for the first fragment of the Kapaa &lt;400&gt; 38 cccagagcag cagtacccat agcaggagtg 30 &lt;210&gt; 39 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Primer sensible for the second fragment of the Kappa leader sequence &lt;400&gt; 39 ggtactgctg ctctgggttc caggttccac 30 &lt;210&gt; 40 &lt;211&gt; 27 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer for the second fragment of the Kappa leader sequence &lt;400&gt; 40 gcgtcaccag tggaacctgg aacccag 27 &lt;210&gt; 41 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Sense primer with NheI restriction site for Kappa leader sequence &lt;400&gt; 41 attgctagca tggagacaga cacactcctg 30 &lt;210&gt; 42 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; EcoR1 antisense primer for Kappa leader sequence &lt;400&gt; 42 aattgaattc gtcaccagtg gaacctgg 28 &lt;210&gt; 43 &lt;211&gt; 21 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; mouse IgK chain leader sequence &lt;400&gt; 43

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Leu Trp Val Pro 15 10 15

Gly Ser Thr Gly Asp 20 &lt;210&gt; 44 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Primer sensible for Kappa leader sequence with SpeI &lt;400&gt; 44 actcactagt gctagcatgg agacagacac 30 &lt;210&gt; 45 &lt;211&gt; 30 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Antisense primer for Kappa leader sequence with MluI &lt;400&gt; restriction site 45 aattacgcgt gaattcgtca ccagtggaac 30 &lt;210&gt; 46 &lt;211&gt; 462 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Kappa fusion sequence fusion protein with sHASEGP with F38 as the first amino acid of the likely secretion form sHASEGP (up to P478) &lt;400&gt; 46

<td>Underworld</td><td>Glu</td><td>Thr</td><td>asp</td><td>Thr</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Trp</td><td>val</td><td>Leu</td><td>Leu</td><td>Leu</td><td>Trp</td><td>val</td><td>Pro</td>

<td>1 Gly</td><td>Cheese</td><td>Thr</td><td>Gly</td><td>5 asp</td><td>phe</td><td>Arg</td><td>ala</td><td>Pro</td><td>10 Pro</td><td>val</td><td>How much</td><td>Pro</td><td>own</td><td>15 val</td><td>Pro</td>

<td>phe</td><td>Leu</td><td>Trp</td><td>twenty ala</td><td>Trp</td><td>own</td><td>ala</td><td>Pro</td><td>25 Cheese</td><td>Glu</td><td>phe</td><td>Cya</td><td>Leu</td><td>thirty Gly</td><td>lys</td><td>phe</td>

<td>asp</td><td>Glu</td><td>35 Pro</td><td>Leu</td><td>asp</td><td>Underworld</td><td>Cheese</td><td>40 Leu</td><td>phe</td><td>Cheese</td><td>phe</td><td>He</td><td>45 Gly</td><td>Cheese</td><td>Pro</td><td>Arg</td>

<td>How much</td><td>50 own</td><td>ala</td><td>Thr</td><td>Gly</td><td>Gin</td><td>55 Gly</td><td>val</td><td>Thr</td><td>How much</td><td>phe</td><td>60 Tyr</td><td>val</td><td>asp</td><td>Arg</td><td>Leu</td>

<td>65 Gly</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>You r</td><td>70 How much</td><td>asp</td><td>Cheese</td><td>How much</td><td>Thr</td><td>75 Gly</td><td>val</td><td>Thr</td><td>val</td><td>own</td><td>80 Gly</td>

<td>Gly</td><td>How much</td><td>Pro</td><td>Gin</td><td>85 lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>90 asp</td><td>His</td><td>Leu</td><td>asp</td><td>lys</td><td>95 ala</td><td>lys</td>

<td>lys</td><td>asp</td><td>How much</td><td>100 Thr</td><td>phe</td><td>Tyr</td><td>Underworld</td><td>Pro</td><td>105 val</td><td>asp</td><td>own</td><td>Leu</td><td>Gly</td><td>110 Underworld</td><td>ala</td><td>val</td>

<td>How much</td><td>asp</td><td>115 Trp</td><td>Glu</td><td>Glu</td><td>Trp</td><td>Arg</td><td>120 Pro</td><td>Thr</td><td>Trp</td><td>ala</td><td>Arg</td><td>125 own</td><td>Trp</td><td>lys</td><td>Pro</td>

<td>lys</td><td>130 asp</td><td>val</td><td>Tyr</td><td>lys</td><td>own</td><td>135 Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>Leu</td><td>14 0 Val</td><td>Gin</td><td>Gin</td><td>Gin</td><td>own</td>

<td>145 val</td><td>Gin</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>150 Thr</td><td>Glu</td><td>ala</td><td>Thr</td><td>Glu</td><td>155 lys</td><td>ala</td><td>lys</td><td>Gin</td><td>Glu</td><td>160 phe</td>

<td>Glu</td><td>lys</td><td>ala</td><td>Gly</td><td>165 lys</td><td>asp</td><td>phe</td><td>Leu</td><td>val</td><td>170 Glu</td><td>Thr</td><td>How much</td><td>lys</td><td>Leu</td><td>175 Gly</td><td>lys</td>

<td>Leu</td><td>Leu</td><td>Arg</td><td>180 Pro</td><td>own</td><td>His</td><td>Leu</td><td>Trp</td><td>185 Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>phe</td><td>190 Pro</td><td>asp</td><td>Cys</td>

<td>Tyr</td><td>own</td><td>195 His</td><td>His</td><td>Tyr</td><td>lys</td><td>lys</td><td>200 Pro</td><td>Gly</td><td>Tyr</td><td>own</td><td>Gly</td><td>205 Cheese</td><td>Cys</td><td>phe</td><td>own</td>

<td>val</td><td>210 Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>own</td><td>215 asp</td><td>asp</td><td>Leu</td><td>Cheese</td><td>Trp</td><td>220 Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>Cheese</td>

<td>225 Thr</td><td>ala</td><td>Leu</td><td>Tyr</td><td>Pro</td><td>230 Cheese</td><td>How much</td><td>Tyr</td><td>Leu</td><td>own</td><td>235 Thr</td><td>Gin</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>240 val</td>

<td>ala</td><td>ala</td><td>Thr</td><td>Leu</td><td>245 Tyr</td><td>val</td><td>Arg</td><td>own</td><td>Arg</td><td>250 val</td><td>Arg</td><td>Glu</td><td>ala</td><td>How much</td><td>255 Arg</td><td>val</td>

<td>Cheese</td><td>lys</td><td>How much</td><td>260 Pro</td><td>asp</td><td>ala</td><td>lys</td><td>Cheese</td><td>265 Pro</td><td>Leu</td><td>Pro</td><td>val</td><td>phe</td><td>270 ala</td><td>Tyr</td><td>Thr</td>

<td>Arg</td><td>How much</td><td>275 val</td><td>phe</td><td>Thr</td><td>asp</td><td>Gin</td><td>280 val</td><td>Leu</td><td>lys</td><td>phe</td><td>Leu</td><td>285 Cheese</td><td>Gin</td><td>asp</td><td>Glu</td>

<td>Leu</td><td>290 val</td><td>Tyr</td><td>Thr</td><td>phe</td><td>Gly</td><td>295 Glu</td><td>Thr</td><td>val</td><td>ala</td><td>Leu</td><td>300 Gly</td><td>ala</td><td>Cheese</td><td>Gly</td><td>How much</td>

<td>305 val</td><td>How much</td><td>Trp</td><td>Gly</td><td>Thr</td><td>310 Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>Arg</td><td>315 Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>Cys</td><td>320 Leu</td>

<td>Leu</td><td>Leu</td><td>asp</td><td>own</td><td>325 Tyr</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>How much</td><td>330 Leu</td><td>own</td><td>Pro</td><td>Tyr</td><td>How much</td><td>335 How much</td><td>own</td>

<td>val</td><td>Thr</td><td>Leu</td><td>340 ala</td><td>ala</td><td>lys</td><td>Underworld</td><td>Cys</td><td>345 Cheese</td><td>Gin</td><td>val</td><td>Leu</td><td>Cys</td><td>350 Gin</td><td>Glu</td><td>Gin</td>

<td>Gly</td><td>val</td><td>355 Cys</td><td>How much</td><td>Arg</td><td>lys</td><td>own</td><td>360 Trp</td><td>own</td><td>Cheese</td><td>Cheese</td><td>asp</td><td>365 Tyr</td><td>Leu</td><td>His</td><td>Leu</td>

<td>own</td><td>370 Pro</td><td>asp</td><td>own</td><td>phe</td><td>ala</td><td>375 How much</td><td>Gin</td><td>Leu</td><td>Glu</td><td>lys</td><td>380 Gly</td><td>Gly</td><td>lys</td><td>phe</td><td>Thr</td>

<td>385 val</td><td>Arg</td><td>Gly</td><td>lys</td><td>Pro</td><td>390 Thr</td><td>Leu</td><td>Glu</td><td>asp</td><td>Leu</td><td>395 Glu</td><td>Gin</td><td>phe</td><td>Cheese</td><td>Glu</td><td>400 lys</td>

<td>phe</td><td>Tyr</td><td>Cys</td><td>Cheese</td><td>405 Cys</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>410 Cheese</td><td>Cys</td><td>lys</td><td>Glu</td><td>lys</td><td>415 ala</td><td>asp</td>

<td>val</td><td>lys</td><td>asp</td><td>420 Thr</td><td>asp</td><td>ala</td><td>val</td><td>asp</td><td>425 val</td><td>Cys</td><td>How much</td><td>ala</td><td>asp</td><td>430 Gly</td><td>val</td><td>Cys</td>

<td>How much</td><td>asp 450</td><td>435 ala</td><td>phe</td><td>Leu</td><td>lys</td><td>Pro 455</td><td>440 Pro</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>Glu 460</td><td>445 Glu</td><td>Pro</td><td></td><td></td>

&lt;210&gt; 47 &lt;211&gt; 40 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; 3 'Antisense primer with BamHI restriction site containing stop codon, for sHASEGP sequence with GPI anchor up to position L509 &lt;400&gt; 47 aattggatcc ctacagaaga aatgataaga aacaaaatac 40 &lt;210&gt; 48 &lt;211&gt; 1449 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 48 atgggagtgc tcccagatag cctcctatcta gtagagacta tttccggatt taaaattcaa ttttcacctt ttccaaatgt ttgatgagcc ccgggcaagg caatcacagg tggacaaagc ttattgactg acaagaatag ccactgagaa taaaattggg gttacaacca gcaoatgtcctcctt taagaaagac ggaagaatgg gtctattgaa agcaaaacaa aaaattactt tcactataag ttcagaagct ccatgttgct tgggcctgga agcctcttct ttttatgttg aatggaggaa attacatttfc agacccactt ttggttcagc gaatttgaaa cggccaaatc aaacccggtt ttgttaaatc tgactctgaa atgccccaag ctttcatagg atagacttgg tcccccagaa atatgccagt gggcaagaaa aacaaaatgt aggcagggaa acttgtgggg acaatggaag aagtggagta tttcagagca tgaattttgt aagcccccga ctactatcct gatttcctta agacaatttg ctggaaacct acaacttagt ggatttcctg ttattatctt ttgcttcaat

120

180

240

300

360

420

480

540

600

660

720 · "Gtagaaataa aaagaaatga tgatctcagc tggttgtgga atgaaagcac tgctctttac 780 ccatccattt atttgaacac tcagcagtct cctgtagctg ctacactcta tgtgcgcaat 840 cgagttcggg aagccatcag agtttccaaa atacctgatg caaaaagtcc acttccggtt 900 tttgcatata cccgcatagt ttttactgat caagttttga aattcctttc tcaagatgaa 960 cttgtgtata catttggcga aactgttgct ctgggtgctt ctgg &amp; attgt aatatgggga 1020 accctcagta taatgcgaag tatgaaatct tgcttgctcc tagacaatta catggagact 1080 atactgaatę cttacataat caacgtcaca ctagcagcea aaatgtgtag ccaagtgctt 1140 tgccaggagc aaggagtgtg tataaggaaa aactggaatt caagtgacta tcttcacctc 1200 aacccagata attttgctat tcaacttgag aaaggtggaa agttcacagt acgtggaaaa 1260 ccgacacttg aagacctgga gcaattttct gaaaaatttt attgcagctg ttatagcacc 1320 ttgagttgta aggagaaagc tgatgtaaaa gacactgatg ctgttgatgt gtgtattgct 1380 gatggtgtct gtatagatgc ttttctaaaa cctcccatgg agacagaaga acctcaaatt 1440 ttctactaa 1449 &lt;210&gt; 49 &lt;211&gt; 467 &lt;212&gt; PRT &lt; 213&gt; Homo sapiens &lt;400&gt; 49W

<td>Underworld</td><td>Gly</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>lys</td><td>His</td><td>How much</td><td>phe</td><td>phe</td><td>Arg</td><td>Cheese</td><td>phe</td><td>val</td><td>lys</td>

<td>1 Cheese</td><td>Cheese</td><td>Gly</td><td>val</td><td>5 Cheese</td><td>Gin</td><td>How much</td><td>val</td><td>phe</td><td>10 Thr</td><td>phe</td><td>Leu</td><td>Leu</td><td>How much</td><td>15 Pro</td><td>Cys</td>

<td>Cys</td><td>Leu</td><td>Thr</td><td>twenty Leu</td><td>own</td><td>phe</td><td>Arg</td><td>ala</td><td>25 Pro</td><td>Pro</td><td>val</td><td>How much</td><td>Pro</td><td>thirty own</td><td>val</td><td>Pro</td>

<td>phe</td><td>Leu</td><td>35 Trp</td><td>ala</td><td>Trp</td><td>own</td><td>ala</td><td>40 Pro</td><td>Cheese</td><td>Glu</td><td>phe</td><td>Cys</td><td>45 Leu</td><td>Gly</td><td>lys</td><td>phe</td>

<td>asp</td><td>50 Glu</td><td>Pro</td><td>Leu</td><td>asp</td><td>Underworld</td><td>55 Cheese</td><td>Leu</td><td>phe</td><td>Cheese</td><td>phe</td><td>60 How much</td><td>Gly</td><td>Cheese</td><td>Pro</td><td>Arg</td>

<td>65 How much</td><td>own</td><td>ala</td><td>Thr</td><td>Gly</td><td>70 Gin</td><td>Gly</td><td>val</td><td>Thr</td><td>How much</td><td>75 phe</td><td>Tyr</td><td>val</td><td colspan="2">Asp Arg</td><td>80 Leu</td>

<td>Gly</td><td>Tyr</td><td>Tyr</td><td>Pro</td><td>85 Tyr</td><td>How much</td><td>asp</td><td>Cheese</td><td>How much</td><td>90 Thr</td><td>Gly</td><td>val</td><td>Thr</td><td>val</td><td>95 own</td><td>Gly</td>

<td>Gly</td><td>How much</td><td>Pro</td><td>100 Gin</td><td>lys</td><td>How much</td><td>Cheese</td><td>Leu</td><td>105 Gin</td><td>asp</td><td>His</td><td>Leu</td><td>asp</td><td>110 lys</td><td>ala</td><td>lys</td>

<td>lys</td><td>asp</td><td>115 How much</td><td>Thr</td><td>phe</td><td>Tyr</td><td>Underworld</td><td>120 Pro</td><td>val</td><td>asp</td><td>own</td><td>Leu</td><td>125 Gly</td><td>Underworld</td><td>ala</td><td>val</td>

<td>How much</td><td>130 asp</td><td>Trp</td><td>Glu</td><td>Glu</td><td>Trp</td><td>135 Arg</td><td>Pro</td><td>Thr</td><td>Trp</td><td>ala</td><td>140 Arg</td><td>own</td><td>Trp</td><td>lys</td><td>Pro</td>

<td>145 lys</td><td>asp</td><td>val</td><td>Tyr</td><td>lys</td><td>150 own</td><td>Arg</td><td>Cheese</td><td>How much</td><td>Glu</td><td>155 Leu</td><td>val</td><td>Gin</td><td>Gin</td><td>Gin</td><td>160 own</td>

<td>val</td><td>Gin</td><td>Leu</td><td>Cheese</td><td>165 Leu</td><td>Thr</td><td>Glu</td><td>ala</td><td>Thr</td><td>170 Glu</td><td>lys</td><td>ala</td><td>lys</td><td>Gin</td><td>175 Glu</td><td>phe</td>

<td>Glu</td><td>lys</td><td>ala</td><td>180 Gly</td><td>lys</td><td>asp</td><td>phe</td><td>Leu</td><td>185 val</td><td>Glu</td><td>Thr</td><td>How much</td><td>lys</td><td>190 Leu</td><td>Gly</td><td>lys</td>

<td>Leu</td><td>Leu</td><td>195 Arg</td><td>Pro</td><td>own</td><td>His</td><td>Leu</td><td>200 Trp</td><td>Gly</td><td>Tyr</td><td>Tyr</td><td>Leu</td><td>205 phe</td><td>Pro</td><td>asp</td><td>Cys</td>

<td>Tyr</td><td>210 own</td><td>His</td><td>His</td><td>Tyr</td><td>lys</td><td>215 lys</td><td>Pro</td><td>Gly</td><td>Tyr</td><td>own</td><td>220 Gly</td><td>Cheese</td><td>Cys</td><td>phe</td><td>own</td>

<td>225 val</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>230 own</td><td>asp</td><td>asp</td><td>Leu</td><td>Cheese</td><td>235 Trp</td><td>Leu</td><td>Trp</td><td>own</td><td>Glu</td><td>240 Cheese</td>

<td>Thr</td><td>ala</td><td>Leu</td><td>Tyr</td><td>245 Pro</td><td>Cheese</td><td>How much</td><td>Tyr</td><td>Leu</td><td>250 own</td><td>Thr</td><td>Gin</td><td>Gin</td><td>Cheese</td><td>255 Pro</td><td>val</td>

<td>ala</td><td>ala</td><td>Thr</td><td>260 Leu</td><td>Tyr</td><td>val</td><td>Arg</td><td>own</td><td>265 Arg</td><td>val</td><td>Arg</td><td>Glu</td><td>ala</td><td>270 How much</td><td>Arg</td><td>val</td>

<td>Cheese</td><td>lys</td><td>275 How much</td><td>Pro</td><td>asp</td><td>ala</td><td>lys</td><td>280 Cheese</td><td>Pro</td><td>Leu</td><td>Pro</td><td>val</td><td>285 phe</td><td>ala</td><td>Tyr</td><td>Thr</td>

<td>Arg</td><td>290 How much</td><td>val</td><td>phe</td><td>Thr</td><td>asp</td><td>295 Gin</td><td>val</td><td>Leu</td><td>lys</td><td>phe</td><td>300 Leu</td><td>Cheese</td><td>Gin</td><td>asp</td><td>Glu</td>

<td>305 Leu</td><td>val</td><td>Tyr</td><td>Thr</td><td>phe</td><td>310 Gly</td><td>Glu</td><td>Thr</td><td>val</td><td>ala</td><td>315 Leu</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Gly</td><td>320 How much</td>

<td>val</td><td>How much</td><td>Trp</td><td>Gly</td><td>325 Thr</td><td>Leu</td><td>Cheese</td><td>How much</td><td>Underworld</td><td>330 Arg</td><td>Cheese</td><td>Underworld</td><td>lys</td><td>Cheese</td><td>335 Cys</td><td>Leu</td>

<td>Leu</td><td>Leu</td><td>asp</td><td>340 own</td><td>Tyr</td><td>Underworld</td><td>Glu</td><td>Thr</td><td>345 How much</td><td>Leu</td><td>own</td><td>Pro</td><td>Tyr</td><td>350 How much</td><td>How much</td><td>own</td>

<td>val</td><td>Thr</td><td>355 Leu</td><td>ala</td><td>ala</td><td>lys</td><td>Underworld</td><td>360 Cys</td><td>Cheese</td><td>Gin</td><td>val</td><td>Leu</td><td>365 Cys</td><td>Gin</td><td>Glu</td><td>Gin</td>

<td>Gly</td><td>370 val</td><td>Cys</td><td>How much</td><td>Arg</td><td>lys</td><td>375 own</td><td>Trp</td><td>own</td><td>Cheese</td><td>Cheese</td><td>380 asp</td><td>Tyr</td><td>Leu</td><td>His</td><td>Leu</td>

<td>385 own</td><td>Pro</td><td>asp</td><td>own</td><td>phe</td><td>390 ala</td><td>How much</td><td>Gin</td><td>Leu</td><td>Glu</td><td>395 lys</td><td colspan="2">Gly gly</td><td>lys</td><td>phe</td><td>400 Thr</td>

<td>val</td><td>Arg</td><td>Gly</td><td>lys</td><td>405 Pro</td><td>Thr</td><td>Leu</td><td>Glu</td><td>asp</td><td>410 Leu</td><td>Glu</td><td>Gin</td><td>phe</td><td>Cheese</td><td>415 Glu</td><td>lys</td>

<td>phe</td><td>Tyr</td><td>Cys</td><td>420 Cheese</td><td>Cys</td><td>Tyr</td><td>Cheese</td><td>Thr</td><td>425 Leu</td><td>Cheese</td><td>Cys</td><td>lys</td><td>Glu</td><td>430 lys</td><td>ala</td><td>asp</td>

<td>val</td><td>lys</td><td>435 asp</td><td>Thr</td><td>asp</td><td>ala</td><td>val</td><td>440 asp</td><td>val</td><td>Cys</td><td>How much</td><td>ala</td><td>445 asp</td><td>Gly</td><td>val</td><td>Cys</td>

<td>How much 465</td><td>450 asp</td><td>ala</td><td></td><td></td><td></td><td> 455</td><td></td><td></td><td></td><td></td><td> 460</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 50 &lt;211&gt; 1536 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 50 atgggagtgc taaaattcaa gcacatcttt ttcagaagct ttgttaaatc aagtggagta 60 tcccagatag ttttcacctt ccttctgatt ccatgttgct tgactctgaa tttcagagca 120 cctcctgtta ttccaaatgt gcctttcctc tgggcctgga atgccccaag tgaattttgt 180 cttggaaaat ttgatgagcc actagatatg agcctcttct ctttcatagg aagcccccga 240 ataaacgcca ccgggcaagg tgttacaata ttttatgttg atagacttgg ctactatcct 300 tacatagatt caatcacagg agtaactgtg aatggaggaa tcccccagaa gatttcctta 360 caagaccatc tggacaaagc taagaaagac attacatttt atatgccagt agacaatttg 420 ggaatggctg ttattgactg ggaagaatgg agacccactt gggcaagaaa ctggaaacct 480 aaagatgttt acaagaatag gtctattgaa ttggttcagc aacaaaatgt acaacttagt 540 ctcacagagg ccactgagaa agcaaaacaa gaatttgaaa aggcagggaa ggatttcctg 600 gtagagacta taaaattggg aaaattactt cggccaaatc acttgtgggg ttattatctt 660 tttccggatt gttacaacca tcactataag aaacccggtt acaatggaag ttgcttcaat 720 gtagaaataa aaagaaatga tgatctcagc tggttgtgga atgaaagcac tgctctttac 780 ccatccattt atttgaacac tcagcagtct cctgtagctg ctacactcta tgtgcgcaat 840 cgagttcggg aagccatcag agtttccaaa atacctgatg caaaaagtcc acttccggtt 900 tttgcatata cccgcatagt ttttactgat caagttttga aattcctttc tcaagatgaa 960 cttgtgtata catttggcga aactgttgct ctgggtgctt ctggaattgt aatatgggga 1020 accctcagta taatgcgaag tatgaaatct tgcttgctcc tagacaatta catggagact 1080 atactgaatc cttacataat caacgtcaca ctagcagcca aaatgtgtag ccaagtgctt 1140 tgccaggagc aaggagtgtg tataaggaaa aactggaatt caagtgacta tcttcacctc 1200 aacccagata attttgctat tcaacttgag aaaggtggaa agttcacagt acgtggaaaa 1260 ccgacacttg aagacctgga gcaattttct gaaaaatttt attgcagctg ttatagcacc 1320 ttgagttgta aggagaaagc tgatgtaaaa gacactgatg ctgttgatgt gtgtattgct 1380 gatggtgtct gtatagatgc ttttctaaaa cctcccatgg agacagaaga acctcaaatt 1440 ttctacaatg cttcaccctc cacactatct gccacaatgt tcatttggag gctggaagtc 1500 tgggatcaag gtattagcag aattggtttc ttctga 1536 &lt;210&gt; 51 &lt;211&gt; 6630 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; plasmid vector ΗΖ24 &lt;400&gt; 51 tcaatattgg ccattagcca tattattcat tggttatata gcataaatca atattggcta 60 ttggccattg catacgttgt atctatatca taatatgtac atttatattg gctcatgtcc 120 aatatgaccg ccatgttggc attgattatt gactagttat taatagtaat caattacggg 180 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc 240 gcctggctga ccgcccaacg acccccgccc attgaćgtca ataatgacgt atgttcccat 300 agtaacgcca atagggactc tccattgacg tcaatgggtg gagtatttac ggtaaactgc 360 ccacttggca gtacatcaag tgtatcatat gccaagtccg ccccctattg acgtcaatga 420 cggtaaatgg cccgcctggc attatgccca gtacttgacc ttacgggact ttcctacttg 480 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacac 540 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt 600 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaataaccc. 660 cgccccgttg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagągc 720 tcgtttagtg aaccgtcaga tcactagaag ctttattgcg gtagtttatc acagttaaat 780 tgctaacgca gtcagtgctt ctgacacaac agtctcgaac ttaagctgca gaagttggtc 840 gtgaggcact gggcaggtaa gtatcaaggt tacaagacag gtttaaggag accaatagaa 900 actgggcttg tcgagacaga gaagactctt gcgtttctga taggcaccta ttggtcttac 960 tgacatccac tttgcctttc tctccacagg tgtccactcc cagttcaatt acagctctta 1020 aggctagagt acttaatacg actcactata ggctagcatg ggagtgctaa aattcaagca 1080 catctttttc agaagctttg ttaaatcaag tggagtatcc cagatagttt tcaccttcct 1140 tctgattcca tgttgcttga ctctgaattt cagagcacct cctgttattc caaatgtgcc 1200 tttcctctgg gcctggaatg ccccaagtga attttgtctt ggaaaatttg atgagccact 1260 agatatgagc ctcttctctt tcataggaag cccccgaata aacgccaccg ggcaaggtgt 1320 tacaatattt tatgttgata gacttggcta ctatccttac atagattcaa tcacaggagt 1380 aactgtgaat ggaggaatcc cccagaagat ttccttacaa gaccatctgg acaaagctaa 1440 gaaagacatt acattttata tgccagtaga caatttggga atggctgtta ttgactggga 1500 agaatggaga cccacttggg caagaaactg gaaacctaaa gatgtttaca agaataggtc 1560 tattgaattg gttcagcaac aaaatgtaca acttagtctc acagaggcca ctgagaaagc 1620 aaaacaagaa tttgaaaagg cagggaagga tttcctggta 'gagactataa aattgggaaa 1680 attacttcgg ccaaatcact tgtggggtta ttatcttttt ccggattgtt acaaccatca 1740 ctataagaaa cccggttaca atggaagttg cttcaatgta gaaataaaaa gaaatgatga 1800 tctcagctgg ttgtggaatg aaagcactgc tctttaccca tccatttatt tgaacactca 1860 gcagtctcct gtagctgcta cactctatgt gcgcaatcga gttcgggaag ccatcagagt 1920 ttccaaaata cctgatgcaa aaagtccact tccggttttt gcatataccc gcatagtttt 1980 tactgatcaa gttttgaaat tcctttctca agatgaactt gtgtatacat ttggcgaaac 2040 tgttgctctg ggtgcttctg gaattgtaat atggggaacc ctcagtataa tgcgaagtat 2100 gaaatcttgc ttgctcctag acaattacat ggagactata ctgaatcctt acataatcaa 2160 cgtcacacta gcagccaaaa tgtgtagcca agtgctttgc caggagcaag gagtgtgtat 2220 aaggaaaaac tggaattcaa gtgactatct tcacctcaac ccagataatt ttgctattca 2280 acttgagaaa ggtggaaagt tcacagtacg tggaaaaccg acacttgaag acctggagca 2340 attttctgaa aaattttatt gcagctgtta tagcaccttg agttgtaagg agaaagctga 2400 tgtaaaagac actgatgctg fctgatgtgtg tattgctgat ggtgtctgta tagatgcttt 2460 tctaaaacct gggatagaga cagŁagaHcc tcaaattttc tagtgaaaat ccatagctaa 2520 cgcccctctc ggtgggcgcg gggtaagatt agtggccaaa agggcttaga ataaggcgag 2580 tgtacgtttg tctatatgtt attttccacc atattgggat cttttggcaa tatgaagggg 2640 gggaaaggta gcgctatgtt cttgaggaac attcctaagg g ^ t ^ cccc tctcgccsaa 2700 agaatggaag gtctgttgaa tatcatgaag gaaggaattg ctgtggaagg ttcttgaaga 2760 gaaagaagat ctataagaag ggtttggaag gaggggaacg gcccaectga caacagatgg 2820 ctcŁgcggcc aaaagggacg tgtataagat acaggtggaa aagggagaga aggcgagtgg 2880 gaggttgtga gttggatagt tataaaaaaa gtcaaataag tgtggtgaaa cgŁattcHac 2940 aaggggctaa agaatgggga aaaaataggc cattgtatgg gatgtaatgt aaaggc^tggg 3000 tgcacatgct ttacatgtgt ttaatcgaag ttaaaaaaac atgtagggcg ggggaaccag 3060 aggaaggtgg ttttggttta aaaaacacga tgataagctt gggagaacgg acagcggccg 3120 gtaccatcat gattggagca ttgaactgca tggtcgcgat gtcccaaaat atggggattg 3180 agaagaaggg agacctaccc tgacctgcag tgaggaacga attcaaatag ttggaaaaaa 3240 tgaggagaac gtgttgaata aaaaataaag agaatctggt aattataag^t aaaaaaactg 3300 agttctggat tcctgagaag aatcgacctt taaaggacag aattaatata gttctcagŁa 3360 gagaactcaa agaHccacca ggaaaaggtg attttcttgc caaaagtttg gatgatgcct 3420 taagacttat tgaagaagcg gaattggcaa gtaaagtaga catgatttga atagtcggag 3480 gcagttctgt ggatcatgca aacttctccc ataagtttga gcggccgctt aatgcagtga cattataagc tcagggggag cgataaggat gttgcgcagc gtggtggtta gctttcttcc gggctccctt tagggtgatg ttggagtcca atctcggtct aatgagctga tcctgatgcg ctctcagtac ccgctgacgc ccgtctccgg gaaagggcct agacgtcagg aaatacattc attgaaaaag cggcattttg aagatcagtt ttgagagttt gtggcgcggt attctcagaa tgacagtaag tacttctgac atcatgtaac agcgtgacac aactacttac caggaccact ccggtgagcg gtatcgtagt tcgctgagat atatacttta tttttgataa accccgtaga gcttgcaaac caactctttt tagtgtagcc ctctgctaat tggactcaag gcacacagcc tatgagaaag gggtcggaac gtcctgtcgg ggcggagcct ggccttttgc ttaccaggaa ggaatttgaa agaataccca agtctacgag cgagcagaca aaaaaatgct tgcaataaac atgtgggagg ccgggctggc ctgaatggcg cgcgcagcgt cttcctttct tagggttccg gttcacgtag cgttctttaa attcttttga tttaacaaaa gtattttctc aatctgctct gccctgacgg gagctgcatg cgtgatacgc tggcactttt aaatatgtat gaagagtatg ccttcctgtt gggtgcacga tcgccccgaa attatcccgt tgacttggtt agaattatgc aacgatcgga tcgccttgat cacgatgcct tctagcttcc tctgcgctcg tgggtctcgc tatctacacg aggtgcctca gattgattta tctcatgacc aaagatcaaa aaaaaaacca tccgaaggta gtagttaggc cctgttacca acgatagtta cagcttggag cgccacgctt aggagagcgc gtttcgccac atggaaaaac tcacatggct gccatgaatc agtgacacgt ggcgtcctct aagaaagact tgataagata ttatttgtga aagttaacaa ttttttaaag gtaatagcga aatggacgcg gaccgctaca cgccacgttc atttagtgct tgggccatcg tagtggactc tttataaggg atttaacgcg cttacgcatc gatgccgcat gcttcgzctgc tgtcagaggt ctatttttat cggggaaatg ccgctcatga agtattcaac tttgctcacc gtgggttaca gaacęctttc attgacgccg gagtactcac agtgctgcca ggaccgaagg gtagcaatgg cggcaacaat gcccttccgg ggtatcattg acggggagtc ctgattaagc aaacttcatt aaaatctctt ggatcttctt ccgctaccag actggcttca caccacttca gtggctgctg ccggataagg cgaacgacct cccgaaggga acga ^ gagc ctctgacttg gccagcaacg cgacagatct aa ^ c ^ ^ a ttttccc ^^ g cagcagccca atatacaCcc cctccctcgt atαcggtgct ccatcatCgc cctaCcataa aactccccac c ccccC ^ cc ct ^ cc ^ cg ^ c ^ Ctc tCctcgCatc c cccc ^ cc ttgCCcccca cccccc tccacacgat g ^ c ^^ ac a ^ t ^ cCtC cctcttaaca tgactαCggc gcccttctca cctCcatatc tcttaatgct aacacataac c ca ^ aac ^ ttttttcacg c cggccggcc a ^ cca ^ ta a ^ tCCca ^ t Ctccccccc ccc gcc ^ acc ^ aacttCtagC c cc ^ c ^ cc c cccaccg ^ atataacttC gcatagcagc tttccagcgg aacatcaatc ccgcc ^ c ^ ccatatgtCc aaaccgaccg gcctgaatta tcacccccac gttggaαcat tatccattcc tCcactatat ttcacttacc ^ ccc ^ c ^ tcccc ^ cccc tccaccaatc tcagtaccaa tcc ^ cc ^ ct ^ cc ^ tc ^ tccc ^ tc ^^ c ^ gc ac taccccccat tgCgcatatc ttacatαctc tctCtttcta cccc ^ ^ c ^ c ^ taa ATCC tggac ^^^^ c ^^ gtttgtgcat tgcaaccatc tcc acc gctgcctgca tcttgcCggt gccatcαggt tg ^ cc ^ c gtcatgacaa tαtgctcCct gttatCccat tttCtCgcac ttggccaggt gctctaaαat ggcatcacca ^ a ^ tcCcC gggcagcaaa tatgCgacaa gαtαaccCgc ttcgcgcagc tacc ^ cc ^ ttCgCgcaca ggggtatcag tgcaataaaC ccga ^ ^ C ^^ tC tccaaatact tcttCgtcat CaCO gCggCctaαα gcgcccaata gacagcaggc gacCccCtgc gcagααtcag the ac ^ ^ ^ ga tccCCtgcta the ccc tc ^ c ^ ^ caca ttctggcgca gcaccaaαat gaatcagCgc gacacactcα gtcgCtttat tcc tc ^ ^ c gCgcaαtcat tccccttttg taa. tc ^ cCc ^ c gCcatcagca tc ^ cc ^ ac gtacgatggg gccctccgcc

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6600

6630 &lt;210&gt; 52 &lt;211&gt; 2009 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 52 • atgtggctca cataaattca gaaagtatga tagcagtgta ggtggttagc agcacctcat 60 aaggtccttc ctagcaaggc aaagggatgc taatgactag ccaatgctct aggaagacat 120 tgagaccagc caacttcttg ccttgataac tactgaagag acattgggtg gctggatttt 180 gaaagcagac ttctggttat aggtgatgca acttgaaaaa caatcctgaa acatgaaaca 240 agaataataa tatttaaatg taacttaatc attatacctc tttatccatc aaagtgaatt 300 cattccattc cctttcatct gtgctcatac tttgcatcag atattgggta aaccaaagtg 360 tgtaggaaga aataaatgtt ttcatagtca ttactcttta caatgggagt gctaaaattc 420 aagcacatct ttttcagaag ctttgttaaa tcaagtggag tatcccagat agttttcacc 480 ttccttctga ttccatgttg cttgactctg aatttcagag cacctcctgt tattccaaat 540 gtgcctttcc tctgggcctg gaatgcccca agtgaatttt gtcttggaaa atttgatgag 600 ccactagata tgagcctctt ctctttcata ggaagccccc gaataaacgc caccgggcaa 660 ggtgttacaa tattttatgt tgatagactt ggctactatc cttacataga ttcaatcaca 720 ggagtaactg tgaatggagg aatcccccag aagatttcct tacaagacca tctggacaaa 780 gctaagaaag acattacatt ttatatgcca gtagacaatt tgggaatggc tgttattgac 840 tgggaagaat ggagacccac ttgggcaaga aactggaaac ctaaagatgt ttacaagaat 900 aggtctattg aattggttca gcaacaaaat gtacaactta gtctcacaga ggccactgag 960 aaagcaaaac aagaatttga aaaggcaggg aaggatttcc tggtagagac tataaaattg 1020 ggaaaattac ttcggccaaa tcacttgtgg ggttattatc tttttccgga ttgttacaac 1080 catcactata agaaacccgg ttacaatgga agttgcttca atgtagaaat aaaaagaaat 1140 gatgatctca gctggttgtg gaatgaaagc actgctcttt acccatccat ttatttgaac 1200 actcagcagt ctcctgtagc tgctacactc tatgtgcgca atcgagttcg ggaagccatc 1260 agagtttcca aaatacctga tgcaaaaagt ccacttccgg tttttgcata tacccgcata 1320 gtttttactg atcaagtttt gaaafctcctt tctcaagatg aacttgtgta tacatttggc 1380 gaaactgttg ctctgggtgc ttctggaatt gtaatatggg gaaccctcag tataatgcga 1440 agtatgaaat cttgcttgct cctagacaat tacatggaga ctatactgaa tccttacata 1500 atcaacgtca cactagcagc caaaatgtgt agccaagtgc tttgccagga gcaaggagtg 1560 tgtataagga aaaactggaa ttcaagtgac tatcttcacc tcaacccaga taattttgct 1620 attcaacttg agaaaggtgg aaagttcaca gtacgtggaa aaccgacact tgaagacctg 1680 gagcaatttt ctgaaaaatt ttattgcagc tgttatagca ccttgagttg taaggagaaa 1740 gctgatgtaa aagacactga tgctgttgat gtgtgtattg ctgatggtgt ctgtatagat 1800 gcttttctaa aacctcccat ggagacagaa gaacctcaaa ttttctacaa tgcttcaccc 1860 tccacactat ctgccacaat gttcattgtt agtattttgt ttcttatcat ttcttctgta 1920 gcgagtttgt aattgcgcag gttagctgaa atgaacaata tgtccatctt aaagtgtgct 1980 ttttcgacta attaaatctt tgaaaagaa 2009 &lt;210&gt; 53 &lt; 211&gt; 2395 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;400&gt; 53 atgtggctca cataaattca gaaagtatga tagcagtgta ggtggttagc agcacctcat 60 aaggtccttc ctagcaaggg atgctaatga ctagccaatg ctctaggaag acattgagac 120 cagccaactt cttgccttga taactactga agagacattg ggtggctgga ttttgaaagc 180 agacttctgg ttataggtga tgcaacttga aaaacaatcc tgaaacatga aacaagaata 240 ataatattta aatgtaactt aatcattata cctctttatc catcaaagtg aattcattcc 300 attccctttc atctgtgctc atactttgca tcagatattg ggtaaaccaa agtgtgtagg 360 aagaaataaa tgttttcata gtcattactc tttacaatgg gagtgctaaa attcaagcac 420 atctttttca gaagctttgt taaatcaagt ggagtatccc agatagtttt caccttcctt 480 ctgattccat gttgcttgac tctgaatttc agagcacctc ctgttattcc aaatgtgcct 540 ttcctctggg cctggaatgc cccaagtgaa ttttgtcttg gaaaatttga tgagccacta 600 gatatgagcc tcttctcttt cataggaagc ccccgaataa acgccaccgg gcaaggtgtt 660 acaatatttt atgttgatag acttggctac tatccttaca tagattcaat cacaggagta 720 actgtgaatg gaggaatccc ccagaagatt tccttacaag accatctgga caaagctaag 780 aaagacatta cattttatat gccagtagac aatttgggaa tggctgttat tgactgggaa 840 gaatggagac ccacttgggc aagaaactgg aaacctaaag atgtttacaa gaataggtct 900 attgaattgg ttcagcaaca aaatgtacaa cttagtctca cagaggccac tgagaaagca 960 aaacaagaat ttgaaaaggc agggaaggat ttcctggtag agactataaa attgggaaaa 1020 ttacttcggc caaatcactt gtggggttat tatctttttc cggattgtta caaccatcac 1080 tataagaaac ccggttacaa tggaagttgc ttcaatgtag aaataaaaag aaatgatgat 1140 ctcagctggt tgtggaatga aagcactgct ctttacccat ccatttattt gaacactcag 1200 cagtctcctg tagctgctac actctatgtg cgcaatcgag ttcgggaagc catcagagtt 1260 tccaaaatac ctgatgcaaa aagtccactt ccggtttttg catatacccg catagttttt 1320 actgatcaag ttttgaaatt cctttctcaa gatgaacttg tgtatacatt tggcgaaact 1380 gttgctctgg gtgcttctgg aattgtaata tggggaaccc tcagtataat gcgaagtatg 1440 aaatcttgct tgctcctagą caattacatg gagactatac tgaatcctta cataatcaac 1500 gtcacactag cagccaaaat gtgtagccaa gtgctttgcc aggagcaagg agtgtgtata 1560 aggaaaaact ggaattcaag tgactatctt cacctcaacc cagataattt tgctattcaa 1620 cttgagaaag gtggaaagtt cacagtacgt ggaaaaccga cacttgaaga cctggagcaa 1680 ttttctgaaa aattttattg cagctgttat agcaccttga gttgtaagga gaaagctgat 1740 gtaaaagaca ctgatgctgt tgatgtgtgt attgctgatg gtgtctgtat agatgctttt 1800 ctaaaacctc ccatggagac agaagaacct caaattttct acaatgcttc accctccaca 1860 ctatctgcca caatgttcat ttggaggctg gaagtctggg atcaaggtat tagcagaatt 1920 ggtttcttct gagagtcatg agggaaaaat gtgtttcagg cctcttccct tggcttacag 1980 gaaatgaaaa aaccatgact atcatcacca acatccttgg gtattaagtg cagtcactct 2040 cctagatgct gtggggagaa ggcaagttac aaagatagac cttccctcaa gataatcaga 2100 ttttcatggt attatcctta acctttttga catcatggag gctttgggaa tctgatgaag 2160 cctatcaatt ttcttccaga agatatttat ataagattat aagaaaaatt atgtacacag 2220 cttattttat tgcattggat caaaatgcca tttataaaga attatgcctt ttccatcaat 2280 tttagcatgg aaaaataatt tcaggcaata tgcttaaaaa ttgggggaag acaaaagaaa 2340 tccatatcgt gtaaataaaa ataaattttg gttttgctca aaaaaaaaaa aaaaa 2395